-# :j/pi>L f^^ C E E [I II II C c E C C E C C E E K 3S^SBSS^^^SSS^^SE Marine Biological Laboratory Library Woods Hole, Mass. .^=>#<'=s. Presented by Dr. Philip Person Feb. 26, 1962 s 3ES^^^^^^^^^^^^^83E := i-n (_) WHO nj D i_) lAI r^ LJ m LJ HAEMATIN ENZYMES I.U.B. Symposium Series Volume 19 INTERNATIONAL UNION OF BIOCHEMISTRY SYMPOSIUM SERIES Vol. 1. The Origin of Life on the Earth — A. I. Oparin ?/ a/. (Editors) Vol. 2. Enzyme Chemistry: Proceedings of the International Symposium in Tokyo- Kyoto PROCEEDINGS OF THE FOURTH INTERNATIONAL CONGRESS ON BIOCHEMISTRY VIENNA, 1958 Vol. 3. (I) Carbohydrate Chemistry of Substances of Biological Interest Vol. 4. (II) Biochemistry of Wood Vol. 5. {l\\) Biochemistry of the Central Nervous System Vol. 6. (IV) Biochemistry of Steroids Vol. 7. (V) Biochemistry of Antibiotics Vol. 8. (VI) Biochemistry of Morphogenesis Vol. 9. (VII) Biochemistry of Viruses Vol. 10. (VIII) Proteins Vol. 11. (IX) Physical Chemistry of High Polymers of Biological Interest Vol. 12. (X) Blood Clotting Factors Vol. 13. (XI) Vitamin Metabolism Vol. 14. (XII) Biochemistry of Insects Vol. 15. (XIII) Colloquia Vol.16. (XIV) Transactions of the Plenary Sessions Vol. 17. (XV) Biochemistry Vol. 18. Biochemistry of Lipids — G. Popjak (Editor) Vol. 19. Haematin Enzymes (Parts 1 and 2) — J. E. Falk, R. Lemberg and R. K. Morton Vol. 20. Report of the Commission on Enzymes, 1961 (I.U.B.) PROCEEDINGS OF THE HFTH INTERNATIONAL CONGRESS ON BIOCHEMISTRY MOSCOW, 1961 {Provisional titles) Vol. 21. (I) Biological Structure and Function at the Molecular Level Vol. 22. (II) Functional Biochemistry of Cell Structures Vol. 23. (Ill) Evolutionary Biochemistry Vol. 24. (IV) Molecular Basis of Enzyme Action and Prohibition Vol. 25. (V) Intracellular Respiration: Phosphorylating and Non-Phosphorylating Systems Vol. 26. (VI) Mechanism of Photosynthesis Vol. 27. (VII) Biosynthesis of Lipids Vol.28. (VIII) Biochemical Principles of the Food Industry Vol. 29. (IX) Transactions of the Plenary Sessions Vol. 30. (X) Abstracts of Papers and Indexes to the Volumes of the Proceedings The building of the Australian Academy of Science, Canberra, where the Symposium was held. HAEMATIN ENZYMES A SYMPOSIUM OF THE INTERNATIONAL UNION OF BIOCHEMISTRY ORGANIZED BY THE AUSTRALIAN ACADEMY OF SCIENCE CANBERRA 1959 Edited by J. E. Falk, R. Lemberg and R. K. Morton PART 1 (Pages 1 to 362) SYMPOSIUM PUBLICATIONS DIVISION PERGAMON PRESS OXFORD • LONDON • NEW YORK • PARIS 1961 PERGAMON PRESS LTD. Headington Hill Hall, Oxford 4 & 5 Fitzroy Square, London W.I PERGAMON PRESS INC. 122 East 55th Street, New York 22, N. Y. 1404 New York Avenue N.W., Washington 5 D.C. Statler Center 640, 900 Wilshire Boulevard, Los Angeles 17, California PERGAMON PRESS S.A.R.L. 24 Rue des £coles, Paris V^ PERGAMON PRESS G.m.b.H. Kaiserstrasse 75, Frankfurt am Main Copyright © 1961 Pergamon Press Ltd. LIBRARY OF CONGRESS CARD NUMBER 60-53463 Set in Monotype Times 10ll2pt and Printed in Great Britain by the Pitman Press, Bath PREFACE This volume contains the papers and discussion material presented at the Symposium on Haemotin Enzymes. It was held at Canberra between 31st August and 4th September, 1959, and was organized by the Australian Academy of Science for the International Union of Biochemistry. The Symposium was arranged for the Academy by a Committee com- prising A. H. Ennor, J. E. Falk, R. Lemberg and R. K. Morton (Convener). The Committee is grateful to several organizations, cited in the address by Dr. R. Lemberg, President of the Symposium, for financial and other assistance. The titles and addresses of participants are given on pp. xvii-xx. For convenience, titles have been omitted from the scientific communications. It is with profound regret that we record the untimely death in October, 1960, of Professor Enzo Boeri, one of the distinguished participants in the Symposium. He made many notable contributions to our knowledge of haematin enzymes and he will be remembered with admiration, respect and affection by all who were privileged to know him. R. K. Morton ^ f L ! S .*? A -^ S '" CONTENTS ^ PAGE Participants ........... xvii Presidential Address ......... xxi The Electronic Structure and Electron Transport Properties of Metal Ions Particularly in Porphyrin Complexes by L. E. Orgel 1 Discussion Terminology in Ligancl-Field Theory . . . . . .13 Spin States of Haem Compounds . . . . . . .15 Electron Transport . . . . . . . . .16 Mechanism of Oxidative Phosphorylation . . . . .18 The Role of the Metal in Porphyrin Complexes by F. P. DwYER 19 Discussion Higher Oxidation States ........ 27 Effects of Metal on Reactivity at Periphery . . . . .28 The Physico-Chemical Behaviour of Porphyrins Solubilized in Aqueous Detergent Solutions by B. Dempsey, M. B. Lowe and J. N. Phillips . . .29 Discussion Cations of Porphyrins and Their Spectra . . . . .37 The Reactions Between Metal Ions and Porphyrins by J. H. Wang and E. B. Fleischer ...... 38 Some Physical Properties and Chemical Reactions of Iron Complexes by R. J. P. Williams 41 Discussion Oxidation-Reduction Potentials of Haem Compounds . . . .53 vii 79501 via CONTENTS PAGE Spectra and Redox Potentials of Metalloporphyrins and Haemoproteins by J. E. Falk and D. D. Perrin 56 Discussion Correlations between Structure and Physical Properties. . . .71 Models for Haemoproteins ....... 74 Some New Compounds of Haems with Bases by J. E. Falk 74 Carbon Monoxide-Pyridine Complexes with Haems by J. H. Wang 76 Equilibrium Constants for Reactions of Haems with Ligands by J. N. Phillips 79 Modification of the Secondary Structure of Haemoprotein Molecules by K. Kaziro and K. Tsushima 80 Discussion The Haem-Binding Groups in Haemoproteins . . . . .94 The Nature of Haem-Binding, and the Bohr Effect by J. H. Wang and Y. N. Chiu 94 Models for Linked Ionizations in Haemoproteins by P. George, G. I. H. Hanania, D. H. Irvine and N. Wade . . .96 On the Stability of Oxyhaemoglobin by J. H. Wang 98 Discussion Oxygenation of Haemoglobin . . . . . . .102 Ferrihaemoprotein Hydroxides: A Correlation between Magnetic and Spectroscopic Properties by P. George, J. Beetlestone and J. S. Griffith . . . 105 Discussion Spin States and Spectra of Haemoproteins . . . . .139 The Electronic Origins of the Spectra by J. S. Griffith and P. George 139 Analysis and Interpretation of Absorption Spectra of Haemin Chromoproteins by D. L. Drabkin 142 Discussion Interpretation of Absorption Spectra of Haemoproteins . . .170 The Bands in the Region of 830 and 280 m/t . . . . .171 CONTENTS IX PAGE The Haem-Globin Linkage. 3. The Relationship between Molecular Structure and Physiological Activity of Haemoglobins by J. E. O'Hagan 173 Discussion Native globin . . . . . . . . .190 The Linkage of Iron and Pi otein in Haemoglobin . . . .190 Early Stages in the Metabolism of Iron by J. B. Neilands 194 The Enzymic Incorporation of Iron into Protoporphyrin by R. A Neve 207 Discussion The Formation of Metal-Porphyrin Complexes . . . . .211 Co-ordination of Divalent Metal Ions with Porphyrin Derivatives Related to Cytochrome c by J. B. Neilands . .211 Metal Incorporation in Model Systems . . . . , .214 On the Enzymic Incorporation of Iron . . . . . .215 Biosynthesis and Metabolism of Cytochrome c by D. L. Drabkin 216 The Location of Cytochromes in Escherichia coli by A. TissiERES . , 218 Discussion The Origin of the Respiratory Granules of Bacteria .... 223 On the Cytochromes of Anaerobically Cultured Yeast by Paulette Chaix 225 Discussion The Lactate Dehydrogenase of Yeast . . . . . .233 Components of the Respiratory Chain in Yeast Mitochondria . .233 On the '^ Haemoglobin' Afjsorption Bands of Yeast .... 233 X CONTENTS PAOB Irreversible Inhibition of Catalase by the 3-Amino-l: 2:4-Triazole Group of Inhibitors in the Presence of Catalase Donors by E. Margoliash and A. Schejter 236 Catalase Oxidation Mechanisms by M. E. WiNFiELD 245 Discussion Oxidation States of Haemoproteins ...... 252 Peroxide Compounds of Catalase and Peroxidase .... 254 The Nature of Catalasc-Peroxide Complex I by B. Chance 254 Studies on Problems of Cytochrome c Oxidase Assay by LuciLE Smith and Helen Conrad 260 Discussion Assay of Cytochrome c Oxidase ...... 275 Inhibition of Cytochrome c Oxidase by Cytochrome c ... 275 Interaction of Cytochrome c with Other Compounds .... 276 The EflFect of Cations on the Reactivity of Cytochrome c in Heart Muscle Preparations by R. W. Estabrook . 276 Composition of Cytochrome c Oxidase by W. W. Wainio 281 Discussion Function of Copper in Cytochrome Oxidase Preparations . . . 301 Cytochrome Oxidases of Pseudomonas aeruginosa and Ox-Heart Muscle and Their Related Respiratory Components by T. HoRio, I. Sekuzu, T. Higashi and K. Okunuki . . 302 Discussion Properties and Nomenclature of Cytochromes di and 3.^ . . .311 The Prosthetic Groups of Pseudomonas Cytochrome Oxidase by T. Horio and M. D. Kamen 314 The Reaction of Cytochrome c Oxidase with Oxygen . . . .316 The Oxygen-Reducing Equivalents of Cytochromes a and 03 by B. Chance 316 CONTENTS XI PAGE The Isolation, Purification and Properties of Haemin a by D. B. MoRELL, J. Barrett, P. Clezy and R. Lemberg . 320 Discussion Model Systems for Cytochrome Oxidase ..... 330 Absorption Spectra of Ferro- and Ferri-Compounds of Haem a by R. Lemberg ......... 330 Cryptohaem a ........ . 333 Mitochrome in Relation to Cryptohaem a .... . 334 Cytochrome Oxidase Components by M. Morrison and E. Stotz 335 The Structure of Porphyrin a, Cryptoporphyrin a and Chlorin «2 by R. Lemberg, P. Clezy and J. Barrett .... 344 Discussion The Structure of Haem ?i and Haem a.2 . ..... 358 The Structure of Porphyrin a by M. Morrison ......... 358 The Properties of Haem o^ and Cytochrome a.^ by J. Barrett and R. J. P. Williams . . . , . .360 Extractability of Ferro- and Ferricytochrome c . . . . .361 A Haemopeptide from a Tryptic Hydrolysate of Rhodospirillum rubrum Cytochrome c by S. Palpus and H. Tuppy 363 Electrometric and other Studies on Cytochromes of the C-Group by R. W. Henderson and W. A. Rawlinson .... 370 Discussion Protein Configuration and Linkage to the Prosthetic Group in Cytochrome c 382 Studies of the Haemochrome-forming Groups in Cytochrome c by E. Margoliash 382 The Amino Acid Sequence in Horse Heart Cytochrome c by E. Margoliash and R. Hill 383 Comments on the Structure of Cytochrome c . . . . . 384 Structure and General Properties of Cytochrome c . . . .385 XU CONTENTS Comparative Properties of Cytochrome c from Yeast and Heart Muscle by J. McD. Armstrong, J. H. Coates and R. K. Morton Properties of Native Cytochrome c . . . . Reactivity of Native Cytochrome c in Oxidative Phosphorylation Structure of Bacterial Cytochromes of c-Type . Structure and Redox Potentials of Cytochrome c 385 388 389 389 390 The Electron Transfer from Cytochromes to Terminal Electron Acceptors in Nitrate Respiration and Sulphate Respiration by F. Egami, M. Ishimoto and S. Taniguchi .... 392 Cytochrome Cg by J. PosTGATE 407 Discussion Nature and Properties of Cytochrome C3 . . . . . .414 Functional Aspects of Cytochrome c^ ■ • • • • .415 Evolutionary Aspects of the Sulphate-reducing Bacteria and of Cytochrome C3 416 The Atypical Haemoprotein of Purple Photosynthetic Bacteria by M. D. Kamen and R. G. Bartsch 419 Discussion The Functions of Cytochrome h^ and of Cytochrome c^** (Halotolerant Coccus) ....... 432 Nomenclature of CO-hinding Pigments ...... 432 Cytochrome o by B. Chance 433 On the Oxidase Function of RHP . . . . . . .435 Spectrophotometric Studies of Cytochromes Cooled in Liquid Nitrogen by R. W. EsTABROOK 436 Discussion 'Trapped' Steady-states by B. Chance ... 457 Low-temperature Absorption Spectra of Cytochromes in Relation to Structure . . ...... 458 CONTENTS Xlll PAGE Studies on Microsomal Cytochromes and Related Substances by C. F. SlRlTTMATTER 461 Discussion On the Nature of Cytoplasmic Pigments of Liver Cells by B. Chance 473 Possible Functions of the Cytochromes of the Endoplasmic Reticuhun of Animal Cells 476 The Significance of Eq Values of Cytochromes in Relation to Cellular Function ......... 477 The Cytochromes of Plant Tissues by W. D. Bonner, Jr . . .479 Discussion Cytochromes Ci and 63 of Particulate Components of Plants by R. K. Morton 498 The Cytochromes of Roots ....... 499 The Chemical and Enzymic Properties of Cytochrome Z^g of Bakers' Yeast by R. K. Morton, J. McD. Armstrong and C. A. Appleby . 501 Conditions for the Autoxidation of Flavocytochrome Z>2 by E. Boeri and M. Rippa 524 Kinetic Studies on the Action of Yeast Lactate Dehydrogenase by H. Hasegawa and Y. Ogura 534 Various Forms of Yeast Lactate Dehydrogenase by A. P. Nygaard 544 Studies on Bakers' Yeast Lactate Dehydrogenase by T. Horio, J. Yamashita, T. Yamanaka, M. Nozaki and K. Okunuki 552 Discussion The Problem of Cytochrome bg . ...... 558 Properties of Intact and Modified Cytochrome h.^ .... 560 XIV CONTENTS PAOB Nature of Bakers' Yeast Lactate Dehydrogenase by T. Horio 560 Nomenclature of Cytochrome h^ and Derived Proteins . . . 562 The Substrate Specificity of Cytochrome b2 . .... 563 On the Cytochrome b Components in the Respiratory Chain in Yeast . 564 The Kinetics of Reactions Catalysed by Cytochrome h^ ■ • . 565 The Kinetics of Reduction of Cytochrome b^ by B. Chance 565 The Absorption Spectrum in Relation to the Structure of Cytochrome hz 565 The Contribution of the Prosthetic Groups to the Absorption Spectrum of Cytochrome 62 by R. K. Morton 567 The Oxidation-reduction Changes in the Reaction of Lactate with Cytochrome 62 by E. Boeri and E. Cutolo ........ 568 The Function and Bonding of the Flavin Group of Cytochrome bg . . 569 The Bonding between the Flavin Group and Apoprotein of Cytochrome 62 by J. McD. Armstrong, J. H. Coates and R. K. Morton . . . 569 Possible Free Radical Formation in Flavoproteins byG.D. Ludwig 572 Autoxidation of Cytochrome h^ ...... . 573 The Role of Cytochrome b in the Respiratory Chain by E. C. Slater and J. P. Colpa-Boonstra .... 575 Discussion The Cross-over Theorem and Sites of Oxidative Phosphorylation . . 592 The Oxidation-reduction Potential of Cytochrome b . . . . 593 On the Redox Potential of Cytochrome b, the Kinetics of Reduction of Cytochrome h and the Existence of Slater' s Factor . . . 593 The Influence of Cyanide on the Reactivity of Cytochrome b . . . 596 Energy Transfer and Conservation in the Respiratory Chain by B. Chance 597 Discussion Mechanism of Oxidative Phosphorylation ..... 622 CONTENTS XV PAGE The Significance of Respiratory Chain Oxidations in Relation to Metabolic Pathways in the Cell by F. Dickens 625 Discussion On 'Additional DPN' of Incubated Mitochondria .... 636 Possible Structure of Complexes of DPN or of DPNH involved in Oxidative Phosphorylation ........ 637 Author Index .....,,... 641 Subject Index .......... 653 PARTICIPANTS Dr. C. a. Appleby Mr. J. McD. Armstrong Mr. J. Barrett Dr. N. K. Boardman Professor E. Boeri* Dr. W. D. Bonner Mme. Professor P. Chaix Professor B. Chance Dr. p. S. Clezy Dr. E. Cutolo Professor F. Dickens Professor D. L. Drabkin Professor F. P. Dwyer * Died, 1960. H.E. — VOL. I — B Biochemistry Section, Division of Plant Industry, C.S.I. R.O., Canberra, Australia. Department of Agricultural Chemistry, Waite Agricultural Research Institute, University of Adelaide, Adelaide, Australia. Institute of Medical Research, Royal North Shore Hospital, Sydney, AustraUa. Biochemistry Section, Division of Plant Industry, C.S.I.R.O., Canberra, Austraha. Institute of Human Physiology, University of Ferrara, Ferrara, Italy. Johnson Research Foundation, University of Pennsylvania, Philadelphia, U.S.A. Laboratory of Biological Chemistry, Univer- sity of Paris, Paris, France. Johnson Foundation for Medical Physics, University of Pennsylvania, Philadelphia, U.S.A. Institute of Medical Research, Royal North Shore Hospital, Sydney, Australia. Itahan Serum Research Institute, Naples, Italy. Courtauld Institute of Biochemistry, Middle- sex Hospital Medical School, University of London, London, England. Department of Biochemistry, Graduate School of Medicine, University of Pennsylvania, Philadelphia, U.S.A. The John Curtin School of Medical Research, Australian National University, Canberra, Australia. xvii PARTICIPANTS Professor F. Egami Dr. R. W. Estabrook Dr. J. E. Falk Professor P. George Dr. R, W. Henderson Dr. T. Horio Professor M. D. Kamen Professor K. Kaziro Mr. J. W. Legge Dr. R. Lemberg, Prof. a. D. (Heidelberg) Mr. W. H. Lockwood Dr. G. D. Ludwig Dr. E. Margoliash Dr. D. B. Morell Dr. M. Morrison Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Tokyo, Japan. Johnson Foundation for Medical Physics, University of Pennsylvania, Philadelphia, U.S.A. Biochemistry Section, Division of Plant Industry, C.S.I.R.O., Canberra, Austraha. John Harrison Laboratory of Chemistry, University of Pennsylvania, Philadelphia, U.S.A. Department of Biochemistry, University of Melbourne, Melbourne, Australia. Graduate Department of Biochemistry, Brandeis University, Waltham, U.S.A. Graduate Department of Biochemistry, Brandeis University, Waltham, U.S.A. Biochemical Laboratory, Nippon Medical School, Tokyo, Japan. Department of Biochemistry, University of Melbourne, Melbourne, Australia. Institute of Medical Research, Royal North Shore Hospital, Sydney, Australia. Institute of Medical Research, Royal North Shore Hospital, Sydney, Austraha. Hospital of the University of Pennsylvania, Philadelphia, U.S.A. Laboratory for the Study of Hereditary and Metabolic Disorders, College of Medicine, University of Utah, Salt Lake City, U.S.A. Institute of Medical Research, Royal North Shore Hospital, Sydney, Australia. Department of Biochemistry, School of Medicine and Dentistry, University of Rochester, Rochester, U.S.A. PARTICIPANTS Professor R. K. Morton Dr. F. J. Moss Professor J. B. Neilands Dr. R. a. Neve Dr. a. p. Nygaard Dr. Y. Ogltra Dr. J. E. O'Hagan Dr. L. E. Orgel Dr. S. Paleus Dr. D. D. Perrin Dr. J. N. Phillips Dr. J. Postgate Assoc. Professor W. A. Rawlinson Professor E. C. Slater Dr. L. Smith Dr. C. F. Strittmatter Department of Agricultural Chemistry, Waite Agricultural Research Institute, University of Adelaide, Adelaide, Australia. School of Biological Sciences, University of New South Wales, Sydney, Australia. Department of Biochemistry, University of California, Berkeley, U.S.A. Department of Biochemistry, University of California, Berkeley, U.S.A. Nutrition Institute, Blindern, Oslo, Norway. Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Tokyo, Japan. Red Cross Blood Transfusion Service, Brisbane, Austraha. University Chemical Laboratory, University of Cambridge, Cambridge, England. Biochemistry Department, Nobel Institute of Medicine, Stockholm, Sweden. Department of Medical Chemistry, John Curtin School of Medical Research, AustraHan National University, Canberra, Australia. Biochemistry Section, Division of Plant Industry, C.S.I.R.O., Canberra, Australia. Microbiological Research Establishment, Porton, Wiltshire, England. Department of Biochemistry, University of Melbourne, Melbourne, Australia. Laboratory of Physiological Chemistry, University of Amsterdam, Amsterdam, The Netherlands. Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire, U.S.A. Department of Biological Chemistry, Harvard Medical School, Boston, U.S.A. PARTICIPAhfTS Dr. a. Tissieres Dr. p. Trudinger Professor S. F. Velick Professor W. W. Wainio Professor J. H. Wang Dr. R. J. P. Williams Dr. M. E. Winfield The Biological Laboratories, Harvard Univer- sity, Cambridge, U.S.A. Biochemistry Section, Division of Plant Industry, C.S.I.R.O., Canberra, Australia. School of Medicine, Washington University, St. Louis, Missouri, U.S.A. Bureau of Biological Research and Depart- ment of Physiology and Biochemistry, Rutgers, The State University, New Brunswick, New Jersey, U.S.A. Department of Chemistry, Yale University, New Haven, U.S.A. Inorganic Chemistry Laboratory, and Wad- ham College, Oxford, England. Division of Physical Chemistry, Indus- trial Chemical Laboratories, C.S.I.R.O., Melbourne, Australia. PRESIDENTIAL ADDRESS I DECLARE open the Symposium on Haematin Enzymes of the International Union of Biochemistry and welcome all who attend it. It is a great pleasure for me to welcome the many distinguished scientists from overseas who have come to Austraha to discuss with us the problems which interest us all. I hope that the stimulation which you may receive will repay you for your long and strenuous journeys to our distant shores, that you may carry back happy memories of this week spent in Australia — and that you wiU return! Your visit will certainly be a stimulus to Australian science. I am particularly happy to receive you in this home of the Australian Academy of Science.* When we discussed plans for its erection in the Council of the Academy only a few years ago, I little dreamt that I should have the honour of opening the first International Conference in it. Our thanks are due to the International Union of Biochemistry, not only for accepting our invitation to hold the Symposium, but also for financial support; to the AustraUan Commonwealth Government; to the Wellcome Trust ; and to the various Academies and national bodies, particularly to the National Science Foundation of the United States and to the Royal Society. The support of these various organizations made this meeting possible. We decided that we wanted an intimate symposium in which all participants could be rehed upon to make valuable contributions. We wanted to discuss our problems critically, but with some degree of leisure. This is a Symposium and even if we cannot do as the Greeks did, having a meal, wine and dancers in this hall, the comfortable lounge chairs are the nearest approach to it possible in these hurried times. There you may recline for a little snooze if the richness of the intellectual feast endangers your mental digestion. Our Symposium has a pecuhar note in that it calls together scientists of different branches, from quantum mechanics to microbiology, and asks them to direct the spotlights of their knowledge on to a comparatively narrow field, but a field of great biological importance and chemical interest. After reading the prepublished papers I am convinced that we were right in assuming that the difficulties of finding sufficient common ground for our various denomin- ations are no longer insuperable. Still, we shall have to exert some patience and forbearance. I ask the theorist not to be impatient with the experimenter, if he asks questions which reveal his lack of knowledge of theory, but to answer them in * See frontispiece {Editors). XXII PRESroENTIAL ADDRESS brotherly love ; I ask the experimenter not to be shy to ask such questions, for they may turn out to be quite searching and may indeed enforce modifications of theory. Again, I ask the theorist not to be shy to apply his theories to facts with which he may become familiar only at this meeting; and the experi- menter to try to enlighten the theorist about these facts, again in brotherly love. Finally, I am convinced that quite apart from the direct scientific results of the Symposium, our living together here for one week will cement bonds of friendship and comradeship which will remain a force long after the Symposium. R. Lemberg THE ELECTRONIC STRUCTURE AND ELECTRON TRANSPORT PROPERTIES OF METAL IONS PARTICULARLY IN PORPHYRIN COMPLEXES By L. E. Orgel Department of Theoretical Chemistry, University Chemical Laboratory, Cambridge INTRODUCTION The electronic structure of metal-porphyrins has often been discussed in terms of the valence-bond theory as developed by Pauling (1940). In recent years a related but more quantitative theory of metal complexes has been developed and is known as ligand-field theory (Griffith and Orgel, 1957; Moffitt and Ballhausen, 1956). The first part of this paper attempts to give an elementary account of this theory insofar as it is of interest to biochemists working with haem compounds. In particular I shall discuss the relevance of magnetic susceptibility and magnetic resonance data. In the later part of my paper I shall discuss some recent work on electron-transfer processes involving metal ions, and also show how the electronic structures of the haem enzymes may be relevant to the types of electron-transfer which can take place. LIGAND-FIELD THEORY OF REGULAR OCTAHEDRAL COMPLEXES The five 3- Distortion >- (a) (b) E^., ' Fig. 5. Energy level diagram showing the effect of tetragonal distortions; (a) electrostatic theory; (b) a possible consequence of double bonding. z axis are gradually removed. (This is formally equivalent to replacing two ligands by groups which produce smaller crystal fields.) In Fig. 5a we illus- trate the results of calculations based on the electrostatic theory, and in Fig. 5b the way in which these calculations might be modified by covalent bonding effects. The principal features, namely the splitting far apart of the d^i_y2 and d^i orbitals and the maintaining of the degeneracy of the d^.^ and dy^ orbitals are unaffected by covalent bonding, but the order of the d^^y orbital and the d^.^ and dy^ orbitals might be altered. If the d^i. orbital becomes much more stable than the dj,2_yi orbital all the electrons may crowd together in the bottom /ow orbitals. Then we would get 0, 1, 2 and 3 unpaired electrons in d^, d'', d^ and d^ ions, respectively. These extreme conditions seem to apply in nickel phthalocyanine (diamag- netic), cobalt phthalocyanine (one unpaired electron) and ferric phthalo- cyanine chloride (three unpaired electrons). Electronic Structure and Electron Transport Properties of Metal Ions 7 In the biologically important haem compounds the environment seems more nearly octahedral and no "intermediate-spin" derivatives are known. (Magnetic resonance shows conclusively that ferrihaemoglobin hydroxide exists in a mixture of high- and low-spin configurations.) Paramagnetic resonance absorption has not been detected in ferrous com- pounds and it is probable that even in the paramagnetic compounds it is outside the range of normal experiments. Paramagnetic resonance experi- ments, however, are perhaps the most sensitive tool at present available for the study of the detailed energy-level scheme both in high- and low-spin Fe+++ complexes. Once this is known inferences can be made about the detailed geometrical structure, and correlations established with other properties. PARAMAGNETIC RESONANCE EXPERIMENTS Here I can deal only with the energy-level diagrams deduced (mainly by Griffith, 1956, 1957, 1958) by an analysis of the experimental data of Ingram and co-workers (Gibson and Ingram, 1957; Gibson et al, 1958). The Table 2. Resonance characteristics of some haemoglobin derivatives Compound Spin-type ^-values Ferrihaemoglobi n High .-. = 2 ?, - 6 Ferrihaemoglobin fluoride High <5-l- " 0)5 t " t Weak bond Strong bond OMe O \v_C > ^ \.. transfer c \ O— Cr++^(HoO)5 O— Cr(H20)4(MeOH2)+ (ii) O— Cr+^+(H20)4(MeOH) + H+ In this system the reaction achieved is hydrolytic, that is, the redox reaction catalyses stoichiometrically the hydrolysis of the ester, but I shall now show that this is not an essential feature of the process. In order to bring the discussion closer to the subject of the cytochromes, I shall replace the reactants by ones which may be biologically important, but only for illustrative purposes : PO,H- / A Fe+++— O— (/ \)— O ^ \ Fe++ADP Weak bond Fe++— O— (/ \>— O PO.H- t Fe+++— ADP strong bond ^-^ > Fe++— O— M+++— Rj > M+++— Rx— Ra \ \ the "electron mediator" and the transferred group are part of the same molecule. The advantage of this in ensuring the efficient coupling between the redox and transfer processes is obvious. If mediated electron transfer is important in oxidative phosphorylation, then the details of the electronic configurations of the metal enzymes con- cerned will be critical to the understanding of the process. Here I can summarize only a few of the most important considerations : 1 . The effect of a metal ion in inducing the hydrolysis of a bond as in the reactions (ii) and (iii) will be large if the metal has a maximum number of spins paired after reaction, e.g. if the Fe^++ ion produced is low- spin rather than high-spin (of course it will also be greater for a trivalent than for a divalent ion, other things being equal). 2. The rate of transfer will be greatest if there is no need for a change of spin configuration during the process, i.e. if it occurs between two high-spin or two low-spin complexes. 3. The rate of transfer also depends on the degree of overlap between metal orbitals and mediator orbitals. This is greatest for low-spin complexes. 4. The most effective mediator molecules are likely to be ones which can act either as good electron donors or as good electron acceptors. In conclusion I should like to note that even if oxidative phosphorylation does not involve steps which have an obvious appeal to the theoretician, but rather a sequence of conventional coupled redox reactions, both the redox potentials and the rates of reaction will certainly depend critically on the spin-states of the metal ions and hence on the details of their interaction with their environments, REFERENCES George, P. & Griffith, J. S. (1959). The Enzymes, 2nd edition, Ed. Boyer, Lardy and Myrbiick, Chap. 8. Gibson, J. F. & Ingram, D. E. (1957). Nature, Lond. 180, 29. Electronic Structure and Electron Transport Properties of Metal Ions 13 Gibson, J. F., Ingram, D. J. E. & Schonland, D. (1958). Disc. Faraday Soc, No. 26, 72. Grifhth, J. S. & Orgel, L. E. (1957). Quart. Rev. XI, 381. Griffith, J. S. (1956). Proc. Boy. Soc. A. 235, 23. Griffith, J. S. (1956). J. Inorg. Nucl. Chem. 2, 229. Griffith, J. S. & Orgel, L. E., Unpublished calculations. Griffith, J. S. (1957). Nature, Lond. 180, 31. Griffith, J. S. (1958). Disc. Faraday Soc. 216,91. MoFFiTT, W. & Ballhausen, C. J. (1956). Ann. lier. phys. Chem. 7, 107. Orgel, L. E. (1956). Proc. lOtli Soliay Conference in Chemistry, Brussels. Paultng, L. (1940). The Nature of tiie Chemical Bond, Cornell University Press. Taube, H. (1959). Advances in Inorganic and Radio Chemistry 1, 1, Academic Press, London, New York. Eraser, R. T. M., Sebera, D. K. & Taube, H. (1959). /. Amcr. chem. Soc. 81, 2906. DISCUSSION Terminology in Ligand-Field Theory Williams: Dr Orgel's paper contains a discussion of the quantity. A, which is used by him both in thermodynamic and spectroscopic calculations. In one place it is sug- gested that there is a critical value of A such that if it is exceeded a change of paramagnetic mom.ent would be observed. A is here used in a thermodynamic argu- ment and is an indication of the field strength in the ground state of a complex. At another place it is stated that extensive studies of spectra have led to the relative values of A ; A is related to an excitation energy difference between two states, the ground and the excited states. What is A ? Is it a parameter of the field or is it only to be correlated with differences in character between excited and ground states, or does it represent a confusion of these two factors ? We shall now indicate why we consider the last statement to be true. The order of A given by Orgel for a series of ligands is part of the spectro-chemical series I- < Br- < CI- < F- < OH- < H2O < carboxylate- < SCN" < pyridine < NH3 < NOr < CN-. This series is often confused with a series of increasing field strength (Williams, J. chem. Soc. 8 (1956)). The effect of ligands in reducing the paramagnetic moment of ferric complexes (Scheler, Schoffa & Jung, Biochem. Z. (1957) 329, 232) follows the different order F- < CI- < H2O < carboxylate- < OCN" < SCN- < OH- < NOr < NH3 < pyridine < N3- < CN-; note particularly the underlined ligands. Again there is no doubt that the stability of metal complexes with different ligands does not follow a given order of ligands. With some cations an order somewhat like the spectrochemical series is observed, with others the order is almost completely reversed. Thus experiment shows that A, obtained from spectra, is not to be used as a guide in discussions of thermodynamic quantities (see Jorgenson, Orgel, Williams, Disc. Faraday Soc, 26, 1958, p. 123-130, 110-115, 180-187). However, theory (same references as above) also shows why this is so dangerous. The correlation of A with field strength is only apparent in a first order perturbation treatment of a simple electrostatic crystal field model. A second order perturbation treatment introduces polarization of the ligand dependent upon the cation or, if one wishes to put it this way, covalency, and changes in the radial as well as the angular dependences of the d wave-functions aftect the energies of states. This was pointed out by Owen {Disc. Faraday Soc, 19 (1955)). 14 Discussion This covalency is in part independent of field symmetry and is often called central field covalency. It is best explained by saying that not only is there a tendency for the d electrons of the cation to go into particular directions in space when a field is applied but that they spread out radially in space over the ligands also. The stabihty of a complex depends not only on the ability of a ligand to polarize the d electrons into given directions, where they get out of the way of the ligand electrons, but also on the ability of the ligands to allow the d electrons to spread out over them and the ability of the cation to allow the ligand's electrons to spread over it. Jorgensen has shown that the series of ligands which allow increasingly the d electrons to spread out, is F- < H2O < NH3 < L^^^^'g) < SCN- < Pyridine < Cl^ < CN- < Bi- < (I-?); Note the underlined Hgands. This series is different again from the spectrochemical series. Thermodynamic properties such as the stabilities of complex ions and the relative stability of two spin states are just as likely to follow either one of the two series, spectrochemical and nephelauxetic, which are both derived from spectra. The order of ligands which will bring about a change of spin state is even dependent upon the valency state of the cation. Orgel: 1. The procedures used to derive A, the spectroscopic ligand-field strength, and to determine the "nephelauxetic" series are well defined. To suppose that a single A suffices for ground and excited states of a complex is an approximation, but one which is justified by the success of the simple theory in interpreting spectra. If, as Williams suggests, A varies greatly from state to state, the ligand-field theory would never have been adopted, since it would have failed to accommodate even the simplest observa- tions on the spectra of high-field complexes. 2. The approximations implicit in the treatment of spin-pairing are more serious since (a) The internuclear distances decrease on spin-pairing and so the ligand-field increases (this is in contrast to the situation encountered in spectroscopy, where all energies are determined for the same internuclear distance). (See, for example, Orgel, 10th Solvay Conference Proceedings, Brussels, 1955.) (b) Increased delocalization in the low-spin state facilitates spin-pairing. (See, for example, the many papers on Co+++ in the spin-paired state.) It may well be that in special cases these effects can change slightly the ligand-field order; I would only question whether any experimental evidence for this is available in the regular octahedral complexes to which the theory applies. 3. The series of ligands of Scheler, Schoff"a and Jung may reveal a reversal of the ligand-field parameter. Before concluding that this is so Williams should establish: (a) That detailed calculations show that the conclusions of the theory of regular octahedral complexes can be taken over for non-regular complexes. (This is plausible but by no means obvious or easy to demonstrate.) (b) That the NOg" group is present as a nitro group (not a nitrito group) in both high- and low-spin forms of ferric haem complexes. (c) That the addition of ligands to haems (and changes of pH, etc.) do not affect the protein-metal interactions. 4. Williams' identification of electrostatic (as contrasted with covalent) theories with first order perturbation theories, and the latter with ligand-field theory, is incorrect. In conclusion, I should like to say that I usually find myself less in disagreement with Williams' view than with those which he attributes to others. In presenting material in reviev.'s or introductory articles it is normal to omit the many qualifications which appear in the detailed literature, and to indicate that this has been done. That, for example, is why I say that the theory of spin-pairing as presented is "much- oversimplified" and why I placed pyridine and ammonia, for the purposes of an elementary treatment, together in the ligand series as amines. (The ligand-fields of the compounds are very similar and the order probably does change from one com- pound to another, both for electronic and stereo-chemical reasons.) A useful purpose Electronic Structure and Electron Transport Properties of Metal Ions 1 5 may be sened by drawing attention from time to time to the well-recognized approxi- mations of a theory; an even more useful purpose is served by doing something about them (see, for example, Hush and Pryce, J. chein. Phys. (1958), whose work could well be extended to cover the transition from high-spin to low-spin states). Williams: It is not suggested that A, the field strength in any one state, varies greatly from every state to every other state. The variation of A will depend upon the character of the different excited and ground states. The spectrochemical series was observed to be a series roughly independent of whether one is dealing with low or high spin complexes or with metals from different transition metal series. Will Orgel state whether he believes this series to be also the series of the heats of interaction of ligands with a given cation, independent of cation ? The case of the octahedral complexes could be taken as an example. Orgel has made this assumption himself in his discussion here (3a) and elsewhere. .o- o- Under 3 (b); if the NOg- group changes from ^N^' to -<-0— N^ then this is but an indication of a change in ligand character with cation or spin state and/or valence state which I wish to demonstrate. This will be described shortly, not only for this case, but for the SCN~ complexes also. Under 3 (c), the hydroxide ion pro- duces spectroscopically the same effect in complexes of iron porphyrins where there are no proteins. (4) needs amplification before I can discuss it. I agree with Orgel's conclusion. My criticisms stand if the over-simplifications of theory lead to inconsistency with experi- ment. I say that they do. Spin-states of Haem Compounds Falk: I agree with Orgel that the relatively easy transition of many haemoproteins between the low- and high-spin states is very interesting. This phenomenon has fascinated me for some years, and Falk & Nyholm {Current Trends in Heterocyclic Chemistry (1958), p. 130) have discussed it briefly. But I think it is pertinent to remark that this phenomenon is established only for compounds of the haemoglobin, catalase and peroxidase types, and not for haemoproteins which are electron-transport agents in the classical cytochrome c fashion. I am not aware of any evidence, from magnetic susceptibility m.easurements, of high-spin low-spin changes in cytochromes of c, b or a types. If I may be allowed to guess, I would suggest that of these cyto- chromes, the properties of cytochromes a point to them as the most likely of the three types to shov/ this phenomenon. Orgel mentioned at one point the old observation of three unpaired electrons in ferric phthalocyanine chloride. In this context I think it is interesting to draw attention to the unpublished investigations of Nyholm and myself, mentioned in Falk and PeiTin (this volume, p. 56) on ferriprotoporphyrin chloride ("haemin chloride"). We found no conductivity in nitrobenzene solutions, indicating that the compound is not an electrolyte, and in view of the 5 unpaired spins, reported in the literature and confirmed by us, have suggested that it must be a square pyramidal complex with AsApHd'^ hybridization. Orgel: The observations of Falk and Nyholm are very relevant here. I v/onder whether the ferric protoporphyrin chloride has the same structure both in the solid and in nitrobenzene solution. Perhaps it would have the 3-spin ground state in nitrobenzene corresponding to a pyramidal structure, but have five unpaired electrons and an octahedral structure (with shared chloride ions) in the solid. George: I think there is some doubt whether the paramagnetic resonance absorption measurements at pH 7 and 8-5 carried out by Gibson, Ingram and Schonland {Disc. Faraday Sac, 26, 72 (1958)) prove that ferrihaemoglobin hydroxide is a mixture of high- and low-spin forms. First, the pK of the ionization is about 8 at room temperature so that at pH 7 only about 10% of the ferrihaemoglobin would be present as the hydroxide. Secondly, Keilin and Hartree {Nature Land., 164, 254 (1949)) have shown that on cooling the conjugate acid is favoured in the dissociation equilibrium, as 16 Discussion would be expected for an endothermic ionization process. Hence, if the measurements were carried out at low temperatures (liquid air or liquid hydrogen) as is normally the case, without suitable control experiments it is not certain how much hydroxide was present. Thirdly, unless ferrihaemoglobin is carefully freed from ammonium salts there is a marked tendency for the ammonia complex to be formed in alkaline solution : so, if the experiments were made with either single crystals or a microcrystalline paste from an ammonium sulphate mother liquor, in the absence of suitable controls a contribution to the observed signal from the ammonia complex cannot be disregarded. In the case of measurements at low temperatures this would be more serious since there is good reason to believe that the formation of the complex would be exothermic. The experiments reported in our present paper are not subject to these uncertainties, and they provide equally direct evidence for the existence of a thermal mixture. Electron Transport Lemberg: The interesting theory of oxidative phosphorylation involving a quinone- hydroquinone system (Todd and others) appears less likely in the phosphorylation step connected with the oxidation of cytochrome c, although Glahn and Nielsen {Nature, Lond., 183, 1578 (1959)) have recently suggested that this step involves binding of the phosphate to the formyl group of haem a. Orgel's explanation still leaves us with the difficulty that we do not know conjugated systems which might take up phosphate groups, except perhaps the histidine imidazoles bound to haem iron. The electron transport through a respiratory chain of several cytochromes makes it appear sterically unlikely that electron transport through the imidazoles as postulated by Theorell can be a sufficient explanation; similar difficulties exist with regard to haem-haem inter- actions in haemoglobin. I therefore ask whether the physicochemists consider it impossible that electron transfer may occur through "aliphatic" portions of a protein, or possibly through a chain of water molecules bound in the protein. Winfield: The example of electron transfer given by Orgel (terephthalic acid complex) is one which can readily be demonstrated experimentally. But may there not be some kinds of conduction which are important in biology and yet not readily demonstrable? If one were able to remove an electron from one end of a paraffin chain simultaneously with addition of an electron at the other end, would not the resulting electron move- ment along the chain take place with negligible activation energy? It seems possible that conducting chains of this kind could be interposed between conjugated con- ducting groups of the type described by Orgel. In other words, conduction of electrons between the prosthetic groups of adjacent enzymes {in vivo) may not require a path which is conjugated throughout its length. In the passage of electrons through a series of cytochromes in the living cell, I think that the individual enzymes are joined by metal bridges or hydrogen bonds. If the metal atom were calcium, one might expect that the electrons would pass across the bridge either not at all or with no pause. But with a metal atom such as iron or copper acting as bridge, I think that the electron would reside for a finite time in the metal ion and that there would be an activation energy required to move an electron across such a bridge. The pause might well be of biological significance. A small activation energy for the transfer of electrons between an interconnected series of cytochromes would restrict "hunting" in a system which would otherwise be uncon- trollably sensitive to transient fluctuations in the environment. In addition the metal bridge could provide for by-passing part of the electron flow along paths which branch from the main respiratory chain. Lockwood: In proposing models for electron transport two essentially diff"erent ones have been given. There is one in which electrons can be put in at one end of the chain and taken out at the other and that can be repeated an indefinite number of times. The comparison to a piece of copper wire is convenient. I take it that the model given in reaction (1) of Orgel's paper is an example of this type of conductor. In other models that have been proposed an electron can be put in at one end of the chain and taken out at the other but this produces an alteration of the configuration and the process cannot be repeated till the electron transport has been reversed. An Electronic Structure and Electron Transport Properties of Metal Ions 17 example of this is electron transport transverse to the polypeptide chains of protein where the transport occurs through the CO and NH group via a hydrogen bond. Orgel: This could be compared to a condenser. LocKWOOD : Yes. The picture of the cytochrome change in particular preparations where the cytochromes are situated spatially side by side is a legitimate one and the distinction between the two types of models becomes important. The transport of the electrons through the members of the cytochrome chain is a process which is repeated an indefinite number of times and the model, to be satisfactory, should belong to the copper wire type. It appears to me that the condenser type of model would be useless to explain the transport of electrons through the cytochrome chain. Orgel: We cannot be sure that electron transport will never take place through aliphatic side chains. However, I myself would be very surprised to find transport through more than at most three or four carbon atoms. We are currently investigating this problem by magnetic resonance methods. Transport through the a helix or similar protein structure via a long series of hydrogen-bonded C=0 and NH groups is more problematical; again I suspect that this process is not favoured except in systems which have been excited optically. Chance: Although we have been discussing in some detail the mechanisms by which electrons might be transferred through the peptide chain of the protein, an experi- mental test of this possibility suggests that an insufficient conduction rate would occur at least in the case of cytochrome c. Experiments carried out by my collaborator Patrick Taylor on dried cytochrome c in an atmosphere of nitrogen, show that less than 1 ,000th of the conductivity w ould be obtained when compared with the rate at which electrons are transferred in the cytochrome chain. While this experiment may not be conclusive it is certainly indicative of the difficulty of applying this approach. Our early experiments on the reaction of cytochrome c and the peroxidase intermediate have been reviewed and considerably extended by John Beetlestone. He finds that an active centre of the size of 5 A would be adequate to explain the observed kinetic data. This size is larger than that of the iron atom but would fit nicely with the idea that a histidine group is involved. Thus to within the accuracy that is possible with this determination, some group on the outside of cytochrome c may be responsible for the interaction. George: I would like to add a few comments to those of Chance on the subject of kinetic data for haemoprotein reactions. Even though some of the velocity constants are quite low, i.e. 10^ to 10^ M~^ sec^^ in comparison with high values of 10® to 10* M~^ sec~^, these low values are often found to originate in large (unfavourable) activation energies E, so that when the temperature independent factor A in the Arrhenius equation, k = A e-^l^^'^, is evaluated it is found to have remarkably high values of the order lO^^ to lO^'. Now in terms of the simple collision theory for bimolecular reactions A is equated to PZ, where Z is the collision frequency, 10^\ and P is the steric factor. Considering "target areas" for haemoprotein reactions one would expect P to be a fraction, yet it is apparent that P can in fact be several powers of ten. It would seem that other features are extremely important in these reactions of which we know very little at present. For example, the haem plate is hydrophobic in nature and undoubtedly alters the structure of the liquid water in its vicinity. In addition, around tlie haem plate, there is a constellation of ionic charges on the protein, which may be very important when a reaction between two haemoproteins occurs. Chance: I agree with George that temperature-independent factors in haemoprotein interactions are high and variable and thus the accuracy with which one can determine tlie size of the active centre is definitely limited. However, the results are useful indicators nevertheless. In this connexion, increasing knowledge of cytochrome c structure is of importance and the apparent inaccessibility of the haematin, due to the surrounding structures, provides independent support for the idea that the active centre of cytochrome c in the peroxidase reaction may have to exceed the size of the iron atom. 18 Discussion Margoliash: Orgel's idea of the importance of the native configuration of the protein of haemoproteins in determining the closeness of attachment of the haem-iron hgands and hence the nature of the complex formed, fits well with the results of our study of the denaturation of cytochrome c. With this haemoprotein it appears that denatur- ation probably does not change the haem iron bound groups but rather has a quanti- tative effect on the haem iron-ligand bonds resulting, as denaturation proceeds, in the gradual disappearance of the specific properties of cytochrome c and its trans- formation into a normal chemical haemochrome (Margoliash, Frohwirt & Wiener, Biochem. J., 71, 559, 1959). Mechanism of Oxidative Phosphorylation George : Lemberg has raised the question of the kind of mechanism by which the oxida- tion-reduction of cytochrome c can be coupled with phosphorylation, since, for structural reasons, it is difficult to see how an electron mediator can be involved like terephthalic acid or its ester in the oxidation of Cr" by Co"^ Arising from our studies of the opening of the crevice in ferricytoclirome c, which happens when the azide and cyanide complexes are formed, Glauser and I have suggested that a conformational change in the protein may be involved as a conse- quence of a switchover to a different bonding group during the oxidation-reduction cycle. For example, if the most stable crevice structures for the ferric and ferrous forms, at the pH at which oxidation-reduction occurs, differ in having a primary amino group and a histidine group respectively coordinated to the iron as in A and C. I I I red I II (A) Prot— Fe"'— NH2 imid > Prot— Fe"— NHg imid (B) I 1 I oxid I I I (D) Prot— Fe"i— imid NHj < " ' Prot— Fe"— imid NHg (C) then upon reduction of ferricytochrome c (A) a metastable, "energy-rich" form of ferrocytochrome c (B) would be produced, reverting to the stable form (C) with a release of energy. Likewise on oxidation of ferrocytochrome c (C) an "energy-rich" form of ferricytochrome c (D) would be produced, reverting to the stable form (A) with a release of energy. The switchover of the crevice group in the reactions (B) -* (C) and (D) -> (A) would entail a conformational change in protein structure which could conceivably be linked in some way to a phosphorylation step (George, P., & Glauser, S. C. Abstracts Third Meeting Biophysical Soc, Pittsburgh, April 1959, D4). Chance : I should like to ask Orgel for more information on equation (iii) of his paper. At first I thought that you wished to distinguish between electron transfer reactions and the coupling to phosphorylation. However toward the end of your paper you show them to be intimately associated, unless I have misunderstood you. Further, is the iron atom to which ADP is linked a haematin or a non-haematin iron? Would you be willing to indicate to me arguments in favour of one or the other alternative? Orgel : I should like to make clear that in this paper I have tried to describe a very general scheme for preserving the energy of oxidation-reduction reactions. I had no particular chemical system in mind. If the Fe+++ is part of a haem compound then the ADP or other acceptor could not be attached to the metal atom but would have to be held in position by attachment to the protein; if the Fe+++ atom is not in a porphyrin ring, then the ADP could be attached to the metal directly. I have no view on the relative likelihood, if any, of the possible alternatives. The main idea is that if an electron is extracted from a metal ion which is weakly associated with a ligand then the metal in its new valency may decompose the ligand in such a way as to preserve the redox energy. One illustration is given. THE ROLE OF THE METAL IN PORPHYRIN COMPLEXES By F. P. DwYER John Curt in School of Medical Research, Australian National University INTRODUCTION The more obvious implications of the co-ordination of organic molecules to metal ions : the effective charge reduction of the metal ion, and the polariza- tion of the organic moiety, have tended to become obscured by the wide interest in ligand-metal bond theories. In as much as these of their nature incline to emphasize the separate entities of metal and ligand, attention has been directed away from the properties of the complex as a whole. Sugges- tions that many reactions can occur at the periphery of the metal-porphyrin molecule rather than exclusively at vacant or labile sites on the metal (Williams, 1956a; Chance, 1951; King and Winfield, 1959), deserve more serious consideration. The purpose of this paper is to direct attention to properties which are those of the whole complex unit rather than the ligand and metal components. The metal-porphyrins are derived from a planar di-acid molecule which differs from the usual planar quadridentates such as 1 : 2-bis(a-pyridyl- methyleneaminoethane) (Fig. 1), by implication of the metal in a closed-ring system which probably contributes considerably to the stability of the complex structure, since the organic molecule cannot be detached point by point and hence unwrapped from the metal. Recent exchange work with the sexadentate molecule 1 : 2-propanediaminetctraacetic acid has shown that the six points of attachment to a metal can be broken progressively ^^ ^ in this way (Dwyer and Sargeson, 1960). Co-ordination proceeds with the extrusion of two protons and the metal complexes have zero or a small overall positive charge. As a result, since it is obviously easier to detach electrons from complexes with zero or a small positive charge, oxidation is facilitated. This is shown by the redox potential shift on passing from the simple hydrated ions to the iron and manganese complexes. Silver(I) acetate and protoporphyrin react, with the extrusion of a single proton, to yield a silver(I) complex, which spontaneously oxidizes with the extrusion of a second proton. The rather rare formal Ag(II) complex is favoured by the low charge and the planar arrangement of the bonds (Falk and Nyholm, 1958). 19 20 F. P. DWYER The two co-ordination positions at right angles to the plane of the quad- ridentate ring can be occupied by a variety of ligands: water, halide or cyanide ion, organic bases, the histidine anion, carbon monoxide. There is a good deal of still somewhat empirical evidence, e.g. labihty, that these out of plane bonds are rather long, and the geometry is therefore tetragonal. The bonds may be so long that the complex is essentially planar. Long bonds in the polar, (1:6), positions of many copper, nickel and palladium complexes are well known (Nyholm, 1953; Nyholm et al, 1956). In cytochrome c the interaction of the imidazole groups is sufficient to promote the maximum electron pairing in both oxidation states and the geometry must be octahedral. The higher oxidation states favour co-ordination of anions because of the greater polarizing power of the metal and also the greater electronegativity. In the oxidized forms of the metal-porphyrins there is thus a stronger tendency to co-ordinate OH' and CI' to available sites or by displacement of another ligand. The ionic structure ascribed usually to haemin chloride is unlikely. The 'Vra«5'-effect" may be of considerable significance in the 6-co-ordinate metal-porphyrins. The effect, which has been extensively studied in planar complexes (Chatt et al, 1955), refers to the labilizing effect of groups, e.g. CI, CN, CO, on other groups or ligands attached in the opposite {trans) position. In octahedral complexes, though the chemistry is more compli- cated, the "/ra/75-eflFect" has been fruitful in elucidating substitution reaction mechanisms (Quagliano and Schubert, 1952; Basolo and Pearson, 1958). Strongly trans influencing groups : CO, CN, or the thiol anion, should modify the strength of attachment or even the properties of other ligands in the polar position. The imposition of a fixed spatial arrangement on groups attached in the 1 : 6 co-ordination positions is an important function of the metal atom, especially when it is realized that donor atoms of the protein itself are often linked in this way. Part of the functional role of the cobalt atom in vitamin Bi2 is the rigid and unique conformation imposed on the large organic moiety. Another part is probably the lability of the sixth co-ordination position normally occupied by cyanide ion, water or hydroxyl, but which can be used to attach a donor atom from protein. CHARGE DISTRIBUTION IN COMPLEXES The fundamental principles involved in the formation of metal complexes, first enunciated by Pauling (1938) have been elaborated by numerous authors (Martell and Calvin, 1952; Basolo and Pearson, 1958). Co-ordination of a Hgand to a metal ion decreases the charge on the ion and makes the donor atom more positive. Since donor atoms are amongst the most electronegative of the elements, part or most of the positive charge spreads over the ligand molecule. In effect, this means that the ligand molecule is polarized, with the withdrawal of peripheral electrons, or electrons from electron donating The Role of the Metal in Porphyrin Complexes 21 groups. It is well known that the stability of metal complexes is usually related directly to the strength of the ligand as a base, and this is merely another way of expressing this concept. Electron withdrawing substituents in the ligand molecule promote polarization in the wrong sense : compete with the metal atom for electrons. These ideas have been expressed succinctly in the "principle of essential neutrality" (Pauling, 1948). The pronounced curariform activity of complex cations containing phenanthroline, and bipyridine [M phengj^+j [M bipy3]++ in which the characteristic biological response must be due to distributed charge, supports the principle (Dwyer et al, 1957). The extrusion of protons during the formation of porphyrin metal com- plexes reduces the charge by two units but, even in the reduced form, the zero charge does not necessarily imply electrical neutrality of either the H Oij — C- H.C- C C — CH, m-i .0 0, ,0 NHj ~-o cr ^0 1^1 II T '^'"'^*^'^^« HjC-C^^i— CH, ° H Fig. 2 Fig. 3 metal or the ligand. Apart from the electrical capacities of the substituent groups, the transition metals are moderately electronegative. This property may well be enhanced by the spin-paired electronic situation existing in strongly interacting complexes. Recently, it has been shown that the methylene groups in the neutral complexes Z?/.s(glycine)copper (Fig. 2) and rr/j'(glycine)cobalt are sufficiently activated in this environment to enable Knoevenagel type condensations to be performed with acetaldehyde (Sato, Okawa and Akabori, 1957; Ikutani, Okuda, Sato and Akabori, 1959). A mixture of threonine and allothreonine was obtained from the cobalt complex in the presence of sodium carbonate. Djordjevic, Lewis and Nyholm (1959), found that nitrite ion and nitrogen dioxide attacked the neutral complexes 6/Xacetylacetone)nickel (Fig. 3) and /)/5(acetylacetone)copper, with the formation of complex organic nitrogen compounds, as yet unidentified. It is probable that the sites of attack are the activated resonating — CH — groups, which may carry a small positive charge. In common with phthalocyanine, phenanthroline and bipyridine, metals are bound more firmly in the porphyrins than might be anticipated from the base strength of these ligands. The donor power of the ligand, concerned primarily with the primary co-ordination or a bond, is responsible for the dissipation of charge from the metal atom. It is believed that much of the bonding strength of these molecules derives from at least two tt bonds in which the d electronic orbitals of the metal overlap the vacant/? orbitals of the donor atoms. These bonds tend to make the metal more positive. In the ferrocyanide ion 22 F. P. DwYER [Fe(CN)6]*" the excess negative charge conferred by six negatively charged donors is supposed to be nearly off-set by three tt bonds from iron to carbon (Pauling, 1938). The charge interaction picture of donor atom and metal is thus quite complex. The molecules 4:7-dihydroxy-l :10-phenanthroline (Fig. 4a) and 4:4- dicarboxy-2 : 2'-bipyridine (Fig. 4b) exist normally in the zwitterion forms. In neutral solution very little reaction occurs with iron(II) salts, but in alkaline solution very strong co-ordination occurs not only because the nitrogen atoms are now free, but because at least two protons have been Fig. 4 detached and neutralized. The tris-chelatQ iron complexes have therefore zero charge or are anions depending on the pH. At the biological pH the carboxylic acid side-chains in many porphyrins, e.g. protoporphyrin, haemato- porphyrin, etc., make some contribution to the stability of the complex. Quite independently of other factors, such groups when sufficiently acidic promote oxidation by reducing the overall positive charge. OXIDATION STATE OF THE METAL IN COMPLEXES In the absence of obvious oxidizing or reducing conditions, the co-ordina- tion of a ligand to a metal ion is taken to involve no change in the oxidation state. The number of unpaired electrons, but not necessarily their location, can be obtained from magnetic moment measurements. The validity of Hund's Rule, which usually needs to be invoked to translate magnetic data into the oxidation state, has been frequently questioned when the magnetic evidence is at variance with the chemical properties. 5/5'(dimethylglyoxime)- copper has the moment characteristic of one unpaired electron, but from the absence of metal-metal interaction in the crystalline state, it has been deduced that the unpaired electron is mostly located on the ligands (Rundle, 1954). It would not be unreasonable to think of the Cu atom as in the -f3 diamag- netic state and the ligands as reduced. The observation that the 1 -electron oxidation of copper phthalocyanine removes an electron from the ligand and not the metal suggests a similar disposition of the unpaired electron (Cahill and Taube, 1951). The elaborate system of conjugated double-bonds in iron protoporphrin makes it feasible that oxidation could yield stable seini-quinone structures without affecting the oxidation state of the iron. Recently, Gibson and Ingram (1956), using the electron spin resonance method, showed that the The Role of the Metal in Porphyrin Complexes 23 oxidation of methaemoglobin removed an electron from peripheral carbon atoms and not from the metal, which was taken as formally remaining in the +3 state. Some sim.ple metal complexes containing nitric oxide provide examples of where chemical, magnetic and electronic structure considerations fail to establish the oxidation state of the metal. The ions [Fe(CN)5N0]-~ and [RuCljNO]^" are both obtained by boiling salts of the tervalent metal ions [Fe(CN)6]^~ and [RuCIgHaO]^^ with concentrated nitric acid. They are diamagnetic and hence the oxidation state is assumed to be +2. It is proposed that nitric oxide co-ordinates as N0+ following the loss of its odd electron to the metal which is thereby reduced. A tt bond also is formed between a d orbital of the metal and the vacant p orbital of the nitrogen. Exactly the same ultimate electronic structure would result had the nitric oxide formed the usual a bond, and the tt bond had come about by pairing the odd d electron of the metal with the odd p electron of the nitrogen, or had the metal lost an electron to nitrogen, which then utihzed four electrons to form a double bond. It is questionable whether the donation of four electrons by N0~ is more objectionable electronically than of two electrons by NO+. The metals should then be considered in the +4 state, which is certainly more consistent with the method of preparation and the resistance of the iron complex to oxidation. Metal complexes are generally regarded simply as Lewis acid-base entities but it is possibly more fruitful, especially in their oxidation-reduction reactions, to regard some of them as integral internal redox systems in which the metal alone is not the sole electron source or sink. Certain band spectra of the strongly interacting transitional metal complexes with the porphyrins, phenanthroline and bipyridine have been assigned to the transfer of negative charge from the metal to the ligands, or in highly oxidized states of the complex, in the opposite sense. In the similar activated states in which reac- tion occurs we are, in effect, dealing with an oxidized or reduced ligand. Rapid racemization of both species has been found to occur when aqueous solutions of d[Os bipy3]"'"+ and /[Os bipy3]+++ are mixed. This must proceed through a peripheral electron, located most likely on a carbon atom in the 4-position to the nitrogen atom, leaking across to the oxidized form. Because of the large organic molecules and the octahedral geometry, the metal atoms themselves are inaccessible for direct electron transfer, even through a water bridge (Dwyer and Gyarfas, 1952): J[Os bipy3]++ -^ ^[Os bipy3]+++ -f e /[Os bipy3]+++ + Q-> /[Os bipy3]++ If we think of the oxidized complex as having an electron deficiency, i.e. a positive charge, localized on a similar carbon atom, which is then solvated, a water bridge is provided for electron transport (Fig. 5). 24 F. P. DwYER This mechanism, which is similar to that proposed by Wilhams (1956b) for the haemin catalysed oxidation of cysteine by molecular oxygen, is applicable also to the remarkable reaction first discovered by Blau (1889). The oxidized forms of the tris complexes of Fe, Ru and Os with phenanthroline, bipyridine and terpyridine undergo spontaneous reduction when the pH of the aqueous solutions is raised. Hydroxyl radical has been detected (Uri, 1952). This may reoxidize the complexes if the pH is lowered soon enough, or decompose to ozone (Blau, 1898) or hydrogen peroxide (Brandt, Dwyer II2. II2* Fig. 5 and Gyarfas, 1954). The polarization of the carbon atom (Fig. 6) may be sufficient to lead to dissociation of a proton, as happens with simple hydrated cations, and the electron then is captured from the attached OH group. Reaction mechanisms of this kind could well be applied to oxidation- reduction reactions in the cytochrome systems, but may have applications to many synthetic processes involving activated — CH — and — CH2 groups, as discussed previously. Undue attention seems to have been paid in metal porphyrins to the formal oxidation state of the metal in relation to possible oxidation states as H^ /^OH H^ deduced from simple compounds or salts. As a result, jf 1 ► r "jj + OH there has been much hesitation in invoking otherwise II 1^ feasible reaction mechanisms involving, for instance, formal Fe(IV), Fe(V) or Mg(I). In such strongly interacting systems both the metal and the ligand are in unique electronic states because of their combination. The relevant fact is the number of electrons that can be added to or detached from the complex unit. The source or fate of the electrons is immaterial. Often this informa- tion can be obtained by electrolytic methods or simple chemical reagents. REDOX POTENTIALS OF RUTHENIUM COMPLEXES Simple model metal-complex systems offer much promise in elucidating problems in metal-porphyrin chemistry (Williams, 1956a, b). This is especially so when considering redox potentials. Much useful information on the effect of substituents has been obtained from the iron /m(phenanthroline) and bipyridine complexes. In general, the results parallel those obtained with various porphyrins (Martell and Calvin, 1952). Electron attracting substituents (NO2, Br, CI) render oxidation of the complexes more difficult (potentials are more positive than in the unsubstituted complexes), whilst The Role of the Metal in Porphyrin Complexes 25 electron donating groups cause the opposite effect (Brandt, Dwyer and Gyarfas, 1954). Because of the non-equivalence of the electronic states in the oxidized and reduced forms of many metal-porphyrins true equilibrium is not attained on an electrode. This raises the question of the applicability of redox potential results obtained from truly reversible model systems to the metal-porphyrins. The effect of substitution in the ligand molecule itself is but one aspect of the problem. There is little precise information available from models on the effect of the overall charge upon the redox potential, or of the effects that might be anticipated from various ligands when added to a basic planar complex. Recently, we (Dwyer and Goodwin, 1959) have prepared a large number of mono- and Z?/5(bipyridine) and phenanthroline ruthenium(II) and (III) complexes which serve as better models for the metal-porphyrin systems than the //•/5(chelate) iron complexes. The Z?/5(chelate) complexes which evidently are the more appropriate, however, always have the labile two groups in the cis{\ : 2) position instead of the desirable trans{\ : 6) position. Ruthenium is the heavier analogue of iron in Periodic Group 8, and unlike iron, the mono- and bisichdaie) complexes do not disproportionate. The complexes are spin-paired in both oxidation states and reversible redox potentials can be obtained. By suitable replacement of the labile positions, anions, cations and neutral complexes can be prepared. The effects of overall charge and of the nature of the ligand are shown in Table 1 . Oxidation is greatly facilitated by lowering the positive charge. The replace- ment of bipyridine, (pK^ = 4-33) by two molecules of the stronger base pyridine (pA:„ = 5-20), also facilitates oxidation, but slightly. Large poten- tial changes are associated with the co-ordination of ammonia, ethylene- diamine and water. These seem much too large with the basic ligands to be ascribed wholly to their greater strength as bases. Water, of course, is a much weaker base than pyridine. The implied enhanced stability of the oxidized state can be related in considerable part to the capacity of the ligands to dissipate positive charge to their hydrogen atoms. The latter are then more strongly solvated or can form hydrogen bonds to the solvent water. At the acid concentrations used, dissociation of a proton from the aquo groups is unlikely, though this would stabilize the oxidized form most effectively by reducing the overall charge. There are still insufficient data to make much of a comparison between the ruthenium systems and the metal-porphyrins containing various co-ordinated addenda. The potentials of the latter systems certainly cover a much narrower range, possibly because of the smaller overall charge. The replacement of the water molecules (or water and hydroxyl) in protoporphyrin, for instance, by pyridine only changes the potential from —0-14 V to -f 0-107 V. A much more positive potential might have been anticipated. Similarly, the potential of the dicyano-protoporphrin couple (—0-183 V) would be expected to be 26 F. P. DWYER more negative. In some of these couples, however, the electronic states are different. It is questionable whether comparisons between the spin-free protoporphyrin and the spin-paired Z)/5(pyridine) and dicyano complexes can be made on the same basis as the electronically equivalent ruthenium complexes. Table 1, Redox potentials of ruthenium complexes in SULPHURIC ACID (1 N) Couple £0 [Ru bipy3]++ -[Ru bipy3]+++ 1-257V [Ru b!py2py2]++ ^[Ru bipyjpyal''""''"'" 1-25 [Ru bipy py4]++ — [Ru bipy py4]"'"'"*' 1-246 [Ru bipy py3Cl]+ — [Ru bipy py3Cl]++ 0-894 [Ru bipy CI4]— — [Ru bipy CI4]- 0-35 [Rubipypy3-H201++ -[Rubipypy3-H20]+++ 1-041 [Ru bipy py2-(H20)2]++-[Ru bipy ^y^-{H^O)Y++ 0-782 [Ru bipy.,(NH3)2]++ -[Ru bipy2(NH3)2]+++ 0-875 [Ru bipy2-en]++ — [Ru bipy2-en]+++ 0-74 (py = pyridine, en = ethylenediamine). SUMMARY The metal-porphyrins have been discussed as typical strongly interacting metal complexes in respect to such properties as the vacant co-ordination positions about the metal, the peripheral charge distribution and the oxidation state. A series of mono- and Z)zXbipyridine) ruthenium complexes has been proposed as model systems. The importance of the whole complex unit is emphasized in opposition to the concept of metal with attached ligand. REFERENCES Basolo, F. & Pearson, R. G. (1958). Mechanisms of Inorganic Reactions, 34-90; 177-210 John Wiley, New York. Blau, F. (1889). Mh. Chem. 10, 367. Brandt, W. W., Dwyer, F. P. & Gyarfas, E. C. (1954). Chem. Rev. 54, 959. Cahill, a. E. & Taube, H. (1951). /. Amer. chem. Soc. 12,, 2847. Chance, B. (1951). The Enzymes, 2, 428, Sumner and Myrback, Academic Press, New York. Chatt, J., Duncanson, L. H. & Venanzi, L. M. (1955). J. chem. Soc, 4456. Dwyer, F. P. & Goodwin, H. (1959). Unpublished work. Dwyer, F. P., Gyarfas, E. C, Shulman, A. & Wright, R. D. (1957). Nature, Lond. 179, 452. Dwyer, F. P. & Gyarfas, E. C. (1950). Nature, Lond. 166, 1181. Dwyer, F, P. & Sargeson, A. M. (1960). Nature, Lond. 186, 966. Djordjevic, C, Lewis, J. & Nyholm, R. S. (1959). Chem. and Ind. 4, 122. Falk, J. E. & Nyholm, R. S. (1958). Current Trends in Heterocyclic Chemistry, 130-139, Butterworths, London. Gibson, J. F. & Ingram, D. J. (1956). Nature, Lond. 178, 871. Itukani, Y., Okuda, T., Sato, M. & Akabori, S. (1959). Bull. chem. Soc. Japan 32, 203. The Role of the Metal in Porphyrin Complexes Tl King, N. K. & Winfield, M. E. (1959). Aiist. J. Chem. 12, 47. Martell, a. E. & Calvin, M. (1952). Chemistry of tlw Metal Chelate Compounds, 101 237; 373-375, Prentice-Hall, New York. Nyholm, R. S. (1953). Chem. Rev. 53, 263. Nyholm, R. S., Harris, C. M. & Stephenson, N. C. (1956). Rec. trav. chim. 75, 687. Pauling, L. (1938). The Nature of the Chemical Bond, Cornell University Press, Ithaca, N.Y. Pauling, L. (1948). J. chem. Soc. 1461. QuAGLL\NO, J. V. & Schubert, L. (1952). Chem. Rev. 50, 201. RuNDLE, R. E. (1954). Conference on Coordination Compounds, Indiana University, Bloomington, 25. J. Amer. chem. Soc. 76, 3101. Sato, M., Okawa, K. & Akabori, S. (1957). Bull. chem. Soc. Japan 30, 937, Uri, N. (1952). Chem. Rev. 50, 375. Williams, R. J. P. (1956a). Nature, Lond. Ill, 304. Williams, R. J. P. (1956b). Chem. Rev. 56, 299. DISCUSSION Higher Oxidation States Lemberg : While it is certainly correct that we have strongly interacting systems in which both the metal and the ligand are in unique electronic states because of their combina- tion, I feel that Dwyer has somewhat overstated his case. In such instances as the RO2H or HjOa-complexes of peroxidase, catalase and ferrimyoglobin, one may well be in doubt about the exact valency state of the iron, but in most other haemoprotein compounds there is little doubt about the valency of the iron. George: Gibson and Ingram {Nature, Lond. 178, 871, 1956) demonstrated the presence of a free radical in the oxidation of ferrimyoglobin by H2O2 by paramagnetic resonance absorption measurements, and identified this with the higher oxidation state, Mb'^. However, in more recent experiments, Gibson, Ingram and NichoUs {Nature, Lond. 181, 1398, 1958) have shown the radical to be present in much lower concentration than the oxidation state IV of the prosthetic group, invalidating the previous conclusion. It is not unexpected that radicals can be detected in such a system because there is already ample chemical evidence for the production of a radical in the formation reaction, i.e. Mb"' -h H2O2 -> Mb^ -f- radical and furthermore, in the reduction of Mb"", which occurs spontaneously and more rapidly the higher the concentration, radical species must again be formed, since Mb'^ is a one-equivalent oxidation product of Mb"'. (George and Irvine, Biochem. /. 52, 511, 1952.) Some years ago it was shown that a simple radical structure of the type that was proposed by Gibson and Ingram would not account for the hydrogen ion participation in Mb'^ reactions, whereas the "ferryl ion" structure or an isomer of this structure is in accord with the experimental data (George and Irvine, Sympos. on Coordination Compounds, Danish chem. Soc, p. 135, 1954; Biochem. J. 60, 596, 1955). It should be emphasized that although paramagnetic resonance absorption provides an excellent technique for the detection of free radicals, other evidence must also be considered in discussing possible structures for intermediates in oxidation-reduction reactions. The same is true of the mechanism of oxidation-reduction reactions, since radical species could be formed in side reactions, and not necessarily be involved in the principal reaction path. George: Another example where higher oxidation states are formed, somewhat similar to that of the Cu", Co", Zn" and Al"' phthalocyanines, is that of a-/3-}'-5-tetraphenyl- porphin (TPP). Whereas with the metal-free phthalocyanine the one-equivalent H.E. — vol. 1 — D 28 Discussion higher oxidation state is very unstable, TPP yields both a one-equivalent and a two- equivalent higher oxidation state that are appreciably more stable, i.e., one one TPP ^ ^ TPP^ ^ ^ TPP" equiv. equiv. In phosphoric acid solution TPP is bright green, TPP' and TPP" are dull violet and orange-brown respectively. These oxidations are completely reversible Uke the one- equivalent oxidation of copper phthalocyanine, the addition of ascorbic acid, hydro- quinone or ferrous salts regenerating the TPP. The structure of TPP" probably corresponds to the removal of the two pyrrole hydrogen atoms with the introduction of a new double bond into the ring system, as in the oxidation of reduced flavin. The copper salt of TPP yields a one-equivalent higher oxidation state Uke the phthalocy- anine derivative, but on addition of more oxidant a whole series of highly coloured products are formed from which the original compound can no longer be recovered by the addition of reducing agents (George and Goldstein, Abstracts \19th Meeting Amer. chem. Soc, Dallas, K 16, p. 13, 1956; George, Ingram and Bennett, J. Amer. chem. Soc. 79, 1870, 1957). Effects of Metal on Reactivity at Periphery Barrett: Concerning Dwyer's remarks on the effect of the introduction of metals into porphyrins, and the consequent events occurring at the periphery of the molecule, I would like to make this comment. Fischer and Bock (Hoppe-Seyl. Z. 255, 1, 1931) exposed protoporphyrin in pyridine solution to light and obtained a substance with a chlorin-like spectrum. The substance is not a true chlorin, or dihydroporphyrin, but carries two or possibly three oxygen atoms. The addition of these oxygen atoms results in the formation of a hydroxy group and a carbonyl group (Barrett, Nature^ Loud. 183, 1185, 1959). A vinyl group is necessary for the formation of dioxy- protoporphyrin. Pertinent to Dwyer's remarks is the observation that photo-oxidation of the tetrapyrrole does not occur when complexed with a metal, e.g. Cu++, Fe+++, or if irradiated in 1-10% hydrochloric acid. Could Dwyer comment on these effects: the suppression of photo-oxidation by (1) the formation of a metal complex and (2) the formation of the di-hydrochloride ? DwYER : One can anticipate a common effect as far as the peripheral charge is concerned by either protonation or the formation of a metal complex. However, I feel that the altered charge distribution is not per se the reason for the inhibition of photo- oxidation, but rather the effect of the proton or the metal is on the fluorescence of the protoporphyrin, and hence its ability to form active oxygen (or hydroxyl) which is presumably the attacking agent. THE PHYSICO-CHEMICAL BEHAVIOUR OF PORPHYRINS SOLUBILIZED IN AQUEOUS DETERGENT SOLUTIONS By B. Dempsey,* M. B. LowEf and J. N. PHiLLiPsf Department of Chemistry, Royal Military College, Duntroon, and Division of Plant Industry, C.S.I.R.O., Canberra The Tetrapyrroles, and in particular the haem pigments, occur in a biologi- cal environment which is essentially aqueous. It is therefore desirable to determine their physico-chemical properties in an aqueous medium. Unfor- tunately, the information available, particularly as regards their ionization and co-ordination behaviour, is meagre (Phillips, 1960). The major experi- mental obstacle to obtaining such data has been the very low solubility of these compounds in water. Some measurements have been carried out in non-aqueous and mixed solvent media (Conant, Chow and Dietz, 1934; Aronoff and Weast, 1941; Aronoff, 1958; Barnes and Dorough, 1950; Caughey and Corwin, 1955; Corwin and Melville, 1955). However, such systems are unsuitable for electrochemical studies because of unknown ionic activity effects. This situation led us to explore the use of aqueous detergent solutions as solvent media (Pliillips, 1958). Studies have been carried out using fully esterified porphyrin derivatives to avoid electrostatic effects arising from the ionized carboxylic acid groups on the periphery of the nucleus. A wide variety of porphyrin esters has been shown to disperse molecularly in a number of detergent solutions, presumably by solubilization within the lipid micelle. This phenomenon is analogous to a phase distribution equilibrium in which one of the phases is of molecular dimensions. Macroscopically, such a system would behave as a single phase and equilibration between the phases would be expected to be extremely rapid. The distribution of the porphyrin molecules between the aqueous and micellar phases can be repre- sented by a simple equilibrium of the type : (PHg)^ ^ (PH2)(j the solubilization constant K^ being defined by [«(PHjJM * Royal Military College. t C.S.I.R.O. 29 30 B. Dempsey, M. B. Lowe and J. N. Phillips where the subscripts d and vi' refer to the micellar and water phases respec- tively, and Tij. is the number of molecules of species x\ Na is the number of detergent molecules in the micellar phase ; and A'^ is the number of water molecules in the system. When an ion, e.g. a hydrogen ion H+ or a metal ion M++, is introduced into such a system it will presumably prefer the aqueous environment exclu- sively to that of the lipid micelle. Accordingly, any reaction involving such an ion and the porphyrin molecule must take place within the aqueous phase, the detergent micelles acting as a readily available reservoir for the porphyrin molecules. Typical reactions which may be studied in this way are : PH2 + 2H+ PH, + M++ PH3+ + H+ MP -f- 2H+ PH. ionization chelation (1) (2) The products of such reactions may or may not be solubilized depending on their nature. For example, one might expect the nonionic metal complex // y—NH =N HC H NH N-< to) (b) (c) Fig. 1 (a) Dimethyl protoporphyrin ester (DMPP) (b) Dimethyl mesoporphyrin ester (DMMP) (c) Tetramethyl coproporphyria III ester (TMCP) M = — CH3 V = — CH=CH2 E = — CH2— CH3 P = — CH2— CH2— COOCH3 (MP) but not the ionic porphyrin species (PH3+ and PH4++) to be readily solubilized. The purpose of this paper is to indicate the type of data that may be obtained using the solubilization technique and to suggest how such data may be interpreted. The following discussion is primarily concerned with the ionization (as in equation (1)), co-ordination (as in equation (2)) and spectro- scopic behaviour of porphyrins. In particular, the discussion will refer to the behaviour of the fully esterified derivatives of mesoporphyrin IX (DMMP), protoporphyrin IX (DMPP) and coproporphyrin III (TMCP) (see Fig. 1). The detergent solutions used Physico-Chemical Behaviour of Porphyrins in Aqueous Detergent Solutions 31 were either 2-5% (w/v) sodium dodecyl sulphate (SDS) or 0-25% (w/v) cetyltrimethyl ammonium bromide (CTAB). Ionization Behaviour The ionization of porphyrins solubilized in aqueous detergent solutions can readily be studied spectroscopically. Two general types of behaviour occur, the one with cationic and non-ionic and the other with anionic detergent solutions. In the former case two species only, the neutral porphyrin (PHo) and the dication (PH4++) are observed upon spectroscopic titration within the pH range 0-12. The variation in optical density {E) with pH at a given wave- length is such as to indicate that the reaction involves two protons : PH2 + 2H+ ^ PH4++ (3) the overall dissociation constant (A'obs) of the conjugate acid PH4++ being given by _[PHJ[H+P ^^^'^ ~ [PH,++] • ^^^ This does not necessarily imply the simultaneous addition of two protons in the kinetic sense. A more likely explanation is that under these conditions the monocation behaves as a stronger base than the free porphyrin. The equihbria postulated to account for this behaviour are illustrated below. ^, K,= [(H+)J[(PH,)J (PH,).;.'(PH,), "" [(PH3+)J +°" ^^ ^^ ^ [(H+),][(PH3+)J (PH3+), A4 = -——_——, (PH4++), ^ _ [(H+)J^[(PH,), + (PH,),] [(PH,++)J ^obs = In terms of the scheme outUned the observed constant (ATobs) would corres- pond to K^K^{\ + K,). The pXobs (= - logio/sTobs) values for DMMP and TMCP in 0-25% CTAB were found to be 2-08 ± 0-05 and 2-24 ± 0-05 respectively. Unfor- tunately the corresponding value for DMPP was too low (< -f-O-S) to be determined with any certainty. It is clear, however, that the observed values will depend on the nature and concentration of the detergent, and that com- parisons of pAT values for different porphyrins in the same detergent solution will reflect differences both in their aqueous basicities and in their solubiliza- tion constants. In anionic detergent solutions three species, corresponding to PH,, PH3+ and PH4++, may be observed upon spectroscopic titration, each ionization 32 B. Dempsey, M. B. Lowe and J. N. Phillips step corresponding to a one-proton addition. It has not been possible fully to interpret this behaviour theoretically, although it is certain that both the ionic porphyrin species PH3+ and PH4++ are being stabilized by some type of solubilization process. This leads to the observed pK values being a function of the intrinsic aqueous basicity constant and of the ratio of the solubilization constants of the species concerned in the equilibrium. Such a ratio would tend to eliminate specific porphyrin solubilization effects and hence one would expect the relative p^ values for a series of porphyrins in anionic detergent solutions to parallel their values in water. In the case of DMMP, TMCP and DMPP, the ipK^ values observed in 2-5% SDS were 5-94, 5-58 and 4-88, and the piQ values 2-06, 1-80 and 1-84 respectively. The pATg values appear to reflect the relative electrophilic character of the side chains, and this effect has been confirmed with a number of other porphyrin derivatives. The pK^ values appear less sensitive to the nature of the side chain. It is of interest to note that the pK^ of 1-84 for DMPP in 2-5 % SDS is equal to the value for DMPP in water (pK^ = 1-8) as estimated indepen- dently from solubility measurements (Dempsey and Phillips, unpublished). This suggests that the solubilization constants for the species PH3+ and PH4++ not only parallel each other but are in fact very similar in magnitude. Co-ordination Behaviour The interaction between porphyrin molecules and divalent metal ions can be represented by an equilibrium of the type shown in equation (2), There are little quantitative kinetic or thermodynamic data available about such reactions, and it was therefore thought desirable to explore them using the solubilization technique. It has been found that in cationic detergent solutions the reactions between porphyrins and metal ions are markedly dependent on temperature and also on the nature of the metal ion. At 20°C in 0-25 % CTAB no reaction has been observed with Co++, Ni++, Cu++, Zn^, Cd++, Mg++, Mn++, Pb++, or Fe++, over a period of weeks. On the other hand, at 100°C very rapid reac- tions occurred with Cu++ and Zn++ under suitable pH conditions, though not with any of the other metal ions studied. Accordingly the reactions involving Zn++ and Cu++ were investigated in greater detail. These reactions were shown to conform to equation (2), the apparent equilibrium constant (K'g) being given by: [MP][H+]^ ' [M++][PH2]' ^ ^ Such constants were evaluated by determining spectroscopically the ratio MP/PH2 at equilibrium, for a range of hydrogen and metal ion concentrations. Typical formation curves are shown in Fig. 2 for the zinc-mesoporphyrin Physico-Chemical Behaviour of Porphyrins in Aqueous Detergent Sohttions 33 reaction at 60°C. The time required to attain equilibrium at this temperature in the presence of IQ-i m zinc sulphate and 5 X 10"' M DMMP in 0-25% CTAB was approximately 48 hr. The reversibility of the equilibrium was demonstrated by studying both the forward and backward reactions as expressed by equation (2). The KJ value determined for the zinc mesoporphyrin equilibrium at 80°C, extrapolated to zero ionic strength is 6-0 X 10~^; the corresponding value at IrO n 05 i / / i y / p / / 20 30 40 PH Fig. 2. Formation curves for the zinc mesoporphyrin complex at 60°C Forward reaction equilibrium points O Zn++ + PHg ^ ZnP + 2H+ Backward reaction equilibrium points • ZnP + 2H+ ^ Zn++ + PHj ZnP n = , where TpHo is the total concentration of mesoporphyrin present. TPH, 60°C is 2-5 X 10"^. The relationship between the observed equilibrium constants {K^) and their value in water {K^ is given by : f(l +^,^)\ (6) where K^ is the solubilization constant of species x. If both species PH2 and MP were equally solubilized then the observed equilibrium constant would correspond to the water value. It seems likely that the solubilization constants will be of a similar order of magnitude, and in any event K^ values are likely to parallel K^ values either when comparing the one porphyrin with different metals or different porphyrins with the same metal. 34 B. Dempsey, M. B. Lowe and J. N. Phillips Preliminary results suggest : (i) that Zn++ reacts faster and forms a more stable complex with DMMP and TMCP than DMPP, as might be expected from the greater electro- philic character of the unsaturated vinyl side-chain compared with its saturated analogues; (ii) that Cu++ reacts faster and forms more stable complexes than Zn++ with DMMP, in accord with the normal relative chelating ability of the two ions (see Bjerrum, Schwarzenbach and Sillen, 1956-57); and (iii) that Co++ and Ni++ react infinitely more slowly than Cu++ or Zn++, although there is evidence (Caughey and Corwin, 1955) to indicate that in general Co++ and Ni++ form the more stable porphyrin complexes. It is suggested that this reluctance on the part of Co++ and Ni++ may be associated with their tendency to form hexaco- ordinate compounds as compared with the tetraco-ordinating tendency of Cu++ and Zn++. The overall kinetics conform to a simple bimolecular reaction involving metal ions and the neutral porphyrin species (PHg). This, and the fact that Zn++ reacts more readily with DMMP and TMCP than DMPP suggests that the reaction mechanism is of the displacement rather than the dissociation type (Basolo and Pearson, 1958). For purposes of comparison it is convenient to express KJ values in terms of the more conventional stability or formation constants {Kf) defined by : _ [MP] ^^"[M++][P=]' ^^ In the absence of acidic ^K data for mesoporphyrin the value for aetio- porphyrin II at room temperature {"pK^ + pA'g '^ 32 (McEwen, 1936)) has been used. This leads to extrapolated logio Kf values for zinc mesoporphyrin at 20°C of '->-' +29. The high stability of porphyrin metal complexes is illustrated by comparing this value with the corresponding figures in water for the zinc complexes of, for example, 8-hydroxyquinoline-5-sulphonic acid ('^16-0), ethylene diamine ('-^ll-O), and glycine ('-^9-5) (Bjerrum et al, 1956-57). Spectroscopic Behaviour Aqueous detergent solutions form useful solvent systems for studying the spectroscopic properties of porphyrin molecules, their salts and metal com- plexes. Anionic detergents are of particular interest in that they permit a study of the spectral behaviour of the monocationic species (PH3+), a species which has proved virtually impossible to obtain in normal solvent media. Much theoretical argument (Piatt, 1956; Kuhn, 1959) concerning electron distribution in the porphyrin nucleus has been based on the absorption spectra of the symmetrical free porphyrin (PHg) and its dication (PH4++). It seems Physico-Chemical Behaviour of Porphyrins in Aqueous Detergent Solutions 35 - /~\ - 1 1 1 1 \ 4, \PH. \ \ \ PH*„ l^\ \ / ^/^ X\PH.\^ / .<^ \\ \ /"y^^. / ^\\ ^^^^^ ^ y^ 1 1 0300 0 200 0100 /'\ / \ V_/ \ 4000 4200 A, A 5500 6000 o' A, A phV 0400 0300 Fig. 3. Soret and visible absorption spectra for the various protoporphyrin species. Table 1. Spectroscopic properties of some PROTOPORPHYRrN DERIVATIVES IN 2*5% SDS Species Absorption maxima ±5A Fluorescence excitation maxima ±20 A Fluorescence emission maxima ± lOA PHj 4080 Soret 5050 \ 5405 ,,. .^, 5780 ^'^'^'^ 6330 j 4150 5080 5420 5820 6340 6340 PH3+ 3985 Soret 5350] 5680 Visible 6095 ' 4080 5370 5660 6100 6125 PH4++ 4120 Soret 5570] ^,. ... 6020/ ^'^'^^^ 4130 5580 6020 6060 ZnP 4120 Soret 5425] ,,. ... 57901 ^'^''''s 4150 5440 5780 5890 36 B. Dempsey, M. B. Lowe and J. N. Phillips likely that similar information on the asymmetric intermediate species (PH3+) would further facilitate the understanding of this problem. Figure 3 compares the absorption spectra of the species, PHg, PH3+ and PH4++ for DMPP solubilized in 2-5% SDS. Such curves are typical for porphyrins having actio type spectra. It will be observed that in the Soret region the absorption maximum for the monocation is displaced towards the violet relative to both the other species. In the visible region the four-banded spectrum associated with the neutral porphyrin (IV > III > II > I) changes upon ionization to a three-banded type (II > III > I) and finally to the typical two-banded dication spectrum (II > I). Measurements have been made also of the fluorescence excitation and emission spectra of the various protoporphyrin species and of the zinc protoporphyrin complex in 2-5% SDS. The fluorescence excitation and emission maxima are shown in Table 1 along with the absorption maxima. It will be observed that in each case the single fluorescent emission maximum lies at wavelengths 10 to 100 A longer than the a absorption band. In general the fluorescent excitation maxima correspond to the absorption bands. SUMMARY This paper is concerned with physico-chemical studies of porphyrin esters solubilized in aqueous detergent solutions. In particular, quantitative data have been reported for: (i) the relative basicity of proto-, meso- and copro-porphyrins at 20°C ; (ii) the chelation of zinc ions by mesoporphyrin at 60° and 80°C ; and (iii) the absorption and fluorescence spectra of the monocationic species of protoporphyrin at 20°C. Preliminary results have also been reported for the zinc-protoporphyrin and copper-mesoporphyrin reactions at 80°C. The technique could readily be adapted to other physico-chemical studies, e.g. the further co-ordination of metalloporphyrins with other ligands and oxidation-reduction equilibria in metalloporphyrin systems. Such studies, aimed at providing basic information on the physico- chemical behaviour of these pigment prosthetic groups, seem an essential prerequisite to a detailed understanding of the role of such molecules in biological processes. A cknowledgem en t The authors are indebted to Miss I. Verners for her skilled experimental assistance and to Dr. J. E. Falk for providing the purified porphyrin esters. REFERENCES Aronoff, S. (1958). J.phys. Chem. 62, 428. Aronoff, S. & Weast, C. A. (1941). J. org. Chem. 6, 550. Barnes, J. W. & Dorough, G. D. (1950). J. Amer. chem. Soc. 11, 4045. Physico-Chemical Behaviour of Porphyrins in Aqueous Detergent Solutions 37 Basolo, F. & Pearson, R. G. (1958). Mechanisms of Inorganic Reactions, p. 91, John Wiley, New York. Bjerrum, J., ScHWARZENBACH, G. & SiLLEN, L. (1956-7). Stability Constants, Pts I & II, The Chemical Society, London. Caughey, W. S. & CORWIN, A. H. (1955). J. Amer. chem. Soc. 77, 1509. CoNANT, J. B., Chow, B. F. & Dietz, E. M. (1934). /. Amer. chem. Soc. 56, 2185. CoRWiN, A. H. & Melville, M. H. (1955). /. Amer. chem. Soc. 77, 2755. KUHN, H. (1959). Helv. chim. Acta 42, 363. McEwEN, W. K. (1936). J. Amer. chem. Soc. 58, 1124. Phillips, J. N. (1958). Current Trends in Heterocyclic Chemistry, p. 30 (Ed. by A. Albert, G. M. Badger and C. W. Shoppe), Butterworths. London. Phillips, J. N. (1960). Rev. pure appl. Chem. 10, 35. Platt, J. R. (1956). Radiation Biology, Vol. Ill, p. 71 (Ed. by A. Hollaender). McGraw- Hill, New York. DISCUSSION Cations of Porphyrins and Their Spectra Orgel: I should like to ask Phillips why it is that in the detergent system it is possible to measure the addition of a single proton to a metal-free porphyrin, while in the past those measurements which have been done have suggested that two protons are added simultaneously. Phillips: We believe the reason that the monocation is so readily obtained in anionic detergent solution to be due to its stabilization at the negatively charged micelle-water interface. It is of interest to note that no monocationic species can be detected in non-ionic or cationic detergent solutions. Lemberg: It is reassuring that on the whole there seems to be a satisfactory agreement between the conclusion as to basicity of porphyrins derived from the Willstiitter eniM and R I/IV and R III/IV of porphyrins I III Porphyrin £mM i?I/IV fniM R III/IV Deutero 4-33 0-27 8-59 0-54 Aetio 5-18 0-38 9-50 0-70 Copro Meso 5-15 5-41 0-35 0-38 9-97 9-82 0-68 0-69 Proto 5-58 0-38 11-58 0-79 Diacetyldeutero Diformyldeutero 3-52 3-48 0-29 0-29 6-7 8-00 0-55 0-67 Monoacetyldeutero Rhodo 1-56 2-0 0-17 0-17 10-5 15-0 1-14 1-29 Crypto a 2-35 0-21 14-7 1-32 Chlorocruoro 2-25 0-21 15-1 1-40 Acrylic acid 3-01 0-27 16-13 1-46 Phaeo 05 1-88 0-20 16-06 1-71 Formylpyrro Formyldeutero Vinylrhodo Diacetylpyrro Acetylrhodo a 1-86 1-82 1-2 1-42 2-21 1-30 019 0-20 0-115 0-245 0-27 0-15 16-74 15-7 19-8 13-62 21-0 21-0 1-77 1-73 1-89 2-36 2-40 2-40 38 Discussion number, and their basicity now more correctly established by Phillips' interesting method. It is also interesting to note that evidence for a monocation had previously been obtained by Neuberger and Scott for deuteroporphyrin disulphonic ester, somewhat resembling anionic detergents, although Walter had not been able to confirm this. With regard to the spectra of porphyrin dications, it is of interest that on closer observation four, not two, absorption bands can be observed in the visible part of the spectrum. This leads me to doubt the correlations assumed by Piatt for the neutral and acid spectra of porphyrins. In this connexion I should like to point out that the two bands I and III of neutral porphyrin spectra are not at all influenced in a similar manner by the substitution of an electron-attracting group on the porphin nucleus. Thus all formyl and ketonyl-substituted porphyrins have their band III greatly increased, but their band I greatly diminished as the table on p. 37 shows. While it is true that band I is the most variable and band III the second in variability, there is, in fact, little difference between the variability of ^niM of bands II, III and IV in a variety of different porphyrins. Phillips: Walter's failure {J. Amer. chem. Soc. 75, 3860, 1953) to detect the monocationic species observed by Neuberger and Scott {Proc. Roy. Soc. A. 213, 307, 1952) was due to an unfortunate choice of experimental conditions. This aspect has been discussed by Scott (/. Amer. chem. Soc. 77, 325, 1955). The Reactions between Metal Ions and Porphyrins By J. H. Wang and E. B. Fleischer (Yale) Wang : Phillips and his co-workers suggested that the combination of porphyrin esters with Zn++ to form Zn++-porphyrin derivatives takes place through a displacement rather than a dissociation type of mechanism. I would like to report some work which not only confirms his suggestion but also gives a more detailed understanding of this displacement mechanism. We found that the rates of the successive steps in the combination of a metal ion and a porphyrin derivative can be markedly affected by varying the solvent composi- tion, presumably due to the change in solvation of the metal ion. In acetone solutions some metal ions, Cu++, Bi+++, Hg++, Cd++, etc., react readily with the dimethyl ester of protoporphyrin even at room temperature to form the corresponding metallo- porphyrin, whereas other metal ions, Fe++, Fe+++, Cr+++, Pt+++"'", Sn++, Zn++, etc., form a new type of complex with absorption spectra markedly different from that of the corresponding metallo-porphyrins; only upon heating do their spectra change to those of the metallo-protoporphyrins. The spectra of some protoporphyrin derivatives in chloroform solution are shown in Fig. 1 . The spectra are respectively (A) protoporphyrin dimethyl ester, (B) haemin dimethyl ester, (C) the new type of complex formed between ferric chloride and proto- porphyrin dimethyl ester, and (D) the dihydrochloride of protoporphyrin dimethyl ester. If alcohol or pyridine is added to the chloroform solution of this new complex formed between ferric chloride and protoporphyrin dimethyl ester, the spectrum changes immediately to that of protoporphyrin dimethyl ester, (A) in Fig. 1 . This shows that the binding between Fe"''++ and protoporphyrin in the new complex is quite weak, since the protoporphyrin can readily be displaced by other ligands. We suggest that this new complex has a sitting-atop type of structure as illustrated in Fig. 2. It is also of interest to note that the spectrum of the sitting-atop complex of Fe+++ bears striking resemblance to that of protoporphyrin dication, PH4++, as shown by diagram (D) in Fig. 1. This observation suggests that the observed absorp- tion in (C) is probably due to electronic transitions in the protoporphyrin rather than the metal part of the complex. Similarly, if acetone solutions of the sitting-atop complexes of Fe++, Fe+++, Cr+++, Pt++++, Sn++, Zn++ respectively are diluted with water the spectrum immediately changes Physico-chemical Behaviour of Porphyrins in Aqueous Detergent Solutions 39 o to that of the dimethyl ester of protopoq^hyrin itself, {A) in Fig. 1, as water molecules rapidly displace the organic ligand from the complex. We have isolated the ferric sitting-atop complex in pure crystalline form. The structure suggested in Fig. 2 was confirmed by the infra-red spectrum of this complex dissolved in deuterated chloroform, 99% CDCI3. In protoporphyrin dimethyl ester the N-H groups are responsible for three distinct infra-red absorption peaks at 3-05 fx (stretching), 6-15 /< (deformation), and 9-06 // (rocking) respectively. In the infra-red spectrum of haemin dimethyl ester all of the above three peaks disappear as expected. 20 15 10 05 0 20 15 10 OS ■t ° c -o 20 8 15 " aA yvTV^^ B ■^ 1 1 1 D 1 r 1 C,H COoCH, COXH, Fig. 2. Proposed sitting-atop type of structure for the reaction intermediate. 650 600 550 500 Wavelength, m/i Fig. 1 450 We found that in the infra-red spectrum of the ferric sitting-atop complex the 3-05 and 6-15 [i peaks shifted to 3-11 and 6-64 /t respectively, and that the 9-06// peak is no longer easily detectable. This observation confirms our proposed sitting-atop structure, since it shows that the corresponding N — H bonds still exist in the said complex. The observed frequency shift of the 3-05 and 6-15 /< peaks is presumably due to the polarizing influence of the ferric ion. Phillips: Wang's suggestion that the sitting-atop complex is involved in metal porphyrin formation is both novel and interesting. However, I find the suggestion that the dication structure involves two nitrogens each with two hydrogens and two nitrogens without hydrogens rather disturbing for a number of reasons, viz. (i) the spectra of the dication and dianion are identical, which would seem to support the symmetrical dication structure (i.e. 1 hydrogen per nitrogen atom); 40 Discussion (ii) energetically one would expect the symmetrical structure to be favoured because of its greater conjugation; and (iii) Wang's postulatedfstructure would imply that porphyrin basicity should be comparable to pyrrole itself whereas in fact it is many p^ units stronger. In our experience the reaction between anhydrous ferric chloride and dimethyl- protoporphyrin ester led either to the mono- or di-cationic species, probably due to the difficulty of removing the last trace of water from the system. It would be inter- esting to have the infra-red spectrum of the dihydrochloride for comparison with the other species. SOME PHYSICAL PROPERTIES AND CHEMICAL REACTIONS OF IRON COMPLEXES By R. J. P. Williams Inorganic Chemistry Laboratory and Wadham College, Oxford This article is intentionally speculative in that it will use a comparison between known properties and reactions of some simple iron complexes with chelating and conjugated ligands on the one hand, and of the wide range of haem-containing compounds of biology on the other, in order to make comments about the structure and mechanism of reactions of the latter molecules. First we restrict our attention to simple iron complexes. There are three classes of these. In class I all the properties and reactions are consistent with the iron (Fe++ or Fe+++) being in a high-spin state (ionic complexes). In class III the properties are consistent with the cation (Fe++ or Fe+++) being in a low-spin state (covalent complexes). In the intermediate group, class II, the properties and reactions are consistent with the cation being present in the complexes as an equilibrium mixture of high- and low- spin states. Table 1, which hsts the properties and reactions of the groups, is a summary of evidence presented in detail elsewhere (Williams, 1955, 1956, 1958, 1959). The change in the character of the iron complexes (from one group to another) is to be associated with the change in the strength of the effective ligand field increasing from class I through class II to class III. The porphyrin unit supphes a ligand field which, together with groups above and below the porphyrin plane, places its iron complexes in either class II or class III, although the extreme weak-field members in class II can have properties very like complexes in class I. The evidence for this statement is collected and discussed elsewhere (Williams, 1955-59). Here we take this position as estabUshed. However we will illustrate it by reference to some physical properties of porphyrins. SPECTRA The absorption bands in the spectra of iron porphyrins have been discussed (Williams, 1956) except for the Soret band. Here we will examine the shifts in the position of this band ignoring the others. Some band positions are given in Table 2. In Table 2 wavelengths are in m^. The description 'ionic' and 'covalent' is only applicable to the first three rows of data. 41 42 R. J. P, Williams Table 1. The three classes of iron complexes Class I Class II Class III Physical Properties Magnetic moment (+ +) 4-90 4-9O-000 0-00 (+ + +) 5-90 5 •90-2-00 2-00 Visible spectra (++) weak emax < 10^ £max ^ 10^ strong fmax = 10* (+ + +) strong £max ~ 10* intermediate weak fmax < 10^ Stability (++) weak intermediate very high (+ + +) quite high high high Chemical Properties Metal and ligand exchange (++) rapid rapid-slow slow (+ + +) rapid rapid-slow slow Autoxidation (++) rapid intermediate or rapid slow Reaction with H2O2 rapid usually rapid slow Electron transfer reactions rapid rapid rapid Examples of ligands in complexes (++) H2O, NH3, aT>MGUU^O\, pMG)2(NH3)2, and (+ + +) oxalate. oxine. (DMG)2(pyridine)j,, Enta. ^ I CN-. (DMG is dimethylglyoxime.) In the table £max is the molar extinction coefficient. Table 2 Ferrous Ferric Ionic - > Covalent Ionic - > Covalent H2O CO CN- Pyridine H2O OH- CN- Peroxidase 438 425 420 404 418 425 Myoglobin 435 424 420 405 414 425 Haemoglobin 430 418 420 404 409 419 Cyt. ^3 448 432 430(?) 420(?) Cyt. a 444(?) 430 440 430 420 428 Cyt. 6 425 no effect 416 no effect Cyt. c 415 no effect 410 no effect Cyt. ^ 423 415 420 415 390 407 404 Data taken from Lemberg and Legge (1949) and Morton (1958). Some Physical Properties and Chemical Reactions of Iron Complexes 43 Tbeorell (1942), Williams (1955, 1956) and Scheler, Schoffa and Jung (1957) have drawn attention to the band-shifts as the magnetic moment of the ferric complexes changes. The Soret band moves to longer v^avelengths in the lower moment complexes. Here we point out that in ferrous complexes the band moves in the opposite direction. The lower the moment of a Fe++PX2 complex (P is porphyrin and Xg the further co-ordinating ligands) the shorter the wavelength of the absorption band. Now if we plot the difference in Soret band position Fe++-Fe+++ (AA) against the sum of the magnetic ^ ^ 30 X" ^ y ^^"h.o x y y 20 ^^0H~ y"^ y y - y y y y y ■^ m 10 /^ ®co y y y ® y _ y* • _ 0 ^ , , , 20 70 12-0 Fig. 1 . The relationship between the sum of the magnetic moments of the ferrous and ferric haemoglobin complexes in the presence of the additional ligands shown and the difference in position of the Soret band in the two complexes. moments (/fFe++ -f ^Fe+++) we can obtain a qualitative guide to the character of iron porphyrin complexes from their spectra (Fig. 1). A large value of AA implies ionic complexes. On this basis we have the series of complexes of decreasing ionic character: peroxidase = myoglobin = haemoglobin > cytochrome a^ > cytochrome d > cytochrome a > cytochrome h > cyto- chrome c. This series was devised by entirely different reasoning in earlier papers (Williams, 1958, 1959). The extreme members are ionic and covalent respectively (from magnetic observations), whereas the intermediate members are apparently mixtures of two spin forms in equilibrium (from spectroscopic evidence). A possible explanation of the opposed band shifts in the ferrous and ferric series is that the two cations are differently affected in their character on going from the high-spin to the low-spin state. If we take the Soret transition to be either an « -> tt or a tt' -> tt transition in which the electrons are more concentrated on the nitrogen in the ground state than in the excited state, H.E. — VOL. I — E 44 R. J. P. Williams this transition is naturally more difficult in ionic ferric than in ionic ferrous complexes on account of the charge difference. The band is at shorter Amax in the ferric complex. In the low-spin states both ferrous and ferric ions become better cr-acceptors and on these grounds the Soret transitions should both be more difficult in the promoted state (low-spin) complexes and a shift of Amax to shorter wavelengths would be expected. Now the ferrous ion also becomes a stronger tt donor in the low-spin state which again makes the transition of the electrons concentrated on the nitrogen more difficult (Note 1). Thus in the case of Fe""^ both a and tt interactions of the cations in the ligand change so as to shift Amax (porphyrin) to shorter wavelengths when the complex becomes of low spin. On the other hand, the ferric ion in the promoted state is a tt electron acceptor with one hole in the de shell. This property makes the Soret transition of the porphyrin easier. We conclude that the increased stabilization of tt electrons in the excited state overrides the increased stabilization of the electrons in the ground state on change from the high- to the low-spin ferric state. Thus we can see that the difference in TT-bonding characteristics of the cations can explain the opposed shifts. A similar explanation has been advanced in discussing the phenanthroline series of ferric and ferrous complexes where spectral changes in opposed directions are observed in a series of ferrous and ferric complexes which we have considered as models for the study of the physical properties of iron porphyrins (Williams, 1955, 1956). A good illustration of the opposed shifts of the Soret band in the Fe++ and Fe+++ states is that observed in the study of the effect of pH upon the spectra of Rhodospirillum cytochrome (Morton, 1958). At pH 7 the band positions are: Fe++, 424 m/t, and Fe+++, 390 m/<, with a band at 640 m/< in the ferric spectrum indicating an ionic complex. At pH 11-8 the bands are: Fe++, 413 m/i, and Fe+++, 407 m/.<, with no band at longer wavelengths than 565 m/i in the Fe+++ spectrum indicating a covalent complex. Thus at pH 7-0 this cytochrome is very largely ionic but at pH 11-8 it is largely covalent and the value of Am/t has changed from 34 m// to 6 m/i. The suggested change of magnetic moment is in keeping with the observed fall in the ratio of the y to a peak intensities of the reduced cytochrome on increasing pH. The observations are also very suggestive with regard to the group of the protein which is responsible for the pH dependence. As both the ferric and ferrous complexes show changes it can not be the reaction HoO -> 0H~. Imidazole groups are completely ionized at a pH just greater than 7-0 and we are left with the strong impression that the change involved is — NH3+ -^ NHg (see pp. 46, 49, 50). It will be observed that from the above analysis we consider that cytochrome ^3 is largely ionic. This tentative conclusion is supported by the following evidence. (1) The intensity ratio of the a to the y peak is 1:11. The usual ratio in covalent complexes is 1 : 6 as in the pyridine complex of haemoglobin Some Physical Properties and Chemical Reactions of Iron Complexes 45 while in ionic complexes it is approximately 1:12, as in haemoglobin. (2) The reaction of cytochrome a^ with carbon monoxide is rapid and only ionic forms of ferrous complexes can undergo rapid substitution reactions of this kind (Table 1). All ferrous complexes which react rapidly with carbon monoxide also react rapidly with oxygen. (3) The ferric form of cytochrome ^3 has a weak absorption maximum at '^ 650 m/^, which only appears when at least some of the ferric couple is in the ionic state. A similar discussion of the same spectroscopic features would lead us to suppose that cytochrome a is largely covalent with but a small percentage of the ionic form. The correlations between Soret band position and the magnetic moments of the ferrous complexes permit comment on the interaction between the ferrous ion and the protein. For example, Gibson (1959) observed that on intense illumination of carboxyhaemoglobin (HbCO) a short-lived species Hb* was produced. This species has its Soret band at slightly longer wavelengths than Hb itself. The discussion presented here would lead us to conclude that the protein groups interacting with the ferrous ion must be more weakly bound in Hb* than in Hb. In keeping, the reaction of Hb* with CO is forty times as fast and of lower activation energy. Again, the Soret band of myoglobin (438 m/<) is at a longer wavelength than that of haemoglobin (431m//) which are to be compared with MbCO 424 mju, Mb02 416 m/^ and HbCO 418 m//, HbOa 414 m/< (Lemberg and Legge, 1949). The values suggest that the protein groups are less strongly bound to myoglobin than to haemoglobin. This is in keeping with the more rapid reactions of myoglobin. Before leaving this point it is clear that the spin state of a ferrous or ferric complex is very sensitive to environment. We must expect that extraction of a cytochrome will sometimes alter its properties either through a minor denaturation of the protein or even through a change of the medium di-electric constant. OXIDATION-REDUCTION POTENTIALS We have made several comments upon the redox potentials of ferrous/ferric couples (Williams, 1959; Tomkinson and Williams, 1958). The general impression of the variations in the FePX, potential with change of X is that either by (1) a continuous adjustment in the nature of X from water to increasingly improved donors such as ammonia or (2) a gradual reduction in the Fe-X distance for a group X which is a good donor, the redox potential can be made to go through a continuous series of values which show a maximum, see Fig. 2. The maximum is reached because the ferrous as opposed to the ferric ion undergoes electron rearrangement at lower effective electronegativities of the group X. At low electronegativities, increase in electronegativity favours ferrous over ferric, while at higher effective electro- negativities increase in donor properties of the ligand favours ferric over ferrous. The maximum will be most accentuated for ligands which are 46 R. J. P. Williams TT-electron acceptors (Note 2). In order to develop further the discussion of iron-porphyrin redox potentials we present some new data, Table 3, on the redox potentials of Fe(NiOx)2X2 couples in water where NiOx is cyclo- hexanedionedioxime. From the table we see that the potential of the complex with pyridine is higher than that with imidazole which is higher than that with ammonia. All these complexes except the hydrates in both ferrous and ferric forms are covalent (of low spin). The values fall with the increasing donor character of the groups as shown by the p^ values, pyridine 5-2, imidazole 7-1 Table 3. Redox potentials of some iron nioxime complexes Ligand X in Fe(NiOx)2A'2 HaOCpH —3 0) pyridine imidazole ammonia Redox potential (mV) Ease of autoxidation + 180 slow very rapid at pH > 40 + 130 very slow + 30 very slow -370 slow rapid at pH < 80 The redox potential in water alone is pH-sensitive, falling to about —250 mV at pH approximately 100. NiOx is cyclohexanedionedioxime. and ammonia 9-1. Now the difference between the redox potential of Fe(NiOX)2, (NH3)2 and Fe(NiOx)2 (imidazole)2 is +400 mV. This difference is close to that between cytochrome c and cytochrome h, +200 mV, but cytochrome Z) is a protoporphyrin complex whereas cytochrome c is a meso- porphyrin complex. The redox potential difference produced by the different porphyrins is '-^ +80 mV in their pyridine forms (Lemberg and Legge, 1949). This factor should be added to the observed difference between cytochromes h and c giving +250 — 300 mV difference between the h and c type cyto- chromes had their porphyrins been identical. We conclude that if cytochrome c is an imidazole (histidine) complex, cytochrome h is likely to be an amine- imidazole complex. We will show later that the reactions of Z>-type cyto- chromes are in accord with this hypothesis as are their spectra (WiUiams, 1958). We now turn to cytochromes d (using the nomenclature of Morton, 1958) which appear to us to be related to h cytochromes structurally in much the way haemoglobin is related to cytochrome c. For although the cytochromes h and d have similar redox potentials they have very different chemical properties. The cytochromes d are very readily autoxidized for example. As we have noted in Fig. 1 the value of AA (Soret) between the Fe++ and Fe+++ forms of cytochromes d suggests that they are largely ionic complexes. This is supported by the easy autoxidation, by the uptake of carbon monoxide in the reduced state, and by the position of the Soret band of the Fe+++ form Some Physical Properties and Chemical Reactions of Iron Complexes 47 (>^max !^ 400 m-fi). Again there is evidence of bands at about 640 m/t in the Fe+++ complexes, the band expected for ionic Fe+++ haems. On the basis of this analysis of the properties of the cytochromes we need to modify the picture which we have given previously for the relationship between redox potential and the basicity of the co-ordinating groups amongst these compounds. Figure 2 gives our present impression. For all groups of haem-proteins there is a range of redox potentials and a range of otiier Ligand basicity (effective) Fig. 2. The suggested relationship between redox potentials and ligand basicity for the cytochromes. The top three curves are for haem a, protohaem and mesohaem complexes in descending order of potential. All these complexes are assumed to be imidazole-coordinated. The lowest curve is for either protohaem or mesohaem complexes where the further binding of the haem from the protein is assumed to be through amino-nitrogen. By effective ligand basicity we imply that basicity which obtains under the conditions of steric hindrance realized in the protein and we suggest that steric hindrance increases from right to left in all the cases. physical properties, such as magnetic moments and absorption spectra, as well as a range of chemical properties such as affinity for oxygen and carbon monoxide and rate of autoxidation. There is no reason to think that these groups of haem proteins can be rigidly differentiated, in fact. The variation in combination between porphyrins and the type of group X so far suggested would then be (see Williams, 1958), as shown in table on page 48. Inspection of these combinations leads to two immediate comments. Far from every possible intercombination between different porphyrins and different groups X or between different combinations of the two X groups for any one porphyrin has been postulated, let alone demonstrated. Second, where it is stated in the table that only one group is involved, say under 48 R. J. P. Williams myoglobin, and the other group is water we imply that there is no other co-ordinating group very near to the iron. Between this extreme and the case where there are two groups equally and strongly co-ordinated, as in cyto- chrome c, every possible intermediate may arise through the inability of the protein to satisfy simultaneously the stereochemical requirements of the iron and those of hydrogen-bonding in its own structure. Haem protein Porphyrin substituent Group X* Cyt. fl3 Aldehyde (hydroxyU?)) Imidazole HgO Cyt. fl Aldehyde (hydroxyU?)) Two imidazoles (one weakly held) Myoglobin Vinyl Imidazole HjO Haemoglobin Vinyl Two imidazoles (one weakly held) Cytochrome b Vinyl Two amino Peroxidase Vinyl Carboxylate HjO or — NH2 Catalase Vinyl Two carboxylates Cytochrome d no unsaturation One amino HgO Cytochrome c no unsaturation Two imidazoles * See also Williams, p. 72 of this volume. The 'Imidazole' Hypothesis Pauling and Coryell (1936) considered that the two dissociation constants of haemoglobin in the pH range 5-8 could be accounted for by assigning one ^K to a dissociation of type (1), of an imidazole group which was at a con- siderable distance from the iron atom and a second to the reaction (2). Basicity Fig. 3 This hypothesis is readily tested by examining the ionization of Fe(DMG)2 (imidazole)2. We find that the imidazole ionizes as in reaction (1) but that reaction (2) does not occur up to a pH of 11-0 (Croft and WiUiams, unpub- lished). We add the following evidence against any such ionization. Some Physical Properties and Chemical Reactions of Iron Complexes 49 (1) The absorption spectrum of Fe(DMG)2 (imldazole)2 does not change with pH from 6-11 except in intensity (Croft and WilUams). (2) The complex Fe++(DMG)2 (imidazole)2 is extractable into /j'o-amyl alcohol (Croft and WilUams). (3) The complexes Cu++(histidine) and Cu++(histidine)2 show no ioniza- tion of the type (2) (Leberman and Rabin, 1959; James and Wilhams, unpublished). (4) Amongst biological molecules, Fe++-cytochrome c has no ionization in the expected range (Lemberg and Legge, 1949). In the presence of oxygen it is observed that the lower ^K is raised. We accept Pauling and Coryell's (1936) explanation, that this implies that the oxygen inserts itself between the imidazole and the Fe++ ion. It would appear that the second p^is not due to the imidazole groups at all. The ^K shift could be due to the ionization of an — NH3+ group in the protein, the basicity of which was altered by the change in protein stereochemistry on deoxygenation of haemoglobin. Again, we have no evidence to show that Fe+++(NiOX)2 (imidazole)2 undergoes any ionization up to pH 10-0. The titration of the complex with alkali, its insolubility in organic solvents, and its absorption spectrum all indicate that the ligand is imidazole and not the imidazole anion. CHEMICAL REACTIONS OF IRON COMPLEXES It has always been our intention to proceed from a detailed study of the physical properties of ferrous and ferric complexes to a study of their chemical reactions. We have now made a start with the latter phase of this work. Reactions of Molecular Oxygen Oxygen can either combine with ferrous complexes (oxygenation) or oxidize them (autoxidation). In biological systems both reactions occur. We have observed both reactions also in the chemistry of the complexes Fe++(DMG)2X2 and Fe++(NiOX)2X2, where DMG is dimethylglyoxime. Our studies show that in the model systems the oxygenated complexes Fe(DMG)2X02 are not stable if X is readily exchanged for water or if X is a group containing labile hydrogen. The reactions can be illustrated by examples. When X is imidazole or pyridine the oxygenated complex is stable with certain qualifications. The replacement of the ligands X in the imidazole and pyridine complexes is much slower than in other complexes. When X is water, hydrazine, ammonia, aniline or other substituted amines, or sterically hindered pyridines or imidazoles (e.g. histidine) the oxygenated complex is not as stable but undergoes autoxidation. The replacement of ligands in these complexes is more rapid. Autoxidation is different in different cases giving oxidation of the group X in some cases (e.g. N2H4) and not in 50 R. J. P. Williams others. In both cases the same ferric complex is always obtained, Fe+++(DMG)2(H20)OH. We suggest the mechanisms: Fe+ ^(DMG)2X2 Fe++(DMG)2(H20)02 -> Fe+++(DMG)2(H20)OH Fe++(DMG)2X02 Fe++(DMG)2X+02- -> Fe+++(DMG)2(HoO)OH m^ (Reaction (2) is a catalysed autoxidation of X.) No complex of a saturated base X is known which carries oxygen (cf. cytochromes b and d) ; however, all the complexes of unsaturated bases X can carry oxygen (cf. myoglobin, haemoglobin, cytochrome a). It is of great importance here to note that Fe(DMG)2 (imidazole)2 can even pick up molecular oxygen in the presence of borohydride, sodium formaldehyde sulphoxylate, or sodium dithionite. This reaction is common to cytochrome a (Sekuzu, Takemori, Yonetani and Okunuki, 1959) but not to haemoglobin. It implies that the iron complex undergoes slow dissociation of its ligands as free oxygen reacts rapidly with borohydride or sulphoxylate. Haemoglobin undergoes very rapid de-oxygena- tion under these circumstances. We can now make some comments about the binding in haemoglobin. We note first that covalent ferrous complexes (Class III, Table 1) undergo slow ligand exchange. Magnetic data show oxyhaemoglobin to be covalent, yet it takes part in fast reactions involving ligand replacement. The iron must be in an energy state which is only slightly more stable than its high-spin states. This is in agreement with spectroscopic evidence as well as with the value of its redox potential. Now if the oxygen is labile in HbXOg the group X, the histidine, must be labile also. What is it then that prevents the autoxidation of haemoglobin in accord with equation (1) above? The answer which we suggest to this question is that it is the high activation energy of the rearrangement of the protein which prevents a water molecule replacing X and thus prevents autoxidation. On the other hand, the cytochrome a oxygen complex dissociates slowly to a covalent haemocliromogen (judged by the spectra) whence there is little danger of dissociation of groups X leading to autoxidation. In cytochrome a we suggest iron is more strongly bound to imidazole than in haemoglobin. Elsewhere (WiUiams, 1958) we have reached this conclusion from a very different argument. Amongst cytochromes some of the cytochromes b appear rapidly autoxidiz- able. We believe that this observation is irrelevant to biological function. If, as we suppose, cytochromes b (d) are amine ( — NH2 -^ Fe) complexes, then bringing them out of a cell environment to a pH of about 7-0 in free solution may well dissociate the NHg -^ Fe link. We can show this easily with Fe(DMG)2(NH3)2 which is fairly stable to autoxidation at pH 100 but Some Physical Properties and Chemical Reactions of Iron Complexes 51 rapidly autoxidized at pH 7-0. The behaviour is also very similar to that of Rhodospirilhim haemoprotein (cytochrome d) and could well arise in both cases through the high acid dissociation content of the — NH3+ group. It seems to us that some lower organisms may not have suitable histidine-con- taining proteins to give rise to Fe-histidine cytochromes but must be content with Fe-amine cytochromes. If this is the case and our discussion is valid then these organisms are unlikely to be able to store or transport molecular oxygen. Their cytoclirome oxidases and electron-transporting cytochromes are amine complexes whereas those of higher organisms are both amine and histidine complexes. One such conjecture about these compounds leads immediately to another. The development of histidine cytochromes a and c in a cell gives the organism the advantage over cells containing only amine cytochromes b and d that the energy of the oxygen molecule can be more efficiently used. Some 300 mV more energy (the difference in redox potentials) can be stored chemically for each electron transported. ELECTRON TRANSPORT There are two reasons for thinking that electron transport occurs across the porphyrin of the cytochromes. If catalytic activity resided in the imidazole-Fe-imidazole bonds then it should be demonstrable in Fe(DMG)2 (imidazole)2. The model complexes do not have the electron transporting properties of cytochromes as far as we can discover. Again using the models we have shown that although there is strong charge transfer interaction be- tween Fe++ and (DMG) there is no evidence for it in Fe++-imidazole. On the other hand, there is good evidence in Fe++(DMG)2 (pyridine)2. In this complex there is a band (absent in other Fe++(DMG)2X2 complexes) at '^ 400 m^a. For differently substituted pyridines it moves in the following manner (Jillot and Williams, 1958): Substituent None 4-bromo 3-cyano 4-cyano Maximum Absorption 385 385 460 475 (m/<) If it is assumed that this band is due to a partial charge transfer of an electron from the ferrous atom to the pyridine, the band positions are explicable in terms of the electron-acceptor properties of the substituents. The band position is solvent-dependent, again suggesting a charge transfer band. The absence of such a band in the imidazole complexes would suggest that charge transfer and therefore electron-transport across the imidazole is not facile. Finally, if we are correct in saying that cytochromes b and d are amine complexes then as amines are not unsaturated systems and presumably could not carry out electron transport we must assume in these cytochromes that the electron moves through the porphyrin. If this is so then it is very likely that electrons are mobile in the porphyrin of FeP (histidine)2, but of 52 R. J. P. Williams course this by itself does not eliminate the possibility that electron transport occurs across both the porphyrin and the imidazole in cytochrome c. NOTES 1. The position of the Soret band moves in the order of increasing wavelength with ligand ammonia, pyridine, carbon monoxide and oxygen, cyanide, water. This order, saturated bases < unsaturated bases < water is similar though not exactly the same as that found for the a and |3 bands and the explanation which we offer is that given earlier (Williams, 1956). 2. We imply here that the ferrous ion is more stabilized than the ferric ion by 7r-electron acceptors. The evidence is given elsewhere (Tomkinson and Williams, 1958). Thus in Fig. 2 we expect that the maximum will be more sharply defined in a series of pyridine complexes of increasing pyridine basicity than in a series of ammines. Imidazoles will occupy an intermediate position while for a series of oxygen anion-donors there need be no maximum as the ferric ion may well go over into the strong-field complex the more readily. SUMMARY An account is given of the properties of iron-porphyrin complexes of biological interest which is largely developed from a consideration of the properties of simpler iron complexes. Spectroscopic criteria for distinguishing between high- and low-spin complexes are suggested. New features of the inter-relationship of different cytochromes are proposed, based upon their redox potentials and their chemical reactions. Some comments are made upon the reactions of haemoglobin and their pH dependences. A discussion of the model iron complexes which are autoxidizable as opposed to those which can carry oxygen leads to a discussion of autoxidation and oxygenation of haem complexes. A cknowledgement I would like to acknowledge the help of the late B. A. Jillot, and of J. M. F. Drake and D. Croft, who have done all the experimental work connected with this paper. REFERENCES Coryell, C. D. & Pauling, L. (1936). Proc. nat. Acad. Sci. Wash. 22, 159. Croft, D. & Williams, R. J. P. Unpublished observations. Gibson, J. F. (1959). Disc. Faraday Soc. 29. James, B. R. & Williams, R. J. P. Unpublished observations. Jillot, B. A. & Williams, R. J. P. (1958). J. chem. Soc, A61. Leberman, R. &. Rabin, B. R. (1959). Nature, Lond. 183, 746. Lemberg, R. & Legge, J. W. (1949). Hematin Compounds and Bile Pigments, Chap. 5 & 6, Interscience, New York. Morton, R. K. (1958). Rev. pure appl. Chem. 8, 161. Scheler, W., Schoffa, G. & Jung, F. (1957). Biochem. Z. 329, 232. Sekuzu, I., Takemori, S., Yonetani, T. & Okunuki, K. (1959). J. Biochem. Tokyo 46, 43. Theorell, H. (1942). Ark. Kemi. Min. Geol. 16A, No. 3. Tomkinson, J. & Williams. R. J. P. (1958). J. chem. Soc, 2010, Some Physical Properties and Chemical Reactions of Iron Complexes 53 Williams, R. J. P. (1955). Special Lectures in Biochemistry, University College, London. H. K. Lewis & Co., London. Williams, R. J. P. (1956). Chem. Rev. 56, 299. Williams, R. J. P. (1958). Disc. Faraday Soc. 26, 123. Williams, R. J. P. (1959). TIw Enzymes (Ed. by P. D. Boyer, H. Lardy & K. Myrbiick), vol. I, p. 391, Academic Press, New York. DISCUSSION Oxidation-reduction Potentials of Haem Coinpounds Perrin : I should like to ask Williams what evidence he has for believing that with increasing ligand basicity the redox potentials of ferrous/ferric couples pass through a maximum. The only experimental evidence that I have so far found suggests, on the contrary, that in a related series of ligands there is a roughly linear dependence of redox potential on the pA^ of the ligand: the potential decreases continuously as the ligand pAT increases. This is true, for example, of iron complexes with a number of 5-substituted- o-phenanthrolines (Brandt and GuUstrom, 7. Amer. diem. Soc. 74, 3532, 1952). Other systems where linearity is found include the 1 : 1 iron-amino-acid complexes (Perrin, J. chem. Soc. 290, 1959) and the iron complexes of 8-hydroxyquinolines and polyaza- 1-naphthols (Albert and Hampton, /. chem. Soc. 505, 1954; Albert, Biocliem. J. 54, 646, 1953). The reported potential of 0-7 V for the iron complex of 4-hydroxy-3- carbethoxy-o-phenanthroline (Hale and Mellon, /. Amer. chem. Soc. 72, 3217, 1950) appears at first sight to be anomalously low but I think it can be readily explained. In heterocyclic compounds a hydroxyl group in a gamma position relative to a nitrogen makes two tautomers possible — an enol form, where the H is on the oxygen, and an amide form where the H is on the nitrogen. Contrary to the way the formulae are generally written, the amide form is greatly favoured relative to the enol form (for example, in 4-hydroxyquinoline the ratio is 24,000 to 1 (Albert and Phillips, J. chem. Soc. 1294, 1956) and it would be expected to be even higher for a 1-hydroxy- o-phenanthroline). I suggest that this effect, together with the sparing solubility of the substance, leads to insufficient complex formation to prevent extensive hydrolysis of ferric ion and this is what causes the potential to be so low. It should be pointed out (1) that this system did not behave reversibly and (2) that the corresponding substance without the carbethoxy group was more soluble and gave a higher and reversible potential that was closer to the expected value (Hale and Mellon, loc. cit.). In the metalloporphyrins and related substances also there seems to be, in all cases where both are known, a continuing decrease in redox potential with increasing pA' of the ligand. Some examples are given in Tables 5 and 6 of the paper of Falk and Perrin (this volume, p. 69). It seems reasonable to suppose that the change from high-spin to low-spin in a complex does not result in any great alteration in the nature of the metal-ligand bonds. The main differences would lie in the extent to which the metal's Id orbitals are made more or less available to take part in bond formation. As discussed more fully elsewhere (Perrin, Rev. pure andappJ. Chem., 9, 257, 1959) I believe these and ligand field stabilization energy changes for any series of iron complexes would make only a slight contribution to change in their overall stabilities and hence their redox potentials. Could Williams give any examples of iron complexes where the redox potentials in any series do, as he suggests, increase with the pA" of the ligand ? Williams: The arguments I use in discussing redox potentials are set out in full in my papers. There is as yet no direct evidence for the maximum Perrin discusses. On the other hand for the series of ligands H2O, pyridine, histidine, ammonia, there is good evidence that ^ohas little relationship to pA: of the base (see Dwyer, this volume, p. 25, and Falk and Perrin, this volume, p. 69). In both cases E^ goes through a maximum with pAT. I do not accept either the discussion of iron phenanthroline or iron 8-hydroxy- quinoline complexes given by Falk and Perrin {loc. cit.) and in the question, but prefer 54 Discussion our own interpretation of the data (Tomkinson and Williams, /. chem. Soc. 2010, 1958) for the reason given in that paper. I do not agree with Perrin's interpretation of ligand field theory as presented in the question and in his paper (see discussion of Orgel's paper, p. 13). I think that bonds, both in energy and in length, undergo considerable changes on change of spin type and that in biological systems these changes are of greater importance than almost any other factor. I was well aware of the data on the redox potentials of iron porphyrin complexes and have tried to use them properly and with due reservation. Perrin : The experimental values of E^ for metal porphyrin complexes with bases which Falk and I list represent probably the most complete series available in the literature. They show a general decrease with ^K of the ligands and so cannot be quoted as support for Williams' suggestion that they should pass through a maximum. I think it is dangerous to attempt to argue too finely from redox potential differences. For example, the porphyrin in cytochrome c differs from mesoporphyrin (with which it was compared) by having two CH3CHSR groups instead of ethyls. It would seem to be better to take haematoporphyrin, with two CH3CHOH groups, rather than meso- porphyrin, as an approximation to porphyrin c. The difference in redox potentials is then much smaller for the reactions : Fe++ (porph)-Py2 + OH- = Fe+++ (porph)-Py-OH + Py + e at pH 9-6, £'0 is + 15 mV for protoporphyrin, + 4 mV for haematoporphyrin, and — 63 mV for mesoporphyrin. (Lemberg and Legge, Haematin Compounds & Bile Pigments, 1949.) Potentials are even more different depending on whether the reaction is for the loss of a molecule of base from the ferric complex and its replacement by OH", as in the example just quoted, or simply for the loss of an electron from the Fe(porph)-B2 complex. The difference between the pyridine and the histidine complex of iron-pro toporphyrin is < 80 mV in the first case and about 210 mV in the second case (calculated from Barron, /. biol. Chem. Ill, 285, 1937, and Shack and Clark, /. biol. Chem. 171, 143, 1947). In the absence of other evidence as to the nature of the extra ligands in cytochromes and other metalloporphyrins, any suggestions from redox data must be almost entirely speculative. It should also be pointed out that there is not one, but many, members of each of the cytochrome families. There are, for example, many cytochromes c which, although similar in absorption spectra, do not have the same redox potential — compare cytochromes Cj and c^ where the Eo-values differ by 0-545 V (Morton, Rev. pure appl. Chem. 8, 161, 1958). Which of these should we assume from spectra or redox potentials to contain two imidazole groups bound to the metal, and how are mixed ligands to be ruled out? Williams : In all my discussions of redox potential data I have been fully aware of Perrin's points. I therefore restate that (i) All conclusions about structure are made taking into account spectra, redox, and magnetic properties (see Chem. Rev. 56, 299, 1959 for the way in which I do this). (ii) A maximum in redox potential is only expected on change of spin type. None is expected for the compounds in the series of Falk and Perrin (p. 69) except in the sequence water, pyridine, imidazole, NHg. (iii) Mixed complexes are treated later in this discussion (p. 55). The cytochromes c of different kinds are discussed by myself and Chance in Disc. Faraday Soc. 27, 269, 1959. Mixed complexes are included in that discussion. George: In answer to Williams' question about the £'0 for the Fe+++/Fe++ tetrapyridyl couple, I would like to report on the results of an investigation recently carried out in collaboration with G. Haight and A. Bergh, We thought originally, following Morgan and Burstall, that both ferrous and ferric derivatives were square planar complexes containing one tetrapyridyl molecule, somewhat analogous to haem and haemin. But while the ferric complex has the composition Fe(tetrapy)i+++, two ferrous complexes are formed with K^ >-^2» Fe(tetrapy)i++ and Fe(tetrapy)2++. K for the ferric complex is greater than K^ for the first ferrous complex, so that upon the addition of tetrapyridyl, E'q first falls below the value of 0-77 V for the Fe+++/Fe++ aquo-ion couple. But, in principle at least, as Some Physical Properties and Chemical Reactions of Iron Complexes 55 the tetrapyridyl concentration is increased Eq will eventually increase again when the oxidation-reduction reaction becomes Fe(tetrapy)i+^ + + tetrapy + t'~ -;-=^ Fe(tetrapy)2++ bright yellow reddish-violet Using Courtauld atomic models we found that co-ordination of all four TV-atoms to give square planar complexes is not possible. In all probability the structure of the Fe(tetrapy)2++ complex is that of a distorted octahedron with only three of the four nitrogens of each tetrapyridyl molecule co-ordinated to the iron. Its absorption spectrum resembles that of Fe(tripy)2++ very closely, which supports this hypothesis. The 1 : 1 complexes probably have only three bonded N-atoms, and we suppose that steric hindrance prevents the formation of the ferric complex corresponding to Fe(tetrapy)2++. Margoliash: For cytochrome c the evidence we have obtained from a study of the chemical and physico-chemical properties of the denaturation products, as well as of those of the pepsin digested 'core', indicates that cytochrome c is probably not a di-imidazole haemochrome, but probably a mixed haemochrome with a primary amino-group and an imidazole group bound to the haem-iron. Moreover, by denatur- ation it is possible to obtain products having Eq values ranging from that of native cytochrome c down to not far from 0 V. In cytochrome c the E^ value seems to be an expression of the effect of the protein configuration on the haem iron-ligand bonds rather than an intrinsic property of the particular groups involved. I should therefore think it would be difficult to ascribe specific ranges of E'q values to specific haemo- chrome-forming ligands in haemoproteins. Williams : I consider that Margoliash has studied a series of complexes, often mixtures varying from di-imidazoles through mixed complexes, to di-amines. No simple explanation of his results is possible. SPECTRA AND REDOX POTENTIALS OF METALLOPORPHYRINS AND HAEMOPROTEINS By J. E. Falk* and D. D. PERRiNf Division of Plant Industry, C.S.I.R.O., Canberra and Department of Medical Chemistry, Australian National University, Canberra Why is it that there is no relationship between the oxidation-reduction potential of the cytochromes a, b and c and their absorption spectra (Table 1)? The porphyrin side-chains of these cytochromes increase in electron-attracting power in the order c, b, a. This sequence is reflected in the spectra of the Table 1 Cyto- chromes Side-chains in positions: Fe++- cytochrome Absorption maxima (m/ii) £•0'* PH 7 (V) Pyridine haemochromes Absorption maxima (m/j) c b a 2 4 8 — CHSRCH3 — CHSRCH3 — CH==CH2 — CH=CH2 — CHOHCH2R2 — CH=CHRi — CHO 550 564 603 -i-0-255 +0077t 4-0-29 551 557 587 Here and throughout this paper, the data, for the cytochromes of animal mitochondria, are taken from Morton (1958). Side-chains other than those shown are methyl and propionic acid groups, which have little effect upon the properties discussed. For side- chains in haem a, see Lemberg, Clezy and Barrett, this volume, p. 344. * The Eo' for a reaction is the electrode potential for 50% oxidation at a stated pH. t From Colpa-Boonstra and Holton (1959). cytochromes and of the pyridine haemochromes of their prosthetic groups. But while the pyridine haemochrome of haem c has a spectrum very like that of the cytochrome itself, the spectra of cytochromes b and a are displaced far to the red of their respective haemochrome spectra. The redox potentials of the three cytochromes follow no sequence whatever in relation to their spectra, or the chemistry of their haem prosthetic groups. In the absence of protein, however, the electron-attracting power (Falk and Nyholm, 1958) of porphyrin side-chains is correlated with changes of both spectrum (Table 2) and redox potential (Table 3). * C.S.I.R.O. t Australian National University. 56 Spectra and Redox Potentials of Metalloporphyrins and Haemoproteins 57 Table 2 Suljctitiipntc in Porphyrin absorption Pyridine haemochrome 0...^. ... ... maxima, band I absorption maxima a-band deuteroporphynn IX (dioxan) at positions: (m//)* (m/i) 2 4 — H — H 618 545t — C2H5 -QHs 620 547t — CH=CH2 — CH=CH2 630 558* — H — COCH3 634 571* — COCH3 — COCH3 639 575* — H —CHO 640 578* — CHO — CH=CH, 644 583* — CHO —CHO 651 584* * From Lemberg and Falk, 1951. t This study. Table 3 Haems Side chains in positions: £0 of the (CN-)2 derivatives* of the Pyridine2 derivatives! -CH2CH2COOH -C2H5 -CHOH— CH3 -CH=CH2 -CHO -CH2CH2COOH -C2H5 -CHOH— CH3 -CH=CH2 -CH=CH, -0-247 -0-229 -0-200 -0-183 -0-113 -0-04 +0-107 * From Martell and Calvin (1952). t Vestling, 1940. Among those cytochromes b for which it has been estabhshed that the prosthetic group is protohaem, there is again no correlation between spectrum and redox potential; the same is true for those cytochromes c for which it has been established that haem c is the prosthetic group (Morton, 1958). These anomalies can be due only to some particular properties of the proteins. If we regard these biological compounds as further co-ordination complexes of haems, to what extent can these differences from model com- plexes be explained in terms of the nature of the protein-haem iron bonds ? It is possible that, as ligands, the proteins may have properties difficult or impossible to reproduce in model systems. Thus, the stereochemical environ- ment of the protein ligand atom may influence the way in which this atom co-ordinates. The protein, and in intact tissue perhaps other macromolecules, 58 J. E. Falk and D. D. Perrin may well create a specific micro-environment about the haem molecule. The evidence v/hich Dr. Wang has found (Wang, Nakahara and Fleischer, 1958) that the dielectric properties of the medium affect some co-ordination properties of haem may be an example of such an effect. Nevertheless, we believe that it is important to see how much or how little can be learned from the study of model systems. The haemochromes which have been studied in any detail that bear any likely relationship to natural haemo- proteins are, practically without exception, complexes between haems and ligands containing unsaturated nitrogen atoms (=N — ). But complexes of protohaem with such ligands do not have the range of absorption maxima of the cytochromes b (which are protohaem complexes), nor do they have comparable redox potentials. We wish to outline here some theoretical aspects of porphyrins and metalloporphyrins and to draw some inferences from existing data. We discuss below (p. 74) some studies we are making of complexes formed by several different haems with ligands of a variety of chemical types. SOME THEORETICAL ASPECTS OF PORPHYRINS AND METALLOPORPHYRINS It is convenient to picture the porphyrin molecule as a framework of atoms held together by ordinary, two-electron, single {g) bonds, while the remainder of the valence electrons occupy molecular orbitals which extend over the whole of this framework. The strong delocalization of these mobile (tt) electrons confers considerable stability and 'aromatic' character on the porphyrins. Electron-withdrawing substituents on the peripheral carbon atoms reduce the 77-electron density on the pyrrole nitrogens, so that it becomes easier for the protons to dissociate from the two pyrrole N — H groups which make the porphyrin molecule a weak dibasic acid. Though little precise data exist, this clearly increases the acid strength of the porphyrin (lowers its pi^„) and, as discussed later, raises the oxidation-reduction potential of metalloporphyrins. The Absorption Spectra of Porphyrins Another consequence of the extensive 7r-electron delocalization is that the highest of the occupied molecular orbitals and the lowest of the vacant orbitals differ in energy by an amount small enough for transitions between them to give rise to absorption bands in the visible and near ultra-violet. In the porphyrins themselves in neutral solvents there are four bands in the visible in addition to the Soret band at about 400 m^. There appears to be good reason to believe (Piatt, 1956) that the four visible bands are really two pairs of bands which would be superimposed if the porphyrin nucleus were strictly square and uniformly substituted. X-ray analysis of the closely similar phthalocyanine molecule has shown that its structure is slightly Spectra ami Redox Potentials of Metalloporphyrins and Haemoproteins 59 distorted from square (Robertson, 1936), probably because the hydrogens bound on opposite nitrogen atoms each form hydrogen bonds with an adjacent nitrogen atom. This distortion probably occurs in the porphyrins also (Mason, 1958; Piatt, 1956), leading to the fairly constant difference of 6-7 kcal between the energy levels of corresponding absorption bands (I and III, II and IV). This difference is removed in the di-anion, the di-cation and the metal complexes, and in all these cases only two of these bands are found. By making some simplifying assumptions Longuet-Higgins, Rector and Piatt (1950) and Seely (1957) have carried out molecular orbital calculations to find the nature of the transitions giving rise to the observed porphyrin spectra. Essentially the same conclusions are reached using an electron-gas model (Kuhn, 1959). The visible bands all arise in a similar manner, as transitions between filled orbitals of /IgM'typ^ symmetry and vacant ^'^-type orbitals (Fig. 1). In all cases these bands are associated with an electronic displacement towards the periphery and this may be either along (bands III and IV) or perpendicular to (bands I and II) the axis tlirough the two H's which are on opposite nitrogens (Piatt, 1956; Mason, 1958). Because bands I and III are for a 0-0 vibrational transition which is classed as forbidden, their intensities will depend very much more on any loss of symmetry in the poi-phyrin molecule than will bands II and IV which are interpreted as 0-1 vibrational bands (Piatt, 1956). This symmetry, which is to be thought of in terms of possible pathways for the mobile electrons, will not be much affected by substituents such as alkyl groups, or carboxyl groups which are insulated by at least two CHg's as in propionic acid side-chains. Much greater distor- tion would be expected from substitution of a peripheral hydrogen by a group such as — CHO, — COCH3, — COOCH3 and COCgHj, and it is among such porphyrins that 'oxorhodo' (III > II > IV > I) (Lemberg and Falk, 1951) and 'rhodo' (III > IV > II > I) type spectra are found rather than the 'actio' (IV > III > II > I) type which is the usual one with alkyl and similar substituents (Stern and Wenderlein; for references see Lemberg and Falk, 1951). Extension of tliis generalization to porphyrins containing more of the symmetry-disturbing groups is difficult because of the necessity to allow for the vector directions of the moments of the substituents, but if this is done there is a reasonable correlation between observed and predicted spectra (Piatt, 1956). The a- (long wavelengths) and /S-bands of metal-porphyrin complexes seem to be related to bands I and III, II and IV, respectively. Thus bands II, IV and /5 are little affected by substituents, while bands I, III and a vary considerably. It has also been shown that the intensity of the a-band in some copper-porphyrin complexes varies with the intensity of band III of the corresponding porphyrins (Williams, 1956). The Soret band is attributed to the transition to an ^^-type orbital of an electron in an y4i„-orbital (Fig. 1) in which it was confined to the carbon atoms H.E. — VOL. I — F 60 J. E. Falk and D. D. Perrin of the pyrrole rings. The ^'^-orbital is one of a pair of equal energy strongly polarized along and perpendicular to the axis of the two NH's. Although the overall movement of electronic charge towards the periphery is negligible, the increase of electron density on the non-pyrrole carbons and a pair of Fig. 1. Nature of transitions giving rise to porphyrin spectra. The signs + and — indicate wave functions. The areas of the circles give electron densities in the vicinities of atoms ; the atomic distances are the same as in phthalocyanine (Robertson, 1936). opposite nitrogens (Fig. 1) affects the absorption spectrum of porphyrins and metalloporphyrins. Co-ordination of Porphyrins with Divalent Metals The property that distinguishes transition metals from other elements in the Periodic Table is that their 6?-orbitals are incompletely filled with electrons. These orbitals have the directions in space shown in Orgel's paper {loc. cit.). Depending on whether electrons occupy these orbitals singly or in pairs, complexes will be para- or dia-magnetic. This difference in magnetic properties is interpreted in the Valence Bond Theory as distinguishing two types of complexes. The main concept underlying Spectra and Redox Potentials of Metalloporphyrins and Haemoproteins 61 this theory, as applied to co-ordination complexes, is that suitable vacant orbitals of the metal are hybridized, and these hybrid orbitals are filled by electron pairs 'donated' by ligand atoms with the formation of cr-bonds. To be suitable for hybridization in this way an orbital must have an appreciable component in the directions finally occupied by some or all of the ligands. The diamagnetism of pyridine haemochromes is interpreted to mean that two of the 3i/-orbitals of Fe++ {d^i^yi and d^^ are used in this hybridization and are occupied by two pairs of ligand electrons ; the electrons already in these orbitals are forced to pair up in the remaining 3c/-orbitals. Such com- plexes have long been called 'covalent' and, more recently, 'inner-orbital', 'spin-paired', or 'low-spin'. Their formation is favoured by ligands of low electronegativity and in octahedral complexes such as pyridine haemochrome they are described as being ZdHsAp^, or d'^sp'^, types. On the other hand, haemin chloride, like the Fe+++ ion itself, has 5 unpaired electrons. Such paramagnetic complexes ('semi-ionic', 'outer-orbital', 'spin- free', 'high-spin') are generally formed by ligands of high electronegativity which, in addition, have little or no d acceptor capacity for double bonding (e.g. F" as against pyridine-N). It is now believed that high-spin complexes do have some degree of covalent bonding (cf. Craig et al., 1954) and it is convenient to regard haemin chloride, for example, as a hybrid of the type AsApHd"^. There is little doubt that in 'haemin chloride' (ferriprotohaem chloride), traditionally regarded as a square-planar complex with the Cl~ ionically associated, the Cl~ is 'co-ordinately' bound. Falk and Nyholm (unpublished) have found a 0-001 m solution in nitrobenzene to be a non- conductor of electricity. Under these conditions, univalent electrolytes have conductivities of 20-30 r.o. It appears likely that ferriprotohaem hydroxide ('haematin') is a similar complex. A more recent and more satisfying interpretation of the magnetic and other properties of complexes is provided by the Ligand Field Theory (Griffith and Orgel, 1957). In essence, this theory says that as co-ordinating groups, or ligands, approach a metal ion to form a complex, ^-orbitals pointing towards the ligands are raised in energy and electrons in them become less stable, while J-orbitals pointing away from the ligands become more stable. Bonding molecular orbitals are formed by suitable electron-filled orbitals on the ligands with the metal's vacant s- and /7-orbitals and the ^/-orbitals which point tov/ards the ligands; in octahedral complexes, the c^-orbitals involved are d^2_y2 and d^2. Any electrons already in these ^/-orbitals are removed by promoting them into antibonding orbitals, but their presence reduces the stability of the final complex. In any complex the magnitude of the differences in the energy levels of the various J-orbitals is a function both of the ligand and of the geometrical shape of the complex itself. The electrostatic effect of ligands in splitting these levels is enhanced by *back double bonding', which arises from the ability of suitably placed, 62 J. E. Falk and D. D. Perrin occupied ^-orbitals on the metal to form molecular orbitals with vacant 7T-orbitals on the ligand, so that these electrons gain in stability. (If the 77-orbitals are already filled the interaction is repulsive.) Similar interactions can take place between vacant orbitals on the metal and filled 7r-orbitals on the ligand. Effects such as back double bonding cannot be separated from True octahedral complex ZA ----B- I-9J 0=0 /^/ N — i-N Globin 10 Square planar complex HjO / Pe / N— |-N Globin 05 Ratio of ligand field in z direction to ligand field in x or y direction Fig. 2. Ligand field splitting of ^-orbitals in Metalloporphyrins (qualitative only). purely electrostatic contributions and they are probably important factors in unsaturated ligands, including porphyrins, oxygen, carbon monoxide, cyanide ion and unsaturated heterocyclic bases, all of which exert strong ligand fields. The porphyrin nucleus confers a planar configuration on its metal com- plexes, and any additional co-ordination sites on the metal are perpendicularly above and below this plane (i.e. along the z-axis). This leads with most transition metals to a more or less vertically distorted octahedral structure and, in the metalloporphyrins, the effect of ligands occupying these positions is to split the ^^-orbital energy levels as shown qualitatively in Fig. 2. If the energy separations are greater than the energy needed to pair electrons in the lower energy levels, diamagnetic or low-spin complexes will be formed; Spectra and Redox Potentials of Metalloporphyrins and Haemoproteins 63 otherwise there will be as little spin pairing as possible. For example, in haemoglobin the situation is probably something like that shown at A in Fig. 2. The diflference in ligand field stabilization energy (L.F.S.E.) between the low-spin and the high-spin alternatives is not great enough to prevent the ^/-electrons of the ferrous iron from spreading over all five of the 3fif-orbitals, four of which are occupied singly while the fifth, and lowest, holds a pair of electrons. Replacement of the water molecule in the sixth co-ordination position by oxygen, to give oxyhaemoglobin, displaces conditions towards B in Fig. 2, where the L.F.S.E. difference is sufficient to make the filling of the three lowest orbitals with pairs of electrons the more energetically-favoured process, giving a diamagnetic complex. For theoretical reasons complexes intermediate between high-spin and low-spin are unlikely. It is unnecessary to postulate any sudden changes in the nature of the ligand-metal bonds and, in fact, it is an important consequence of this theory that the magnetic behaviour of complexes does not provide a means of classifying complexes into 'outer-' and 'inner-orbital' or 'ionic' and 'covalent'. THE SPECTRA OF SOME PORPHYRINS AND THEIR METAL COMPLEXES Because the visible absorption spectra of porphyrins are associated with a displacement of electrons towards the periphery of the porphyrin nucleus, any effect which results in an extension of the distance the electrons can move in this direction reduces the energy required for these transitions, so that visible absorption maxima move to longer wavelengths. Any effect operating in the opposite direction moves these absorption maxima to shorter wave- lengths. A number of examples illustrate this. The Ejfect of Porphyrin Side-chains As has already been seen (Table 2) the spectra of a series of free porphyrins, and of the pyridine haemochromes of their Fe '^ "^ complexes, move to longer wavelengths stepwise as the electron-attracting power of the porphyrin side- chains increases. This porphyrin side-chain effect operates similarly in the simple (square) porphyrin complexes with a variety of divalent metals (cf. Table 2 of Falk and Nyholm, 1958), and indeed throughout the metallo- porphyrins of all types, including, in a broad sense, the haemoproteins (cf. Table 1). Among the latter, replacement of the protein with pyridine is a convenient way to obviate effects on spectrum peculiar to the protein; pyridine haemochrome spectra reflect accurately the effects of electron- attracting side-chains on the haem nucleus. The Effect of Co-ordinated Metal Ions Falk and Nyholm (1958) have compared the protoporphyrin complexes of a number of different divalent metal ions. It was found that the following 64 J. E. Falk and D. D. Perrin complexes fell into three classes, according to magnetic susceptibility, spectroscopic and other properties : A Co++, Ni++ B Cu++, Ag++ C Zn++, Cd++ From the point of view of valence-bond theory, the conclusion was reached that class A were spin-paired (3d4s4p^), class B spin-free (4s4pHd, 5s5p^5d respectively) ; both A and B have great (qualitative) stability. Class C is also spin-free {4s4pHd, 5s5p"5d) but of great (qualitative) instability. Within the three classes the spectra were very similar, but from ^4 to 5 to C the visible absorption moved to longer wavelengths. From Fig. 2, the essential difference between classes A, B and C is that they have 0, 1 and 2 electrons, respectively, in the d^i_y2 antibonding orbital. These electrons are favourably placed for strong repulsive electrostatic interaction with electrons on the pyrrole-N's, so that the more electrons in d^2_y2 the easier it is to displace 7T-electrons towards the periphery. Hence, in agreement with experiment, the visible absorption maxima move to longer wavelengths in passing from yl to jB to C. In the transition which gives rise to the Soret band there is a displacement of some of the electron density to the non-pyrrole (methene bridge) carbons, i.e. in a direction away from d^2_y2 (Fig. 1). This transition should be facilitated in the same way and the Soret maximum should shift to longer wavelengths in a similar sequence, as observed by Falk and Nyholm (1958) for the protoporphyrin complexes in benzene, which at the time appeared to be an inert solvent. It has recently been found (J. N. Phillips, unpublished) that in fact this solvent modifies the spectra of certain of the metalloporphyrins. The measurements have been repeated in CCI4, which shows no evidence of interaction, and the follov/ing maxima have been found : Co Ni Cu Ag Zn Cd a-band, m/< Soret, m/.i 561-5 403 561 403 573 409 570 417-5 579 411-5 5875 414 The a-bands have virtually identical maxima in benzene and in CCI4, as do the Soret bands of the Co, Ni, Cu and Ag complexes. The Soret bands of the Zn and Cd complexes in benzene, however, were at 415 and 423 m/< respectively. In the square planar, low-spin, cobalt (d"^) and nickel (d^) complexes, since there are the same number of electrons in d-^y (=2) and none in dj.2_y2, similar Soret and visible maxima would be predicted and have been found. In the same way the ferrous 6/>pyridine complexes which are diamagnetic Spectra and Redox Potentials of Metalloporphyrins and Haemoproteins 65 with an octahedral distribution of ^-orbital splittings, have no electrons in d^2_yi and two in d^y. From a spectroscopic point of view they therefore approach conditions for square planar nickel and cobalt complexes. Back double bonding should shift the visible bands to slightly shorter wavelengths and the Soret band to slightly longer wavelengths. Thus, for the proto- porpiiyrin metal complexes the predicted order of the visible wavelength maxima is: Co c;^ Ni > FePyg, and the observed values are 561, 561 and 558 m/< respectively. Although the stability of porphyrin metal complexes is due in large measure to the difficulty of providing enough energy to rupture four bonds, whether electrostatic or covalent, simultaneously, an additional effect in transition elements is the ligand field stabilization energy. Considerations of L.F.S.E. would suggest that the stability of these metalloporphyrins should lie in the series, A> B> C. This is because electrons occupying low lying ^-orbitals increase the stability of a complex, while those in higher (antibonding) orbitals will remove some of this stability. The additional stabilization becomes zero when all five of the J-orbitals are equally occupied. We estimate the L.F.S.E. for the bivalent Co, Ni, Cu and Ag complexes of protoporphyrin dimethyl ester to be at least 40 kcal, and this may be one factor contributing to the quahtative differences in the difficulty of dissociation of the metal from these complexes, as against the Zn, Cd and Pb complexes (which have no L.F.S.E.) (Falk and Nyholm, 1958). For ferro- and ferrihaemoglobin the estimated L.F.S.E.s are 20 kcal and zero, respectively. The great stability of ferric iron in complexes is probably mainly due to the electrostatic forces of attraction between opposite charges. The Effect ofLigands in the 5th and 6th Co-ordination Positions We restrict our discussion here to further complexes of iron-porphyrins, i.e. the haemochromes. The 5th and 6th positions on these complexes, above and below the plane of the haem molecule, correspond to positions 1 , 6 in octahedral complexes in general co-ordination chemistry. We discuss the spectra of the Fe++ complexes only, since, as is commonly recognized, their spectra are not complicated by the large component which has been attributed (Williams, 1956) to charge-transfer from the ligand to the metal in the Fe+++ complexes. Ligands to haem iron such as pyridine and chemically similar bases, and CN~ ion, allow back-double-bonding of the electrons in the dy^ and d^^ orbitals of the metal. The time these electrons spend in the plane of the porphyrin molecule, and hence the average electron density in this plane are reduced, so that it is harder for electrons to move towards the periphery, and the visible absorption maxima move to shorter wavelengths. That is, there is less electrostatic repulsion in the ground state if double bonding can occur. 66 J. E. Falk and D. D. Perrin Ligands such as ROH (including HOH), RS", RCOO-, HQ- and others, by their dipolar or electrostatic interactions with the metal ion, should facilitate the movement of the mobile porphyrin electrons away from the metal, and shift absorption to longer wavelengths. Among the haemoproteins, one clear example of these two effects is seen when Fe++ cytochrome c and Fe++ peroxidase are compared. The former is low-spin (diamagnetic) and the latter is high-spin (4 unpaired electrons found). The visible absorption maxima lie at 520, 550 m/^ and 558, 594 m/< respec- tively (Lemberg and Legge, 1949). In cytochrome c one, and possibly both, protein groups bound to the haem iron are histidine nitrogens, and in peroxidase the groups are a — COO on one side of the haem and probably a water molecule on the other (Chance, 1952; Theorell and Paul, 1944). Among Fe++ haemoglobin derivatives a similar change from high- to low-spin (para to diamagnetism) is found on substitution of water (Hb itself) by the double-bonding ligands O2 or CO. Similarly, when the 6th position on the haem of Fe+++ haemoglobin is occupied by HgO, F~, 0H~, EtOH, high-spin complexes, and by the double-bonding ligands CN~, — SH, Ng" or imidazole, low-spin complexes are found (for references see Falk and Nyholm, 1958). Similar examples are to be found among peroxidase derivatives (Lem- berg and Legge, 1949). Complexes of the haems with non-protein ligands have been studied extensively, but many of the data which may be very relevant to understanding of the haemoproteins are lacking. For comprehen- sive reviews of this subject, see Lemberg and Legge (1949) and Martell and Calvin (1953). As might be expected, with ligand atoms of high field strength and also capable of double bonding, such as =N — (in pyridine, nicotine, a-picoline, imidazole, etc.), low-spin complexes are formed. Thus all the complexes of protohaem listed in Table 5 are diamagnetic. Though there must be variations in complex-forming ability between these ligands, as indicated by their pA!' and Eq values (Table 5), the spectra of the complexes are very similar; this is probably because of comparable back-double bonding ability. In these hexaco-ordinate complexes, the wavelengths would be expected to be modified by the ligands on the 5th and 6th co-ordination positions only if these hgands alter the spatial distribution of the electrons round the central metal ion in such a way as to affect electronic transitions in the plane of the porphyrin molecule. Such effects would be expected if spin-free and spin-paired com- plexes were compared, or even in complexes with very different amounts of back-double-bonding, spin-paired and least back-bonded having maxima displaced towards longer wavelengths. However, especially with unrelated ligands, there is no reason why the factors that govern their complex-forming ability with two different valence states of a metal should bear any relation to the effective component of the metal's c?-electrons at right angles to the direction of bond formation, Spectra and Redox Potentials of Metalloporphyrins and Haemoproteins 67 as would be required for any general correlation of spectra with redox potential. OXIDATION-REDUCTION POTENTIALS Factors affecting the oxidation-reduction potentials of metal complexes (Perrin, 1959b) include: (i) Purely electrostatic effects of attractions between ions or dipoles of opposite charge. Thus if the ligand is an anion this will always favour the higher valent state of the metal and the potential will be less than for the corresponding free metal ions in water. This is because the standard oxidation-reduction potential, E^, of any pair of ligand- metal complexes is related directly to the stabiUty constants of the complexes by the identity, 2'3036RT^, ,, , .. ^ E1 = Em- 7 7^ (log ^M"^ - log Kj^m+) (n — m)r where E^ is the potential of the free metal ions. (ii) Back-double-bonding. Because this involves the removal of electrons from the vicinity of the positively charged metal ion, the effect is always greater for the lower valent state of the metal, so that it tends to raise the oxidation-reduction potential. (iii) Ligand field stabilization energies. The difference in L.F.S.E. varies with the particular pairs of cations and the ligands, as discussed in Dr. Orgel's paper (loc. cit.). For example, in weak ligand fields ferrous ion, but not ferric ion, is stabilized in this way : this raises the potential. On the other hand, in manganous, manganic systems manganic ion is stabilized but not manganous ion, so the potential is lowered. (iv) The acid dissociation constants (p/sTa's) of the ligands. In many series of closely related ligands the stabiUty constants of metal com- plexes vary with p^^ ^^ ^^ approximately linear fashion : log K ^^ apAr„ •\- c. Such a relation would be expected if the factors governing the binding of protons and cations by ligands were similar. From a simple electrostatic model it has been predicted that a should increase with increasing cationic charge (Jones et al., 1958). As a direct consequence, the oxidation-reduction potentials of metal complexes in which the ligands are sufiiciently similar should decrease linearly with increasing ipK^. For several series of iron complexes this has been found to be the case (Perrin, 1959b). In the porphyrins, electron-withdrawing substituents at the peripheral carbons increase the acid strength of the porphyrin (lower the pATJ and hence raise the oxidation-reduction potential of the metal complex. Martell and 68 J. E. Falk and D. D. Perrin Calvin (1953) have discussed this correlation between electron-withdrawing effect and Eq; potentials of iron complexes rise as the electron-attraction of side-chains increases (Table 3). The differences, although rather small, lie in the expected order. In suflEiciently alkaline solutions, complexes of nitrogenous bases with iron- porphyrins give oxidation-reduction potentials which vary Unearly with pH. The reaction can be written: Fe+++B2 + OH- ^ Fe++B . OH + B + e although the ferric complex may be present mainly as an easily split dimer (Shack and Clark, 1947). Such potentials should therefore be compared at approximately constant base concentration and pH. Around pH 9-6 reported potentials are as shown in Table 4, Table 4 Haems Side-chains in positions: £■0 at pH 9-6* (Pyr.)2 complex Eo at pH 9-6* (a-Picoline)2 complex 2 4 — CgHj — C2H5 — CH2CH2CO2H — CH2CH2CO2H 2 6 4 7 — C2H5 — C2H5 — C2H5 — C2H5 2 4 — CHOHCH3 — CHOHCH3 — CH=CH2 — CH^=Cri2 —CHO — CH=CH2 -0-063 -0-036 -0-029 +0-004 +0-015 -0-099 -0-033 -0010 Data from Martell and Calvin (1952). * For the reaction Fe+++B2 + OH- ^ Fe++BOH + B + e. A different kind of comparison can be made by keeping the porphyrin nucleus constant and varying the base co-ordinating with the iron complexes. Taking the data of Barron (1937) for protoporphyrin-iron we find that at pH 9-2 nicotine, pyridine and a-picoline give oxidation-reduction potentials showing the expected pH-dependence — A^"/ ApH = 0-06. On the other hand, the slope for histidine and pilocarpine is much less, indicating that the complexes Fe+++B2 are also present in significant amounts. To minimize this interference the Eq values listed in Table 5 have been calculated from the data using as low a concentration of bases as possible. The series shows a roughly linear dependence on pK^, with a slope of the order of — AEfApK <^ 0-04 V. This slope is similar in magnitude to that found for 1 : 1 iron Spectra and Redox Potentials of Metalloporphyrins and Haemoproteins 69 complexes with amino-acids, 8-hydroxyquinoline and o-phenanthroline (Perrin, 1959a). Table 5. Complexes of protohaem with bases Base ^Ka [B] M E',* pH 7 Haemochrome a-band, m/< Nicotine 3-15 0-04 0-200 5581 Pyridine 5-2 0-7 0-158 558t a-Picoline 6-2 0-5 0-115 558t Histidine 6-0 0-03 > 0-079 Pilocarpine 7-0 002 > 0-052 556-2t * Fe+++B2 + OH- t This study. + Barron (1940). Fe++BOH + B + e. Much less information is available for reactions of the type Fe++B2 ^ Fe+++B2 + e which become important in neutral solutions. For iron-protoporphyrin we find the values shown in Table 6. As would be expected the anion, CN"", Table 6 Complexes of protohaem with: ^Ka p * Pyridine Histidine Pilocarpine Water Cyanide ion 5-2 6-0 7-0 +0-107t -0-1051: -0-13t -0-14t -0-183t * For the reaction Fe^+Bg t Shack and Clark (1947). t Barron (1937). Fe+++B2 + e. stabilizes the ferric state to a greater extent than any of the neutral ligands : among the latter the ferric state is favoured the higher the p/f^ of the ligand. Compared with the cyanide complexes, potentials for these reactions appear to be much more sensitive to change in the porphyrin in the complex if the ligand is neutral (pyridine. Table 3). SUMMARY AND CONCLUSIONS We have outlined some theoretical aspects of the absorption spectra of porphyrins and metalloporphyrins, of the co-ordination chemistry of metallo- porphyrins, and of the redox potentials of haems and haemochromes. 70 J. E. Falk and D. D. Perrin It has been shown that the following firm correlations exist among the non-protein complexes : (a) The more electron-attracting the side-chains on the porphyrin nucleus, the greater the shift to longer wavelength of the visible absorption maxima of porphyrins, their square metal complexes, and their haemochromes with double-bonding ligands. (b) The more electron-attracting the porphyrin side-chains, the more positive the redox potential of the haemochromes formed by double- bonding ligands. (c) The greater the electron-donation (higher pK^) of double-bonding ligands, the more negative the redox potential of the haemochromes formed by them. (d) For ligands, in the above group, of ipK^ from about 3 to 7, although Eq varies by about 150 mV, the spectra of the haemochromes hardly differ. Reasons for this have been discussed. Among the haemoproteins : (a) The effects of porphyrin side-chains upon both spectrum and redox are obscured and overweighed by the effects of the proteins. (b) The haemochromes which have been studied in any detail are mainly those formed by double-bonding ligands, and their spectroscopic and redox properties may have little value as models for haemoproteins, even in cases hke cytochromes c, which resemble haemochromes in some ways. REFERENCES Barron, E. S. G. (1937). J. biol. Chem. Ill, 285. Barron, E. S. G. (1940). J. biol. Chem. 133, 51. Chance, B. C. (1952). Arch. Biochem. Biophys. 40, 153. CoLPA-BooNSTRA, J. & HoLTON, F. A. (1959). Biochem. J. 72, 4P. Craig, D. P., Maccoll, A., Nyholm, R. S., Orgel, L. E. & Sutton, L. E. (1954). J. chem. Soc. 332. Falk, J. E. & Nyholm, R. S. (1958). Current Trends in Heterocyclic Chemistry, p. 130. Butterworths, London. Griffith, J. S. & Orgel, L. E. (1957). Chem. Revs. 11, 381. Jones, J. G., Poole, J. B., Tomkinson, J. C. & Williams, R. J. P. (1958). J. chem. Soc. 2001. Kuhn, H. (1959). Helo. chim. Acta 42, 363. Lemberg, R. & Falk, J. E. (1951). Biochem. J. 49, 674. Lemberg, R. & Legge, J. (1949). Haematin Compounds and Bile Pigments, Interscience, New York. Longuet-Higgins, H. C, Rector, C. W. & Platt, J. R. (1950). J. chem. Phys. 18, 1174. Martell, a. E. & Calvin, M. (1952). Chemistry of the Metal Chelate Compounds, Prentice-Hall, New York. Mason, S. F. (1958). /. chem. Soc. 976. Morton, R. K. (1958). Rev. pure appl. Chem. 8, 161. Perrin, D. D. (1959a). J. chem. Soc. 290. Perrin, D. D. (1959b). Rev. pure appl. Chem. 9, 257. Platt, J. R. (1956). Radiation Biology, 3, 101. McGraw-Hill, New York. Spectra and Redox Potentials of Metalloporphyrins and Haemoproteins 71 Robertson, J. M. (1936). /. chem. Soc. 1195. Seely, G. R. (1957). J. chem. Pliyi. 27, 125. Shack, J. & Clark, W. M. (1947). J. biol. Chem. 171, 143. Theorell, H. & Paul, K.-G. (1944). Ark. Kemi. Min. Geol. 18A, No. 12. Vestling, C. S. (1940). J. biol. Chem. 135, 623. Wang, J. H., Nakahara, A. & Fleischer, E. B. (1958). J. Amer. cliem. Soc. 80, 1109. Williams, R. J. P. (1956). Chem. Rev. 56, 299. DISCUSSION Correlations between Structure and Physical Properties George : With regard to the correlations that are being sought between cheniical reactivity, physical properties and structural factors in co-ordination chemistry, and their exten- sion to haemoprotein compounds, I would call attention to the more detailed and revealing information that can often be obtained from A/f" and AS" data, which it is not possible to get if only AG" data (e.g. E'q and pAT values) are considered. In many cases, notably when ligand field effects are being investigated, A/f" is the significant thermodynamic quantity: and the successful correlations based on AG" probably result from the values of A^" remaining relatively constant throughout a series of compounds. But in some instances apparently valid conclusions based on AG" turn out to be rather misleading. For example there is the well-known correlation between the affinity of structurally similar ligands for a metal ion and for the hydrogen ion; linear plots are obtained for log (stability constant) against ^K. This suggests that the stronger the bond to hydrogen, the stronger is the bond to the metal. Yet an examination of the rather scanty thermodynamic data which are available indicates that the correlation is only determined by the A//" values, which contain the bond energy terms, for certain families of ligands, and that for others the AS" values dominate the relationship. Similarly oxidation-reduction potentials are often regarded as a relative msasure simply of the energy required to remove an electron from the reduced form of the couple. But this is not always so. For example, the Eq values for the Fe(dipy)3^+/^+, Feaci^+/^+ and Fe(CN)6=^-/*- couples are about 1-0, 0-77 and 0-36 V respectively. Yet this sequence is not determined by the electron-donating property of the ligands following the sequence CN" < HjO < dipyridyl. The values of Ai/" for the cell reaction in the presence of these ligands Fe'" + iH2^Fe" + H+ are about —30, —10 and —26 kcal/mole respectively, which show that the contri- bution from the ionization potential of the Fe++ compounds is more nearly the same for the dipyridyl and the cyanide complexes, and that entropy changes play a very dominant role in determining the magnitude of Eq. This is not unexpected in view of the entirely different charge changes in the cell reactions, -1-3 to -1-2 in contrast to —3 to —4 respectively. Somewhat more surprising is the unfavourable entropy contribution for the corresponding couples of haemoglobin and myoglobin as com- pared to the favourable entropy contribution for the aquo-ion couple. The apparent charge changes are -f-l to zero and -1-3 to +2 respectively, both of which should make a favourable contribution to AS'". A//" is nearly the same as that for the aquo- ion couple, and it is the entropy change which is responsible for the Eq values for the two haemoproteins being some 0-6 V lower, i.e. at about 0-2 V compared to 0-77 V. In setting up correlations, therefore, it can be extremely important to determine whether A//° or A5" is the dominant factor. Spin Type and Spectra of Haem Compounds Williams: The spectra of metal porphyrins have recently been analysed by Gouterman (/. chem. Phys. 30, 1 139, 1959). The suggestion that the band positions are solely due 72 Discussion to the CT-bonding power of the metal is, I believe, incorrect. The band positions are partly affected by metal acceptor power through a-bonds and partly through the 77-bond system (see Williams, Cliem. Rev. 56, 299, 1956). However the total analysis of the spectra of porphyrins given by Gouterman is the best available to date. The diagram in my paper. Fig. 1 (this volume, p. 43), is based on relatively little evidence for protoporphyrin and mesoporphyrin complexes. Lemberg has pointed out to me that it is inadequate for haem a complexes. While I deal with his complexes later I should like now to state the general case (see Fig. 1). In the region of high-spin complexes in both oxidation states Amax of both ferrous and ferric complexes moves to longer wavelengths with increasing a-donor power of the ligands perpendicular to the porphyrin plane. This is also true for the band positions of completely covalent complexes. Both these statements have theoretical and practical support. In the region of mixtures of high- and low-spin complexes the Soret band of Fe++ complexes moves to shorter wavelengths while that of Fe+++ does not. The reasons I give for these shifts remain unaltered but here the discussion is empirical first and theoretical afterwards. In this region all the spectra are composite, part being due to low-spin and part due to high-spin forms. Unlike Falk, I consider that some correlation between spectra and spin form must be established by us for otherwise there is little hope of knowing in what state the cytochrome is in the cell. It may be that the correlations I suggest are only partially true but I know of few exceptions to them. In order to avoid terminological difficulties could I point out the equivalent definitions: ionic = high-spin = weak field covalent = low-spin = strong field The discussion of redox potentials given in my paper needs modification. In an exchange with Chance (Faraday Society Discussion on Energy Transfer, 1959) I was led to the conclusion that £)-type cytochromes are mixed imidazole, amine porphyrin complexes. The diagram of redox potential against ligand basicity now should be as in Fig. 2 of my paper (this volume, p. 47). I have attempted to show the relationships to simple haem complexes at the risk of over-simplification. My table of conclusions on the basis of spectra and redox poten- tials (this volume, p. 48) is incorrect now and should read: Cytochrome b, one amino, one imidazole. From some work we have done recently (Brill and Williams, un- published) we have good reasons for supposing that peroxidase is an amine carboxylate complex rather than a simple carboxylate complex. The chemical reactions of model compounds with oxygen have recently revealed a possible cause of the differences between cytochrome 03, uptake of oxygen and rapid autoxidation, and haemoglobin, uptake of oxygen only. The reactions we have ob- served are that whereas Fe (dimethylglyoxime)2 (imidazole)2 picks up oxygen reversibly, Fe (cyclohexane dione-dioxime)2 (imidazole)2 first picks up oxygen and is then slowly oxidized to a conjugated hydrocarbon. The oxidation is also possible with ferri- cyanide, and iridichloride. Schematically we have: A B Fe++A'(imidazole)2 <^ Fe++A' (imidazole)02 ^ Fe+++A' (imidazole)2 The reversible reaction. A, goes if X has no hydrogens which can react with O2 of the complex, while B follows if X contains suitable hydrogen. If analogies are helpful, the oxidation of cytochrome a^ by O2 is due to the removal of a hydrogen from a sensitive group interacting with the porphyrin and the electron transport is initiated by hydrogen oxidation. Falk : In the paper with Perrin (this volume, p. 56), the haemochromes in our Table 5 are low-spin by magnetic susceptibility measurements; the ligands forming these compounds are =N — atoms of pAT values varying from 3 to 7. The visible spectra hardly differ from each other. I do not have Soret band measurements. In the com- pounds with primary and secondary aliphatic amines described a little later in this discussion, changes directly related to ^K occur in both X and e of the visible bands. Spectra and Redox Potentials of Metalloporphyrins and Haemoproteins 73 Soret band data are not available; magnetic susceptibility measurements have not yet been made on these. Because I suspect that there may be surprises in store in the magnetic properties of some of these new compounds, I suggest that we should be careful to refer to low-spin and high-spin compounds only when definitive magnetic susceptibility measurements have been made. If inferences are being made from spectra only, pending magnetic measurements, perhaps we could say 'low-spin spectral type', and so on. With this reservation, I do agree with Williams that we must continue to develop correlations between spectrum and spin-type. Perrin : I suggest that the electronegativity of the central metal is of less consequence in determining spectral shifts in porphyrin complexes than are the number of its d- electrons and the directions of the orbitals they occupy. Falk and I discussed this. In the case of ferrous ion the change from a high-spin to a low-spin configuration means that the unpaired electrons in the d^^_y''- and d^^ antibonding orbitals vacate these positions and all six of the rf-electrons fill, in pairs, the d^y, dy^ and d^.^ orbitals. From the spatial distribution of these orbitals it is easy to see that this leads to an increase in electron density along the xy axes (i.e. through opposite methene carbon atoms) and a reduction in density along the x and y axes; the net increase in electron density in the plane of the porphyrin ring is probably small. If the transitions giving rise to porphyrin spectra are of the types shown in Fig. 1 of our paper (based on Longuet- Higgins, Rector and Piatt, 1950; Seely, 1957; and Kuhn, 1959; see Falk and Perrin, p. 56, for references), what effects have the change from a high-spin to a low-spin ferrous complex on these transitions? The important thing to remember, and what distinguishes low-spin ferrous complexes from low-spin ferric complexes, is that in the former the dx^_y''- electron of the high-spin state has gone into the d^y orbital, whereas in the latter it is in the dy, or d^^ orbital. In ferrous complexes this makes the Soret transition more difficult because in the excited state there is increased electro- static repulsion with electrons on the methene carbon atoms. This is diminished by back-double-bonding whh ligands in the 5th and 6th co-ordination positions and this might be expected to displace Amax to longer wavelengths. On the other hand, for the visible bands in low-spin ferrous complexes there is decreased electrostatic repulsion in the excited state but this effect is reduced by back-double-bonding, so here Amax moves to shorter wavelengths the greater the back-double-bonding. If the electronic transition which gives rise to the Soret band does not involve a net movement of electronic charge away from the metal in the high-spin ferric and ferrous complexes (which have symmetrical d electron distributions in the plane of the porphyrin) Amax would be expected to be at shorter wavelengths for ferric than for ferrous, irrespective of whether electron density on the pyrrole nitrogens is decreased as suggested by Williams or increased as suggested by Falk and myself. In the ferric haemoproteins the Soret band shifts to longer wavelengths in the low- spin complexes. This is in line with expectation. The change from high-spin to low-spin includes taking an electron from d^.-^^y"- and putting it in dy,, d^^ (or possibly dxy): this makes it easier to put more 7r-electron density on the pyrrole-nitrogens in the excited state and hence lowers the energy needed for the transition if it is of the form Falk and I suggest in our Fig. 1 . The opposing effect of increasing the electrons in d^y, dy, or d,j. would be diminished where back-double-bonding is possible and, in fact, one might expect, other things being equal, that the longest wavelengths for Soret maxima will be given by the best back-double-bonding ligands. Williams : As far as I can see Falk and Perrin used the same theory as I do, that of Piatt, in the discussion of the spectra of porphyrins (see Chem. Rev. 56, 299, 1956). There is then the question of how metals affect the spectra. I appear to agree with the treat- ment given recently by Gouterman (J. chem. Phys. 30, 1139, 1959), and which is the best I know, while Falk and Perrin's discussion would say something different. I say that the Soret band shifts to longer wavelengths with decrease of effective electro- negativity of the central metal. From what Perrin says I feel he must conclude that the opposite is true. Lemberg : I am not convinced that there is a general relationship between the Soret band positions and the spin type. The Soret band of high-spin ferrohaemoglobin lies at 74 Discussion longer wavelengths than that of the low-spin pyridine protoferrohaemochrome, but that of high-spin protohaem lies at still shorter wavelengths. Nor does there seem a clear relationship between bond-type and autoxidizability. We find autoxidizable haem compounds both with high-spin (haem) and low-spin (pyridine and imidazole haemochromes), and compounds slowly or not at all autoxidizable also both with high-spin (haemoglobin) and low-spin (cytochrome c). Williams: I have discussed above the shift in Soret band with ligand (Fig. 1, p. 43). Only for haemoproteins will the Soret band shift to shorter wavelengths (with change of spin-type) as ligand basicity increases. For the change haem to haemoprotein (haemoglobin) there is no change of spin-type and the Soret band moves to longer wavelength with basicity. This is also likely to be true for all low-spin complexes. I believe bond-type and autoxidizability to be clearly related. I wonder whether the compounds (pyridine and imidazole haemochromes), which Lemberg speaks of as low-spin, are in equilibrium with amounts of dissociated complexes (high-spin). It does seem that imidazole and pyridine reduce the rate of autoxidation of haems; thus imidazole and pyridine complexes of haems in strong solution pick up oxygen reversibly without being autoxidized (Corwin and Bruck, /. Amer. chem. Soc. 80, 4736, 1958). I think oxidation occurs only on addition of water in this case. Models for Haemoproteins Some New Compounds of Haems with Bases By J. E. Falk (Canberra) Falk : The properties of some new complexes of meso-, proto- and 2 : 4-diformyldeutero- haems are indicated in Figs. 1 and 2 and Table 1. These complexes were made in 0-01 N NaOH. The ligands hydrazine (pA'a8-l), ethanolamine (9-5), n-propylamine (10-5) and dimethylamine (10-6) form an interesting series of compounds with these haems. As shown in Fig. 1 both Amax and cmax of the meso- and proto-haem com- plexes increase with pKa of the ligand, and the effects are very similar for both haems. With the diformyl haem, the e of the dimethylamine complex is sim.ilar to that found with meso- and proto-haems. It was not possible to measure A values with the other amines because of spectral shifts associated with Schiff's base formation between the ligands and the formyl side-chains. This reaction was slow enough, however, to permit measurement with the reversion spectroscope of the position of the absorption bands of the initial complexes, and as shown in Table 1, the a-bands of all these complexes, from hydrazine to dimethylamine, were at the same wavelength as the a-band of the pyridine haemochrome. This lack of influence of the ligands upon the spectrum, as com.pared with meso- and proto-haems, is apparently related to the much greater electron-attraction in the side-chains of diformyldeuterohaem. We were able to obtain interesting complexes of protohaem and the diformylhaem with 2-mercaptoethanol (Table 1 and Fig. 2) ; the a-band of the protohaem complex is displaced 17 m/« to longer wavelength than that of pyridine protohaemochrome — the greatest shift we have yet encountered. Ethanol did not react in the same way under these conditions, so that as a first presumption, it appears that the thiol-group of mercaptoethanol is complexing with the haem iron. The electrophilic side-chains of proto- and diformyl haems appear to have some influence, since mercaptoethanol reacted poorly with mesohaem (Fig. 2). The Srnu of the pyridine haemochrome of 2 : 4-diformyldeuterohaem at the a maxi- mum was assumed to be the same as that of protohaem. As shown in Table 1 , the a-bands of these compounds of meso- and proto-haems more than cover the ranges of a-band positions (cf. Morton, Rev. pure appl. Chem. 8, 161, 1958) of the known cytochromes of types c and b respectively. Absorption spectra were determined with a Beckman DU Spectrophotometer, except compounds marked *, which were measured with a Beck-Hartridge reversion spectro- scope (see text). The fmM=31 for the pyridine haemochrome of 2: 4-diformyl- deuterohaem is assumed, and the £niM for the other ligands calculated on this basis. 1 1 Protohaem 4 A / '. t; 1 ■'/ ^\ r\ 1 / \\ iA 2\1 I " 2 /, \ 1^^ T > 1 ^ J ^ ^ 1 1 1 ■■ 1 1 2 4-Diformyl deuterohaem '\ / p 1 1 4 i \, I P 510 530 550 520 540 560 A, m// 540 560 580 600 X, xw/x Fig. 1, Spectra of compounds of haems with bases. The haems (2 X 10~^m) were dissolved in 0-01 n NaOH; samples were reduced with dithionite imme- diately before adding the ligands and measuring the absorption spectra on the Beckman DU Spectrophotometer. Ligands were added in excess, higher concentrations causing no further change in absorption, in the following molarities : P. pyridine 1. hydrazine 2. ethanolamine 3. «-propylamine 4. dimethylamine Mesohaem 3-41 0-488 0-655 0-282 15-06 Proto- and 2 : 4-diformyl haems 3-41 0-195 0-301 0-159 15-06 HK) Mesohoem 1 P 30 A \ \ lac 10 1 1 \ \ \ 1 \ \ \ \ / / / / / / I \ \ ME ^^^ ^s 0 S \ Pr otoha am P / \ / / \ \ ME A / J \ 1 \ 1 ^\ P \ \ \ ME / ^ K / \ \ ^ -^ E 510 530 550 520 540 560 580 A, m// A, m^ Fig. 2. Spectra of mercaptoethanol-haem compounds. The haem solutions and the procedure were as described in Fig. 1, The ligand concentrations were(M): pyridine (P), 3-41 ; ethanol (E), 8-5; mercaptoethanol (ME), 3-45. H.E. — VOL. I — G 76 Discussion Table 1. a-BANDS of some haem complexes 2:4-Di- Ligand Meso A (rcifi) fmM Proto A (m/<) ^mM formyldeutero A (m/i) f mil Pyridine 547 33-2 558 31 584 31 pKa 8-1 Hydrazine 545 17-2 556 17-3 584* 9-5 Ethanolamine 546 24-2 557 22-2 584* 10-5 rt-Propylamine 550 35-7 562 28-0 584* 10-6 Dimethylamine 550 38-8 563 37 584 36-4 2-Mercaptoethanol 560 9-8 575 25-7 595 21-2 Lemberg: There is considerable difficulty in finding suitable models for haemoproteins among simpler haem compounds. Thus amino acids at neutral pH are zwitterions and therefore unsuitable. Research with amino acids at a physiological pH should therefore be carried out with amino acid esters or poly-aminoacids, but such data are missing. Moreover, even then the affinity to haem may be far smaller than if the combining group is held in the protein in a restrained position close to the haem iron. In this regard Kaziro's approach offers perhaps more hope than model experiments. I was interested to note, in Falk's discussion (this volume, p. 74), the lack of vari- ability of the diformyldeuterohaem spectrum with different ligands. This is quite different from the great variability of band position of nitrogenous compounds of monoformyl haems such as haem a. Falk: Lemberg reinforces the point I have made — that relatively few model haem com- pounds have been studied; those which have been are largely of one class, in which the ligand atom is — N^. The reasons for this are partly the difficulties, as Lemberg points out — we are, in fact, studying amino acid esters and small peptides — and partly, perhaps, that we have all been over-impressed with the suspected role of histidine, and have modelled our models upon it. I agree that model studies can never entirely solve the problems of haemoprotein structure, but I believe that there still remains a great deal to discover about the chemistry of the combination of haem with new types of ligands, and that knowledge on this level will be a prerequisite for our eventual understanding of the haemoproteins. Not only do we need data on compounds of haems with primary and secondary nitrogen atoms, with thiols, etc., but as Perrin and I have pointed out, there is a par- ticular need for studies of mixed compounds, with one ligand of these types and the other of the — N== type. In this regard Wang's model (this volume, p. 98), in which the haem is held down on one side to a — N= bond, should be particularly valuable. Phillips: It is interesting to note that although free amino acids form haemochromes only with great difficulty, the corresponding amino acid esters react readily. The reluctance of the free amino acids to react is not simply due to electrostatic repulsion betv^een the amino acid anion and the carboxylate anions on the porphyrin side chains, since a similar behaviour is observed in detergent solutions using porphyrin esters. Carbon Monoxide-Pyridine Complexes with Haems By J. H. Wang (Yale) Wang: In connexion with Falk's remarks on the special significance of mixed haemo- chromes, I should like to discuss some interesting results obtained in our equilibrium studies on the combination of carbon monoxide with haem in aqueous solutions containing small amounts of pyridine. Spectra and Redox Potentials of Metalloporphyrins and Haemoproteins 77 Our results show that as the concentration of pyridine in the solution increases, the affinity of haem for carbon monoxide also rapidly increases (Nakahara and Wang, J. Anier. chem. Soc. 80, 6526, 1958). This observation shows that in dilute solutions the mixed complex pyridine-haem-CO has greater thermodynamic stability than both the complex HaO-haem-CO and the complex pyridine-haem-pyridine. Indeed the affinity of haem for carbon monoxide is so high that when the latter is bubbled through a solution of dipyridine haemochrome in pure pyridine, some mixed complex pyridine-haem-CO is formed. On the other hand no detectable amount of dicarbon- monoxyhaem, OC-haem-CO, was found when aqueous haem solutions, whether with or without added pyridine, were equilibrated with carbon monoxide at even 1 atm. pressure (Wang, Nakahara and Fleischer, /. Amer. chem. Soc. 80, 1109, 1958). O 0 III 12 II 9 I c :Fe' 810 :Fe: -I,^Z *■ Fe r 49 671 P) ^ Identical J'^P^S^ ligands (c) i) Fig. 5. Visible spectra of ferrimyoglobin, ferrihaemoglobin and ferriperoxidase fluoride (Keilin and Hartree, 1951; Hanania, 1953). 12 10 mM 8 6 4 2 650 + + + Fe CN /'^X />" x\ / A J \ ^ yf \ A^ // y ^Per. - 1^/ Hb^ ..— y^ '^ 600 550 500 A(mjj) Fig. 6. Visible spectra of ferrimyoglobin, ferrihaemoglobin and ferriperoxidase cyanide (Keilin and Hartree, 1951 ; Hanania, 1953). 114 P. George, J. Beetlestone and J. S. Griffith band at about 500 m.ii ; and all the low-spin ferric complexes have spectra like the cyanide derivatives, with a very pronounced absorption band at about 540 m/j, and a shoulder, or second band, at about 580 m/« (Theorell, 1942). The high-spin complexes have additional minor bands of lower intensity, at about 580 and 540 m/<, but for the present it is the positions of 1.2 1.0 'mM 0.8 0.6 0.4 0.2 0 + + + ■ ^^f + + + Mb / \ / N / \ 900 800 ;\(m;j) 700 Fig. 7. Near infra-red spectra of ferrimyoglobin fluoride, hydroxide and cyanide, and ferrihaemoglobin hydroxide (Hanania, 1953). the major bands which differentiate the two types of complex that are important. Similar contrasting features appear in other regions of the absorption spectrum. In the near infra-red the fluoride complex has a v/ell-defined absorption band at about 850 m/( with a shoulder at about 750 m//, whereas the cyanide complex has remarkably low absorption throughout the whole range 700 to 950 m// as shown in Fig. 7 (George and Hanania, 1955). In the ultra-violet, from 280 to 450 m/<, there are three regions to consider. The very intense Soret band lies between 405 and 410 m/t for the acidic ferri- haemoproteins and the fluoride complexes, the latter having lower absorption in the case of myoglobin and haemoglobin but higher in the case of peroxidase. On the other hand, the low-spin derivatives have the band shifted towards the red in the neighbourhood of 418 to 425 m^a (see Fig. 8). Minor bands occur at about 350 m/u. These are unresolved in the case of the acidic ferri- haemoproteins and the fluoride complexes, but two distinct bands at about Fenihaemoprotein Hydroxides 115 345 and 360 mfi can be distinguished in the case of the cyanide complex. At shorter wavelengths, from 260 to 300 m/i, absorption due to both the ferri- porphyrin prosthetic group and tyrosine and tryptophane residues in the protein occurs, as evidenced by the greater absorption of the ferrihaemo- proteins as compared to their apo-proteins. As shown in Fig. 9 the low-spin 450 400 350 300 7\(m>i) Fig. 8. Ultra-violet spectra of ferrimyoglobin and ferrihaemoglobin cyanide and fluoride (Keilin and Hartree, 1951; Hanania, 1953). cyanide derivative has greater absorption than the high-spin fluoride deriva- tive throughout this region, although the band at 290 m/( is less well resolved. While the spectra of the high- and low-spin derivatives exhibit these characteristic distinguishing features, which as far as can be judged are common to myoglobin, haemoglobin and peroxidase, the spectra of the hydroxides vary a great deal as shown in Figs. 7, 10, 1 1 and 14. Moreover these variations are not haphazard, but appear to be related to the change in magnetic moment, i.e. FerriMb -^ FerriHb -> FerriPer (5) 5-11 B.M. 4-45 B.M. 2-66 B.M. To take but one example, in the region of 600 m/<, the extinction coefficients follow the order as shown in Fig. 10, 'Mb > £Hb > e Per (6) 116 P, George, J. Beetlestone and J. S. Griffith 50i — 10 300 250 A(nnjj) Fig. 9. Ultra-violet spectra of ferrimyoglobin fluoride, hydroxide and cyanide in the region of tyrosine and tryptophane absorption (Hanania, 1953). 'mM 650 600 550 500 7\imp) Fig. 10. Visible spectra of ferrimyoglobin, ferrihaemoglobin and ferriperoxidase hydroxide (Keilin and Hartree, 1951 ; Hanania, 1953). Fenihaemoprotein Hydroxides 117 Now the regular and systematic differences between the spectra of high- and low-spin complexes suggest very strongly that if the hydroxides are mixtures of high- and low-spin forms their spectra and magnetic moments should conform to a certain pattern. (a) For the same haemoprotein, the extinction coefficients for the hydroxide should be intermediate in value between those for typical high- and low-spin complexes in the regions where the major absorption bands occur. (b) For a series of hydroxides, there should be a regular trend in the extinc- tion coefficients in the region of the major absorption bands, such that the 450 400 350 300 A(mjj) Fig. 11. Ultra-violet spectra of ferrimyoglobin and ferrihaemoglobin hydroxide (Hanania, 1953). higher magnetic moment hydroxides resemble more closely the high-spin complexes, and the lower magnetic moment hydroxides resemble more closely the low-spin complexes. In the case of ferrimyoglobin, the only haemoprotein for which complete data are available at present, the first criterion is found to hold throughout the entire range of wavelength, 250 to 950 m/<. For the visible region the myoglobin curve in Fig. 10 is to be compared with those in Figs. 5 and 6; Fig. 7 covers the region 700 to 950 m/t ; Figs. 8 and 1 1 give the Soret bands, and the smaller bands in the region 330 to 370 m/i ; and Fig. 9 covers the region of composite absorption, 250 to 300 m//. The second criterion is borne out by a comparison of the spectra of ferrimyoglobin and ferrihaemoglobin hy- droxides in Figs. 7 and 10, where the extinction coefficients follow the sequence Fluoride Complex -» FerriMb Hydroxide -* FerriHb Hydroxide -> Cyanide Complex (7) high spin 5-11 B.M. 4-47 B.M. low spin 118 P. George, J. Beetlestone and J. S. Griffith in order either of increasing or decreasing magnitudes, depending on the particular wavelength. In the ultra-violet region, 330 to 450 m//, the trend is not so clear-cut, but, as will be shown in the next section, this can be attributed to the small but significant shift of all the ferrimyoglobin band maxima relative to those for ferrihaemoglobin, together with systematically 300 250 A(rTVj) Fig. 12. Ultra-violet spectra of ferrimyoglobin and ferrihaemoglobin hydroxide in the region of tyrosine and tryptophane absorption (Hanania, 1953). lower extinction coefficients (see Fig. 8). In the region 250 to 300 mft no strict evaluation is possible because myoglobin and haemoglobin are not alike in tyrosine and tryptophane content. Nevertheless it is interesting that the curve for ferrimyoglobin hydroxide, which has higher moment, in contrast to that for ferrihaemoglobin with the lower moment, has a well-defined shoulder at 290 m/u Hke the high-spin fluoride complex (see Figs. 9 and 12). The data for ferriperoxidase are not quite suflficient for it to be included in the sequence in equation (7), although there are ample indications that it would fit into the pattern and come between ferrihaemoglobin hydroxide and the low-spin cyanide complex. The magnetic moment has been determined for horseradish peroxidase, 2-66 B.M. (Theorell, 1942), but the absorption spectrum, recorded by Keilin and Hartree (1951) and reproduced in Fig. 10, refers to a pH of 11-4, which, judging from the pK of 10-9-1 1-3, would give Ferrihaemoprotein Hydroxides 1 19 only about 60-75 % hydroxide formation. It is already evident from Fig. 10, however, that the hydroxide has a pronounced peak at about 540 mn with a second peak at about 575 ra^i, and no peaks either in the region 600 to 640 m/i or at about 500 m,a. Spectroscopically, as well as magnetically, it can safely be classified as a low-spin complex. The spectroscopic type is fully substantiated by the corresponding spectrum for Japanese root peroxi- dase (Morita and Kameda, 1958), which has absorption bands at 548 and 578 ma, with £niM = 12-3 and lOT respectively, together with relatively lower absorption in the region 620 to 650 m//, compared to horseradish peroxidase in Fig. 10. But its magnetic moment has not yet been measured. The data for two other haemoglobins may be considered at this point. The first, Chironomus haemoglobin (Scheler and Fischbach, 1958), presents some anomalous features. The visible spectrum of the hydroxide is most like that of ferrimyoglobin, and the shape of the curve in the region of 600 m,a suggests that it should come between ferrimyoglobin and ferrihaeraoglobin in the sequence in equation (7), but on a quantitative scale nearer the former. However its magnetic moment is 4-45 B.M., a little less than that of ferri- haemoglobin hydroxide (Scheler, Schoffa and Jung, 1957). No explanation can be offered for this discrepancy, although it is to be noted that the magnetic moment of the acidic ferrihaemoglobin is appreciably lower than the values determined for ferrimyoglobin and erythrocyte ferrihaemoglobin, namely 5-68 and 5-80 B.M. respectively (Theorell and Ehrenberg, 1951; Coryell, Stitt and Pauling, 1937). The second, root nodule haemoglobin (leghaemoglobin) is particularly interesting. The visible spectrum of the hydroxide, reproduced in Fig. 16 (Sternberg and Virtanen, 1952), is almost the same as that of Japanese root peroxidase, which indicates that it is a low-spin complex. Furthermore, preliminary spectroscopic observations by George, Hanania and Thorogood (1959) in the near infra-red have shown it to have significantly lower absorp- tion in the region 700 to 900 m,a than the myoglobin and haemoglobin derivatives, which is in keeping with the trend in extinction coefficients from high- to low-spin complexes (see Fig. 7). The magnetic moment however still remains to be determined. Hence, provided it is appropriate to regard leghaemoglobin as a true haemoglobin*, the haemoglobins themselves, without recourse to peroxidase, furnish a series of hydroxides covering almost the whole range of spectroscopic characteristics. There is thus a substantial body of evidence to suggest that the hydroxides, especially of ferrimyoglobin and ferrihaemoglobin, are mixtures of high- and * This classification is based on the ability of ferroleghaemoglobin to form an oxygen complex, and it is further substantiated by the reaction of ferrileghaemoglobin with hydrogen peroxide. An intermediate compound is formed with absorption bands at 550 and 575 m//, resembling the ferrimyoglobin and ferrihaemoglobin derivatives, in contrast to ferriperoxidase and ferricatalase, which give two such compounds neither having bands at these wavelengths. 120 P. George, J. Beetlestone and J. S. Griffith low-spin forms, and in the next section this hypothesis will be put to a quantitative test. QUANTITATIVE CORRELATION BETWEEN THE MAGNETIC MOMENTS AND THE SPECTRA OF FERRIHAEMOPROTEIN HYDROXIDES Making the assumption that the hydroxides are mixtures of high- and low- spin forms, the magnetic moments and extinction coefficients at each wave- length should be interrelated in the following way. Denoting the moments of the high- and low-spin forms by /^^ and i^ii, the moments of, say, ferrimyoglobin and ferrihaemoglobin hydroxide, /^ji,, and / / / \ S. / / \ \ n > ^---''''^^^---O^ 450 400 350 300 7^(mjj) Fig. 17. Ultra-violet spectra of the high- and low-spin hydroxides calculated from the data for ferrimyoglobin and ferrihaemoglobin hydroxides. The absorption curve of ferrimyoglobin hydroxide has been corrected by a 5 m/i displacement toward the red, and all extinction coefficients multiplied by 1-08: /i) 700 Fig. 18. Near infra-red spectra of the high- and low-spin hydroxides calculated from the data for ferrimyoglobin and ferrihaemoglobin hydroxide. No A or fniJVl corrections: /tj = 2-24, n^ = 5-92. throughout this region like the cyanide derivative (see Fig. 7). The small negative extinction coefllicients actually obtained for the low-spin form can be attributed to the uncertainties in the calculation procedure when the correction factors are unknown, and also to experimental error. Precise extinction coefficients are very difficult to determine in this region because the magni- tudes are so small, and errors introduced by extraneous background absorp- tion, which is hard to remove completely, become significant. Summary Assuming that the ferrihaemoprotein hydroxides are thermal mixtures, and adopting /Uf^ = 5-92 and /tj = 2-24 as the magnetic moments of the 128 P. George, J. Beetlestone and J. S. Griffith high- and low-spin forms, calculations give the following percentages for the various haemoproteins : Myoglobin : High - 70 %, Low - 30 % Haemoglobin : High - 50 %, Low - 50 % Peroxidase: High- 7%, Low -93% From the extinction coefficients of the hydroxides the spectra of the high- and low-spin forms have been obtained over the range 250 to 950 m^. Major absorption bands, or shoulders, occur at about the following wave- lengths, with extinction coefficients having the approximate values given in brackets : High-spin form: 830 (1-2), 740 (0-9), 600 (11), 540 (8), 490 unresolved band (10), 405 (116), 350 shoulder (44). Low-spin form: 575 (10), 545 (12), 417 (104), 360 (20), 340 (14). The calculation procedure has certain inherent limitations, nevertheless these absorption bands correspond so closely to those which distinguish high- from low-spin derivatives that the assumption of a thermal mixture can be regarded as entirely consistent with the spectroscopic and magnetic data. THE EFFECT OF TEMPERATURE ON THE SPECTRUM AND ON THE MAGNETIC MOMENT OF FERRIMYOGLOBIN HYDROXIDE As suggested previously, if the ferrihaemoprotein hydroxides are thermal mixtures of high- and low-spin forms, then changing the temperature would be expected to influence the equilibrium. High-spin hydroxide ^^ Low-spin hydroxide (12) and a change should therefore be observable in the magnetic moment and in the absorption spectrum. The magnitude of the change would depend on the value of AT/ for reaction (12), since this determines the variation of equili- brium constant with temperature according to the van't Hoff Isochore. But, since a close balance between the energies of the two forms is to be anticipated, A// is likely to be small, and, as a consequence, K^ to have a small temperature dependence resulting in only slight changes in magnetic moment and absorp- tion spectrum. This is borne out by the observation that there were no noticeable variations in the optical densities of ferrimyoglobin or ferri- haemoglobin hydroxide solutions at 582 m// and 578 m/t respectively over the temperature range 7-5° to 37°C in experiments carried out to obtain thermo- dynamic data for the ionization reactions (George and Hanania, 1952, 1953). However, further experiments have now been made using a sensitive recording spectrophotometer, and a temperature effect has been detected. The absorption spectrum of a concentrated solution of ferrimyoglobin Ferrihaemoprotein Hydroxides 129 hydroxide at pH 11-0 and 5°C was recorded from 470 to 650 m,a, the optical density at 540 m/t being 0-7411. The reference cuvette, which previously contained buffer, was then filled with more of the hydroxide, and a baseline was recorded over the wavelength range with both solutions at 5°C. The solution in one cuvette was then rapidly warmed to 35°C, and maintained at +0.02 — Fig. 19. The difference spectrum in the visible region for 8-4 x 10~^ m ferrimyoglobin hydroxide at 5° and 35°, plotted as id^° - ^35°). this temperature, by the insertion of a specially constructed hollow metal heating unit through which water from a thermostat was circulated. A difference spectrum was recorded, from which the curve illustrated in Fig. 19 was obtained after correction for the baseline. As can be seen, the effect of a 30° alteration in temperature is rather small. The change in optical density is at the most 2-9% at 542 m/^, while at 582 m/i it has dropped to 1-6%, which accounts for the effect escaping notice in the earlier investigations. Control experiments using the cyanide and fluoride derivatives showed no similar effect. The negative regions from 480 to 520 m// and above 600 m/<, together with the positive region in between which shows two well-defined bands, are qualitatively consistent with an increase in the fraction of the high-spin form as the temperature is increased. Some indication of its magnitude can be obtained from the difference between the extinction coefficients of the 130 P. George, J. Beetlestone and J. S. Griffith high- and low-spin forms calculated in Section IV. At 540 m/< the difference in e^^ is about 4, which gives an increase of between 0-05 and 0-07, i.e., between 5 and 7 %. However, in order to obtain the individual spectra of the high- and low-spin forms from the difference spectrum, an independent determination of the fractions present at the two temperatures is required. This can be seen from the equations, fg = agSj -}- (1 - a5)£^ (13) £35 = aaaCj + (1 - a35)e;, (14) where £5 and £35 are the extinction coefficients of the hydroxide at 5° and 35°, a5 and cc^^ are the fractions of the low-spin form at the two temperatures, and £f^ and £j are the extinction coefficients of the high- and low-spin forms. Since £5 is known and £35 can be obtained from the difference spectrum, provided cc^ and cc^^ can be determined, jUj^ and jUi can be evaluated from the equations rearranged in the form, ^3b'^5 ~ ^5*^35 "■35 (15) €5(1 - ^35) - ^35(1 - 0^5) .... El = (16) 0^5 - ^35 The variation of a with temperature has been obtained in the following way. Using a sensitive Gouy balance, constructed from a Varian electro- magnet V4004 and a Sartorius Microbalance MPR 5 II, and equipped with a coaxial glass thermostat surrounding the sample tube and suspension fibre, the change in Aw was measured as a function of temperature over the range 1° to 30°C for the fluoride, cyanide and hydroxide derivatives of ferrimyo- globin. Calibration with nickel chloride solution enabled these changes in Aw to be converted into changes in molar susceptibility, Xu- The value of Xu obtained by Theorell and Ehrenberg (1951) for the three derivatives at 20°C were adopted, namely, 14,790, 2,340 and 11,040 x 10-^ c.g.s. units respectively, and hence values of^Xu o^^^ the temperature range were obtained. The variation of Xu for the fluoride and cyanide derivatives was found to follow very closely the simple Curie law, x = constant/r. The magnitude of the change is illustrated by the following data: from 20° to 1°, Xu for the fluoride and cyanide derivatives increases by 1,046 x 10~^ and 82 x 10~^ c.g.s. units respectively. On the other hand, Xu for the hydroxide, although it has a high value approaching that of the fluoride, only increases by 145 X 10~^ c.g.s. units for the same decrease in temperature. As a conse- quence, the values of Xm do not follow the Curie Law, and the type of devia- tion is just what would be expected if, on lowering the temperature, the fraction of the high-spin form decreases. Ferrihaemoprotein Hydroxides 131 The simplest method by which the fractions of the high- and low-spin forms can be calculated, throughout the temperature range, is to use the experi- mental values of x^si for ^^'^ fluoride and cyanide derivatives at various temperatures as the values appropriate to the high- and low-spin forms, and substitute in the equation, yfM(hydroxide) "" °^XM(cyanide) + (^ ^)ZM(fluoride) (17) In practice, this is equivalent to taking // j = 2-34 B.M., i.e., the value for the cyanide derivative, instead of 2-24 B.M., as in the majority of calculations in 1 1 1 p V-^ low 10 fo ^^^ \ y iJyQ~K mM high-W^ / TJ— cr^ ^"""^ \^ J 5 Jo ■ 1 ( / 1 Ferrimvoqlobin Hydroxide 650 600 550 A(mjj) 500 Fig. 20. The visible spectra of the high- and low-spin hydroxides calculated from the difference spectrum in Fig. 19, and the corresponding change in the fraction of the low-spin form, 0055, as obtained from the variation of magnetic suscepti- bility with temperature. Section IV. Such a slight change in Hi only affects the value of a to a negli- gible extent, i.e., from 0-300 to 0-304. Values of a and (1 — a), obtained in this way, are listed in Table 5 for temperatures from 0° to 30°, together with values for K^, the equilibrium constant for the conversion reaction (12). Interpolation and extrapolation for the temperature interval 5° to 35° gives 0-055 for the corresponding change in a. The spectra of the high- and low-spin forms of ferrimyoglobin hydroxide were then obtained by calculating Ey^ and £j throughout the wavelength range according to equations (15) and (16). The similarity between these spectra, shown in Fig. 20, and those in Figs. 13, 14 and 16 is very gratifying. But it must be remembered that the previous spectra, calculated from data for pairs of haemoproteins, are 132 P. George, J. Beetlestone and J. S. Griffith approximations, consisting of average values of the extinction coefficient appropriate to the two individual high-spin forms and the two individual low-spin forms. The new spectra in Fig. 20, based entirely on data for one haemoprotein, are therefore more valid. THE SPECTRA OF FERRIMYOGLOBIN DERIVATIVES IN HEAVY WATER The interplay of structural and electronic factors necessary for a ferri- myoglobin derivative to exist as a mixture of high- and low-spin forms is evidently so critical that the ligands most closely related to the hydroxyl group in chemical type give predominantly, or entirely, high-spin or low-spin complexes. On the basis of spectroscopic data, or magnetic data, or both, it is clear that the complexes with phenol, and presumably ethanol, i.e., Fejib+++ — OCgHg and Fe]^+++ — OC2H5 come in the former category; whereas the complexes with the sulphur analogues, hydrogen sulphide, ethyl mercaptan and thiophenol, i.e., Fejib'^++ — SH, Fe]yii,+++ — SC2H5 and Fejnj+++ — SCgHg, come in the latter (George, Lyster and Beetlestone, 1958; Coryell and Stitt, 1940; Keilin, 1933; Heussenstam and Coryell, 1954). The least drastic of all substitutions that can be achieved, with the exception of employing HaO^^, is the replacement of hydrogen by deuterium, and the spectrum of the alkaline form in heavy water, which should accordingly have the structure Feniij+++ — OD, has therefore been studied. In the prelimi- nary experiments, reported below, the highest mole ratio of DgO to H2O that could be attained was 134: 1. Hence, although the affinities of the iron atom for OH~ and 0D~ also have to be taken into consideration because they determine the relative amounts of Fe]ynj+++ — OH and FejyQj+++ — OD formed, it is unlikely that in pure D2O the effect observed would be very much enhanced. A very concentrated solution of acidic ferrimyoglobin in ordinary water was used, so that only 0-02 ml in a total of 3 ml was required to give optical density values of about 0-7 at the band maxima in the visible region. Solutions of the alkaline form were prepared in the following way. Tiny quantities of caustic soda solution were added to acidic ferrimyoglobin (0-02 ml stock solution -f 2-98 ml ordinary water) from a micro-syringe until the pH was 11-0. The same volume of caustic soda was added to a corresponding solution of acidic ferrimyoglobin made up in heavy water. Difference spectra were then recorded with the heavy water solution in the reference cuvette for the alkaline form, and, as controls, for the acidic form and the cyanide derivative, which was prepared by adding a little solid KCN. With the cyanide derivative no difference could be detected and with the acidic form there was scarcely any change. But with the alkaline form a well- defined difference spectrum was obtained, very similar to that in Fig. 20, and the optical density differences were about the same in magnitude. The Ferrihaemoprotein Hydroxides 133 simplest interpretation of this result is that for the Fej]^+++ — OD formed in heavy water the fraction of the low-spin form is about 6 % higher than the fraction for Fejj^+++ — OH under similar conditions. Using the data given Table 5. The fractions of the low-spin and high-spin forms of ferrimyoglobin hydroxide, a and (1 — a) respectively, at different temperatures, calculated from the temper- ATURE VARIATION OF Xm FOR FERRIMYOGLOBIN HYDROXIDE, FLUORIDE, AND CYANIDE Kg is the equilibrium constant for the conversion high-spin form ^^ low-spin form and is given by a/(l — a). TCO a (1 -a) Ke 0 0-34 0-66 0-52 10 0-32 0-68 0-47 20 0-30 0-70 0-43 30 0-285 0-715 0-40 in Table 5 for the hydroxide, an approximate value of the equilibrium con- stant for the reaction high-spin deuteroxide ^^ low-spin deuteroxide (18) is found to be 0-55 at 25°C, compared to 0-41 for the hydroxide. Substitution of hydrogen by deuterium thus favours the conversion by about 0-2 kcal/mole in units of free energy. It is probable that the bulk of this difference arises via the water of solvation and not from any effect on the ligand field. The change of mass of the hydrogen nucleus affects the free energy of 'crystalliza- tion' around the iron ion directly but the ligand field only very indirectly through the effect of the change of vibrational amplitudes for the OH group on the mean ligand field. GENERAL REMARKS The experiments with heavy water offer particularly direct evidence for the existence of a thermal mixture in ferrimyoglobin hydroxide. Further, because the fundamental difference between the high- and low-spin form lies in the electronic structure of the iron ion, they show that water molecules (or those protons which are exchangeable with those of water) play an essential part in determining the free energy change. We would naturally guess that the water molecules which are 'crystallized' around the iron ion (and also the hydrogen of the OH~ group) are the ones concerned here. 134 P. George, J. Beetlestone and J. S. Griffith Although this is far from direct evidence for the existence of the hydroxide structure it is at least thoroughly consistent with it. The hypothesis of a thermal mixture is also fully borne out in the case of ferrimyoglobin hydroxide by the temperature variation of its spectrum and magnetic moment as described in Section V; and, in view of the self-consistent results of the calculations using magnetic and spectroscopic data in Section IV, it can be concluded that ferrihaemoglobin hydroxide is also a mixture of high- and low-spin forms. This accounts equally well for its apparently anomalous magnetic moment as the explanation in terms of the electronic configuration with three unpaired electrons, which was shown to be unlikely on theoretical grounds (see Section II). Thermodynamic data for the conversion of the high-spin to the low-spin form can be obtained from the values of K^ for ferrimyoglobin in Table 5. A plot of log Kg against \jT gives A// = — 1-5 ± 0-2 kcal/mole, and from the equation AG° = A// - T^S°, with ^G° equal to 0-5 kcal/mole at 25°C, AS" is found to be — 6-7 ±0-7 e.u. The conversion is thus favoured by the enthalpy change, but is appreciably hindered by the entropy change to such an extent that the resulting free energy change has a small positive value. In other words, with respect to their heats of formation the low-spin form is the more stable, whereas in terms of their entropies the high-spin form is the more stable. The favourable value of A// may be regarded as purely fortuitous, because, although the conversion to the low-spin form implies an increase in the value of A, and hence extra stabilization, pairing energies have also to be taken into consideration and in addition solvent interaction effects may be important (see below). In order to discuss the entropy change accompanying the conversion, it is convenient to distinguish the contribution arising from the degeneracy of the electronic state of the iron in the two forms from the remainder. The following estimate shows that this contribution is unlikely to be more than about — 2 e.u. In the high-spin form the ferric ion has a ground term which is spatially non-degenerate but has a sixfold degeneracy due to the spin 5* = 5/2. The ligand field combined with the spin-orbit coupling lifts the degeneracy into three Kramers doublets. If this splitting is large compared to kT, only one Kramers doublet is occupied and the effective degeneracy of the ferric ion is 2. If it is small then the degeneracy is 6. This means that the entropy associ- ated with the degeneracy of the electronic state of the iron in the high-spin form lies between the two limits of i? log^ 2 = 1-38 e.u. and R log^ 6 = 3.56 e.u. The actual magnitude of the splitting is unknown. If we assume that it may be represented in a spin-Hamiltonian for the ground term with S = 5/2 by the quadratic expression Ferrihaemoprotein Hydroxides 135 then electron resonance measurements show that D can hardly be less than 4°K (Bennett and Ingram, 1956; Griffith, 1956c). With D in these units, the partition function, Z, is given by the equation, IQD 2D 8D Z = 2e sr +2e3r + 2e3r (19) from which the entropy follows from the formula S = 5(/?nogg Z)ldT. For D/r small we deduce S = 3-56 — (28D-i?/9r^). The second term is inappre- ciable (< 0-01) at room temperature for D < 12°K, i.e., an overall splitting of 48 cm~i. Therefore it seems likely, although not certain, that at room temperature this contribution to S is close to 3-56 e.u. In the low-spin form we have a spatial degeneracy of three and a spin degeneracy of two. Here, however, it is probable that the three Kramers doublets have a separation large compared with kT at room temperature (Griffith, 1957) so that the contribution to S from the degeneracy is close to 1-38 e.u. This means a contribution to A^ for the conversion of the high-spin to the low-spin form of 1-38 — 3-56 = —2-18 e.u. If our assumptions are incorrect the numerical value of this contribution will almost certainly be lower. It is much more difficult to obtain any a priori numerical estimate for the remainder of the entropy change, which, using the value obtained in the last paragraph for the degeneracy contribution, is seen to amount to about —5 e.u.* We should expect it to be negative, however, for the following reason. In the high-spin form the overall distribution of the five ^-electrons about the iron has nearly spherical symmetry, thus producing no orientating effect on the environment. On the other hand, in the low-spin form the five electrons are in the three orbitals away from the bond directions, thus im- posing an extra rigidity on the environment of the iron. This would result partly in a more rigid ferrimyoglobin molecule, and partly in a more rigid arrangement of water molecules around the Fe — OH group. Just as A// for the conversion is determined by other energy terms besides the electronic stabilization energy arising from the splitting of the ^-orbitals, so the values of A// for the formation of complexes with different ligands cannot be taken as an accurate indication of the variation in A. From one extreme to the other, however, a rough correlation would be expected, with the high-spin complexes having the less favourable values of AH. This trend, which has also been discussed by Havemann and Haberditzl (1958), is illustrated by the data in Table 6. The values of AS° become progressively more negative from fluoride to cyanide, but they are not amenable to any straightforward correlation because the entropies of the ligands themselves vary so much, with S"" for F~, 0H~ and CN~ having the values —2-5, —2-3 and -|-28 e.u. respectively. Some allowance for this can nevertheless be made * The assumed additivity of entropies is equivalent to a factorization of the partition function, which is probably a good approximation here at room temperature or below. 136 P. George, J. Beetlestone and J. S. Griffith by comparing the differences in partial molal entropies of the complexes and the parent haemoprotein (George, 1956). Table 6. IS.H and A^" values for the formation of ferrimyoglobin FLUORIDE, HYDROXIDE AND CYANIDE: AND THEIR MAGNETIC MOMENTS (GEORGE AND HANANIA, 1952, 1956: THEORELL AND EHRENBERG, 1951) Ligand Aiykcal/mole ^S° e.u. /f B.M. Type of complex F- OH-* CN- -1-5 -7-65 -18-6 + 1-8 -2-6 -24 5-75-5-92 5-11 2-35 high-spin 70% high, 30% low-spin low-spin * See footnote to Table 7. A more rigorous correlation can be sought, if, for the same ligand, data are available for closely related haemoproteins. But if the whole range from high- to low-spin complexes is to be covered, this would clearly be restricted to those ligands capable of giving theimal mixtures in some cases. For example, the data in Table 7 for the various haemoglobin hydroxides show that the increase in the fraction of the low-spin form is accompanied by more negative (i.e., favourable) values of Ai/, which was to be anticipated from the overall trend illustrated in Table 6. Furthennore, with a series of derivatives of this type, where the ligands are identical and the structure of the complex in the immediate neighbourhood of the iron is presumably very similar, the values of AS* can be taken as a true indication of a general trend paralleling the trend in Ai/. As the fraction of the low-spin form increases, IS.S assumes more negative (unfavourable) values, while A// assumes more negative (favourable) values. This trend in AS* is entirely in accord with the entropy change obtained above for the conversion of the high-spin to the low-spin hydroxide in the case of ferrimyoglobin, and it can likewise be associated with a greater structural rigidity in the vicinity of the iron atom of the low-spin form. The T^K values for the ionization of ferriperoxidase and ferricytochrome c are so much higher than those for the haemoglobins (see Table 1) that inevitably either the A// values, or the A^* values, and very probably both, would show marked deviations from the correlation set out in Table 7. This is not unexpected because the acidic forms of these haemoproteins have different structures, and as a consequence the formation of the hydroxide is a different type of chemical reaction. In the case of the haemoglobins, the reactions of the acidic form can be very adequately expressed by the hydrate structure, e.g., Prof. — Fegb"'^++(H20), and the ionization is accordingly the simple dissociation of a proton. Prot.— FeHb+++(H20) ^ Prot.— Fc Hb -OH + H+ (20) Ferrihaemoprotein Hydroxides 137 Table 7. A^ and A5° values for hydroxide formation* Haemoprotein \H kcal/mole ^S° (e.u.) Type of hydroxide Ferrimyoglobin Ferrihaemoglobin Ferrileghaemoglobin -7-65 -9-5 -11-0 -2-6 -7-9 -13-0 70% high, 30% low-spin 50% high, 50% low-spin approaches 100% low-spin * These values have been calculated from the corresponding data for the ionization reaction, and for the ionization of water (A// = -1-1 3-4 kcal/mole and AS^ = —19-2 e.u.). The references for ferrimyoglobin and ferrihaemoglobin are given in Table 1 ; for ferrileghaemoglobin, see George, Hanania and Thorogood (1959). On the other hand, in the acidic form of ferricytochrome c the iron is bonded in an intricate crevice structure to nitrogenous base groups of tlie protein at both the fifth and sixth co-ordination positions. One of the crevice bonds must be broken if 0H~ is to replace one of the groups, and the ionization reaction therefore takes the form, Prot. - Fe+++cyt.c - N(base) + H2O v^ Prot. - Fe++cyt.c - OH N(base) + H+ (21) With ferriperoxidase the nature of the reaction is rather more obscure, because in less alkaline solution the pH variations of the equilibrium con- stants for complex formation with cyanide, fluoride, azide, etc., differ systema- tically from the corresponding variations for ferrimyoglobin, ferrihaemo- globin and ferricytochrome c. The difference lies in the consumption of a proton accompanying the formation of the complex. George and Lyster (1958) have discussed various explanations that have been advanced, and suggested as a further possibility that acidic ferriperoxidase also has a crevice structure but with the labile bond, which is broken in complex formation and upon ionization, to some group other than a nitrogenous base, with a ^K of about 10 in horseradish peroxidase. Whatever the true explanation may be, it is clear that no strict comparison can be made between thermodynamic data for the ionization of ferriperoxidase and ferricytochrome c and the corresponding data for the haemoglobins. Finally, the question naturally arises as to whether any other haemo- protein derivatives are mixtures of high- and low-spin forms, Scheler, Schoffa and Jung (1957) and Havemann and Haberditzl (1958) have suggested that this may be the case for several other derivatives where the magnetic moments differ appreciably from the usual high or low values. However, no quantitative correlation of magnetic and spectroscopic properties, of the 138 P. George, J. Beetlestone and J. S. Griffith kind used in Section IV to calculate the individual spectra of the high- and low-spin hydroxides, was considered. Since azide gives a high-spin complex with ferricatalase and a low-spin complex with ferrihaemoglobin, it is quite likely that the ferrimyoglobin derivative would contain a significant fraction of the high-spin form. Preliminary calculations tend to confirm this. Never- theless, until temperature variations of the magnetic moment and absorption spectra have furnished direct experimental evidence for the existence of a thermal mixture, as in the case of ferrimyoglobin hydroxide, it is perhaps better to leave it as an open question with regard to the other derivatives. Acknowledgements The work reported above forms part of a research programme on haemo- proteins supported by grants from the National Science Foundation (G2309 and G7657). We wish to thank Dr. Britton Chance for making the facilities of the Department of Biophysics and Physical Biochemistry available to carry out the spectrophotometric study described in Section V. REFERENCES Basolo, F. & Pearson, R. G. (1958). Mechanisms of Inorganic Reactions — A Study of Metal Complexes in Solution, John Wiley, New York. Bennett, J. E. & Ingram, D. J. E. (1956). Nature, Lond. Ill, 275. Coryell, C. D. & Stitt, F. (1940). J. Amer. chem. Soc. 62, 2942. Coryell, C. D., Stitt, F. & Pauling, L. (1937). /. Amer. chem. Soc. 59, 633. Deutsch, H. F. & Ehrenberg, A. (1952). Acta chem. Scand. 6, 1522. Gamgee, a. (1868). Phil. Trans. {London) 158, 589. George, P. (1956). Currents in Biochemical Research, p. 338 (Ed. by D. E. Green), Interscience, New York. George, P. & Hanania, G. I. H. (1952). Biochem. J. 52, 517. George, P. & Hanania, G. I. H. (1953). Biochem. J. 55, 236. George, P. & Hanania, G. I. H. (1955). Disc. Faraday Soc. 20, 293. George, P. & Hanania, G. I. H. (1956). Currents in Biochemical Research, especially Table 5, p. 353. George, P., Hanania, G. I. H. & Thorogood, E. (1959). Unpublished results. George, P. & Lyster, R. L. J. (1958). Proc. nat. Acad. Sci. Wash. 44, 1013. George, P., Lyster, R. L. J. &. Beetlestone, J. (1958). Nature, Lond. 181, 1534. Gibson, J. F. & Ingram, D. J. E. (1957). Nature, Lond. 80, 29. Grifhth, J. S. (1956a). J. inorg. nuclear Chem. 2, 229. Griffith, J. S. (1956b). J. inorg. nuclear Chem. 2, 1. Griffith, J. S. (1956c). Proc. Roy. Soc. A 235, 23. Griffith, J. S. (1957). Nature, Lond. 180, 30. Griffith, J. S. (1958). Biochim. biophys. Acta, 28, 439. Griffith, J. S. & Orgel, L. E. (1957). Quart. Rev. 11, 381. Hanania, G. I. H. (1953). Ph.D. Thesis, The University of Cambridge, England. Havemann, R. & Haberdftzl, W. (1958). Z.phys. Chem. 209, 135. Heussenstam, p. &. Coryell, C. D. (1954). See Coryell, C. D., Chemical Specificity in Biological Interactions (Chap. 8, p. 108 and 113), Academic Press Inc., New York, 1954. Howard, J. B. (1935). J. chem. Phys. 3, 813. Keilin, D. (1933). Proc. Roy. Soc. B. 113, 393. Keilin, D. & Hartree, E. F. (1951). Biochem. J. 49, 88. KoTANi, M. (1949). J. phys. Soc. Japan 4, 293. Fenihaemoprotein Hydroxides 1 39 MoRiTA, Y. & Kameda, K. (1958). Mem. res. Ins fit. Food Science, Kyoto, Japan, No. 14,61. Orgel, L. E. (1955). J. chem. Phys. 23, 1819. ScHELER, W. & FiscHBACH, I. (1958). Acta Biol. Med. Germ. 1, 194, ScHELER, W., SCHOFFA, G. & JuNG, F. (1957). Biocliem. Z. 329, 232. Sternberg, H. & Virtanen, A. I. (1952). Acta. chem. Scand. 6, 1342. Taube, H. (1952). Cliem. Rev. 50, 69. Theorell, H. (1941). J. Amer. chem. Soc. 63, 1820. Theorell, H. (1942). Ark. Kemi, Min. Geol. 16A, No. 3, 1. Theorell, H. & Akesson, A. (1941). /. Amer. ciiem. Soc. 63, 1812. Theorell, H. & Ehrenberg, A. (1951). Acta chem. Scand. 5, 823. DISCUSSION Spin States and Spectra of Haeryioproteins The Electronic Origins of the Spectra By P. George and J. S. Griffith (Philadelphia) George : It is perhaps desirable to say something about the electronic origins of the spectra of the pure high-spin and low-spin compounds (ferrous and ferric), although we have not yet made a detailed analysis of them. Considering first the iron-porphyrin group we may divide the possible electronic transitions into three categories: porphyrin transitions, metal transitions and charge- transfer transitions. Free porphyrin has strong absorption in the visible and also a Soret peak and the intensity associated with these cannot be lost in the metal com- pound. Therefore one naturally supposes the Soret band of the latter and some at least of its visible absorption to be porphyrin transitions. These porphyrin transitions have the characteristic that the electric vector of the light lies in the porphyrin plane so that when it is at right-angles to it the light does not get absorbed. This is not necessarily true of the other transitions discussed later. In thinking about the part of the spectrum which arises from the porphyrin ring one should also remember that the singlet-triplet transitions may enhance their intensity considerably through coupling with the metal ion when the latter has non-zero spin. The metal transitions in the visible and infra-red are d-d transitions which would be of low intensity and probably completely masked by the porphyrin bands. At least they can hardly be responsible for the gross visible structure. The metal 3d-4p transi- tions would probably be in the ultra-violet although it is possible that the 4pz orbital might have its energy lowered sufficiently by interaction with a porphyrin tt orbital to invalidate this view. If this were so, however, the transition would also involve charge-transfer to the ring and so be partly included in our third category. Charge-transfer transitions are of two kinds — to and from the metal ion. Naturally we expect the low energy ones to be to the metal ion for ferric compounds, and from the metal ion for ferrous compounds. In each case, then, they would involve the iron atom commuting between the ferrous and the ferric state. It is natural to suppose that the infra-red bands characteristic of high-spin ferric compounds, oxy-haemoglobin and myoglobin, and the single-equivalent higher oxidation states, are indeed charge- transfer bands. Some components of such transitions are of course fully allowed for electric dipole radiation, and can therefore account for the relatively high intensities. The ligands in the fifth and sixth positions can also have transitions and give charge transfer to and from the iron, and so in a particular compound one or more bands may arise which have no counterpart in other compounds. It would be quite feasible, for example, that the infra-red band of oxyhaemoglobin might be of this type: it could be a transition from a weakly bonding to a weakly antibonding orbital embracing the ferrous ion and the oxygen molecule. We have for simplicity deliberately treated the system as if it can be broken up in a unique and well-defined manner into a number of pieces. This is not strictly H.E. — VOL. I — L 140 Discussion allowable and it is important to remember that interaction may occur among the various types of excited state, resulting in shifts of their positions and donations of intensity from one transition to another. Williams: In his introduction George has stated that the work he described on the equilibrium between two spin states was initiated in 1956. I do not wish to claim any priority in the discussion of the equilibrium between two spin states as it has been mentioned by a large number of authors since 1944 and extensive reference has been made to the factors controlling this equilibrium both by myself and others. In par- ticular, however, I would refer to a three cornered discussion between myself, George and Griffith, in discussions of the Faraday Society, 1955, where I was the only one to maintain this point of view. I have held this point of view consistently since 1953 in discussing the very compounds now examined by George and co-workers. The general idea that equilibrium exists between spin states has had excellent experimental foundation in the study of model complexes for some years past. I should add that I do not wish to detract from the importance of George's contribution which sets my discussion, and that of other earlier authors, on a firm basis. Falk's contribution (see Orgel's paper and discussion, this volume, p. 15) should be read in the light of a summary of my views in Lardy and Myrback, The Enzymes, 1959. There, as previously, I elaborate on spin state equilibria in the cytochromes. Boardman: In their paper, George, Beetlestone and Griflith (this volume, p. 126) correct their calculations for differences in the milliinolar extinction coefficients of the corres- ponding ferrihaemoglobin and ferrimyoglobin derivatives. I feel that these corrections are unnecessary as the extinction coefficients for the myoglobin derivatives appear to be too low by a factor of approximately 1-08. The extinction coefficients for the myoglobin derivatives are taken from the work of Hanania, who assumed a molecular weight of 17,000 for myoglobin whereas now there is evidence to suggest that the molecular weight of myoglobin is above 18,000. A few years ago, Adair and I at Cambridge succeeded in isolating two CO-myo- globins from horse-heart extracts by means of ammonium sulphate fractionation and chromatography on columns of Amberlite IRC-50. The main myoglobin fraction accounted for 90% of the total myoglobin. The molecular weight of the main com- ponent was determined from measurements of osmotic pressure and a figure of 18,400 was obtained. The extinction coefficient of the ferrimyoglobin cyanide derivative at 542 vnn was 0-613 for a 0-1 % solution and this corresponds to a millimolar extinction coefficient of 11-3, if we assume a molecular weight of 18,400. Fig. 6 of the paper by George et al. (this volume, p. 113) shows a millimolar extinction maximum of 10-6 for ferrimyoglobin cyanide. A value of 11-3 agrees closely with the corresponding value for ferrihaem.oglobin cyanide as determined by Drabkin. Theorell and Akeson have concluded also that the molecular weight of horse myoglobin is above 18,000. Their preparation was purified electrophoretically. Three myoglobin components were obtained and the iron content of the main component was 0-297 %. This figure gives a molecular weight of 18,800. George : I would Uke to thank Williams for drawing attention to the early suggestion of Willis and Mellor (/. Amer. chem. Soc. 69, 1237, 1947) that some co-ordination compounds may be thermal mixtures of high- and low-spin forms, and to his own remarks on the subject in the 1955 Faraday Discussion. I agree with Boardman's comments on extinction coefficients. The spectrophoto- metric data employed in the calculations were obtained before the electrophoretic separation of myoglobin into a major and two minor components had been demon- strated. The sample used had been subjected to repeated recrystallizations from ammonium sulphate and treated with strong phosphate buffer, pH 5-7, to remove haemoglobin. For a more exact analysis of the type described in Section IV of our paper it would not only be necessary to have extinction coefficients but also magnetic susceptibility measurements for single components. But an analysis of this kind only gives average spectra of the high-spin form and of the low-spin form for the pair of haemoproteins upon which the calculations are based. Undoubtedly there will be minor variations Ferrihaetnoprotein Hydroxides 141 from one haemoprotein to another as there are for other complexes, e.g. the cyanides and the fluorides. We regard these analyses rather as semiquantitative evidence for the existence of a thermal mixture, in that the calculated band maxima for the two forms occur at appropriate wavelengths with extinction coefficients of the right magni- tude. The spectra of the two forms calculated from the temperature dependence of the spectrum and magnetic susceptibility of a single haemoprotein derivative, e.g. ferrimyoglobin hydroxide in Section V, are however free from this uncertainty. Lemberg: Applying similar spectrophotometric methods to those used by George, Beetlestone and Griffith, we find that in contrast to horseradish peroxidase itself, the compound of haematin a with the apoprotein of horseradish peroxidase has the most 'high-spin type' of spectrum of the haemoproteins a which we studied. There is thus an interesting difference between two compounds of the same protein with the two diff"erent haematins, haematin a and protohaematin. O'Hagan : Preliminary results suggest that aetiohaematin does not attach to apomyoglobin at pH values higher than about pH 8. Holden and Hicks {Aiist. J. exp. Biol. med. Sci. 10, 219, 1932) considered alkaline ferrihaemoglobin to be a mixture of a compound in which linkage was not through the iron atom, and globin ferrihaemochrome. My results would appear to confirm this view and would seem to have some bearing on these results of George. ANALYSIS AND INTERPRETATION OF ABSORPTION SPECTRA OF HAEMIN CHROMOPROTEINS By David L. Drabkin Department of Biochemistry, Graduate School of Medicine, University of Pennsylvania, Pennsylvania The molecular spectra of various common haemin chromoproteins, their derivatives, and such related compounds as the haemochromes (nitrogenous ferro- and ferriporphyrins) exhibit selective absorption over the broad spectral range of 1000 to 200 m/t. The spectrum may be conveniently subdivided into four regions, in which individual maxima have a 500-fold difference in density (see Fig. 1). The most frequently examined region is the visible (2 in Fig. 1), the location of the a and /5 bands, as they have been designated historically. The selective absorption in this region is very different for the different haemin chromoproteins and some of their derivatives, whereas the absorption in regions 3 and 4 (Fig. 1), respectively the location of the y band (Soret, 1878, 1883a and b; Grabe, 1892) and the ultra-violet, is more generally similar for the different chromoproteins. Although the differences in absorption, as in the visible and near infra-red regions, form a very convenient and accurate basis for the quantitative determination of the various pigments (Drabkin, 1950; Gordy and Drabkin, 1957), they yield little obvious information concerning the relationship of the absorption to structures in the complex molecules. Dhere's finding that haemoglobin, like other proteins, had an absorption maximum in the vicinity of 275 m/< led him to conclude that the haemin nucleus v/as responsible for the absorption in the visible region, while the globin caused the absorption in the y band and ultra-violet regions (Dhere, 1906). In an early attempt to analyse the spectra of haemoglobin derivatives. Vies (1914) presumably accepted Dhere's earlier generalization, which has persisted to the present day. An examination of the spectrum curves of several haemoglobin derivatives led me to deduce about a quarter of a century ago that all the maxima (which represent bands) in the ultra-violet and some in the visible region were spaced at approximately equal frequency distances from each other (Drabkin, 1934). This was a potentially important discovery, since it materially simplified the interpretation of the complex spectrum from the viewpoint of its origin in the molecular structure. Before this work such a distribution of integrally related absorption bands, belonging to a spectral 142 Analysis and Interpretation of Absorption Spectra of Haemin Chromoproteins 143 series, had been demonstrated only in relatively simple molecules, such as KMn04 (Hagenbach and Percy, 1922) and CoCU in concentrated HCl (Erode, 1928). It was deduced that the absorption spectra of haemoglobin derivatives were largely an expression of iron in a co-ordination complex, and attention was called to certain similarities in the spectra of K3Fe(CN)g '- ^fh 1 1 / : P ^ 'J ; ^^' 1 H r : i f%\ r : 1 I 1 : HbC 32 r.^' -^^^^ /" ^^i;i>« •"^ / '- J / HbC 0 / "i"iiVli 1 1 1 1 II ll !| M ilLllllll ll 11 lllIM 'Ml III II 1 1 1 1 1 1 1 J 1 iiiiiiiii mil 11 II II 1 ll 1 1 1 1 tOOO 900 800 700 600 500 400 300 200 A, tn/^ Infrared 4" Visible 4*— Ultraviolet " d ore =10-50 d ore =1 d ore =01 Fig. 1. Absorption spectrum curves of oxyhaemoglobin, HbOj, deoxygenated haemoglobin, Hb, and carbonyl haemoglobin, HbCO (Drabkin, 1950; Gordy and Drabkin, 1957). The values of cuvette depth, d, or concentration, c, suggest the relative thicknesses of layer or concentrations required for optimal spectro- photometry in the different spectral regions, due to a 500-fold difference in the densities of the maxima over the spectral range of 1100 to 200 m/<. The use of log £ (the log of the molar or 1-Fe-atom equivalent extinction) permits the portrayal of the maxima in region 1 together with the rest of the spectrum. Many derivatives of the chromoproteins have weak bands in this region (the red and near infra-red). As examples, proto- and mesohaemin hydroxides have maxima in the region 810-820 m/<. Met- or ferrihaemoglobin hydroxide (alkaline methae- moglobin, pH 9), the spectrum of which in the near infra-red was originally studied by Horecker (1943), also has an absorption maximum at 820 m/< {v x 10~^ = 122), with £ (1 mM/1.) = 0-544 and log E = 2-735 (Gordy and Drabkin, 1957). and cyanmethaemoglobin (Drabkin, 1936). The same deduction was later made from the similar paramagnetic susceptibilities of these iron complexes by Coryell, Stitt and Pauling (1937). The absorption spectrum curves were resolved into component bands by means of a novel graphic-mathematical analysis (Drabkin, 1937, 1938, 1940, 1950). Interestingly enough, the analysis indicated that the a and ^ bands did not belong to the main spectral series and that the band at 275 m^< was not primarily owing to globin (Drabkin, 1937, 1938). Furthermore, the analysis predicted the potential occurrence of bands in the neighbourhood of wavelengths 833, 313 and 250 m/<. This was 144 David L. Drabkin verified by the finding of a definite maximum at 820 m/t in the spectrum of methaemoglobin hydroxide and at 314 mjLi in the spectrum of ferrocytochrome c (Drabkin, 1941a). The maximum at 314 m/< was later designated as the 6 band (cf. Theorell and Nygaard, 1954; Tsou and Li, 1956; Morton, 1958). The very brief past reports embracing this phase of our studies have presumably escaped notice by those more recently concerned with the analysis of the spectra of chromoproteins (Williams, 1956; Morton, 1958). In this communication a more complete description of our graphic analytical method will be supplied, together with an assembly of absorption data relevant to an interpretation of the spectra of haemin-protein complexes and possibly to the development of the theory of molecular spectra. MATERIALS AND METHODS Materials The haemoglobins were crystallized preparations (Drabkin, 1946, 1949a), the solutions of which were rendered 'salt-free' either by dialysis or passage through 'Deeminac 16-4' resin (Drabkin, 1954). The cytochrome c was prepared from horse heart by Tint and Reiss (1950), and by our analyses (see below) was 96 % pure, with reference to 0-43 % iron content. Methods Spectrophotometry. Most of the more recent measurements were carried out with the Beckman DU instrument, but in the earlier work the Hilger Spekker photometer and medium quartz spectrograph were used. When other types of instrumentation such as recording spectrophotometers were em- ployed, this will be indicated in the legends to the figures. Table 1 . f max of cyanide derivatives in the direct determination of HAEMIN IRON Compound Wavelength (in m/<) £max Fe-contentt (%) 1-Fe-atom equivalent weight Cyanmethaemoglobin Cyanmetmyoglobin Ferricytochrome c cyanide Ferriprotohaemin dicyanide 540 545 537 545 11-5 11-5 11 •o§ 11-3 0-338 0-339 0-41211 8-5711 16,552t 16,503t 652 * £max, the millimolar extinction referred to 1 milliatom of iron, t Iron determined independently as ferrous 1 : 10-phenanthroline (Drabkin, 1941b). % May be rounded out to 16,500. § Provisional. II Sample was 96% pure on basis of 0-43% of iron, or 91-6% pure on basis of 0-45; of iron. H Iron content of a-chlorohaemin. Analysis and Interpretation of Absorption Spectra of Haemin Chromoproteins 145 Concentration of Reference. The spectrum curves are plotted for a conven- tional depth of 1 cm. The concentration of reference, unless otherwise indi- cated, is 1 mM/L, where 1 niM represents 1 milliatom of iron. The iron content was determined independently (Drabkin, 1941b), but the spectrophotometric determination of the extinction at the maximum in the region of 545 to 170 180 190 200 210 220 - /" ol I - / 1 P\ — \ y / r^^ - // // / / / / X -^~ la - / 1 1 1 1 ! y t 1 1 1 / / !2- '" 1 ^ 1 1 1 1 1 1 K 510 Fig. 2. Absorption spectrum curves of complexes of iron. Curve 1 , ferrous 1 : 10- phenanthroline (ferrous o-phenanthroline). Curve la, ferrous dipyridyl complex. Curve 2, cyanmethaemoglobin (ferrihaemoglobin monocyanide). Curve 2a, haemin dicyanide (ferriprotoporphyrin dicyanide). The e (c = 1 mM/1.) for ferrous 1 : 1 O-phenanthroline is 11 -05 at the maximum of 500 m//, read against an appropriate blank. The open circle corresponds with a value of 11-25 for this complex read against water. For details see Drabkin (1941b). (Absorption spectra closely similar with those of cyanmethaemoglobin and haemin dicyanide are yielded by the cyanide derivatives of the ferrihaemochromes, such as monocyanide monopyridine ferriprotoporphyrin, with e = 11-7 for the maximum at 545 m/t (Drabkin, 1942a) and by mesohaemin dicyanide and coprohaemin dicyanide with maxima respectively at 537 and 535 m/<, displaced about 10m/< toward the shorter wavelengths in comparison with protohaemin derivatives (Drabkin, 1942b).) 535 m/f of corresponding cyanide derivatives of the ferri-complexes served in the direct, unequivocal evaluation of haemin iron (Drabkin, 1942a and b, 1949, 1954). Table 1 and Fig. 2 supply pertinent information. It may be noted that the writer's value of 0-338% for iron in haemoglobin (Drabkin, 1949b) has been tentatively accepted by the Protein Commission of the International Union of Pure and Applied Chemistry (cf. Drabkin, 1957). This iron content appears to be valid on a dry weight basis, and corresponds with a 1-Fe-atom equivalent weight of 16,500 for haemoglobin and 15,850 for globin, the total molecular weight of which may be taken as 15,850 X 4 = 63,400. 146 David L. Drabkin Notation. In the molecular interpretation of spectra, the frequency v (the number of waves passing a fixed point in a unit time, as 1 sec) is of more fundamental interest than the wavelength I. A term closely related to the frequency is the wavenumber v (the number of waves in a unit of length, as 1 cm). The relationship between )' and v is given by v = v x c, where c = 3 X 10^° cm/sec, the speed of light, and the relationship between v in cm~^ and X in vnfx is given by r = (l/A) x 10^. Thus A500 vo-fx corresponds Xov = 20,000 cm~^. It is convenient to use v X 10~^, and most of the graphs are so plotted. Anotherterm, the fresnel,/, has been employed. /= v X 10~^^. Hence, v x 10~^ =fl^- The absorption curves are plotted logically against v in ascending order from left to right, which is in the descending order with reference to A (cf. Drabkin, 1950). The Graphic Analysis of the Absorption Spectrum Curves. In their analysis of the visible absorption spectrum of permanganate Hagenbach and Percy (1922) made the assumption that the component bands (represented by maxima in the spectrum) could be resolved into individual simple curves, all of the same shape, but of different height. The shape of the curves was determined by the curvature of the slope at the lowest frequency end of the absorption curve. The summation of the resolved curves yielded the absorp- tion spectrum curve. Erode (1928) used effectively a similar method of analysis for the spectrum of CoClg. The spectra of these relatively simple molecules could be regarded as possessing essentially one broad band in which the multiple maxima represented a finer structure (Harrison, Lord and Loofbourow, 1948). The general character of the absorption spectra of the haemin chromoproteins is very different from that of KMn04 or CoCL, in HCl (see Figs. 1 and 4), and the spectrum curves could not be shown to be reproduced by a summation of component curves of the same shape, varying only in height. A highly satisfactory resolution of the complex haemin-protein spectrum curves has, however, been attained (Drabkin, 1937, 1938, 1940 and 1941a) by assuming that the component elements (bands) did not have the shape of simple curves but could be described by the 'bell-shaped' normal frequency curves of the form ;; = A: e ^ 2a== ; (1) or y = -tke:^ ^^^ q\ 2o^ ) The summation of unit curves of this type, each with different values for k and a (Fig. 3), reproduced very accurately the original (determined) spectrum curves. The families of curves in Fig. 3 are drawn for orientation to equation (1). In such symmetrical curves the values of j and x on each side of the I Analysis and Interpretation of Absorption Spectra of Haemin Chromoproteins 147 centre of distribution at 0 are the same, y is the height from the base of a point on the curve at a distance .v from the centre, k is the height at the centre of distribution a (usually designated the 'mean') for a particular case (in the diagrams of this figure a = 0). a, the standard deviation, is a measure 1 / \ ^ 3 k. i/ariable 3 , / \ cr, constant y = ke" 20-2 C, varioble / f \ \ 2 1 \ 1/ /- ^ \ \ J \ 1 / ^ / V -s \/ ^ \ 1 ^ y ^ n -\ S >-, \ ^ ■—/ ^ I ^ 0 A /, " ^ ^ y J, 1 I \ ^1 \ -L_±x-0. -i- ± X -Q^ I 2 ^ / VI K \, k, constant ^ Vh ^ .^ 0 FiG. 3. Families of normal frequency curves of the form: _ fix - a)-\ y = kc ^ 2a^ ^ The eflfect of variability in either ^ or c is shown in 1 and 2. In 3, most pertinent to the present analytical assumptions, both k and a are variable. of the spread or variation about the centre of the individual points, over two- thirds of which in such curves lie within the interval Icr and 95% within la. e is the base of natural logarithms, 2-7183. In our application to spectra, y and k are in a units, x and a (the locations of the centres of the curves or bands, assumed or deduced from the locations of the maxima in the absorption data) in units of/ or v. In the construction ot the curves, 2cr^ is conveniently evaluated by a rearrangement of terms in equation (1). (.V- a)^ (3^ 2a2 = loge kly k and y in equation (3) are derived either directly from the absorption data or by adjustment for overlapping of neighbouring component curves. In the case of prominent bands, as the so-called y or Soret band in the spectra of the chromoproteins, or bands at the lower frequency end of spectrum as with 148 David L. Drabkin C0CI2 (Fig. 4), a mean value of la"- is readily obtained from several points along the left contour of the determined absorption curve. In other cases simultaneous equations are useful in deriving appropriate values, aided by suitable processes of curve fitting, details of which cannot be supplied here. Having settled on values for 2a^ and /c, values for y are calculated with Fig. 4. The graphic-mathematical analysis of the absorption spectrum curve of C0CI2 in concentrated HCl (Drabkin, 1940). The continuous solid line with multiple inflections is the absorption spectrum obtained by Erode (1928), and the open circles represent summational points obtained by his method of analysis (see text). The individual curves, numbers 34 to 41, were obtained by the writer's method of analysis with curves of the normal frequency form. The black dots show the summation of these curves, expressed by the equation inserted in the figure. For 734 to J41 the values of k are 0-19, 1-20, 0-75, 1-08, 0-55, 0-80, 0-57, and 0-19. The corresponding values for Id^ are 102-0, 102-0, 102-0, 97-5, 93-8, 92-3, 27-2, and 66-2. equation (2) to yield the curves. Experience will suggest labour- and time- saving devices in this type of graphical analysis. It should be clear that the choice of spectral interval in terms of i^ X 10"" determines the locations (at equal frequency distances from each other) of the values of a. It is of interest that the analysis of the absorption spectra of complex molecules into component bands of the shape of normal frequency curves makes it possible to express the spectra in relatively exact mathematical terms. This cannot be done with the earlier successful analytical procedure used for KMn04 and CoCU. Hence it was desirable to test the applicability of the new method by the analysis of the spectra of the simpler molecules. Figure 4 and its legend give the analysis of the spectrum of CoClg, and the Analysis and Interpretation of Absorption Spectra of Hacmin Chromoproteins 149 conclusion appears justified that practically as good a result is yielded by the new method as by the one used by Brode (1928). The latter's frequency spacing of/= 12-28 was purposely retained in the analysis. It should be pointed out that the writer's method yielded one extra band (number 34) at the low-frequency end of the spectrum. This is explained by the fact that a values, calculated from the left slope of the absorption curve were suflftciently discrepant from each other to denote skewness and suggest the presence of an additional component. Tentative Corollaries or Rules of the Analysis and Notation of Bands. (a) The presence of bands in the absorption curves may be indicated either by well-defined maxima, by inflections ('bumps') in the curve, or by regions of relatively flat absorption. On the other hand, several component bands may be merged together into an apparently single band or may be hidden in the final 'summational' spectrum curve. These propositions can be demonstrated by the summational results obtained with two or more neighbouring (over- lapping) curves of the normal frequency type, (b) The positions of the maxima in the determined absorption spectrum may be displaced from their theoretically correct locations in the resolved components. Indeed, this is an expected consequence of the overlapping of the component elements in the proposed analytical method, (c) The analysis itself best discloses the multiple components, and, as has alieady been stated, 'predicts' the possible presence of hidden bands. Hence, certain component bands are represented by prominent maxima in the spectra of all haemin chromoproteins, others only in the spectra of some of the complexes or their derivatives (see Table 2). The descriptive notation used for a particular band in the complex spectra may prove controversial. This matter need not be debated here. The historical designations a and (i have been retained for the bands in the visible green spectral region. Even this may be illogical, since bands are present in the red and near infra-red spectral regions (Fig. 1). The a and /5 bands are deduced by our analysis to have a structural significance differing from the main frequency distributed series. For the latter, which includes the Soret and ultra-violet bands the designations y and 6 appear inappropriate, and they will be assigned a number, n, which is an integer (3, 4, 5, 6, 7, etc.) based on the frequency spacing v x 10"^ = 40. Thus 6 x 40 = 240, the wavenumber location or a of the y or Soret band; 8 x 40 = 320, the postulated location of the d band (Table 2, Figs. 5 to 10). EXPERIMENTAL Contribution of Haemin to Over-all Spectroscopic Character of the Haemin Chromoproteins. In Tables 1 and 2 and in Figs. 1, 2 and 5 to 11, which with their legends are largely self-explanatory, the basis for the analysis of the spectra and deductions drawn therefrom is furnished. Attention may be directed to several points. 150 David L. Drabkin 150 250 30 — - K3Fe(CN)s,IOOmlVIA - Hematoporphyrin, ImM/l. - Cyanmethemoglobin, ImM/L /" ]■ ■ ■■ III Ferriprotohemin dicyanide, ImM/L \ i V \ ( N k u - f 1 J 1 \' i U v ^ f 1 J 1 * / /^ =^-^_../" Multiples of 40 = 4 5 6 7 9 10 II 600 500 tT\/i Fig. 5. Comparison of absorption spectra of K3Fe(CN)6, haematoporphyrin (in alkaline solution), cyanmethaemoglobin or ferrihaemoglobin monocyanide (from dog haemoglobin), and ferriprotohaemin dicyanide or haemin dicyanide at pH 13. Hogness and colleagues (1937) reported a maximum at 230 m/t (corresponding with V X 10~^ = 435), with e = 32-1, for haemin dicyanide. The abscissal scales indicate the postulated locations of bands in the equally spaced, frequency distributed series (Drabkin, 1936, 1938). ■xlO" 1 ■ - — Cyanmethemoglobin 1 1 1/ ooo Cyonmetmyoglobin ; V / 1 \ / 1 V a^^ J 0 r ' y^ / \ y ^^ Multiples of 40=4 \ n\// Fig. 6. Comparison of spectra of cyanmethaemoglobin (from the haemoglobin of man), ferricytochrome c cyanide at pH 10 to 11 (for preparation see legend to Fig. 7), and cyanmetmyoglobin (from horse heart myoglobin). The remarkable similarity of these spectra is evident. The abscissal scales indicate the postulated locations of bands in the equally spaced, frequency distributed series. Analysis and Interpretation of Absorption Spectra of Haemin Chromoproteins 151 (1) The determination of haemin iron is most unequivocally and quantita- tively accomplished through the characteristic absorption in the visible region of the cyanide derivatives of haemin and the haemin proteins (Drabkin, 1942a, 1949b), and the spectra are remarkably similar in both the visible and ultra-violet regions (Figs. 2 and 6). These findings indicate that a single major molecular characteristic is responsible for the general over-all spectrum. In the writer's analysis for iron by the 1 : 10-phenanthroline method the e value for the maximum of ferrous 1 : 10-phenanthroline was found to be close to identical with that of the cyanide derivatives of the haemin complexes (Table 1 and Fig. 2). Accordingly it was deduced that in the analytical procedure iron was liberated from one complex (hexaco-ordinated haemin iron; Drabkin, 1936, 1938) and bound up in another, diimine iron (Drabkin, 1941b). The similarity of the spectra favoured the idea of a spectroscopically operative structural similarity in these different classes of compounds. The spectroscopic similarity of the cyanide derivatives of the ferrichromoproteins has its counterpart in their low paramagnetic susceptibilities (Coryell, Stitt and Pauling, 1937; Theorell, 1941). It was deduced from their magnetic behaviour that they are essentially (though not fully) octahaedral d'^sp^ co- valent bonded stabilized structures, in essence of the Werner hexaco-ordina- tion type (cf. Pauling, 1940, 1948, 1949). (2) In Table 2 it may be seen that in some haemin complexes only a limited number of the bands, postulated by the analysis, are represented by definite maxima in the absorption spectra. However, considering the data on the different haemin complexes as an interrelated whole, maxima representative of at least eight, possibly nine bands (numbers « = 3 to 1 1) of an equally spaced frequency distributed series are found. As has been stated, the spectrum of ferrocytochrome c (Drabkin, 1941a) proved to be particularly rich in maxima (Fig. 7) and disclosed the presence of bands missing from the earlier examined spectra of haemoglobin derivatives, but 'predicted' by the analysis. The basis for these differences in the spectra of reduced and oxidized cytochrome c and haemoglobin derivatives is not clear, unless it can be attributed to the difference in the bonding of the haemins with the protein (Theorell, 1938, 1941). With the exception of small 'shifts' in the location of some of the maxima, such spectral differences are erased in the spectra of the respective cyanide derivatives of these chromoproteins (Fig. 6). (3) An examination of Table 2 will disclose that bands number 3, 6, 9 and 11 in the series Vq x 10^^ = 40 can also be distributed at regular fre- quency intervals on the basis of Vq x 10"^ = 60. In the latter case the /9 band would be included as number 3 and the bands would be represented by n = 2, 3, 4, 5, 6 and 7, with number 5 in an intermediate position between 7 and 8 of the spacing Vq x 10"^ = 40. The 60 spacing was originally assumed (Drabkin, 1934), and led to the analysis, illustrated for the spectrum of cyanmethaemoglobin, in Fig. 8. In this figure it may be seen that, utilizing 152 David L. Drabkin the frequency interval of 60, the graphic-mathematical analysis into com- ponent bands fails to reproduce by their summation the observed absorption in the regions v X 10^- = 160, 200, 280 and 320. These spectral regions do have representative maxima in the absorption spectra of some of the chromo- proteins (Table 2), and either two or more separate series would have to be 150 250 1 1 1 1 ff] Ferrocytochrome c i Ferrici tochrorr e c, p H8 45 -/ 1 1 -f - 1 1 1 I \ V h ji > J V; >L V Multiples of 40 = 4 » /? 5 400 350 Fig. 7. The spectra of reduced and oxidized cytochrome c from horse heart, pH 8-45. The molecular weight of reference was taken as 13,000 (0-43 % of iron). The preparation had 0-412% of iron by Drabkin's o-phenanthroline method (1941b). Reduction to ferrocytochrome c was by means of NaoSgOi for the data to 380 m/ii and with palladium asbestos and hydrogen for the data in the ultra- violet region beyond 380 m/<. To insure complete oxidation to ferricytochrome c 0-4 of an equivalent of ferricyanide was added to the 60 % oxidized preparation. In the measurements this was balanced out by an equivalent amount ferrocyanide. The abscissal scales indicate the postulated locations of bands in the equally spaced, frequency distributed series (Drabkin, 1941a). assumed or a more appropriate single frequency spacing adopted. The latter alternative was taken, and accordingly the 40 spacing was tested. This spacing was preferred not only because of the locations of the observed maxima, but also intuitively since it excluded both the a and (j bands. The supplemental Figs. 9 and 10 illustrate the analysis of the absorption spectrum of cyanmethaemoglobin, taking Vq x 10~^ = 40. It may be seen that the summation of the analytically resolved ten absorbing units or bands agrees excellently with the observed spectrum, which can be expressed mathematically (Drabkin, 1937) by sj, = ( kQ {x - [n X 40])-\ + S(>'«,J^) (4) Analysis and Interpretation of Absorption Spectra of Haemin Chromoproteins 153 The closeness of fit of the analytically derived spectrum with the experi- mentally determined one is reflected by values of 3-29 for the mean of devia- tions between them, 0-24 for the mean about which the deviations fall, 6-06 for S.D., and 0-47 for S.D. from 151 to 205 i5 x IQ-^ (Fig. 10). All the absorption spectra of the haemin chromoproteins and their derivatives thus far studied by the writer (a partial list of which is given in Table 2) can be similarly analysed into their component bands, and equation (4) may be regarded as generally applicable. (4) The dissection of the broad band of the spectrum of cyanmethaemo- globin at wavelength 540 m// or v X 10-^ = 185 (Drabkin, 1937, 1938 and Fig. 10) merits particular attention. The accurate establishment of the left contour of this band reveals a very slight 'bump' in the vicinity of 570 m/< (see curve 2 in Fig. 2 and the solid line in Fig. 10). There is also the appreciable absorption in the regions of 630-600 and 500 m^w, the locations assigned in the analysis to bands 4 and 5, which are represented by definite maxima in some of the chromoprotein spectra (Table 2). These findings in themselves Table 2. Location of maxima (bands) in chromoprotein spectra, postulated to belong to series « = (jt x 10"^)/(vo x 10~^) Vq X 10~^ is assumed = 40 (where n = 1). The values in brackets give the number n of the band in the series. The values in parentheses give locations of inflections (or bumps) in the absorption curve, as distinguished from obvious maxima. Compound a P f X 10 - observed 173 184 f, Oxyhaemoglobin 109 241 290 362 417* [3?J [6] [71 [9] [11?] Carbonylhaemoglobin 176 186 111-125 [3?1 238 [6] 290 [7] 366 [91 426 [11?] Cyanmethaemoglobin (from 185 239 285 368 437 dog haemoglobin) [6] [7] [91 [11?] Cyanmethaemoglobin (from 185 237 282 366 haemoglobin of man)t [61 [7] [9] Methaemoglobin, pH 5-9 (173) (184) 159 198 246 285 359 435 [4] [5] [61 [7] (91 [11?] Methaemoglobin hydroxide. 173 184 122 167 (206) 241 282 365 431 pH9-2 [3] [4] [5] [6] [71 [91 [11?] Ferrocytochrome c, pH 8-45 182 192 (135) 241 (284) 319 (365) (403) (446) [3?] [61 [71 [81 [91 [101 [111 Ferricytochrome c, pH 8-45 182 191 (143) 244 280 (315) 359 (430) [3?] [6] [71 [81 [91 [11?] Ferriprotohaemin dicyanide. 183 235 278 (360) t pH13 [6] [71 [9] * With the Beckman DU spectrophotometer, measurements in this spectral location are unreliable; with ferrocytochrome c a definite inflection in the curve at v x 10~^ = 446 was obtained with Hilger's Spekker photometer and medium quartz spectograph. t For the spectra of ferricytochrome c cyanide and cyanmetmyoglobin see Fig. 6. X For a maximum reported to be present in this location see the legend to Fig. 5. suggest the composite nature of this band. In the analysis, the order of solution was band 4 first, then 5, then a, and finally /5. The need for a /5 band and the locations of the centroids of a and /5 were consequences of the analytical procedure. The centroids of the analytical a and /? bands are respectively at i^ x 10"- = 179 (559 m//) and 187 (535 m/0 and k is larger 154 David L. Drabkin for a. The locations and relative densities of the bands are very suggestive of those found in the spectra of ferrohaemochromes, like pyridine and globin ferroprotoporphyrins (see Fig. 17 and Drabkin, 1937, 1938). It is difficult to regard this analytical result as pure coincidence. It is at the least highly provocative, revealing as it does fundamental similarities in the spectrally ^ 80 MHbCN (■-;4ov y^= Il5e" 243 I. -3001 yj,= 3IOe -lozo (x-360) yg=3l'3e 360 (x-120)' y^= 100 e 7go~ 500 Vu X 10" Fig. 8. The graphic-mathematical analysis of the absorption spectrum curve of cyanmethaemoglobin (from dog haemoglobin). The continuous solid line is the absoqDtion spectrum obtained experimentally. The broken line represents the summation of the individual component elements or bands (solid dotted lines) of the normal frequency form, derived by the writer's method of analysis. The bands are numbers 3 to 7 in an equally spaced, frequency distributed series, with Vq X 10~^ = 60. The summation of the resolved component bands is given by the equation inserted in the figure. The equations y^ to J7 (insert in figure) are for the respective components, with the applicable values for a and 2a- sub- stituted in equation 1 (see Methods). divergent visible region of haemin-protein spectra. The single band of deoxygenated haemoglobin at 555 m/< can be similarly resolved into bands 4, a, /? and 5. To summarize, it may be concluded from the graphic-mathematical analysis that (1) the absorption spectra of all haemin chromoproteins and their derivatives (both oxidized and reduced) are fundamentally similar, (2) the a and /5 bands have a different origin from bands 3 to 11 of the equally spaced frequency distributed series, and (3) the differences in the spectrum curves (largely evident in the visible spectral region) are an expression of the relative densities or intensities of the absorption bands in the different compounds (Drabkin, 1937, 1938, 1941a; Table 2 and Figs. 10 and 12). W 60 MHbCN / " 2'y„=ke x-tn40l)' r - y4 = 0 80e" 108 "\ 1 - 7^=630 6" 155 1 1 y^=4 32e- <-l87)^ 121 1 y^=5 00e" .-200)^ 320 1 1 y, = ll8e- «-240)^ 236 1 y^ = 270e- >-280)^ 1613 1 !l yg = 14 8e" .-320)^ 1332 1 i ,;/^ \ 1 . y9 = 333e- .-360)2 564 Un ^ I - y,o=2l7e- «- 400)2 1345 i K'^^ /y * \L \ - y,| = 190e- 1 1 «-440)2 B90 /"^^ J^l ^ 1/ ''"^^SK :>< .1. n*-.t. . 250 300 VuXlO" Fig. 9. The graphic-mathematical analysis of the absorption spectrum curve of cyanmethaemoglobin. (This figure and Fig. 10 are supplementary.) The continu- ous solid line is the absorption spectrum obtained experimentally. The broken line represents the summation of the individual components or bands (solid dotted lines) of the form of normal frequency curves, obtained by the writer's method of analysis. In contrast vi'ith Fig. 8, the bands in the equally spaced frequency distributed series are resolved on the basis of Vq X 10"^ = 40. Bands with numbers « = 6 to 1 1 are shown in this figure; numbers 4 and 5 and resolved bands a and /S in Fig. 10. The summation of the bands (4 to 11) in the single series and the mathematical formulation of each unit is given by the equations inserted in the figure. See Fig. 10 and the text. 140 150 160 170 180 190 Fig. 10. The graphic-mathematical dissection of the band in the green spectral region of cyanmethaemoglobin. This figure is supplemental to Fig. 9. See legend to Fig. 9 and the text. H.E. — VOL. I — M 156 David L. Drabkin Contribution of Protein to the Over-all Spectroscopic Character of the Haemin Chromoproteins. From what has already been said, the affect of the metalloporphyrin complex is dominant throughout the spectral range, and the absorption of the haemoglobins and of cytochrome c in the ultra-violet region, even at the location of the so-called protein band (280-270 m/t ; band number 9 in the analysis), cannot be attributed exclusively to the protein moieties. Fig, 11. The contribution of the protein moiety, globin, to absorption in the region 275 to 280 m/t (v x 10"^ = 360; band number 9). Curve 1, heavy solid line, oxyhaemoglobin, 1 him/I. (1 milliatom of Fe); curve 2, heavy broken line, carbonyl haemoglobin, 1 mM/1. (1 milliatom of Fe); curve 3, light solid line with open circles, met- or ferrimyoglobin, 1 mM/1. (1 milliatom of Fe); curve 4, light solid line, globin (from haemoglobin), 0-25 mM/1. (reference mol. wt. = 15,850); curve 5, light solid line with black dots, bovine plasma albumin, 1 roM/l. (reference mol. wt. = 68,000); curve 6, broken line with dots, bovine albumin, 0-25 mM/1. (reference mol. wt. = 17,000); curve 7, broken light line, denatured globin, 0-25 mM/1.; curve 8, light line with crosses, 1 mM/1. with reference to haemin (protohaemin added to amount of globin used for curve 7). Curves 4 and 6 aflFord a comparison of globin and albumin at approximately similar concentrations by weight. Curves 1 and 2 indicate that, if expressed on a molar basis (mol. wt. = 66,000), the absorption in this spectral region would be four times greater for the haemin proteins than for albumin. See the text. The spectroscopic data plotted in Fig. 11 substantiates this conclusion. It may be seen that the bands of haemoglobin derivatives and metmyoglobin, though generally similar yet differ slightly from each other (curves 1 to 3). On the other hand, they are broader and smoother than the band of bovine albumin (curve 5). Misleading deductions as to the molecular origin of this band in chromoproteins may have been drawn because of the convention of expressing information on the chromoproteins on a 1-Fe-atom equivalent basis, whereas for proteins like albumin the molecular weight base has been used for reference. If the absorption for the haemoglobin derivatives were to be referred to 1 mM/l., instead of 1 milliatom of Fe/1., its maximum in this Analysis and Interpretation of Absorption Spectra of Haemin Chromoproteins 157 spectral region would be four times higher than that of albumin (see curves 1 to 6 and the legend, Fig. 1 1). However, denaturation does have an influence on chromoprotein spectra in this region, as it does on the albumin spectrum (compare curves 4 and 7). The influence of haemin in this spectral region is suggested by curves 4, 7 and 8. Contrariwise, if the protein moieties do exert spectroscopic influence, they do so more evidently in the visible spectral region, the spectral changes in which may be deduced to be a function of the nature of co-ordinating groups or ligands bonded to the iron, which is also bonded to the four pyrrolic nitrogens. Thus, the alkaline denaturation of haemoglobin yields globin haemochrome, and the spectrum of globin ferro- protoporphyrin in the visible spectral region is practically indistinguishable from that of pyridine ferroprotoporphyrin (Drabkin, 1942a and Fig. 17). The situation with respect to spectroscopically operative protein structures is presumably different in the case of cytochrome b^. The pronounced maximum at 265 m/i {v x 10^^ = 377) appears to be clearly ascribable to the riboflavin phosphate and a polydeoxyribonucleotide, which are structural components of this complex molecule (Appleby and Morton, 1954; Morton, 1958). Whether the influence of the nucleotide is confined to this spectral region or may be reflected in other spectral regions remains to be ascertained. Thus far no convincing evidence has appeared that the number 9 band in the chromoproteins may be a composite with finer structure attributable to the aromatic amino acids, as has been shown for non-haemin proteins (Holiday, 1936,1937; Lavin, Northrop and Taylor, 1933; Lavin and Northrop, 1935). It may be concluded that from the qualitative viewpoint the protein moiety of the haemin proteins contributes only negligibly to their over-all spectra (see Discussion). The a and (j Bands and the Neighbouring Visible Spectral Regions. Despite the fundamental similarities disclosed by the analysis of the spectra of the haemin proteins, in the past major attention has been devoted to characteristic and pronounced diff'erences in these spectra in the visible spectral region. Such diff'erences have been valuable in the accurate determination by means of spectrophotometry of biologically important derivatives as well as in the study of certain equilibria (Austin and Drabkin, 1935; Drabkin and Singer, 1939; Drabkin and Schmidt, 1945; Drabkin, 1950; Gordy and Drabkin, 1957), but may have directed attention away from the basic similarities. It may be said that the combination of haemoglobin with gases (Og, CO, NO), the oxidation of haemoglobin to ferrihaemoglobin, the alkaline denaturation of haemoglobin to haemochrome, and the pH dependency of ferrihaemoglobin are all spectroscopically operative, and that many of these reactions have parallels in the spectroscopic behaviour of the cytochromes. Figures 12 to 17 and Table 3 are relevant in the interpretation of the diff'erences in the visible spectra of chromoproteins, consonant with the analytical viewpoint that all the spectra have a and /3 components straddled by bands 4 and 5 of the dominant 158 David L. Drabkin Table 3. Correlation of spectroscopic pattern with electronic structure S = strong band; W = weak band; M = moderately strong band; N = negligible band Compound Figure number Spect 4 rum bands a B Number of unpaired Typet r electrons* Ferrihaemoglobin 12 S W w 5 Ionic (1) Ferricytochrome c I and II 7 St W w 5 Ionic (2) Ferrohaemoglobin 1 St MJ Mt 4 Ionic (3) Ferrihaemoglobin hydroxide 12 Mt S s 3 Ionic (1) Ferrihaemoglobin cyanide 10 wt Mt Mt 1 Covalent (1) Ferricytochrome c cyanide 6 wt Mt Mt 1 Covalent (2) Oxyhaemoglobin 1 N S S 0 Covalent (3) Carbonylhaemoglobin 1 N s s 0 Covalent (3) Globin ferroprotoporphyrin 17 N s s 0 Covalent (4) Pyridine ferroprotoporphyrin 17 N s s 0 Covalent (4) * The permanent magnetic dipole moment in these complexes is due almost entirely to Hsy the spin moment of the unpaired electrons, /is is derived from the measured molal paramagnetic susceptibility. Theory for //s for 1 to 5 unpaired electrons is 1-73, 2-83, 3-83, 4-90 and 5-92 Bohr magnetons. t The terms ionic and covalent should be prefaced by 'essentially' to indicate partial ionic and covalent character. Thus, ferrihaemoglobin is more ionic than ferrihaemoglobin hydroxide (Pauling, 1940, 1948, 1949). References: 1, Coryell, Stitt and Pauling, 1937; 2, Theorell, 1941 ; 3, PauUng and Coryell, 1936b; 4, Pauling and Coryell, 1936a. t Deduced from the graphic analysis of the spectrum. series. The relative prominence of these four components resuUs in the spectral differences. This is well illustrated in supplementary Figures 12 and 13, which show the transition with change in pH of the spectra of human met- er ferrihaemoglobin to ferrihaemoglobin hydroxide. The pH dependency of the spectrum of ferrihaemoglobin was originally studied by Hartridge, 1920 and Haurowitz, 1924. A detailed and very careful spectrophotometric study by Austin and Drabkin (1935) of dog ferrihaemoglobin, MHb, with reference to the equilibrium MHb ^ MHbOH revealed a reaction of the first order with OH ion, and pi^g (as it is now usually designated) was accurately established as 8- 12 ± 0-01 at /< (ionic strength) = 0-1 and a = 0-6. This value for p^3 has been confiimed by the independent techniques of magnetometric titration (Coryell, Stitt and Pauling, 1937) and differential acid-base titration (Wyman and Ingalls, 1941). Furthermore, the demonstration of the effect of ionic strength, regarded in that day as unusual for such complex com- pounds, was also apparent in their magnetic behaviour. Thus, the haemin- linked group, responsible for ^K^ (i.e. the release of a proton from a molecule of water co-ordinated with the iron in acid ferrihaemoglobin, MHb • HOH+, to form MHb -OH -H H+) was spectroscopically, magnetometrically and titrimetrically operative. It appeared that at least in this case the electronic structural change involved in MHb ^ MHbOH (MHb, with a magnetic Analysis and Interpretation of Absorption Spectra of Haemin Chromoproteins 159 susceptibility corresponding with 5 unpaired electrons, to MHbOH with 3 unpaired electrons; Coryell, Still and Pauling, 1937) could be correlated with the spectra of the transition of one spectroscopic species to the other. (In ferricytochrome c, appreciably more pH-stable than ferrihaemoglobin, four such spectroscopically operative acid groups have been uncovered (Theorell and Akesson, 1941).) The pA'3 for human MHbOH is 8-15 at /i = 0-1, nearly the same as that of dog MHbOH. This value is derived from the automatically recorded spec- trum curves in Fig. 12 (Drabkin and E. Thorogood, unpublished). That only two species participate in the spectroscopic transition was evident both in the visible and near infra-red spectral regions and was reflected by the presence of 5 isosbestic points (see legend to Fig. 12). However, it may be seen in Fig. 13 that there is a departure from this behaviour in the near ultra- violet region, since band number 6 is appreciably higher and located at a shorter wavelength for MHb than the corresponding band for MHbOH (cf. also Hicks and Holden, 1929). In the present connection, the main point which may be stressed is that bands 4, a, /5 and 5 are evident in the spectra of both MHb and MHbOH, but in the latter a and ^ dominate, whereas in the former a and ^ are only weakly expressed, while 4, particularly, and 5 are relatively dominant (see Fig. 12). Using the spectra of MHb and MHbOH as models, the relation between the quantum mechanical deductions drawn from measurements in Linus Pauling's laboratory of the molal paramagnetic susceptibilities of haemin and its derivatives and the spectra of these compounds may be placed upon a somewhat broader base by a suggestive correlation of the spectroscopic pattern with the corresponding electronic character. In general, all essentially covalent structures have prominent a and ^5 bands and weak number 4 bands, whereas essentially ionic structures may have relatively weak a and /5 bands, but are mainly characterized by strong number 4 bands or marked absorption in the spectral region of the 4 band. This is brought out in Table 3. The spectral patterns in the visible region are not as diverse as may have been supposed (Drabkin, 1942a and b, and Figs. 15 to 17). However, oxy-, carbonyl, cyanide and pyridine derivatives of ferrohaem are spectro- scopically distinguishable from each other, whereas their electronic configura- tion is the same (Table 3). It seems reasonable to infer that both the electronic structure of the haemin iron and the nature of the co-ordinating ligand contri- bute to the spectrum (cf. also Williams, 1956). The co-ordination of haemin iron with OH ion is a general reaction, exhibited also by the ferrihaemochromes (cf. Haurowitz, 1927; Davies, 1940). In Fig. 14, the writer's spectrophotometric measurements of the equilibrium pyridine ferriprotoporphyrin ^ pyridine ferriprotoporphyrin hydroxide are supplied. From these data a pAT value of 9-64 may be derived (see Insert to Fig. 14). However, there is a most interesting difference between these 160 David L. Drabkin D I Fig. 12. Absorption spectrum curves of solutions of crystalline human met- or ferrihaemoglobin, buffered to various pH values. All the buffers had an ionic strength, /<, of 0-1. D is the optical density. The spectra were obtained with the General Electric recording spectrophotometer on solutions with a haemoglobin iron concentration of 0-0584 mw/l. (Figs. 12 and 13 are supplementary.) Curve 1, methaemoglobin at pH 6-20 (taken as species 1). Curve 2, methaemoglobin hydroxide at pH 9-35 (taken as species 2). The nine intermediate curves represent mixtures of the two species at respective pH of 6-61, 7-05, 7-34, 7-7, 8-17, 8-50, 8-60, 8-80 and 9-20. Methaemoglobin hydroxide has an additional maximum at 820 m/< and there is an additional isosbestic point for the two species at 845 m/t (Gordy and Drabkin, 1957). For the earlier work on the equilibrium of methae- moglobin-methaemoglobin hydroxide and the treatment of spectrophotometric data in a two component system see Austin and Drabkin, 1935. Analysis and Interpretation of Absorption Spectra of Haemin Chromoproteins 161 spectra and the corresponding ones for MHb ^ MHbOH. The colours of the spectroscopic species are reversed with reference to pH. MHb is brown, MHbOH red, whereas in the pyridine complexes the colour is olive brown at D I A ^ - n - V - 1 - 1 - J '^Tl 1 1 2 1 1 1 r 1 1 1 1 r -r Fig. 13. Absorption spectrum curves of solutions of crystalline human met- or ferrihaemoglobin, buffered to various pH values. The solutions correspond to those shown in Fig. 12, but the concentration of haemoglobin is 0-01 17 mM/1. Tsee legend to Fig. 12). The e (lmM/1) values for the maximum of methaemoglobin (pH 6-2) at 407 m/< and for the maximum of methaemoglobin hydroxide (pH 9-35) at 415 m/i are 169 and 113 respectively. pH 11-38, red at pH 7-26. Uncertainties still exist as to the structure of pyridine ferriprotoporphyrin (Davies, 1940) and magnetometric measure- ments are available only for the hydroxy form (Rawlinson, 1940), which was deduced to be essentially covalent in contrast with MHbOH (see Table 3). Further information is required for the clarification of this situation, but at present the spectra cannot be easily reconciled with the proposed correlation. 162 David L. Drabkin Contribution of Porphyrin to the Over-all Spectroscopic Character of Haemin Chromoproteins. In the early days of the investigation of the struc- ture of cytochrome c, Theorell (1939) questioned whether the thio-ether linkage he had proposed (Theorell, 1938) for the union of the protein with the haemin at positions 2 and 4 was present in the native compound or was 190 200 Fig. 14. Absorption spectra showing transition, with change in pH, from pyridine ferriprotoporphyrin to its hydroxide. In all solutions the final concentrations of haemin Fe and pyridine were 0-1 mM and 5700 mM/1. respectively. The pH was modified by the inclusion of HCl in all solutions except that represented by curve 9. Curve 1, absorption spectrum of species 1, probably dipyridine ferriproto- porphyrin, pH = 7-26. Curve 9, absorption spectrum of species 2, pH = 11 -38. Curves 2 to 8, absorption spectra of mixtures of species 1 and 2. The pH values of the solutions represented by curves 2 to 8 were 7-62, 8-32, 8-95, 9-45, 9-86, 9-98, and 10-36. The insert in the figure shows the partition of species 1 and 2 against pH (see legend to Fig. 12). The solid circles are from data presented in the figure. The open circles are based upon absorption data not shown. See the text. artifactually obtained by the vigorous hydrolysis conditions employed in the isolation of porphyrin c. This led the writer (Drabkin, 1942b) to investigate the spectra of cyanide, pyridine and carbonyl derivatives of proto-, meso-, and coproferrohaem, and corresponding derivatives of haemoglobin and ferrocytochrome c. In protohaemin positions 2 and 4 are occupied by the unsaturated vinyl group, whereas in meso- and coprohaemin the substituents in this position are respectively ethyl and propionic groups. The spectra of the meso- and copro- derivatives were virtually indistinguishable from each other (Figs. 15 to 17 and see Drabkin, 1942b, for the individual spectra of Analysis and Interpretation of Absorption Spectra of Haemin Chromoproteins 163 carbonyl derivatives), indicating tliat the CoHg and CH3CH2COOH side chains had individually no distinguishable spectroscopic affects, but the maxima in the spectra of all protohaemin complexes were shifted some 10 m^« toward the longer wavelengths. Three distinctive spectroscopic patterns were found for all the ferrohaems, characteristic for the combination with 16 17 18 19 20 : 1 1 1 1 1 1 ' ' T 1 1 1 III' — 1 — '-. 1 2A - r r 2. 12 ': r^ ^ r J \/n \ \ r 1, V ►3 0 rir «(=!»-: ^. 1 1 1 \ 1 1 1 1 1 1 1 I 1 620 580 540 500 A, m/^ Fig. 15. The three distinctive spectral patterns exhibited by ferrohaems co- ordinated with three types of ligands, exemplified by derivatives of ferromeso- porphyrin. Curve 1, cyanide ferromesoporphyrin, representative of Pattern Type 1. Curve 2, pyridine ferromesoporphyrin, representative of Pattern Type 2. Curve 3, carbonyl ferromesoporphyrin, representative of Pattern Type 3. In all cases the NaOH concentration was 0-2 m/1. and the NagSjOi concentration 5 mM/1. The pyridine concentration was 6-19 m/1. and that of cyanide 400 mM/1. The horizontal arrows and appended numbers represent the magnitude in m/i of the shift of maxima towards longer wavelengths in corresponding derivatives of ferroprotoporphyrin (Drabkin, 1942b). cyanide, pyridine and carbon monoxide (Fig. 15). These conclusions were reached: (1) The shape and intensity of absorption in the visible region was a function of the nature of the co-ordinating ligand. (2) The wavelength location of the maxima (of the a and /5 bands) was a function of the haemins (or porphyrins) themselves, and most probably of the groups substituted in positions 2 and 4. The maxima in the spectra of haemoglobin derivatives were in the locations expected for protohaemin derivatives. On the other hand, the 164 David L. Drabkin spectra of derivatives of cytochrome c (Figs. 16 and 17) were in the meso- or copro- locations. Hence, this was offered as evidence that the spectrum itself of cytochrome c revealed a structural difference of its haemin in positions 2 and 4, namely a vitiation of the unsaturated vinyl bond structure, such as would occur in Theorell's thio-ether linkage (Drabkin, 1942b) Fig. 16. Pattern Type 1; absorption spectra of cyanide derivatives of ferro- haems, haemoglobin, and ferrocytochrome c. Curve 1, cyanide ferroprotopor- phyrin. Curve 2, cyanide ferromesopoi"phyrin. Curve 3, cyanide ferrocopropor- phyrin. Curve 4, the reduced cyanide derivative prepared from dog haemoglobin in alkaline solution, probably cyanide ferroprotoporphyrin. Curve 5, the reduced cyanide derivative prepared from cytochrome c in alkaline solution, ferrocyto- chrome c cyanide. See legend to Fig. 15 (Drabkin, 1942b). The spectral displacement of the protohaemin complexes toward the longer wavelengths was ascribed (Drabkin, 1942b) to the increase in the conjugated double bonds in the porphyrin system by the presence of the unsaturated vinyl radicals, analogously to the situation disclosed by the systematic studies of Hausser and Kuhn and their collaborators on polyene dyes of the type R— (CH=CH)„— R' (Hausser, 1934; Hausser et al, 1935a to e). Incidentally, on the basis of the above analysis, it could be prophesied that the haemin of cytochrome b.^ must be ordinary protohaemin with the vinyl residues intact (Morton, 1958). I believe that in the haemin proteins the influence on spectral location of the maxima in the visible region, evident also in ferrihaemin complexes such as the cyanide derivatives Analysis and Interpretation of Absorption Spectra of Haeniin Chromoproteins 165 (Drabkin, 1942a), represents the major contribution of the structure of the porphyrin nucleus to the spectra, although it is tempting to draw analogies between the band distribution and location in the spectra of porphyrins and /"xlO^^ Fig. 17. Pattern Type 2. Absorption spectra of denatured globin (globan) derivatives of ferrohaemins (reduced haemochromogens) and the spectra of haemoglobin and ferrocytochrome c in alkaline solutions. Curve 1, globan ferroprotoporphyrin, prepared from human globin and protohaemin. Curve 2, globan ferromesoporphyrin, prepared from human globin and mesohaemin. Curve 3, globan ferrocoproporphyrin, prepared from human globin and copro- haemin. Curve 4, globan ferroprotoporphyrin, prepared from haemoglobin of man. Curve 5, ferrocytochrome c in alkaline solution. See legend to Fig. 15 and for details see Drabkin, 1942b. their two-banded hydrochlorides and in haemin derivatives (Williams, 1956). Nevertheless, this is a matter of predilection, and Williams' interpretation that in porphyrins (see Fig. 5) the near ultra-violet band ('Soret band') is accounted for by a tt -> 77' electron transition, and the visible bands, I, II, III and IV by a second -n electron transition may be valid for porphyrin spectra (Williams, 1956). DISCUSSION Theory of Absorption Spectra. In general the absorption of light energy by atoms and molecules may be considered to be the reverse of emission. 166 David L. Drabkin Enlargement of this assumption involves the inference that the internal energy of the atom or molecule is increased by the absorption of light, and transfers from lower to higher quantized energy states occur, which give rise to absorption bands. In the ultra-violet and visible spectral regions the bands may represent either transitions of optical or valence electrons (as distin- guished from core electrons) through different quantized energy levels, or internal vibrational phenomena set up in the molecule by the absorption of hght energy. Henri (1919, 1923a and b) and Lifschitz (1920) were among the first to recognize that orderliness exists in the distribution of bands in simple molecules, which is expressed by the spacing of the bands at constant fre- quency distances from each other. Such integrally related bands, forming a spectral series, were early demonstrated in the spectra of KMn04 and C0CI2 (see Fig. 4), and have been interpreted as vibrational fine structure in a broad band of electronic origin (Harrison et al, 1948). The important implication lies in the inference that all bands which are members of a single series probably originate from a common configuration in the molecule, or from the same fundamental molecular disturbance incident to the absorption of light energy. The Spectra of the Haemin Chromoproteins. The finding — extended and supported by a graphic-mathematical analysis — that most of the bands (exclusive of a and (i) in the spectra of haemin chromoproteins and their derivatives are spaced at equal frequency distances allows the interpretation that they originate (as in the simple molecules above) in a common molecular structure and from fundamentally the same dynamic source. This enormously simplifies the interpretation of the complex spectra of these complex molecules. In effect, spectrally they behave like much simpler molecules. The location of the bands in the ultra-violet and visible regions permits the deduction that they represent electron transitions. Furthermore, the total iron-porphyrin structure as a unit is held responsible for the bands in the spectral series. In the resonating conjugated double bond system of the porphyrins, each atom contributes an optical electron to the molecule's collection, but the electrons belong collectively to all the atoms in the complex, not to a particular atom. Such electrons, belonging to several atoms, are characterized by relatively low energy (hence the spectral bands in the near infra-red, visible and ultra- violet regions), and the energy levels associated with them are regarded as closely and regularly spaced (cf. Harrison et al, 1948; Braude, 1945). The iron may be thought of as either facilitating or modifying the movement of the electrons over the atoms of the porphyrin ring. At any rate, the over-all spectrum is viewed as an expression of the spectrum of iron in a hexa- co-ordinated Werner type structure. Supporting evidence for the common origin of the bands in the spectral series may be drawn from Warburg's classical deduction of the photochemical spectrum of cytochrome c oxidase (Warburg and Negelein, 1928; Warburg, 1929). The photochemical spectrum, Analysis and Interpretation of Absorption Spectra of Haemln Chromoproteins 167 which measured the release of the enzyme from its carbonyl derivative (poisoned state) had several maxima in the ultra-violet, including the y band. This would not have been the case unless the bands had a similar molecular origin. The a and (i bands, as disclosed by the analysis, do not belong to the main spectral series, and have been shown to reflect more intimately the effect of different co-ordinating ligands. A broad correlation has been found between the paramagnetic susceptibilities and the corresponding spectral patterns in the area covered by bands 4, a, (i and 5 (Table 3). This correlation would appear to justify the deduction that the visible spectra reflect electron transitions involved in the hybridization of the atomic s, p and d orbitals of the iron cation (Pauling, 1949 ; Williams, 1956). Four major spectral patterns have been uncovered, namely those of ferrihaem and its derivatives with cyanide, and those of ferrohaem complexes with cyanide, pyridine or globin, and carbon monoxide. Identical patterns (see the text and Figs. 15 to 17) are obtained for corresponding derivatives of proto-, meso- and coprohaemin, except for the displacement of the maxima of the protohaemin complexes toward the longer wavelengths. This displacement is also apparent in the spectra of proto- and mesoporphyrin, and is regarded as the main evident contribution of porphyrin />e/-5e to the over-all haemin protein spectra. Since the disclosure of the close similarity of the spectra of ferrous 1 : 10- phenanthroline and the cyanide complexes of ferrihaemin derivatives (Drabkin, 1941b, and Fig. 2), the structure of the ferrous diimine has been shown to be Fe^Pheug (Gould and Vosburgh, 1942; Harvey and Manning, 1952) and the anomalous colours of the metallic diimines have been explained as due to the resonance of 77-electrons (and their increased mobility in the metal complex) over the non-metallic atoms composing the entire chelate ring, assuming that ^/-electrons of the metal are involved in the formation of the co-ordinate bond (Sone, 1952; Krumholz, 1953; see also Pauling, 1940; Calvinand Wilson, 1945; Chatt, 1949). Williams (1956) has used the metallic diimines and the postulated electronic basis for their spectra as models in his interpretation of the visible absorption spectra of the haemin chromoproteins. The contribution of the protein moieties to the over-all spectra of the haemin chromoproteins is not identifiable in any alteration in spectral pattern, nor does it appear to be confined to a particular spectral region such as 280-275 m^w. The role of the protein is postulated to be that of a 'resonator' or 'enhancer'. The relative intensity of the bands, not their pattern, may be influenced, analogously with the effect of the alkaline earth metals on the emission spectrum of copper. The Graphic-mathematical Analysis. It must be frankly stated that this is an empirical approach, and it is recognized that the solutions yielded by the adopted analytical procedure are influenced by underlying assumptions. 168 David L. Drabkin The novel use of curves of the normal frequency form — admittedly agree- able to the writer — need not imply that absorption bands actually possess this shape. Yet, made up as they are (in different spectral regions) of an electronic band, with blurred out vibrational or rotational elements, their shapes may really be similar to those employed in the graphical resolution. It is hoped that methods will be forthcoming which may permit an experimental resolu- tion of the spectra of haemin derivatives at least, if not those of the haemin proteins. Among the possibilities are the spectra at very low temperatures in which interest has been renewed, and which were originally explored in porphyrins by Conant and his colleagues (Conant and Kamerling, 1931). SUMMARY A graphic-mathematical analysis, using curves of the normal frequency form, has been made of the absorption spectra of haemin chromoproteins. The spectra in the near infra-red, visible and ultra-violet regions of all the derivatives examined are fundamentally similar, and represent the summa- tional effect, which can be expressed mathematically, of the a and /5 bands and bands numbers 3 to 11 of an equally spaced frequency distributed series. The bands represent a series of electronic transitions and those in the demonstrated spectral series are inferred to originate from the same molecular structure, the resonating conjugated double bond system of the iron- porphyrin unit. The special significance of the a and /5 bands, the contributions of porphyrin and protein, and the influence of co-ordinating ligands have been discussed. A ckfio 1 vledgemen t The writer's investigations of the chromoproteins have been supported by grants from the Office of Naval Research and the Bureau of Medicine and Surgery of the Navy, and, more recently, by a grant from the National Science Foundation, U.S. REFERENCES Appleby, C. A. & Morton, R. K. (1954). Nature, Lond. 173, 749. Austin, J. H. & Drabkin, D. L. (1935). /. biol. Chem. Ill, 67, Braude, E. a. (1945). Am. Rep. chem. Soc. 42, 105. Erode, W. R. (1928). Proc. Roy. Soc. A, 118, 286. Calvin, M. & Wilson, K. W. (1945). /. Amei: chem. Soc. 67, 2003. Chatt, J. (1949). /. chem. Soc. 3340. Conant, J. B. & Kamerling, S. E. (1931). /. Amer. chem. Soc. 53, 3522. Coryell, C. D., Stitt, F. & Pauling, L. (1937). /. Amer. chem. Soc. 59, 633. Davies, T. H. (1940). /. biol. Chem. 135, 597. Dhere, C. (1906). Compt. rend. Soc. biol. 61, 718. Drabkin, D. L. (1934). Proc. Soc. exp. Biol. Med. 32, 456. Drabkin, D. L. (1936). J. biol. Chem. 114, xxvii. Drabkin, D. L. (1937). J. biol. Chem. 119, xxvi. Drabkin, D. L. (1938). Proceedings of the Fifth Summer Conference on Spectroscopy and Its Applications, p. 94. John Wiley, New York/Chapman & Hall, London. Analysis ami Interpretation of Absorption Spectra of Haemin Chromoproteins 169 Drabkin, D. L. & Singer, R. B. (1939). /. biol. Chem. 129, 739. Drabkin, D. L. (1940). Proceedings of tlie Seventh Summer Conference on Spectroscopy and Its Applications, p. 116. John Wiley, New York/Chapman & Hall, London. Drabkjn, D. L. (1941a). J. opt. Soc. Amer. 31, 70. Drabkin, D. L. (1941b). J. biol. Chem. 140, 387. Drabkin, D. L. (1942a). J. biol. Chem. 142, 855. Drabkin, D. L. (1942b). J. biol. Chem. 146, 605. Drabicin, D. L. & Schmidt, C. F. (1945). /. biol. Chem. 157, 69. Drabkin, D. L. (1946). /. biol. Chem. 164, 703. Drabkin, D. L. (1949a). Arch. Biochem. 21, 224. Drabkin, D. L. (1949b). Haemoglobin, p. 35 (Ed. by F, J. W. Roughton & J. C. Kendrew), Butterworths, London. Drabkin, D. L. (1950). Medical Physics, 2, p. 1039 (Ed. by O. Glasser), Year Book Publ., Inc., Chicago. Drabkin, D. L. (1954). Report to Ad Hoc Panel of the National Research Council (U.S.) on a Standard for Haemoglobin Measurement. Unpublished. Drabkin, D. L. (1957). Fed. Proc. 16, 740. Gordy, E. & Drabkin, D. L. (1957). /. biol. Chem. Ill, 285. Gould, R. K. & Vosburgh, W. C. (1942). J. Amer. chem. Soc. 64, 1630. Grabe, H. (1892). Untersuchungen des Blutfarbstoffes auf sein Absorptionsvermogen fiir violette und ultraviolette Strahlen, Dorpat. Hagenbach, a. & Percy, R. (1922). Helv. chim. Acta, 5, 454. Harrison, G. R., Lord, R. C. & Loofbourow, J. R. (1948). Practical Spectroscopy, Chaps. 10 and 1 1 (pp. 228-299), Prentice-Hall, New York. Hartridge, H. (1920). /. Physiol. 54, 253. Harvey, A. E. Jr. & Manning, D. L. (1952). /. Amer. chem. Soc. 74, 4744. Haurowitz, F. (1924). Hoppe-Seyl. Z. 138, 68. Haurowitz, F. (1927). Hoppe-Seyl. Z. 169, 235. Hausser, K. W. (1934). Z. tech. Physik. 15, 10. Hausser, K. W., Kuhn, R. & Seitz, G. (1935a). Z.phys. Chem. B. 29, 391. Hausser, K. W., Kuhn, R. & Smakula, A. (1935b). Z.phys. Chem. B. 29, 384. Hausser, K. W., Kuhn, R., Smakula, A. & Deutsch, A. (1935c). Z.phys. Chem. B. 29, 378. Hausser, K. W., Kuhn, R., Smakula, A. & Hoffer, M. (1935d). Z.phys. Chem. B. 29, 371. Hausser, K. W., Kuhn, R., Smakula, A. & Kreuchen, K. H. (1935e). Z.phys. Chem. B. 29, 363. Henri, V. (1919). Etudes de Photochimie, Paris. Henri, V. (1923a). C.R. Acad. Sci., Paris, 176, 1142. Henri, V. (1923b). C.R. Acad. Sci., Paris, 111, 1037. Hicks, C. S. & Holden, H. F. (1929). Aust. J. exptl. Biol. med. Sci. 6, 175. Hogness, T. R., Zscheile, F. R. Jr., Sidwell, A. E. Jr. & Barron, E. S. G. (1937). /. biol. Chem. 118, 1. Holiday, E. R. (1936). Biochem. J. 30, 1795. Holiday, E. R. (1937). /. sci. lustrum. 14, 166. Horecker, B. L. (1943). J. biol. Chem. 148, 173. Krumholz, p. (1953). J. Amer. chem. Soc. 75, 2163. Lavin, G. L & Northrop, J. H. (1935). /. Amer. chem. Soc. 57, 874. Lavin, G. I., Northrop, J. H. & Taylor, H. S. (1933). /. Amer. chem. Soc. 55, 3497. Lifschitz, J. (1920). Z.phys. Chem. 95, 1. Morton, R. K. (1958). Rev. pure appl. Chem. 8, 161. Pauling, L. (1940). The Nature of the Chemical Bond, and the Structure of Molecules and Crystals, 2nd ed., Cornell University Press, Ithaca/Oxford University Press, London. Pauling, L. (1948). The Valences of the Transition Elements, Victor Henri Memorial Volume. Lidge: Desoer. Pauling, L. (1949). Haemoglobin, p. 57 (Ed. by F. J. W. Roughton and J. C. Kendrew), Butterworths, London. 1 70 Discussion Pauling, L. & Coryell, C. D. (1936a). Proc. nat. Acad. Sci. Wash. 22, 159. Pauling, L. &. Coryell, C. D. (1936b). Proc. nat. Acad. Sci. Wash. 22, 210. Rawlinson, W. a. (1940). Aust. J. exp. Biol. med. Sci. 18, 185. SoNE, K. (1952). Bull. chem. Sac. Japan, 25, 1. SoRET, J. L. (1878). Arch, sc.phys. et nat. 61, 322. SoRET, J. L. (1883a). Arch, sc.phys. et nat. ser. 3, 9, 513. SoRET, J. L. (1883b). Arch. sc. phys. et nat. ser. 3, 10, 429. Theorell, H. (1938). Biochem. Z. 298, 242. Theorell, H. (1939). Biochem. Z. 301, 201. Theorell, H. (1941). /. Amer. chem. Soc. 63, 1820. Theorell, H. & Akesson, A. (1941). J. Amer. chem. Soc. 63, 1812, 1818. Theorell, H. & Nygaard, A. P. (1954). Acta chem. Scand. 8, 1649. Tint, H. & Reiss, W. (1950). J. biol. Chem. 182, 385, 397. Tsou, C. L. & Li, W. C. (1956). Scientia Sinica. 5, 253. Vles, F. (1914). Compt. rend. Soc. biol. 158, 1206. Warburg, O. (1929), Naturwissenschaften 16, 245. Warburg, O. & Negelein, E. (1928). Biochem. Z. 200, 414. Williams, R. J. P. (1956). Chem. Rev. 56, 299. Wyman, J. Jr. & Ingalls, E. N. (1941). /. biol. Chem. 139, 877. DISCUSSION Interpretations of Absorption Spectra of Haemoproteins Perrin: Would Drabkin please indicate the theoretical significance he attaches to his serial band analysis? In, say. Fig. 9 of his paper (p. 155), how much latitude does his assigning of seven bands, with the resulting sixteen adjustable constants, allow in analysing the spectrum? Drabkin: I have frankly admitted in my paper that my analytical approach was an empirical one. My proposal was originally made a good many years before high- and low-spin complexes were recognized and before it was fashionable to speak of d- and 7T-electrons. It seemed to me very worthwhile, as a first step, to make the most simplifying assumptions. I used Gaussian curves because I hked their shape, and perhaps naively believed that the components (bands) in the complex absorption curves might indeed have such a form. I may say that I anticipated Perrin's searching questions, and posed them to myself, without a wholly satisfactory answer. I recognize that from a rigid physical viewpoint the bands are spaced too far apart to be regarded as electronic in the usual sense, and probably represent a special case. As to the latitude of the analytical procedure, it is probably rather broad. For the pronounced maxima in the absorption spectra they would appear to be unequivocal. For regions of masked absorption the solution may not be unique. However, reference to my Table 2 (p. 153) will disclose that assembling the various derivatives, actual representatives with definite maxima in the postulated locations have been found. It should be stressed that this is the important experimental finding, independent of and, indeed, guiding the analysis. Yet, the proposal of the equally spaced, frequency- distributed series was made before all the data were available. The possible existence of certain maxima was prophesied, a prophecy fulfilled by later finding them in the spectra of oxidized and reduced cytochrome c. Williams: My views and those of Drabkin as to the nature of iron porphyrin spectra are rather different. Drabkin has given us a detailed survey, empirically based, of a large number of absorption bands. This will be most valuable. I have attempted to use Piatt's theory {Radiation Biology, 3, 1956) of the porphyrin spectra to interpret the spectra of metal porphyrin complexes. The analysis led to the conclusion that some bonds in Fe+++ porphyrins were due to the iron and had little to do with the porphyrin, notably at 650 m/f and about 500 m/f. In Fe++ porphyrins there is a band at about 500 m/< due to the iron alone. There is no requirement for a frequency series in Piatt's theory. Analysis and Interpretation of Absorption Spectra of Haemin Chromoproteins 171 Drabkin: Williams has suggested that the band at 500 m/i, which is No. 5 of my frequency distributed series and ascribed by me to originate in the dynamic haemin structure, is rather owing to iron itself. Having earlier discussed this question with him, I believe his deduction originates from finding a maximum at about 510 m/t in such complexes as ferrous orthophenanthroline (see my Fig. 2). This is a very broad maximum and doubtless includes several component bands. Of course my proposal also involves a co-ordination complex. I must respect his theoretical knowledge, but naturally I prefer my own proposal. At any rate, I believe we agree on many other aspects of our individual analytical approaches. Thus, we have brought out somewhat similar correlations between the absorption spectra and corresponding magnetometric data (see my Table 3 and text, p. 158). It may be pointed out that, in Table 3, in the case of ferrihaemoglobin and ferri- haemoglobin hydroxide, one may now substitute the terms 'high-spin' and 'mixture of high-spin and low-spin' in place of the older 'essentially' or 'partially' ionic (see Orgel's, Williams', and George's papers, this symposium). I wonder whether it is possible that the unexpected behaviour in the vicinity of the Soret band (my band No. 6) in the transition spectra of MHb -> MHbOH (i.e. the absence of an isosbestic point) may be related to a change from a high-spin complex to a mixture of high- and low-spin ? Wainio: I wish to ask Drabkin, if the a- and )5-bands of the haem of any one of the haem- proteins is replaced by the bands of the corresponding porphyrin, will the latter fall into the frequency distribution series? Drabkin: In the ultra-violet region they do not fit well. Since I was making the most simplifying assumption, namely that the spectra are an expression of iron in a co- ordinated complex, I was content to start with, and confine myself to iron-porphyrin complexes. I am not at all convinced that there is real validity in drawing parallels between porphyrin bands and correspondingly located bands of haemin derivatives. The bands in the region of 830 and 280 m/x George: I should like to ask Drabkin whether the near infra-red bands of the ferri- myoglobin fluoride complex at 740 and 830 m/t can be fitted into the frequency series with successive values of 'rt'. If the wave-number separation is too small then one or the other would have to be regarded as belonging to another category like the a- and /S-bands. Drabkin: The band at 830 m/i is present in other haemin protein derivatives (as evident in Table 2 of my paper). This band corresponds to v x 10""^ = '-^120, and is No. 3 of my proposed equally-spaced frequency series. The band at 740 m/< may correspond with the at present anomalous band at 760 m/z of deoxygenated haemoglobin (see my Fig. 1), and may in fact belong to a different series, or may be 'odd-man-out' as you have expressed it. Incidentally, in some derivatives (see Fig. 1, p. 143) there are maxima in the vicinity of wavelength 920 m/n which do not fit in the main series. This is brought out in my paper. George: For myoglobin derivatives the millimolar extinction coefficients in the protein absorption region, 260-290 m/<, are about 30 compared to the value of about 13 for apomyoglobin. Similar values have been reported for haemoglobin derivatives and apohaemoglobin, hence there can be no doubt that the haem absorbs significantly in this region. The same is true of vitamin B12 and free benzimidazole, so the eff'ect is not restricted to haemoproteins. Margoliash: In the case of cytochrome c, for which the amino acid composition is reasonably well known, it is easy to calculate the contribution of tryptophane, phenyl- alanine and tyrosine to the 280 m/i band. The results are roughly the same as those quoted by George for metmyoglobin. There is, moreover, a striking difference between the spectra of the ferro- and ferri- forms of cytochrome c in the 'protein' band region, indicating a distinct contribution of haem absorption to this band (Margoliash and Frohwirt, Biocliem. J. 71, 570, 1959). H.E. — ^VOL. I — N 172 Discussion PosTGATE : I think there exists some evidence against Drabkin's view that the 280 m/n peak in haematins is mainly due to some frequency in the iron-porphyrin system and not to residues of aromatic amino-acids. Cytochrome C3, which has a molecular weight closely similar to that of cytochrome c, has two haemins/molecule. Yet in spite of this the absorption of this material at 280 m/i is negligibly small; the 280 m/f peak of cytochrome c is absent. On the other hand, we find from qualitative observations that the aromatic residue content of c^ is very small, which would be consistent with the traditional view of the significance of the 280 m/t band. Morton: As will be seen from the tables in my review (Morton, Rev. pure appl. Chem. 8, 161, 1958), there is a paucity of information concerning the influence of the state of oxidation on the absorption of cytochromes in the ultra-violet region. Our studies with cytochrome b.^ suggest that the position and height of the band in the 260-280 m/i region does change between the oxidized and the reduced compound. Could Postgate comment on the diff"erence between ferricytochrome c^ and ferrocytochrome C3 in the 280 m/< region. Postgate: We have never observed the 280 m/t range of ferrocytochrome c^; because of its low Eq we have found no reducing agent which will maintain the reduced form without absorbing strongly in this range. Drabkin: The situation Postgate describes in cytochrome C3 is certainly most unusual, perhaps unique for haemin chromoproteins. I would not be astonished by the absence of a well-defined maximum, but am puzzled by negligible absorption, particularly since co-ordination complexes such as haemin dicyanide (my Fig. 5) absorb rather strongly in this region. Perhaps the presence of a weak masked band could be brought out by the type of analysis I have employed. What is the nature of the ultra-violet absorption of reduced cytochrome Cg? That approximately 30% only of the total absorption at 280 m/t can be ascribed to the specific absorption of the aromatic amino acids in most haemoproteins is in essential agreement with my own deduction, based upon the content of tyrosine, phenylalanine and tryptophane, as discussed in my paper. I believe that these aromatic amino acids contribute to the absorption, but the main contribution is owing to the haemin structure. It is misleading in the case of the haemin derivatives to speak of the protein band (at 280 m/t). Williams: I wonder should not one re-examine, in respect to the absorption at 280 m/t, the question of energy transfer from aromatic amino-acids to haemoproteins, e.g. in the photochemical decomposition of CO-complexed haemoproteins (Weber, Disc. Faraday Soc. 11, 1959). As I understand Drabkin's remarks, there is a co-operative enhancement of the absorption of the aromatic amino-acids at 280 m/t by the haem unit. Drabkin: I am sorry that I am not acquainted with the actual experimental work of Weber to which you refer. In any event the energy would have to be quantized. In my paper I do refer to Warburg's classical study, and believe that his photochemical spectrum which includes a broad spectral coverage, with several maxima besides the 'protein' band at 280 m/t, must indicate that the same haemin structure is involved and photochemically eff'ective in spectral regions which cannot be ascribed to protein. This appears to support my proposal of a similar origin for the various bands. On the other hand, the total co-ordination complex includes residues from the protein. Hence, the eff"ect of protein cannot be wholly separated from the haemin, and some protein contribution may be present over the whole spectral range (see discussion in paper). Lemberg: Most porphyrins have, indeed, a weak absorption in the region 260-280 m/t. For protoporphyrin in dioxane we have found an £„,« of 14 at 280 m/t. The absorp- tion is thus far weaker than that of the Soret band, whereas many haemoproteins absorb at 280 m/t as strongly or more strongly than in the Soret region. THE HAEM-GLOBIN LINKAGE 3, The Relationship between Molecular Structure and Physiological Activity of Haemoglobins* By J. E. O'Hagan Red Cross Blood Transfusion Service, Brisbane, Queensland, Australia The reactions of haemoglobins have been interpreted in terms of the imidazole, steric hindrance, haem-haem interaction and intermediate com- pound hypotheses, which have had wide acceptance. However, the opinion that the haem iron was linked by groups other than imidazoles has been expressed by Haurowitz (1954, 1959) and by O'Hagan (1959a). Theorell and Ehrenberg (1951) considered that a more acid group was responsible for this linkage in horse myoglobin, while Wyman (1948) in expounding the imidazole hypothesis, made a reservation that the evidence for the identification as imidazole of the more acid of the groups co-ordinating the iron in horse haemoglobin was not completely certain. Recent X-ray studies of ferri- myoglobin by Kendrew, Bodo, Dintzis, Parrish, Wyckoff and Phillips (1958), neither appear to support a hypothesis of co-ordination of the haem iron between two amino acid side-chains of the protein, nor confirm steric hindrance relationships. Roughton (1944), while paying tribute to the value of Wyman's work, pointed out that it did not account for the important carbamino reaction. Strong arguments against the steric hindrance (or embedded haem) concept were presented by Keilin (1953), and these were supported to a certain extent by the preparation of artificial haemoglobins from haems of dimensions larger than protohaem (namely coprohaem III and tetramethyl-coprohaem III (O'Hagan, 1955, I960)). George and Lyster (1958), after a careful analysis of the evidence, considered steric hindrance effects unlikely, at least for small ligands, which, after all, are the physiologically important ones. The haem-haem interaction hypothesis as proposed by Pauling (1935) to interpret the sigmoid dissociation curve of oxyhaemoglobins has not been able to account for a number of apparent exceptions. The addition of oxygen to ferrohaemoglobin is linear when measured by either spectro- photometric (Nahas, 1951) or magnetometric methods (Coryell, Pauling and * Part I, O'Hagan; Part 2, O'Hagan and George; Biochem. J., 74, 417, 424 (1960). 173 174 J. E. O'Hagan Dodson, 1939). Spectrophotometric titrations of the combination of imida- zole (Russell and Pauling, 1939) or hydroxyl (George and Hanania, 1953) with ferrihaemoglobin also show linear relationships. None of the equations proposed, on the basis of haem-haem interaction, to explain the sigmoid oxygen dissociation curves, has been found to be satisfactory, except in a special case at pH 9-1, outside the range of the Bohr effect (Roughton, Otis and Lyster, 1955). There appears to be reliable evidence that specimens of mammalian haemo- globins have at times exhibited hyperbolic dissociation curves (Barcroft, 1928; Hartridge and Roughton, 1925). Takashima (1955) found that at ionic strength 0-03-0-3 the «-value for the Hill equation was about 3, which would not be in accordance with Pauhng's equation. At lower ionic strength, pro- nounced deviation from the Hill equation occurred, especially at the lower portion of the curves. Rossi-Fanelli, Antonini and Caputo (1959) found that human haemoglobin in 2 M sodium or potassium chloride (under which conditions it is dissociated into half molecules), gave an oxygen dissociation curve with slightly increased 'haem-haem interaction'. They did not show that the four haems were divided between the two fragments but if they were, as is most probably the case, their results could mean that the sigmoid curves of haemoglobins were not due to interaction between the haems. Gastrophilus haemoglobin with two haems per molecule has a hyperbolic oxygen dissociation curve, i.e. no 'haem-haem interaction' (KeiUn and Wang, 1946). The sigmoid dissociation curve of diluted chlorocruorin (Fox, 1932) of molecular weight of about 3,000,000, with the possibility of inter- action between about 200 haems (Lemberg and Legge, 1949), and the 'atypical' curves of haemoglobins of species such as the duck and carp (Red- field, 1933), are difficult to reconcile with this hypothesis and have generally been conveniently ignored. Some experimental support for the intermediate compound hypothesis would appear to have been found from the work of Itano and Robinson (1956) who detected intermediates when normal human adult carboxy- haemoglobin was partly oxidized with ferricyanide and the mixture submitted to electrophoretic separation. It does not necessarily follow, however, that their findings are appUcable to the ferrohaemoglobin-oxyhaemoglobin system. The finding by Hill and Holden (1926), Holden (1941) and Granick (1949) of attachment of porphyrins to apohaemoglobins, the instability of aetio- haemoglobin (O'Hagan, 1950, 1955, 1960) and the X-ray studies of ferri- myoglobin (Kendrew et al., 1958) suggested the likehhood of linkages between the haematin propionate side-chains and basic side-chains of the apohaemo- globins and apomyoglobins. While studying the nature of these linkages it appeared that it might be possible to explain more satisfactorily the oxygen dissociation curves, the Bohr effect, the alkali-stabihty, and the differences The Haem-Globin Linkage 1 75 in crystal structure in terms of such linkages. They have therefore been examined with this in view and, as a result, a new interpretation of the structural and functional relationships of haemoglobins and myoglobins is presented. MATERIALS AND METHODS Aetiohaemin III The preparation was as described by O'Hagan (1960). Nickel Mesoporphyrin IX Mesoporphyrin IX was prepared by the method of Grinstein and Watson (1943) from dimethylprotoporphyrin IX (Grinstein, 1947). In 5% w/v HCl the absorption spectrum had bands at I, 590-7; la, 570-5; II, 547-5 m// (order of intensity II > I > la, with the Hartridge Reversion Spectroscope). Bands I and la were 1 m/i lower than those reported by Fischer and Orth (1937). Two methods of conversion to the nickel complex were employed. The first followed the technique used by Fischer and Piitzer (1926) to prepare nickel protoporphyrin, but the yield of crystals, even after standing several days in the refrigerator, was poor. The second, a very simple method of preparation, was as follows. About 0-1 g mesoporphyrin was dissolved in about 5 ml acetic acid, the solution was quickly heated to boiling and nickel acetate in acetic acid (prepared by leaving a piece of pure nickel, just covered with acetic acid, in a beaker for a few days) added drop by drop, with continued boiling, until the fluorescence of the porphyrin under ultra-violet light had disappeared. After cooling and adding \ vol. distilled water, the precipitated nickel mesoporphyrin was centrifuged off, washed with alcohol and ether and dried in a vacuum desiccator over NaOH. It was then dissolved in 0-04 N NaOH, the solution centrifuged to remove a trace of undissolved material, the pigment in the supernatant precipitated with 0-2 N HCl, the precipitate centrifuged off, washed several times with distilled water and dried in a vacuum desiccator over NaOH. The nickel mesoporphyrin prepared by both methods had absorption peaks (Hartridge Reversion Spectroscope) as follows: in dioxane, I, 550-5; II, 513 m/< (order of intensity I> II, cf. Lemberg and Legge, 1949); in pyridine, I, 552-0; II, 514 m/* (order I > II). Proteins The horse apohaemoglobin was prepared by the method previously described for the human material (O'Hagan, 1960) and the apomyoglobin as reported by O'Hagan and George (1959), and both were estimated spectro- photometrically at 280 m// using e^^ = 13 as calculated by Hanania (1953). Before use, the solutions were left for several days at 1°C after adjustment to pH 7-8 to remove as much denatured material as possible. The 25 % human serum albumin was a special batch of salt-poor albumin for transfusion 176 J. E. O'Hagan purposes which had not been subjected to the usual heat treatment to destroy hepatitis virus. Imidazoles Caffeine B.P. and theophyUine B.P. were suppHed by Drug Houses of AustraHa Ltd. They were recrystalhzed from water and used as saturated aqueous solutions. Bujfer Solutions These were prepared with British Drug Houses A.R. grade chemicals from tables calculated by George and Hanania (unpubhshed), and kindly supplied by Professor P. George. The buffers were of constant ionic strength (I = 0.05) and of the following composition: pH 2-0-3-8, HCl + KH phthalate; pH 4-0-6-2, NaOH + KH phthalate + NaCl; pH 5-6-8-0, NaOH + NaH2P04 + NaCl; pH 7- 5-9- 5 HCl + Na4Po04 + NaCl; pH 9-9-1 M, NaOH + glycine + NaCl; pH 11-0-12-0, Na'gHPO^ + NaOH + NaCl; pH 130, NaOH. Dithionite 1 % w/v solutions were prepared immediately before use from sodium hydrosulphite B.D.H., which was taken from a freshly opened ampoule (repacked from a 500 g bottle). Standardization of Instruments The Hilger Uvispek Spectrophotometer, Beck Hartridge Reversion Spectroscope and Jones Electronic pH Meter were standardized as previously described (O'Hagan, 1960). All pH measurements were made with the standard glass electrode, and, although corrections were applied, all readings at high pH should not be regarded as exact. RESULTS Decrease in the Acid Strength of Haematin Propionate Groups on Reduction to Haem It appeared possible that since substituents of R in RCH2CH2COOH could alter the pAT value of the carboxyl by as much as 1-3 units (Edsall and Wyman, 1958), a change in the electronic structure of the haem iron atom producing alterations in the high resonance of the porphyrin ring system might have the same effect as a substitution of R in simple com- pounds, with subsequent change in the acid strength of the propionate groups. To test this hypothesis two reactions known to involve the propionate groups were investigated, those with human serum albumin, and with caffeine. A new reaction with theophylline was found, and prehminary results obtained supported those observed with caffeine. The Haem-Globin Linkage 1 77 Attachment of Haematin and Haem to Human Serum Albumin Haematin combines with human serum albumin to form ferrihaemalbumin (see discussion by Lemberg and Legge, 1949). J. Keilin (1944) considered that the attachment of both haematin and haem to the albumin was through the porphyrin and Lemberg and Legge indicated the haematin carboxyls as being most hkely involved. O'Hagan (1955, 1960) in showing by both spectrophotometric and paper electrophoretic studies that mesohaematin (but not aetiohaematin) combined, demonstrated that the carboxyls were responsible. Keilin (1944) believed it most likely that haem is attached in similar fashion, on account of the spectral differences of ferrohaemalbumin from free haem and from the haemochromes. The extent of the combination of human serum albumin with haematin and haem was investigated by measuring the increment in absorbance in the Soret region on addition of the pigments to excess of the albumin in a series of buffer solutions of constant ionic strength. Tubes of 7 ml capacity were filled with 5 ml buffer (phthalate or phosphate), 1 ml 2-5% human serum albumin (the 25% solution diluted 1:10 with distilled water) and 0-1 ml 2x 10~^M freshly prepared protohaematin solutions (1-30 mg haemin dissolved in 1 ml 005 n NaOH, then 9 ml distilled water added). Other sets of tubes, one containing water in place of albumin and another with albumin but no haematin were set up at the same time. The solutions were stood at 21° for 3 hr, then portions transferred to a 10 mm cuvette and the absorbances read at 404 m/f in the spectrophotometer. Another series of three sets of tubes had 0- 1 ml freshly prepared 1 % w/v dithionite added to each tube and the tubes closed by long stoppers which excluded air almost completely, except for a small bubble which assisted mixing on inversion. After the 3 hr standing, portions of these solutions were carefully transferred with a Pasteur pipette and as little agitation as possible, to the cuvette and the absorbances read at 414 m/i, the Soret peak of ferrohaemalbumin. The curves obtained in Fig. 1 show the increment in absorbance due to the addition of the albumin, i.e. they represent Aj^^ — A^ — A^, where ha = ferri- or ferrohaemalbumin, h = haematin or haem and a = albumin. The difference in the attachment of the haematin and haem, as indicated by the increment in absorbance, is at once apparent. Similar results were obtained for the ferrohaemalbumin when the dithionite was added to the ferrihaemalbumin after it had stood for 3 hr and the mixture stood a further 3 hr. No trace of verdohaem compounds was detected, but these were formed, as expected, when the reduced solutions were reoxidized, so that the reverse reaction (ferro- to ferrihaemalbumin) could not be investigated under these conditions. To check whether the dithionite itself interfered in any way, nickel mesoporphyrin (on which dithionite has no effect) was added to the albumin and the absorbance increment measured before and after addition 178 J. E. O'Hagan of the dithionite. The first curve was obtained after standing 3 hr, dithionite added at the same concentration as for the iron porphyrins, and the solutions stood another 3 hr to give the second curve. The shght difference is due to the Fig. 1 . Attachment of metalloporphyrins to human serum albumin. (a) Soret absorbance increment (A) curves prepared, as described in the text, for haematin + human serum albumin at 404 m/n • — • — •, for haem + human serum albumin at 414 m/n O— O — O. (b) Soret peak absorbance increment curves for nickel mesoporphyrin + human serum albumin at 394 m/t before A — ▲ — ▲ and after ■ — ■ — b adding di- thionite. (Buffers, pH 4-0-6-2 phthalate, pH 6-2-7-2 phosphate, I = 0-05, T = 2rc.) decreased value for the metalloporphyrin (without albumin) which is not in true solution and whose absorbance is decreasing with time. It was concluded that reduction significantly decreased the acid strength of at least one of the haematin propionate groups. Attachment of Haematin and Haem to Caffeine Caffeine was found by J. Keilin (1943) to combine with copper uroporphyrin III and with manganese mesoporphyrin, but she detected no reaction between haematin and caffeine. This seemed unusual and O'Hagan and George (unpubhshed, quoted by O'Hagan and Barnett, 1958) found attachment at pH 11-3 (1-33 mol of caffeine/mol of haematin) and also at pH 7-0 (stoichio- metric relationship not determined). This suggested stronger attachment of haematin than haem to caffeine since J. Keilin had found at least 20 mol of caffeine/mol of haem to be required for caffeine-haem formation at high pH. A saturated solution of caffeine (about 10~i m) was substituted for the albumin in the experiments reported above and the absorption increments plotted as shown in Fig. 2. The peaks for the ferrihaemcaff'eine and ferro- haemcafifeine were 402 and 420 mju respectively. Nickel mesoporphyrin also The Haem-Globin Linkage 179 showed absorption increments on adding caffeine, with Soret peak at 392-5 mn and attachment occurring from about pH 6-0, rising to a maximum at pH 7-0, and being unaffected by addition of dithionite. Fig. 2. Attachment of haematin and haem to caffeine. Soret absorbance incre- ment curves prepared as described in the text, for haematin + caffeine at 402 m/z • — • — •, and haem + caffeine at 420 m/f O — o — o. (Buffers, pH 4-0-6-2 phthalate, pH 6-2-7-5 phosphate, I = 0-05, T = 2rc.) Preliminary Experiments with Theophylline Theophylline has an unsubstituted imino group and therefore resembles more closely the imidazole ring as it would be expected to occur as the side- chain of proteins. In a single series of experiments with theophylHne, attach- ment of haematin occurred from about pH 5, with a pronounced maximum at pH 6-5 and a minimum at about pH 7-3, but no attachment of haem was detected in this range. At lower hydrogen ion concentration, J. Keilin (1943) also had not found combination of haem with theophylline, though she did with caffeine. These experiments with the imidazoles are prehminary; more points should be obtained to plot the increment curves exactly, but if the curves are nearly correct, a mean increase of propionate pA^ value of about 0-9 unit could be indicated on reduction, of the same order as that suggested from the experiments with the albumin compounds. Further studies on the reactions of haematins with imidazoles will be reported later. Haem Propionate - Protein Linkages in Adult Horse Haemoglobin and Horse Myoglobin Nickel porphyrins would appear to be very useful compounds for the study of the attachment of haem propionate groups to the side-chains of proteins. They would be expected to be of almost identical size and shape to the iron porphyrins. Haurowitz and Klemm (1935) found nickel dimethyl- mesoporphyrin and Pauling and Coryell (1936) found nickel protoporphyrin to be diamagnetic and concluded that these porphyrins contained no unpaired 180 J. E. O'Hagan electrons. This means that neither the pyrrole nitrogens nor the metal atom are capable of further combination, only reactive side-chains can effect attachment to other compounds. The covalent linkage of the metal resembles, too, that of the iron in oxy- and carboxyhaemoglobins, and the resonance state of these metalloporphyrins might be expected to be akin to that of the haem in those proteins. In the studies reported here, nickel mesoporphyrin was employed, both because it was found to be more readily prepared in pure form than the corresponding protoporphyrin complex and because its use eliminated con- fusion in interpretation of results, through possible (though unhkely) linkage to protein through vinyl groups. Hill and Holden (1926) had shown that it combined with ox apohaemoglobin, as also did nickel protoporphyrin (Holden, 1941). To investigate attachment, 10 ml of buffer was placed into each of a series of tubes, then 0-1 ml 5x 10"*M apohaemoglobin solution (assumed M.W. = 16,500) and 0-1 ml 1 x 10^^ m nickel mesoporphyrin (1-34 mg pigment + 2 ml 0-05 n NaOH + 8 ml water) added. Other series replacing the apohaemoglobin solution or nickel mesoporphyrin solution with water were prepared at the same time and the tubes containing the three series stood at 1° for 16 hr and then at 21° for 2 hr before reading absorbances at 389 mfi, using a cuvette of 40 mm path length. For the apomyoglobin experiments the undiluted protein solution used was 2 x 10~^ m and the absorbances read at 410 mfi. Excess apoprotein was used because of the likehhood of a small variable quantity of denatured protein being present (O'Hagan, 1960), not removable by any treatment yet described, and likely to precipitate on bringing solutions to room temperature. In order to rule out 'protective colloid' effects or non-specific attachment, carboxyhaemoglobin and ferrimyoglobin were substituted for the apoproteins in the experiments. A small peak centred about pH 9 was found with the haemoglobin, while no attachment was detected with the myoglobin. The results, shown in Fig. 3, indicate that attachment to the apohaemoglobin occurs over the pH range 5-12 with two maxima at about pH 7-4 and 10-0. At pH 9-8 nickel mesoporphyrin had an absorption peak at 380 m/;, intensi- fying and moving to 389 m/t (pH 5-7, 8-0, 9-95) on addition of apohaemoglobin (cf. with caffeine, 392-5 m/u). With apomyoglobin the stability range was wider and the peak of the absorption curve shifted much further to 410 m/<. A difference in the position of the peaks with apohaemoglobin and apomyo- globin is in accord with the finding of differences by Hill (1939) when proto- porphyrin was added to these proteins. The section of the curve with maximum centred at about pH 7-4 for the apohaemoglobin complex is strongly suggestive of imidazolium combination with one or both of the propionates. The other section with maximum at about pH 10-0 varied in height and width with the preparation and is most The Haem-Globin Linkage 181 probably due to a combination with a group in some denatured protein present. Perhaps this group results from unmasking of the one giving the maximum attachment at pH 9 in the unsplit native protein. It might also be the group detected by Theorell (1942) in apohaemoglobin, with pA: of 10 at 0°C, not present in ferrihaemoglobin. The increment curves presented here should not be considered to be exactly reproducible since comparison between solutions and colloidal sus- pensions is being made. The increments are, however, of such magnitude 0-5 - . . - / ^ 0-3 - ^ \ i /"\ l\ \ /\ Y f V > 0-1 - 1; \ \ / ^ ^--\ ^^ V -'r'T:>i L,.X 1 1 1 1 1 pH 10 12 Fig. 3. Attachment of nickel mesoporphyrin to apohaemoglobin, apomyoglobin and carboxyhaemoglobin. Soret absorbance increment curves prepared, as described in the text, for nickel mesoporphyrin + apo Hb at 389 m/i 9 — • — •, + apo Mb at 410 m/i O— O— O, and + CO Hb at 390 m/ii A— A— A. (Buffers, pH 2-6-2 phthalate, 6-2-8-0, phosphate; 8-5-9-5, pyrophosphate; 9-9-11, glycine; 11-5, phosphate; 12-5, NaOH; I = 0-05, T = 2rC.) (e.g. apomyoglobin increased the absorbance of nickel mesoporphyrin at 410 m/t from 0-22 to 0-67 at pH 10-0) that it seems legitimate to make quantitative comparison. The technique should prove useful in the detection and identification of linking groups in other haemoproteins. The Nature of the Acid Groups Linking the Haem Iron The likelihood of combination of haematin propionate side-chains with imidazolium side-chains of horse haemoglobin called for a re-examination of the evidence for the mode of attachment of the iron atom to the protein. Stability curves of ferrihaemoglobin and ferrimyoglobin reported by O'Hagan (1959a) suggested the possibility that groups of ^K value lower than 5-3 were involved. Theorell and Ehrenberg (1951), after their exhaustive study of myoglobin, concluded that a group of more negative character than an imidazole was responsible for iron Unkage, but gave the group a pA^ value of 5-3. Since Coryell, Stitt and Pauling (1937) had shown that in acid ferri- haemoglobin the atom of iron was joined to other atoms surrounding it by 182 J. E. O'Hagan 'essentially ionic' bonds, it could be assumed that as the haematin was being removed at increasing acidity the reaction could, for practical purposes, be regarded as ionic. Since the haematin would be likely to have a tendency to polymerize, with drop in absorbance, it might well act as an 'indicator' of the suppression of the ionization of the group ligating it. To rule out the effects of linkages through the haematin propionate groups, aetiomyoglobin, the properties of which have been described by O'Hagan and 0-5- 0-3- 01 3 4 5 6 7 8 pH Fig. 4. Soret absorbance increment curve for ferriaetiomyoglobin at 393-5 m/n prepared as described in text, o — O — O phosphate buffer, • — • — • phthalate buffer, I ppte., theoretical curve for acid with pK = 5-3, ■ — ■ — H curve for nickel mesoporphyrin + same cone, of apomyoglobin for comparison. (Buffers, I = 0-05, T = 21°C.) .- ; i i / / / / / 7 George (1959) was utilized. It was prepared by adding 0-5 ml of 3 X 10~^ m aetiohaemin in methanol to 5-5 ml of 2-9 X 10~*m apomyoglobin at pH 6-5 (no added buffer) and standing at 1°C overnight. A solution prepared by adding 0-5 ml aetiohaemin solution to 5-5 ml distilled water was treated throughout in the same manner as the aetiohaemin-apomyoglobin solution, to act as a control. Next day the solutions were spun at 15,000^ for 15 min (to remove free aetiohaematin) and the supernatant added in 0-2 ml ahquots to 5 ml portions of buffer solution. After standing 6 hr at 1°C and 3 hr at 21°C the absorbances were read (10 mm cuvette). The curve showing the absorbance increment due to the apomyoglobin is shown in Fig. 4. The nature of the buffer, phthalate or phosphate, made little difference to the shape of the curve in the range pH 5-5-6-8. At about pH 4-95 a discontinuity in the curve appeared, probably due to the taking up of the aetiohaematin by carboxyl groups liberated in the protein. The curve drawn through the points represents a theoretical curve for a group ionizing with pK = 4-95, and for comparison curves for a group with pK = 5-3 and for the attachment of nickel mesoporphyrin to apomyoglobin are given. While by no means The Haem-Globin Linkage 183 conclusive, these studies could indicate the existence of a group of pA!" less than 5-3, if the aetiohaemin is behaving as a weak base (it does not attach to apomyoglobin above pH 8-0) and is acting as an indicator of the ionization of the haem-linked group of the protein. These studies are being extended to apohaemoglobin combination. DISCUSSION That a change occurs in the acid strength of the haematin propionate groups on reduction of the haematin iron is not unexpected, since it has been well established that variation of the side-chains influences the oxidation- reduction potentials of ferro-ferrihaemochrome systems (Lemberg and Legge, 1949). If alterations to the side-chains can affect the reactions of the iron atom, it is not unreasonable to expect that modifications to the electronic structure of the iron could alter the acid strength of ionizable side-chains. The parallelism between changes of the oxidation-reduction potential at 50% oxidation and log/?, where /j is the oxygenation pressure at 50% satura- tion of haemoglobin, with alteration in hydrogen ion concentration, has been clearly demonstrated (Wyman and Ingalls, 1941; see Lemberg and Legge, 1949). We may therefore infer that oxygenation affects the acid strength of the propionate groups in much the same way as does oxidation of the iron. It was first suggested by Altschul and Hogness (1939) that, on oxygenation, a change occurred in the acid strength of groups of the haem rather than of those of the apoprotein. They considered it very probable that the two carboxyl groups of each haem were influenced by oxygenation. They calcu- lated the pAT values of these carboxyl groups as shown in Table 1. The Table 1. Calculated pA" values of haem propionate groups IN FERRO- and OXYHAEMOGLOBIN (AlTSCHUL AND HoGNESS, 1939) Assumed no. of acid groups Calculated pAT values* affected oxygenated reduced ^^K 1 2 5-5 5-8 6-9 6-5 1-4 0-7 * Temperature unspecified, presumably 25°C. actual values may lie somewhere between the extreme values of 5- 5-6-9 since the two acid groups may not be altered in an exactly uniform manner, since the dissociation constants may differ as do those of dibasic acids. Examination of the haem structure shows that the vinyl groups at positions 2 and 4 give asymmetry, which may mean influence of the propionate at 184 J. E. O'Hagan position 6 to a greater or lesser extent than the one at position 7. Considera- tion of the grouping together of rings 1 and 4 and 2 and 3 seems justified, and there may be shared resonance between them as pairs, since on rupture the ring sphts first at the a position to form verdohaems and the tetrapyrrohc ring system of bihrubin sphts at the central methylene group on diazotization (Lemberg and Legge, 1949). This could suggest that one propionate group would be more affected by oxygenation than the other, so that if they were joined by electrostatic hnkages to the apoprotein, one might be a labile link under physiological conditions, while the other only split by such conditions as high ionic strength, high urea or high hydrogen ion concentration, with subsequent parting of the protein molecule into halves. Whatever the actual ipK values, those calculated by Altschul and Hogness show that carbonic acid of pK = 6-352 at 25°C in water (Edsall and Wyman, 1958) could be displaced by a group or groups changing between minimum p^ values of 6-5 and 5-8. The curves of Figs. 1 and 2 could suggest a change of 0-9 unit, compared with a calculated change of 0-7 for two groups and of 1-4 for one group. The asymmetric haem could conceivably be attached to the apoproteins directly or inverted so that structural isomers would be possible unless some orientation by the side-chains occurred. Differences in the acid strength of the two propionates might decide the orientation and also which group could detach from the apoprotein on reduction. The curves obtained for the increment in the absorbance of nickel meso- porphyrin on addition to apohaemoglobin show that a molecule of the size and shape of haem can link by its propionate groups in the pH range 5-9 to a residue in the apohaemoglobin not present in the carboxyhaemoglobin. A specific structure in the proteins binding one or both of the propionates is clearly indicated. That it is not the same as the one binding the iron atom can be deduced from the work with aetiomyoglobin (Fig. 4). The measurements given by Wyman (1948) of the apparent heat of dissociation of horse oxy- haemoglobin and the work reported here, very strongly suggest that in this species the groups binding the propionates are imidazolium side-chains. The shape and range of the curves, and their similarity to the ones obtained for combination with caffeine (Fig. 2), support this. Studies have not yet been extended to haemoglobins of other species; it may be that other amino acid residues are responsible for bonding in these, perhaps explaining the higher heat of dissociation given by Roughton (1944) for ox haemoglobin at pH 6-8, and accounting for his findings in respect to the carbamino reaction. The relationship between the new data, representing the attachment of the propionate groups to the residue in horse apohaemoglobin, and the curve of German and Wyman (1937) is shown in Fig. 5. The top curve of Fig. 5 was obtained by subtracting the increments found for the attachment of nickel The Haem-Globin Linkage 185 mesoporphyrin to apohaemoglobin and to carboxyhaemoglobin. These curves are most probably also related to a differential carbamate equilibrium curve which can be prepared by subtracting the points of the curves of Figs. 5a and 5b of Stadie and O'Brien (1937) for ferro- and oxyhaemoglobin (species unspecified). A new interpretation of the differential titration curve is suggested — (1) that the alkaline loop represents the attachment of propionate groups to imida- zolium side-chains; (2) that the acid loop mirrors the difference between Fig. 5. Comparison of specific increment curve (upper) for attacliment of nickel mesoporphyrin to apohaemoglobin with the differential titration curve (lower) of German and Wyman (1937) for ferroHb-oxyHb (upper curve) prepared by deducting increment with COHb from increment with apoHb). the detachment of the haem iron, in the 'essentially covalent' (and stronger) linked oxyhaemoglobin and the 'essentially ionic' ferrohaemoglobin. As the hydrogen ion concentration increases, the group bonding the iron in ferro- haemoglobin will be liberated, that in oxyhaemoglobin will still be held by the stronger bond. The maximum difference occurs at about pH 5-4 after which the 'covalent' bond begins to break and the extent of ionization of the two groups becomes the same at pH 4-3. Preliminary studies (O'Hagan, unpub- lished) show very marked differences in the stability of carboxyhaemoglobin and ferrihaemoglobin in the pH range 4-6. If the above interpretation is correct, a more pronounced acid loop should be found for the differential titration of carboxyhaemoglobin-ferrohaemoglobin. The experiment with apomyoglobin shows that a much stronger basic group or groups is bonding the propionate(s) as evidenced by the greater height of the curve in the pH range 5-9 and the major shift (30 m/t) in the position 186 J. E. O'Hagan of the Soret peak. This finding of a stronger basic group could explain the considerably lower heat of oxygenation (A//q = — 17-5kcal) found for myoglobin by Theorell (1934), compared with the values of haemoglobins (A/Zq '->-' — 12-5 kcal, Wyman, 1948; see also George, 1956). The greater stabihty of myoglobin towards alkali (Haurowitz and Hardin, 1954) may also be explainable in terms of this basic group. Change in acid strength of the haem propionates would be expected to have less effect on bonds to such stronger basic groups, accounting for the small Bohr effect and minor effect on the shape of the oxygen dissociation curve with a change in the hydrogen ion concentration. Examination of the attachment in the pH range 11-13, using a hydrogen electrode, may give further information on the nature of the linking group. German and Wyman (1937) and Wyman (1948) in discussion of the nature of the group binding the haem iron, pointed out the uncertainty that the group ionizing in the more acid range was imidazole, and that the second carboxyl group of dicarboxylic acids should also be considered. This seems to have been generally overlooked, and the matter calls for more attention in the fight of the observations made here. Even a pK value of 5-3 appears low for an imidazole group by comparison with its value in compounds resembfing those occurring naturally. The strongest evidence against the hypothesis that the haem iron is linked to a group other than imidazole is the ingenious experiment of Wang (1958), who found that diethylprotohaem linked to l-(2-phenyl-ethyl)-imidazole in a film of polystyrene, bound carbon monoxide which could be replaced by oxygen. However, substitution at position 1 on the imidazole ring would appear to change its character, making it unlike that presumed to occur in haemoglobins. It could be that such a substitution makes the free nitrogen acidic and that the acid strength of the linking groups rather than their structure is the important factor. This may apply also to the imidazole haem compound found by Corwin and Reyes (1956) to bind oxygen, though apparently very poorly. Whatever type of linkage exists between the iron and the apoprotein, whether it be imidazole, ^ or y carboxyl or an unusual type not yet detected in proteins, should not materially affect the discussion which follows. The oxygen dissociation curves of the haemoglobins can now be examined in the light of the new findings. It is interesting that Barcroft (1938) likened the curves to oxygen titration curves and we could perhaps visualize them as representing the 'titration' by haem propionates (of changing acid strength) of the weakly basic groups (haemoglobins) or more strongly basic groups (myoglobins). The oxygen dissociation curve of haemoglobin could be considered as representing the titration, from no combination to full combina- tion, of the propionate group with the imidazolium group. The curve for myoglobin would be only the upper section of the curve for the titration of The Haem-Globin Linkage 1 87 a propionate group with a much more basic group, the strength of the com- bination being only shghtly affected on reduction. A weak acid-weak base Hnkage would be expected to be considerably affected by neutral salts and specific ions, as is the oxygenation of haemoglobin, while a linkage to a stronger base would be less affected. This concept could explain the effect known as 'haem-haem interaction', and at the same time account for the Bohr effect. That these effects are apparently interrelated is indicated by the loss of both under certain conditions of reconstitution of haemoglobins (Wyman, 1948). Rossi-Fanelli and Antonini (1959) from a study of human deuterohaemoglobin considered the vinyl groups to be involved in 'haem-haem interaction' and illustrated, but did not explain, the influence of removal of these groups in decreasing the Bohr effect at a given pH. The results seem comparable to the findings of Riggs and Wolbach (1956) on addition of mersalyl to horse haemoglobin. In both cases these effects would appear to be secondary, in the first from a decrease in acid strength of the haem propionates, brought about by removal of the vinyl groups; in the second on account of the propionate binding group of the protein becoming more basic, through combination of a neighbouring sulphydryl group with the mersalyl. A labile electrostatic linkage between at least one of the two haem pro- pionates of each of the four haems and imidazolium side-chains of the apoprotein might explain, better than 'haem-haem interaction', the sigmoid oxygen dissociation curve and the Bohr effect in horse haemoglobin. Linkage to more or less basic groups than the imidazoliums of horse haemoglobin could conceivably explain the curves for other species, including those with 'atypical' curves. The detachment of the propionates in ferrohaemoglobins could explain the change in molecular shape on deoxygenation and also partly account for dry oxyhaemoglobin not releasing oxygen at low oxygen pressures (Haurowitz and Hardin, 1954), the electrostatic bond breaking only in solution. While the exact state of the haemoglobin in the erythrocyte is unknown (see Wintrobe, 1956), the most likely condition — attachment to a framework of stromatin at an equivalent concentration of 34 % haemoglobin — could be conceived as decreasing the velocity of diffusion of oxygen into the interior of the cell. A change in shape of the haemoglobin molecules on breaking the electrostatic link might counteract this to some extent by 'agitation' of the cell contents. SUMMARY 1. Reduction of haematin to haem markedly decreases the acid strength of one or (more probably) both of the propionate side-chains. 2. Nickel mesoporphyrin which can only combine through its propionate side-chains, links with a group or groups in horse apohaemoglobin v/ith imidazolium characteristics. H.E. — VOL. I — o 188 J. E. O'Hagan 3. With horse apomyoglobin nickel mesoporphyrin combines with a more basic group or groups. 4. Fresh evidence is presented that the group in apomyoglobin binding the haem iron is more likely to be of the nature of a /J or y carboxyl than an imidazole group. 5. An interpretation of the sigmoid shape of the oxygen dissociation curve, the Bohr effect, the alkaline stability, and the change of molecular shape on oxygenation, in terms of labile electrostatic linkages between the haem propionate groups and imidazolium side-chains of horse apohaemoglobin is presented. 6. It is suggested that the change in shape of the haemoglobin molecule on oxygenation or reduction may 'agitate' the contents of the erythrocyte and thus assists the exchange of oxygen and carbon dioxide between the cell interior and the plasma surrounding its envelope. Acknowledgement Without the continued encouragement of Dr. A. E. Shaw and generous provision of facilities by the Queensland Division of the Australian Red Cross Society this work could not have been accomplished. ADDENDUM (Note added in proof) The subsequent evidence of Kendrew, Dickerson, Strandberg, Hart, Davies, Phillips and Shore (1960) indicates that the haem iron in myoglobin is attached to imidazole nitrogen, not to a carboxyl group. Their work was carried out with crystalline ferrimyoglobin, mine with this compound in solution, but it is unlikely that the mode of attachment would be essentially different in the crystal and in solution. REFERENCES Altschul, a. M. & HoGNESS, T. R. (1939). /. biol. Chem. 129, 315. Barcroft, J. (1928). The Respiratory Function of the Blood, Pt. 2, p. 102, Cambridge Univ. Press. Barcroft, J. (1938). Features in the Architecture of Physiological Function, p. 71, Cam- bridge Univ. Press. Corwin, a. H. & Reyes, Z. (1956). /. Amer. chem. Soc. 78, 2347. Coryell, C. D., Pauling, L. & Dodson, R. W. (1939). J.phys. Chem. 43, 825. Coryell, C. D., Stitt, F. & Pauling, L. (1937). /. Amer. chem. Soc. 59, 633. Edsall, J. T. & Wyman, J. (1958). Biophysical Chemistry, 1, 558. Academic Press, New York. Fischer, H. & Orth, H, (1937). Die Chemie des Pyrroles, 2, 442. Akademische Verlags- gesellschaft, Leipzig. Fischer, H. & Putzer, B. (1926). Hoppe-Seyl. Z. 154, 39. Fox, H. M. (1932). Proc. Roy. Soc. B. Ill, 356. German, B. & Wyman, J. (1937). J. biol. Chem. 117, 533. George, P. (1956). Currents in Biochemical Research, p. 338 (Ed. by D. E. Green), Inter- science, New York. The Haem-Globin Linkage 1 89 George, P. & Hanania, G. I. H. (1953). Biochem. J. 55, 236. George, P. & Lyster, R. L. J. (1958). Conference on Haemoglobin, May 2-3, 1957, p. 33. Publication No. 557, National Academy of Sciences — National Research Council, Washington. Granick, S. (1949). Harvey Lectures, 44, 228. Grinstein, M. (1947). J. biol. Cliem. 167, 515. Grinstein, M. & Watson, C. J. (1943). /. biol. Cliem. 147, 671. Hanania, G. I. H. (1953). Ph.D. Thesis, University of Cambridge. Hartridge, H. & RouGHTON, F. J. W. (1925). Proc. Roy. Soc. A. 107, 654. Haurowitz, F. (1959). Private communication. Haurowitz, F. & Hardin, R. L. (1954). The Proteins, (Ed. by H. Neurath and K. Bailey), vol. 2, p. 332. Academic Press, New York. Haurowitz, F. & Klemm, W. (1935). Ber. dtscli. cliem. Ges. 68, 2312. Hill, R. (1939). Perspectives in Biochemistry, p. 131 (Ed. by J. Needham and D. E. Green), Cambridge Univ. Press. Hill, R. & Holden, H. F. (1926). Biochem. J. 20, 1326. Holden, H. F. (1941). Aiist. J. exp. Biol. med. Sci. 19, 1, Itano, H. a. & Robinson, E. (1956). J. Amer. chem. Soc. 78, 6415. Keilin, D. (1953). Nature, Lond. Ill, 922. Keilin, D. & Wang, Y. L. (1946). Biochem. J. 40, 855. Keilin, J. (1943). Biochem. J. 2,1, 281. Keilin, J. (1944). Nature, Lond. 154, 120. Kendrevv, J. C, BoDO, G., Dintzis, H. M., Parrish, R. G., Wyckoff, H. & Phillips, D. C. (1958). Nature, Lond. 181, 662. Kendrew, J. C, Dickerson, R. E., Strandberg, B. E., Hart, R. G., Davies, D. R., Phillips, D. C. & Shore, V. C. (1960). Nature, Lond. 185, 422. Lemberg, R. & Legge, J. W. (1949). Hematin Compounds and Bile Pigments, pp. 94, 121, 194, 214, 243, 295 and 458, Interscience, New York. Nahas, G. G. (1951). Science, 113, 723. O'Hagan, J. E. (1950). M.Sc. Thesis, University of Queensland. O'Hagan, J. E. (1955). Abstr. 3rd int. Congr. Biochem., Brussels, p. 10, O'Hagan, J. E. (1959a). Nature, Lond. 183, 393. O'Hagan, J. E. (1959b). Ph.D. Thesis, University of Queensland. O'Hagan, J. E. (1960). Biochem. J. 74, 417. O'Hagan, J. E. & Barnett, C. (1958). Abstr. 3rd Gen. Meeting, Aust. Biochem. Soc. Adelaide, 1958. O'Hagan, J. E. & George, P. (1960). Biochem. J. 74, 424. Pauling, L. (1935). Proc. nat. Acad. Sci. Wash. 21, 186. Pauling, L. & Coryell, C. D. (1936). Proc. nat. Acad. Sci. Wash. 22, 159. Redfield, a. C. (1933). Quart. Rev. Biol. 8, 31. RiGGS, A. F. & Walbach, R. A. (1956). J. gen. Physiol. 39, 585. Rossi-Fanelli, a., Antonini, E. (1959). Arch. Biochem. Biophys. 80, 308. Rossi-Fanelli, a., Antonini, E. & Caputo, A. (1959). Nature, Lond. 183, 827. RouGHTON, F. J. W. (1944). Harvey Lectures, 39, 96. Roughton, F. J. W., Otis, A. B. & Lyster, R. L. J. (1955). Proc. Roy. Soc. B. 144, 29. Russell, C. D. & Pauling, L. (1939). Proc. nat. Acad. Sci. Wash. 25, 517. Stadie, W. C. & O'Brien, H. (1937). /. biol. Chem. Ill, 439. Takashima, S. (1955). J. Amer. chem. Soc. 11, 6173. Theorell, H. (1934). Biochem. Z. 268, 73. Theorell, H. (1942). Ark. Kemi Min. Geol. 16A, No. 14. Theorell, H. & Ehrenberg, A. (1951). Acta chem. Scand. 5, 823. Wang, J. H. (1958). J. Amer. chem. Soc. 80, 3168. Wintrobe, M. M. (1956). Clinical Haemotology, 4th ed., p. 89, Lea & Febiger, Phila- delphia. Wyman, J. (1948). Advanc. Protein Chem. 4, 410. Wyman, J. & Ingalls, E. N. (1941). J. biol. Chem. 139, 877. 190 Discussion DISCUSSION Native Globin Drabkin: I would be glad if O'Hagan could tell us a little more about the state of his apohaemoglobin as against that of his apomyoglobin. I am thinking in terms of the possibility that, since myoglobin is far, far more stable toward alkali than is haemo- globin, some question may be raised as to the strict validity of comparing the two apoproteins. Lemberg: Rossi-Fanelli has found that by his method any irreversible denaturation of globin from haemoglobin can be prevented, and O'Hagan followed his method rather closely. Denaturation will, however, occur if the recombined haemoglobin solutions contain an excess of free globin and are measured in the spectrophotometer at room temperature. O'Hagan: Initially trouble was experienced with these apoproteins due to precipitation of the denatured material at room temperature and neutral pH. The trick is to bring the pH to 7-8, leave at 2rc for 1 hr, leave at O'^C for three days to remove coagulated material, and centifuge for 10 min at 20,000 X g. With one preparation, which I considered to be not as good as the others, on other evidence, the peak at pH 10 (see Fig. 3 of my paper) was higher and broader. George: In our work on reconstituted ferrimyoglobin, O'Hagan and I obtained data similar to that of Rossi-Fanelli on reconstituted ferrohaemoglobin. In a comparison with native ferrimyoglobin we found that the affinity for fluoride is scarcely altered, and that the pK values of the haem-linked ionizing group associated with its Bohr effect are identical to within experimental error. The Linkage of Iron and Protein in Haemoglobin Perrin : In systems such as haem, or haematin plus albumin, we have equilibria such as : Hm COOH ^ Hm COO- + H+ pK^ Alb H+ + Hm COO- ^ Hm COOH Alb K Alb -H H+ ^ Alb H+ pJ^T/ This is certainly a gross over-simplification but will serve to illustrate a difficulty in using O'Hagan's spectral absorption difference approach. At constant albumin and total Hm concentration there are still three light-absorbing species in such a system and their concentrations are governed by three unknown constants, K, K^ and AT/. In addition, each of the absorbing species has its own molecular extinction coefficient, so that the absorbance increment at any given pH is not a simple function of the species. There is no reason to assume the A^'s are the same for haem and haematin, and in fact they are most unlikely to be if any binding through the iron is involved. I should like to ask O'Hagan how he arrives at the conclusion that 'reduction (of haematin to haem) significantly decreases the acid strength of at least one of the haematin propionate groups.' Concerning Fig. 4, 1 should like to point out that stability curves of metal complexes such as ferrihaemoglobin cannot be used to obtain the pATs of the ligands. Falk, Phillips and I have discussed this elsewhere (^Nature, Lond. 184, 1651, 1959). The 'apparent p/Ts' in such cases are, in fact, functions not only of the pAT of the ligand but also of the stability constant of the metal complex and the concentration of the ligand. It is quite erroneous to identify such 'apparent pATs' with the pA:'s of groups such as carboxyl or imidazole. The nature of the metal-to-ligand bond does not affect this conclusion: if a complex is present, there is, of course, a AG = RTXnK which leads to significant changes in the thermodynamics of the system relative to the proton-ligand system. One cannot use the A/f for haem-protein dissociation where the iron-to-protein link is involved as evidence for COO- or imidazole linkage if one takes the data for The Haem-Globin Linkage 191 proton addition to the latter groups. There is no reason why A// for metal-ligand bonding should be the same as for proton addition to the ligand. Phillips: The conclusion reached in part 4 of O'Hagan's summary seems not only un- justified but also contrary to the facts. One cannot deduce the ^K of a ligand from the pH at which the co-ordination complex is half dissociated without knowing the stability constant of the complex and the concentration of the various species: One can be certain, however, that the pA" will be greater than the pH of half-dissociation and all one can deduce from O'Hagan's results is that the pAT of the co-ordinating group is > 5 which could well be histidine (pAT — 7) or even lysine (pAT '~ 9) but is unlikely to be a carboxyl group (pAT — 5). O'Hagan: It is true that the pAT values I have suggested are not exact; they were not intended to be. I was not attempting to obtain an absolute value under these con- ditions, but one which would at least indicate the most likely group ionizing. In regard to Phillips' point, curve (a) shows some preliminary results on the dissociation of horse oxy haemoglobin in acid buffers. There is evidence that at pH 50 groups are dissociating. These cannot be the haematin side-chain carboxyls, the evidence points to the groups linked to the iron. At pH 4-9, Ferry and Green (/. biol. Chem. 81, 175, 1929) found horse haemoglobin stable enough to obtain an oxygen dissociation curve. If we examine the curve for the apparent heat of dissociation of horse oxy- haemoglobin of German and Wyman {J. biol. Chem. 117, 533, 1937), we can see that at pH 5-0 the apparent heat of dissociation is about — I kcal. If there were imidazole groups ionizing we would expect a value of about 6 kcal. The value found would appear to indicate carboxyl groups are linked to the iron atoms. 0 4- 0 3- . 0 The Haem-Globin Linkage (a) Change in the Soret peak absorbance at 410 m/< of horse oxyhaemoglobin in phthalate buffers (pH 4-0-6-2, I = 005). (b) Curve for apparent heat of dissociation of horse oxyhaemoglobin from German and Wyman {J. biol. Chem. Ill, 533, 1937). 192 Discussion Lemberg : Are we really sure that the combination between haematin and serum albumin is only through the haem carboxylic acid groups? This is certainly not so for the combination of haematin a with serum albumin, in which the spectrum clearly indi- cates combination with protein nitrogen. I was inclined to accept the suggestion of J, Keilin for the protohaematin compound, but I feel no longer sure about it now. George: In considering various mechanisms that might account for haem-linked ionization effects, I recently calculated the pH range over which a salt bridge would remain intact, and I think this will clarify the points just raised by Orgel, Perrin, Phillips and Williams. If the equilibrium constant for the formation of a salt bridge between, say, a carboxylate group and a substituted ammonium group is K^, —COO- + NH3+— -COO-NH,+— and the ionization constants for the two separate groups are ATcooh and K-^n^^ respectively, then in acidic solution the pH at which 50% formation occurs is given by /'(A's^cooh), and in alkaline solution the corresponding pH is given by p{K^Yii^lK^. As illustrated in the diagram the formation of the salt bridge results in the 'titration' of the carboxyl group in a lower pH range and the amino group in a higher pH range than usual — the apparent shift in the pA^ values being determined by the magnitude of K^. The shift in the pX^ values is in an opposite sense, because in acidic solution the bridge is broken by combination with H+, I.e. — COO-NH3+— + W — COOH + NH3+- (1) whereas in alkaline solution by the dissociation of H+, i.e. — COO-NH3+— ^ COO- + NHj— + H+ (2) In reaction (1) NH3+ — can be thought of as competing with H+ for the COO- group, thus lowering the 'p^'; in reaction (2) the combination of H+ with NHg — giving NH3+ — is favoured by the formation of the salt bridge; this has the effect of making NH3+ — a weaker acid, and hence raises its pA'. The Haem-Glohin Linkage 193 Falk: It is clear that the basis of the difference which Perrin, Phillips and I have had with O'Hagan is that he seems to consider the protein-haemoglobin bond as something like a simple electrostatic bond, with virtually zero stability constant, where we consider that co-ordination occurs to give a haem-giobin complex with a finite stability constant. Regarding COO~ as the protein-iron bond in haemoglobin, my objection to this is that of all the possible protein ligand groups, — COO" would tend to stabilize the ferric state most. The stabilization of its ferrous state, compared to haem itself, is perhaps the most important and the most outstanding special property of haemoglobin. EARLY STAGES IN THE METABOLISM OF IRON By J. B. Neilands Department of Biochemistry, University of California, Berkeley, California There are certain chemical and biochemical characteristics of iron which place it in a unique category in relation to the other common biocatalytic elements. Both ferric and ferrous ions are quite insoluble in aqueous solution at physio- logical pH; this property is of special significance in the case of ferric ion (solubility product 10^^^) since most of the iron available to living organisms will be encountered, at least initially, in the higher oxidation state. If one considers the very large proportion of iron that takes part in the transport and storage of oxygen as catalytic iron, then the latter element is seen to be quantitatively the single most important biocatalytic element in the entire realm of animal enzymology. Finally, since iron is bound relatively weakly to the usual type of naturally-occurring ligand, it might be expected that living cells, especially those with a high requirement for this metal, would have found it necessary to evolve special complexing agents which have the capacity to overcome such problems that are inherent in the transport and metabolism of this particular element. The complete synthesis of an iron-enzyme involves the ultimate conver- gence of at least two, possibly three, biosynthetic pathways. If the broad definition of an iron-enzyme given above is adopted, it is clear that in many instances the major portion of enzyme iron will occur as the porphyrin chelate, i.e. haem. The early stages in the biosyntheis of the organic part of this pros- thetic group have been thoroughly elucidated at least to the level of porpho- bilinogen (Shemin, 1955; Laver, Neuberger and Udenfriend, 1958) and partially clarified from thence to coproporphyrinogen (Granick and Mauzerall, 1958). Little exact information is available as yet concerning the immediate precursor of protoporphyrin although from the vitamin require- ments for haem synthesis (Lascelles, 1957) it might be speculated that an acrylic acid side chain should occur as an intermediate between the carboxy- ethyl and vinyl side chains. In recent years many investigators have examined the mechanism of uptake of iron by protoporphyrin. The concensus of opinion appears to be that the reaction is enzyme-catalyzed although the specific protein responsible for the observed effect has not been isolated. Until the latter has been achieved, the precise nature of the iron donor in the reaction will remain obscure. 194 Early Stages in the Metabolism of Iron 195 Essentially nothing is known of the structure and origin of the active centres of the non-haem iron enzymes. The special problems which arise in iron metabolism, as contrasted, for example with copper metabolism, can be illustrated by comparison of the characteristics and behaviour of the two elements in question. In the case of copper it is certain that smaller quantities are required by living tissues, the somewhat greater solubility of the hydroxide (saturated water solution of cupric hydroxide is > 10~^ m at 25°, Seidell, 1940) and, finally, the ubiquitous a-amino carboxylic acid structure provides an effective ligand capable of holding the cupric ion in solution at physiological pH. In the present paper, results will be presented for certain experiments dealing with the early stages of iron metabolism in micro-organisms. The latter form of life has been chosen for investigation on account of the well-known metabolic flexibility characteristic of unicellular organisms; however, in spite of this advantage, it must be recognized that micro-organisms as experi- mental subjects suffer from the fact that each species may exhibit certain metabolic variations. This will effectively preclude the formulation of sweeping generalities about the detailed mechanism of iron metabolism in all forms of life. The technique employed in the present instance has been that of cultivation of the aerobic micro-organisms Bacillus subtilis and Ustilago sphaerogena in the presence of diminished levels of iron. Such very aerobic species can be expected to have a reasonably high requirement for iron and a correspondingly well-developed system for the intermediary metabolism of this element. This statement is particularly true for Ustilago sphaerogena since this organism is known to form large quantities of cytochrome c (Grimm and Allen, 1954). The growth of such cells under conditions of iron deprivation provides valuable information about the early stages of iron metabolism. At very low levels of iron there is sparse growth, a feeble metabolism and essentially nothing can be learned about the intimate processes of iron utilization. Similarly, at abnormally high levels of iron, the substances usually involved as intermediates in iron metabolism may be produced in greatly diminished quantities in spite of the excellent cell yields. On the other hand, at inter- mediate levels of iron, three possible metabolic adjustments come into play which lead to the accumulation and excretion of iron-complexing agents : (i) The biosynthesis of specific ferric complexing agents, normally com- petitively inhibited and maintained at a low level by the presence of a variable amount of the ferric chelate, becomes a major metabolic activity of the cell. (ii) The deficiency of iron creates a metabolic block, the latter being manifested by the appearance of iron-complexing products which normally require iron for their further metabolism. 196 J. B. Neilands (iii) The new substances produced in iron deficiency are intended to serve, either as such or as the ferric complex, as a by-pass for electron trans- port around the normal cytochrome system. The isolation, characterization and chemical synthesis of itoic acid (iron- transferring-orthophenol ; 2 : 3-dihydroxybenzoylglycine ; 2 : 3-dihydroxyhip- puric acid) has been described elsewhere (Neilands, 1958; Ito and Neilands, 1958). The following experiments relate to the iron-complexing activity, production and metabolism of the new compound. The isolation and general properties of the ferrichrome compounds have been reviewed previously (Neilands, 1957). The present report will describe recent experiments leading to the identification of the iron-binding centre of the ferrichromes as a polyhydroxamic acid (Emery and Neilands, 1959; Emery, 1960). EXPERIMENTAL Materials Itoic acid was synthesized from 2:3-dihydroxybenzoic acid and ethyl glycinate in the manner previously described (Ito and Neilands, 1958). The preparation, after recrystallization, was chromatographically homogeneous in the following solvent systems {Rf in brackets): «-butanol, 4, acetic acid, 1, water, 5 (0-80); benzene, 2, acetic acid, 2, water, 1 (0-29); methanol, 20, water, 5, pyridine, 1 (0-67); tert. -bwiyX alcohol, 10, methyl ethyl ketone, 10, water, 5, diethylamine, 1 (0-39). The chromatograms were analyzed by ultraviolet illumination or by spraying with either 1 % aqueous ferric chloride alone or acidic sodium nitrite solution followed by dilute NaOH. Ferrichrome and ferrichrome A were prepared by 'low-iron' fermentation with U. sphaerogena and recrystallized by published methods (Neilands, 1952; Garibaldi and Neilands, 1955). Both preparations gave single spots in «-butanol, 4, acetic acid, 1, water, 5 (0-40 and 0-51, respectively) and methanol, 4, water, 1 (0-68 and 0-52, respectively). The ferrichromes can generally be detected visually on paper as tea-coloured spots without the application of a developing spray; alternatively, when only very small amounts of material are present, the chromatograms may be sprayed first with dilute sodium sulphite solution and then with a very dilute solution of 1 : 10-phenanthroline. Under these conditions iron is instantly released from the ferrichromes and appears as the intensely red-coloured ferrous-phenanthroline complex. Methods Ferric Complex of Itoic Acid. The colour reaction of itoic acid with ferric chloride was found to be markedly dependent on pH. A slight excess of itoic acid was added to a dilute solution of ferric chloride. The pH of the solution, initially about 2, was then raised in the automatic titration apparatus (Neilands and Cannon, 1955) by the addition of dilute alkali. The colour of Early Stages in the Metabolism of Iron 197 the solution was noted at various regions of the pH scale. The visible absorp- tion spectrum of the ferric complex in the presence of excess itoic acid was determined in 0-05 M-phosphate, pH 7-0. The method of continuous variation was then applied, in conjunction with spectral analyses, in order to obtain information on the composition of the ferric complex formed in neutral solution. In an attempt to study the reaction with ferric ion quantitatively, 1 3 /<-moles of ferric chloride and 39 /<-moles of itoic acid were mixed in 5 ml of water and subjected to automatic electrometic titration with 1 N-NaOH. Similar titrations were performed with 9-8 //-moles of cupric chloride and 19-6 /i-moles of itoic acid. An estimation of the relative stability of the 3:1 complex was obtained by equilibration of Fe+++ — ItoiCg with free ethylene- diaminetetraacetic acid (EDTA). The surviving phenolic complex was determined spectrophotometrically at 560 m//, a wavelength at which the ferric chelate of EDTA exhibits insignificant hght absorption. The following equilibria were employed : (i) Fe+++— Itoicg = Fe+++ + 3 Itoic; ^ ^ (Fe+++-Itoic3) ^ (Fe+++)(Itoic)=^ (ii) Fe+++ + EDTA = Fe+++— EDTA; _ (Fe+++— EDTA) " ~ (Fe+++)(EDTA) (iii) Fe+++— Itoic3 + EDTA = 3 Itoic + Fe^^-+— EDTA; _ ^ ,^ _ (Itoicf (Fe+++-EDTA) Kui - KnIK, - ^_^-^_-_^^__^ The lability of both free itoic acid and the ferric complex was examined under different laboratory conditions such as in the light and dark and at different temperatures. Production of Itoic Acid as a Function of Iron Concentration. The usual growth medium (Garibaldi and Neilands, 1956) was freed from iron by treatment with 8-hydroxyquinoline followed by extraction with chloroform. The itoic acid production by B. subtilis NRRL B-1471 as a function of added iron and time of incubation was determined by use of the known extinction coefficient of the 3:1 ferric complex in 0-1 m phosphate, pH 7-0. Utilization of Ferric-itoic^ by Bacillus Subtilis. In order to determine whether or not iron complexed with itoic acid is nutritionally available to B. subtilis, the following experiment was carried out. Eight 50 ml growth flasks fitted with side-arms were charged with 8 ml of the usual, i.e. not oxine-extracted, 198 J. B. Neilands medium. The flasks were sterilized by autoclaving and aliquots of sterile solutions of ferric chloride plus itoic acid, itoic acid alone and ferric chloride alone were added to duplicate flasks. The remaining two flasks served as controls. After adjusting the volume to 10 ml with sterile distilled water the flasks were inoculated and incubated at 25° under conditions of vigorous aeration. The turbidity was determined from time to time with a Klett colorimeter equipped with a green filter. Utilization of Itoic Acid by Bacillus Subtilis in the Presence of Iron. Cultures of B. subtilis were grown in duplicate flasks with the usual medium. After 36 hr, when the itoic acid production had reached a maximum value, 1-0 ml of a sterile solution of ferric chloride was added to one flask. To the control flask was added 1-0 ml of sterile distilled water. At suitable time intervals, aliquots of each culture were aseptically withdrawn, the cells removed by centrifugation and the itoic acid determined by the ferric chloride reaction. Release of Hydroxylamine from the Ferrichrome Compounds. Recrystal- lized samples of ferrichrome and ferrichrome A were heated at 100° in 3 N H2SO4 and the hberated "hydroxylamine" determined by the method of Csaky (1948). RESULTS Properties of the Ferric Complex of Itoic Acid The colour reaction with ferric chloride exhibited by itoic acid as a function of pH is illustrated in Table 1. It is apparent from these data that the reaction Table 1. The pH dependence of the colour reaction of ITOIC acid with ferric chloride pH Colour < 2 None 2-0 Light green 3-0-40 Green 4-7 Blue 5-0-5-5 Purple 5-9 Dark purple 7-3 Purple 90 Reddish purple > 100 Wine colour is strongly pH-dependent and that the structure of the complex undergoes several transformations in the region pH 2-10. At neutral pH the visible spectrum of the ferric complex in excess itoic acid showed general absorption in the region 500-700 m/t with a mM extinction coefficient of 3-7 per g atom of iron at 560 m/t (Table 2). Early Stages in the Metabolism of Iron 199 Table 2. The mM extinction coEFPiciENT/g atom of IRON for EXCESS ITOIC ACID IN THE PRESENCE OF FERRIC CHLORIDE IN 0-05 M-PHOSPHATE pH T'O Wavelength Wavelength (m/<) *^ nxM (m/0 *mM 500 2-7 600 3-4 520 3-2 620 31 540 3-5 640 2-7 560 3-7 660 2-3 580 3-6 680 1-7 From the results reported in Table 3 for the continuous variations experi- ment, it seems probable that a 3 : 1 structure is the most favoured combination in neutral solution. Table 3. Determination of the composition of the ferric complex of ITOIC acid by the method of continuous variation All solutes in a final volume of 5 ml of 005 m Na-K-phosphate buffer pH 7-0. Optical density measurements were made after one hour at room temperature H moles itoic acid l-i moles FeClj Ratio itoic acid/FeClg Optical density at 560 m/t 0-524 0-524 1-0 0084 0-734 0-315 2-3 0-128 0-784 0-274 2-9 0-139 0-840 0-209 4-0 0-138 0-890 0-157 5-7 0-109 In the titration experiments referred to above the initial pH of both the ferric chloride and itoic acid solutions was 2-6. On mixing the solutions the pH was depressed still further, to a value of 2-1, thus indicating a strong com- plexing of ferric ion even in very acidic media. Two equivalents of base were consumed below pH 7 and between pH 7 and 8 one additional equivalent of base was taken up. The ferric ion remained in solution throughout the entire course of the titration. In the case of cupric ion, no additional production of acid was observed on mixing the reagents. There was no formation of precipitate in these solutions even at pH 10. The cupric complex was green at pH values of 3-8 or greater and the absorption maximum lay at a wave- length above 600 m/<. As with the ferric ion, two equivalents of acid were titrated below pH 5-5 and one additional equivalent was titrated with a p/<:of 6-6. In the experiments in which the ferric chelate of itoic acid was equilibrated with EDTA, the colour of the solutions was found to reach a stable value several hours after mixing. If an apparent stability constant of 10"^ is selected for Fe+++ — EDTA, the corresponding value measured for Fe+++ — Itoicg is 200 J. B. Neilands 10^^ to 10^^. These data are not considered as highly accurate since con- siderable 'drift' was observed and since the equilibrium was not approached from the reverse direction. Nonetheless it is evident that itoic acid exhibits a very high avidity for ferric ion under approximately physiological conditions. Inasmuch as there was no pH effect on mixing solutions of itoic acid with cupric ion, the complexing of the latter metal is presumably weaker than for ferric ion. Table 4 illustrates the lability of both free itoic acid and the ferric complex under various laboratory treatments. Under the conditions tested, the free substance was stable in both light and dark and was only slightly decomposed, concomitant with browning of the solution, on autoclaving. Solutions which were allowed to stand in dilute alkah, on the other hand, rapidly assumed a coffee-colour that could not be discharged by re-acidiiication. The ferric complex was stable for at least one day at room temperature but was largely decomposed on autoclaving or on standing over a period of several weeks at room temperature. Table 4. Lability of itoic acid and the ferric complex under various conditions The stability of the free substance was determined by use of the absorption maxima at 314 m/t (.^mm — 3 0) and the surviving ferric complex was measured at 560 m/t. The samples in light and dark were allowed to stand at room temperature; autoclaving was carried out at 15 lb for 15 min. The solvent was 0-1 m phosphate buffer, pH 70, and measurements were made 6 hr after mixing the solutions Optical density at 560 m/t of 66 /(M solution of ferric-itoics Optical density at 314 m/t of 100 / \f ^"■"N,^ A ~^^ / S-b, _;3~\ Y a c uced) - a I 550 600 650 Wavelength, m/j Fig. 1. DifTerence spectrum, oxidized minus reduced, of a small granule fraction, The base line, which corresponded to an optical density of 0 at 750 m/<, was somewhat sliifted up at shorter wavelengths. H.E. — VOL. I — Q 220 A. TissiERES A difference spectrum (oxidized minus reduced), of a small granule fraction, taken in the visible region with the Cary Spectrophotometer, is shown in Fig. 1. 2. Relative Amounts of Cytochrome b^ in the Large Cell Debris and in the Small Granule Fraction A rough estimation of the amount of cytochrome h^ present in the large cell debris (sedimented in 15 min at 6000 x g) and in small granules (sedi- mented in 120 min at 100,000 X g) was obtained as follows: the pellets from the centrifuge tubes were resuspended in 3-4 vol. of 50 % (w/v) sucrose solution containing 0-01 m phosphate buffer, pH 7-0, and 0-01 m sodium succinate. These preparations were examined under the microspectroscope with the light path adjusted so that the intensity of the a-absorption band of reduced cytochrome b^ was the same for both large cell debris and small granule fractions. Thus, knowing the hght path, and the volume for each preparation, it was found that the large cell debris contained 75-85% of cytochrome b^, and the small granules 15-25 %. The errors in these measure- ments were probably as great as 20-30%. Grinding the cells with alumina for longer periods decreased the cytochrome content of the large cell debris and increased the number of small particles bearing cytochromes. This is consistent v^ith the finding that large cell debris from Azotobacter can be broken down to yield small granules (Tissieres et al., 1957). 3. Separation of the DPNH Oxidase Activity from Ribonucleoprotein Particles The DPNH oxidase system includes the cytochrome components; thus its activity, under some conditions, can be taken as a measure of the activity of the cytochrome system (Slater, 1950; Tissieres et al., 1957). The DPNH oxidase activity, the amounts of protein and RNA, and the area covered by the RNP particle peaks on the schlieren centrifugation diagrams, were measured on two RNP particle preparations isolated as described previously (Tissieres et al, 1959). Preparation 1 contained RNP particles with sedi- mentation coefficients of 305 and 505; preparation 2, particles of 505. Preparations 1 and 2 were centrifuged, at 25,000 x g for 45 min and at 32,000 X g for 30 min respectively, as shown in Table 1 . The various measure- ments were then repeated on the supernatant in order to find out whether the RNP particles sedimented at the same rate as the DPNH oxidase system. With both preparations, the DPNH oxidase activity in the supernatant after centrifugation decreased 88-90 % from its original value whereas the amounts of protein and RNA, and the area under the schlieren curve, decreased 25% (Table 1). These results indicate that the oxidases are attached not to RNP particles, but to granules which sediment faster. These granules are probably not homogeneous in size, since they did not form a visible peak on the The Location of Cytochromes in Escherichia Coli 221 sedimentation diagram even though they represent 5-10 % of the total fraction (see below). Table I. DPNH oxidase activity, protein, RNA and relative amounts of RIBONUCLEOPROTEIN PARTICLES (aREA COVERED BY PEAKS ON SCHLIEREN DIAGRAMS) IN PREPARATIONS ISOLATED BY CENTRIFUGATION DPNH oxidase activity A f/hr/mg protein Relative DPNH oxidase activity Protein (?„) RNA (%) Relative area covered by ribonucleoprotein particle peaks on schlieren diagrams Preparation 1 14-4 100 100 100 100 Supernatant after centri-| fuging preparation 1 for! 45 min at 25.000 g } 20 10-7 76-5 77-5 85 Pellet 75 (14% loss) 24 - Preparation 2 390 100 100 100 100 Supernatant after centri-1 fuging preparation 2 for! 30 min at 32.000 g J 4-8 12-3 84 76 90 Pellet 81 (7% loss) 16 4. Separation of Granules Containing Cytochromes from Ribonucleoprotein particles (a) Centrifugation in Caesium Chloride Solution with a Density of 1-46- 4-3 ml of a 6% of 70 5 RNP preparation made as described previously (Tissieres et al., 1959) was mixed with 5-7 ml of a 60% caesium chloride solution. The density of the resultant solution was approximately 1-46. The mixture was then centrifuged for 16 hr at 80,000 X ^ in a swinging bucket rotor. A gelatinous, transparent brown layer, about 2 mm thick, formed at the top of the centrifuge tube. This layer could be removed intact and spectro- scopic examination showed strong absorption bands of cytochrome a2, a^ and b^. The rest of the solution was colourless; at the bottom of the tube there was a colourless pellet of RNP particles. No cytochrome could be detected in either of these last two fractions. (b) Centrifugation in Sucrose Solution of Density 1-34. Sucrose (11-2 g) was dissolved in 12 ml of a 2 % of 70 S preparation. The mixture was centri- fuged for 12 hr at 100,000 X o^ in the swinging bucket rotor. A brown layer formed at the top of the centrifuge tube, which showed strong cytochrome absorption bands, as in the preceding experiment. The RNP particles had sedimented to the bottom of the centrifuge tube: it was found that their sedimentation coefficient (70 S) had not been modified by this treatment. Before centrifugation in sucrose, the preparation contained 60% RNA and 40% protein, while after it, there was 65% RNA and 35% protein in the pellet of RNP particles. The amount of protein in the layer containing cytochromes, at the top of the tube, quantitatively accounted for this 222 A. TissiERES difference: it amounted to about 8% (dry weight) of the preparation before centrifugation in sucrose. 5. E. coli 'Ghosts' Containing the Bulk of the Cytochromes of the Cells According to Repaske (1956), E. coli cells are lysed by lysozyme, at pH 8-0, in the presence of ethylenediaminetetraacetate (EDTA). 7 g of cells harvested in the exponential phase of growth and washed twice with 20 vol. of 0-1% NaCl, were suspended in 150 ml of 0-2 m sucrose, containing 0-01 M tris buifer, pH 8-0, and 0-01 m Mg++. Lysozyme (3 mg) and 3 ml neutralized 0-1 m EDTA were then added. The mixture was left at room temperature for 30 min, then kept 12 hr at 4°. By that time 98-99% of the cells had undergone the characteristic transformation into spheroplasts. These spheroplasts were washed twice in sucrose-tris-magnesium mixture containing 1 //g/ml deoxyribonuclease. Finally, they were lysed by re- suspending in 20 ml of 0-005 M tris, pH 7-3, containing 0-01 m Mg++ and 1 //g/ml deoxyribonuclease. Weibull (1956) has shown that when protoplasts from Bacillus megatherium were lysed in the presence of 0-01 m Mg++, the resulting 'ghosts' represented morphologically undamaged cytoplasmic membranes: they were shghtly larger than the protoplasts and there was approximately one ghost per protoplast. In the experiment described here, the number of ghosts obtained was about the same as the number of sphero- plasts from which they had derived. They appeared somewhat larger. The lysate was centrifuged at 10,000 rev/min for 15 min. A large pellet of ghosts was formed, which was washed twice in the centrifuge with tris- magnesium mixture. Spectroscopic examination of the washed ghosts showed strong absorption bands of cytochromes a^, a^ and b^ on addition of succinate or dithionite. No absorption band of cytochrome was seen in the washings. The ratio of RNA to protein in the washed ghosts was found to be 5/100, while it was 50/100 in the supernatant after centrifugation at 10,000 rev/min. This supernatant was examined under the spectroscope in a 70 mm layer, in presence of DPNH, succinate or dithionite. The absorption bands of cytochrome were not visible. The supernatant was next centrifuged for 120 min at 100,000 x ^. A characteristic RNP particle pellet was thus formed, slightly yellow in colour. This pellet, from two 1 1 ml centrifuge tubes was resuspended in 5 ml of sucrose solution with a density of 1-30, and centrifuged for 15 hr at 100,000 x g in the swinging bucket rotor. A very thin yellow layer collected at the top of the tube. It was carefully removed and examined for cytochromes in the presence of dithionite, and also after addition of pyridine. No absorption band could be detected. The colourless solution below the top layer, and the RNP particle pellet were also examined under the spectroscope in the presence of reducing agents, as well as after addition of pyridine. The cyto- chrome components were not detected. / The Location of Cytochromes in Escherichia Coli 223 In conclusion, the ghosts formed under the conditions of this experiment bear all the cytochromes of the cell. This suggests that in E. coli the respira- tory chain is located in the cell membrane. CONCLUSION There is no evidence in bacterial cells for respiratory granules homogeneous in size and shape: the cytochromes are usually attached to granules of widely different sizes in the extracts and these granules probably derive from the breakdown of a larger structure, the cell membrane (Marr and Cota- Robles, 1957). Under some conditions, all the cytochrome system is bound to 'ghosts' or cell membrane preparations, however probably containing still some wall material. The ribonucleoprotein particles, which are uniform in size and seem to fill the bacterial cytoplasm (Tissieres et al., 1959) do not bear any cytochrome pigments. SUMMARY 1. In £". coli extracts granules bearing cytochromes can be separated from ribonucleoprotein particles. 2. 'Ghosts' lysed in the presence of 0-01 m magnesium ions contain all the cytochromes of the cell. Acknowledgement I wish to thank Dr. N. Krinsky for measuring the difference spectrum shown in Fig. 1. REFERENCES Alexander, M. & Wilson, P. W. (1955). Proc. nat. Acad. Sci., Wash. 41, 843. CoTA-RoBLES, E. H., Marr, A. G. & Nilson, E. H. (1958). /. Bad. 75, 243. DisCHE, Z. (1953). /. biol. Chem. 204, 983. GoRNALL, A. G., Bardawill, C. T. & David, M. M. (1949). J. biol. CItem. 177, 751. Keilin, D. & Harpley, C. H. (1941),. Biocliem. J. 35, 688. Keilin, D. & Hartree, E. F. (i946). Nature, Lond. 157, 210. ' . ' Marr, A.'g. & Cota-Robles, t. H. {\951).,J.'Bact. 74, 79.- MiTCHELL, P. (1957). Blochem. J. 65, 44P. Repaske, R. (1956). Biochim. biopiiys. Acta 11, 189. Slater, E. C. (1950). Biochem. J. 46, 484. Smith, L. (1954). Bad. Rev. 18, 106. Storck, R. & Wachsman, J. T. (1957). /. Bact. 12>, 784. Tissieres, A. (1952). Nature, Lond. 169, 880. Tissieres, A., Hovenkamp, H. G. & Slater, E. C. (1957). Biocliim. biophys. Acta 25, 336. Tissieres, A., Watson, J. D., Schlessinger, D. & Hollingworth, B. R. (1959). /. mol. Biol. 1, 221. Weibull, C. (1953). J. Bact. 66, 688. Weibull, C. (1956). Exp. Cell Res. 10, 214. DISCUSSION The Origin of the Respiratory Granules of Bacteria Slater : The respiratory granules derived from the disintegration of the bacterial membrane which can be isolated by the methods described by Tissieres are capable of carrying 224 Discussion out oxidative phosphorylation, as has been shown by several workers. In our labor- atory, Miss Hovenkamp has been continuing the studies, begun by Tissidres and myself in Cambridge, of the system bringing about oxidative phosphorylation in particles isolated from Azotobacter. The P:0 ratios are low compared with those found with isolated mitochondria from animal cells, but the rate of respiration is so high that the rate of synthesis of ATP/mg protein by these bacterial-membrane fragments is the highest ever recorded. Miss Hovenkamp has recently succeeded in dissociating reversibly the phosphoryl- ation from the respiratory chain by suspending the particles in a medium of low ionic strength, following by centrifugation at high speed. Particles obtained in this way oxidize DPNH with little phosphorylation. Oxidative phosphorylation can be re- constituted by incubation of these particles with Mg++ and the supernatant obtained in this centrifugation {Nature, Lond. 184, 471, 1959). It should be noted that, unlike other supernatant factors, Miss Hovenkamp's is derived not from the cell sap but apparently by disintegration of the washed small particles themselves. We do not yet know whether this supernatant factor is an enzyme related to oxidative phosphoryl- ation, or is perhaps a structural factor required to restore the structure of the particles which has been disturbed by incubation in media of low ionic strength. This question is now being studied by Hovenkamp in Pinchot's laboratory. ON THE CYTOCHROMES OF ANAEROBICALLY CULTURED YEAST By Paulette Chaix Laboratoire de Cliimie biologiqiie de la Faculte des Sciences, Paris The first observations of cytochrome spectra, made at ordinary temperature by MacMunn between 1884 and 1886 and by Keilin in 1925, led to the con- clusion that cells grown anaerobically are lacking in these pigments. This point of view appeared later to be confirmed and the term cytochrome, notably because of the intra-mitochondrial localization (Chance and Williams, 1955, 1956) of the components {a + ^3), b, c^ and c of aerobic cells, became synonymous with oxidation-reduction catalysts belonging to the respiratory chain. At the present time, it is known that certain anaerobic cells (Postgate, 1954a, 1954b; Ephrussi and Slonimski, 1950) and certain cellular fractions lacking respiratory activity (Strittmatter and Ball, 1954; Chance and Williams, 1954) of anaerobic organisms may contain spectrographically detectable oxidation-reduction enzymes of haematin type, and the problem arises of trying to distinguish without ambiguity the cytochromes which belong to the respiratory chain from those which do not. Bakers' yeast, which may be cultured aerobically or strictly anaerobically, appeared to us a particularly suitable material to elucidate such problems as these. This work has been done in collaboration with Therese Heyman- Blanchet and Francois Zajdela, with the aid of the spectrographic method for studying the cytochromes in situ at room temperature (Chaix and Fromageot, 1942) and subsequently adapted to low temperature measurements (Chaix and Petit, 1956, 1957). VARIATIONS OF THE HAEMATIN SPECTRUM OF YEAST CULTURED ANAEROBICALLY, AS A FUNCTION OF ITS GROWTH PHASES Saccharomyces cerevisiae ('yeast foam' diploid) may be cultivated under strict anaerobiosis at 25°C either on a Difco yeast extract medium or on a synthetic medium. By adding to these two media Tween 80 and ergosterol, as recom- mended by Andreasen and Stier (1953, 1954) growth rates of // = 0-65 and ^ = 0-45 respectively (Heyman-Blanchet and Chaix, 1959) may be obtained. 225 226 Paulette Chaix If these yeasts are harvested during the exponential phase of growth their reduced haematin spectrum is characterized at low temperature by three a-bands situated at 552-5 mjii, 558 m// and 574-5 m// (Fig. 1, curve 1) (Chaix and Heyman-Blanchet, 1957). 4.2 4.0 3.8 r\y\ 1 y \ V ^^ 4.0 3.8 •^ V 2 \ \ 4.0 3.8 ^^>s. 3 4.0 ^ ^^ y V ^-N 4 \ ^ 3.6 V -% \ 575 600 A. m/^ Fig. 1. Progressive change of the haematin spectrum of yeast cultured anaerobic- ally on a yeast extract medium as a function of growth phase. Optical density of the culture was read at the time of harvesting. Reduction by endogenous substrate. For definition of the CU unit, see text below. (1), 64 CU; (2), 102 CU; (3) 120 CU; (4), 178 CU. If the yeast is harvested after the exponential phase of growth the observed spectra change as the growth rate slows down. When the culture reaches 102 CU (one CU unit means the optical density, measured with the Coleman photometer, which corresponds to 17-1 //g dry weight of yeast/ml), the three a-bands (spectrum 2) present during the expo- nential phase persist, but the band at 574-5-575 m/f has been accentuated; moreover two rather faint new bands have commenced to appear of which one, situated at 585 m//, has already been observed by Lindenmayer and Smith (1957); the other is situated at 630 m^. This last component does not seem to correspond to catalase since the catalatic activity of anaerobic yeast is very low. On the Cytochromes of Anaerobically Cultured Yeast 111 When the culture reaches 120 CU (spectrum 3), the relative strength of the different bands has changed further; the bands located at 552-5 and 558 m/^ have weakened while the three others have strengthened and a new band has appeared at 540 m//. When the culture has reached 178 CU (spectrum 4), the change has pro- gressed further; the bands at 552-5 and 558 m/i have practically disappeared and the four other bands have become sharply accentuated. INDUCTION BY OXYGEN OF RESPIRATORY CYTOCHROME COMPONENTS IN YEASTS CULTURED ANAEROBICALLY In 1950 Ephrussi and Slonimski showed with resting cells from anaerobic cultures that the simple presence of molecular oxygen was able to induce rapidly the synthesis of the classical cytochrome system of aerobic yeast; it was then thought that yeast cultivated anaerobically showed a single type of spectrum only. We have been reinvestigating such induction phenomena, 4.0 3.8 4.0 3.8 c ■D « 4.0 o. O 3,8 3.8 3.4 J ^ t=0 V ~7 \ t=lhr V P J \ t = 3hr \ •^ 7 t = 6hr \ 550 575 600 Fig. 2. Induction by oxygen of the air of the respiratory cyto- chrome system in resting cells of yeast from anaerobic culture on yeast extract medium, harvested in the exponential phase; 32 CU; reduction by endogenous sub- strates. ^^^ 1 3.9 3.7 -A t=0 \ \ V ^ 4.0 3.8 3.6 J \ I = l5min v_> 1 ^ K g 3.9 ■rd int. Congr. Biochem. Brussels, Academic Press, N.Y. Strittmatter, C. F. & Ball, E. G. (1954). J. cell. comp. Physiol. 43, 57. Vanderwinkel, E., De Deken, R. H. &. Wiame, J. M. (1958). Exp. Cell. Res. 15, 418. DISCUSSION The Lactate Dehydrogenase of Yeast Boeri : I should like to ask Chaix for more information about the lactate dehydrogenase in yeast. In our experience, different results are obtained, by varying the electron- acceptor, in aerobic and anaerobic yeast. In aerobic yeast, there is an abundance of a dehydrogenase which reduces cytochrome c in the presence of lactate, while the strong lactate dehydrogenase activity of anaerobic yeast is not revealed by a test with cytochrome c, but instead by one with ferricyanide. Chaix : We have found that lactate dehydrogenase activity in anaerobically grown yeasts is located almost completely in the supernatant. Anaerobic pigments are not reduced by DL-lactate. Morton: A very recent paper of Slonimski and associates reported that anaerobic yeast contains a lactate dehydrogenase which is a flavoprotein (free from haem) specific for d(— ) lactate, whereas the enzyme in aerobic yeast, the haemoflavoprotein, is specific for l(+) lactate. Could Chaix indicate as to where is the enzyme in the aerobic yeast? Chaix: We have used DL-lactate with aerobically and anaerobically grown yeast. Con- cerning your second question, we have found lactate dehydrogenase activity only in the mitochondrial fraction in aerobically grown yeasts. In this case the cytochromes are reduced by DL-lactate. Components of the Respiratory Chain in Yeast Mitochondria Chance: It is important to emphasize the need for distinguishing between pigments of the respiratory chain and accessory pigments. An unusually effective way of doing this is to observe the cytochromes involved in the steady state of aerobic metabolism, since the existence of the steady-state reduction of a cytochrome in intact cells and in phosphorylating mitochondria is acceptable as preliminary evidence for its function in electron transfer. Accurate observation of steady states requires the difference spectrum technique: the oxidized minus the steady-state oxidized spectrum. It has recently been possible in studies with W. Bonner to obtain this steady-state difference spectrum at liquid nitrogen temperatures (see also Estabrook, this volume, p. 436). Thus the method simultaneously gives a clear identification of the kind of cytochrome and its function in electron transfer. Two examples are given, one indicating respiratory enzymes of baker's yeast cells (Fig. 1 (p. 234)), the other of ox heart mitochondria (see Fig. 3, Chance, this volume, p. 607). Both these records show clearly what other methods fail to do: that cyto- chrome Cj is reduced in the steady state and hence functional in electron transfer in the intact cell and isolated mitochondria. Chaix : The experiments mentioned by Chance, which aim to distinguish between pigments which belong to the respiratory chain and the other pigments, are very interesting. But in order to correlate his observations with ours we would first have to agree on the definition of the term 'respiratory chain'. It should be possible, for instance, to postulate that cytochrome c functions as an electron-carrier in both aerobic and anaerobic electron-transfer. Chance considers this question from quite a different point of view from ours. On the ^Haemoglobin' Absorption Bands of Yeast Lemberg: Are the bands at 575 m/t and 630 m/t observed by you in yeast abolished by dithionite? If they are resistant to dithionite, the former may be due to a cryptohaem a-cytochrome, the latter to a cytochrome of type a^. 234 Discussion Chaix: The bands at 575 m/< and 630 mfi do not disappear after incubating the yeast cells for 90 sec at room temperature with dithionite (3 x 10^^ m). Incubation in the same conditions, but with solid dithionite, causes the disappearance of these bands. In the latter case the disappearance may be due to a denaturing effect of dithionite. 500 550 600 A, rr\// Fig. 1. 'Frozen steady-states' of cytochromes c, c^ and b of baker's yeast cells. (A) glucose-treated ; (B) ethanol treated ; (C) oxidized-reduced (Expt. 938-2). Reprinted with permission of the Faraday Society (B. Chance and E. L. Spencer, Jr., Disc. Faraday Soc. 27, 200, 1959). TissiEREs: Keilin and I have described an absorption band at 583 m/t in several moulds including some strains of yeast; this band appears on aeration, disappears on addition of dithionite and is replaced by a diffuse band at 575 m/t on treatment with CO. The band was therefore attributed to an oxyhaemoglobin. In the slow-growing 'petite colonie' strains which have a very low respiration rate, the oxyhaemoglobin absorption On the Cytochromes of Anaerobically Cultured Yeast 235 band is much stronger than in other strains examined. While the properties of the pigment resemble those of haemoglobin, it is conceivable that it has some oxidase activity as well. The study of the isolated pigment would be of considerable interest. Morton: A pigment somewhat resembling that described by Keilin and Tissieres was observed by Martin and myseU (Bioclieni. J. 65, 404, 1957) in plants. Strittmatter : The properties of the 575 m/{ band resemble those of the similar band, which may well be due to adsorbed oxyiiaemoglobin, that is seen in liver microsomes if care is not taken to exclude or remove contaminating blood pigments during prepar- ation of the microsome fraction (Strittmatter and Ball, Froc. nat. Acad. Sci. Wash. 38, 19, 1952; /. ceU. comp. Physiol. 43, 57, 1954; Paigen, Biochem. biophys. Acta 19, 297, 1956). H.E. — VOL. I — R IRREVERSIBLE INHIBITION OF CATALASE BY THE 3- AMINO- 1 : 2 : 4-TRIAZOLE GROUP OF INHIBITORS IN THE PRESENCE OF CATALASE DONORS By E. Margoliash and A. Schejter The Laboratory for the Study of Hereditary and Metabolic Disorders, and the Departments of Biological Chemistry and Medicine, University of Utah College of Medicine, Salt Lake City, Utah, and the Department of Experimental Medicine and Cancer Research, The Hebrew University, Hadassah Medical School, Jerusalem 3-AMiNO-l :2:4-TRiAZOLE (AT) has come into widespread use as a plant growth regulator. When injected into laboratory animals this drug causes a rapid decrease of the catalatic activity of liver or kidney suspensions to low levels but produces no change in the catalatic activity of erythrocyte haemo- lysates (Heim, Appleman and Pyfrom, 1956). 3-Amino-l :2:4-triazole and a number of related substances were shown to cause the irreversible inhibition of purified recrystallized catalase preparations from liver and blood in vitro but only in the presence of a continuous supply of hydrogen peroxide (Margoliash and Novogrodsky, 1958). This reaction parallels the effect of 3-amino-l : 2 : 4-triazole on the liver and kidney catalase activities in laboratory animals, but gives no explanation of the lack of effect of the drug on erythrocyte catalase in vivo. A study of the kinetics of the irreversible inhibition demonstrated that this reaction was second order between the inhibitors and catalase-hydrogen peroxide complex I (Cat. HoOa I), during which the inhibitors become irreversibly bound to the enzyme (Margohash, Novogrodsky and Schejter, 1960). If in addition to catalase, hydrogen peroxide and the inhibitor, the reaction mixture also contains a sufficiently high concentration of a catalase donor, i.e., a substance that can be oxidized by Cat. H2O2 1, the concentration of Cat. H2O2 I available for reaction with the inhibitor will be decreased (Chance, 1953) and the irreversible inhibition reaction will be slower. The purpose of this paper is to develop the kinetic equations that will describe the irreversible inhibition of catalase by the 3-amino-l: 2 :4-triazole group of substances in the presence of catalase donors, and to show that the lack of effect of such inhibitors in blood haemolysates is due to the presence of a naturally occurring catalase donor in erythrocytes. The existence of 236 Irreversible Inhibition of Catalase 237 such a donor substantiates, at least for blood catalase, the ideas of the physiological role of catalase as a peroxidase in tissues rich in the enzyme (Keilin and Hartree, 1945). THEORY Under conditions in which hydrogen peroxide is continuously supplied at a low concentration, to a mixture containing catalase, an irreversible inhibitor of the AT series and a catalase donor, the following reactions occur : (1) The formation of Cat. H^Oa I (Chance, Greenstein and Roughton, 1952): *i E + S ^ ES (a) (E^p—q'—q-) («) *2 (p) in which E represents the enzyme, S the substrate hydrogen peroxide, and ES represents Cat. HgOo I. The expressions in brackets are the concentrations of the reactants and the symbols represent the concentrations of the total original enzyme in terms of catalase haematin (£), of Cat. H2O2 I (/>), of the enzyme haematin bound reversibly by the inhibitor {q), and of the enzyme haemaiin irreversibly bound by the inhibitor {q"). (2) The reaction of the donor with Cat. HgOg I or peroxidatic reaction of catalase (Chance et al., 1952): ES + AH2 -- E + SH2 + A (b) (V) (a) in which AH2 represents the donor and a its concentration. (3) The reversible inhibition of catalase by the inhibitor, first reported for AT by Heim et al, (1956) and shown to be due to a reaction of the inhibitor with the free enzyme (Margoliash and Schejter, unpublished results): E + I 4 EI' (c) (E-^-q'—q") (i)"-' (2C Feiv=o I I I I I I I ! I P- P- P— H (3) Significance of Bond Type The behaviour of the ruthenium complexes mentioned in the previous section resembles in certain respects that of catalase and peroxidase, and is indicative of the kind of kinetic barrier which could limit the oxidation- reduction reactions of the enzymes. Dwyer et al. (1959) suggested that electron transfer between Ru" and Ru^^ could be minimized by preparing a catalyst in which the bond types were different in the two states. Although both Per. H0O2 1 and peroxidase (and Cat. H2O2 1 and catalase) are probably outer orbital complexes, it is possible that their interaction is limited by a kinetic as well as a thermodynamic factor and that the kinetic factor is related to changes in bond type. Hydrogen peroxide is able to carry out the full reduction of Ru^^ to Ru^^ rapidly, probably without passing through an intermediate oxidation state, while most donors yield the relatively inert green Ru^^^ complex. The simplest explanation is that an appreciable activation energy is required for reduction 250 M. E. WiNFIELD in two distinct 1 -electron steps, while little activation is required for the direct addition of two electrons to Ru.^^ In the reactions of catalase and peroxidase the kinetic barriers, namely the activation energies required to transform the complexes from one bond type to another, are supplemented by steric barriers. Chance (1951) has suggested, for example, that in catalase the reaction sites are well below the protein surface. We may perhaps regard the iron-porphyrin as lying at the bottom of a pore whose radius is only a few A units. Thus oxidizing or reducing agents may reach the metal atom without much restriction and yet be hindered from direct reaction with the porphyrin. By contrast the prosthetic group of peroxidase is thought to be relatively unprotected (Chance, 1951). The following scheme is an example of how the barriers mentioned above can lead to apparently unidirectional pathways in electron transfer, and to preferences for 2-electron steps. Elsewhere we have indicated the way in which the 2-electron steps can be thermodynamically favoured (King and Winfield, 1959b). In reaction (4) it is assumed that Per. H.^O.^ I is in equilibrium with a small concentration of a more reactive, free radical form. In (5) the isomer reacts very rapidly with a hydrogen donor to give Per. H2O2 II. More slowly the latter is reduced by a second donor to free peroxidase. \ / \ / C Fe^'=0 :^ ■ C Fei^=0 (4) I ^1 I I ^1 I p- p- Per. HaOa I Isomer of Per. H^Oj I "^ ^ TV +e + nA / ,, .C Feiv=o >C Feiv=0 (5) I I I rapid | j i P- P— H Per. HjOj II \ / +e-fHA / C Feiv=0 >C Fe"i— OH (6) P— H P— H Peroxidase If in catalase the porphyrin molecule is sterically protected from direct attack by H-donors, we have for the O2 liberation reaction : \ / \ / C FeV=0 -h HOOH — -> C Fe"i— OH + O, (7) I ^1 I '^^'^ I ^^ I ' P- P— H Cat. H,0, I Catalase Catalase Oxidation Mechanisms 251 which is virtually a donation of H~ by the peroxide molecule to the FeO ion. A donation of H~ by an alcohol is also conceivable, as described by King and Winfield( 1959a). In the presence of excess H2O2 there is a slow reaction: \ / \ / C Fe^^=0 + HOOH >C Fei^— OH + OOH (8) P- P- Cat. HoGj I Cat. H2O2 II Little HoOo is normally decomposed by the pathway (8) because catalase and Cat. H2O2 1 are predominantly outer orbital while Cat. H2O2 II is assumed to be appreciably inner orbital. The small activation energy introduced by the change in bond type involved in (8), compared with no change in (7), is sufficient to ensure that most of the peroxide molecules give up two electrons simultaneously, provided that the thermodynamics of the reaction are favourable. In the type II complexes the 77 bond from oxygen to metal must be of fractional order, since the reduction of catalase or peroxidase to the ferrous state is known to be difficult. The Fe^^ — O bond is therefore much weaker \ / , \/ than that in C Fe^^O or -C Fe^^=0, and the energy of the complex is I ^1 I l_ comparable with that of Fe^^ — O (inner orbital). Resonance hybrids are therefore possible (see, for example, Williams, 1956), and we may expect to find among those complexes of catalase and peroxidase which have one oxidizing equivalent, some with predominantly inner orbital characteristics, some largely outer orbital, and some which fit neither category. The latter are apt to have an absorption spectrum which is not obviously related to their magnetic moment. It is possible that they are sensitive to pH. \ / If we assume that Per. HgOo II is predominantly C Fe^^=0 (outer I ^1 orbital) with a fractional -n bond from oxygen to metal, while Cat. H2O2 II contains about an equal contribution from the two forms C Fe^^=0 (outer orbital) and C Fe^^ — O (inner orbital), the comparative sluggishness of Cat. H2O2 II as an oxidant, while having a spectrum little different from that of Per. H2O2 II, is understandable (see Theorell and Ehrenberg, 1952). Peroxidase is a feeble catalyst for H2O2 decomposition for the very reason that it has high 'peroxidatic' activity, namely that Per. H2O2 1 can be reduced very rapidly by a 1 -electron mechanism. In the presence of H2O2 alone, or of H2O2 and a 2-electron donor, reaction will mostly follow the pathway via H.E. — VOL. I— s 252 Discussion Per. H2O2 II. It will be slow because Per. HgOg II is not reduced rapidly by H2O2 or donors such as alcohols. SUMMARY 1. The need for limiting interaction between the normal and the doubly oxidized state of the catalyst during decomposition of H2O2 by a 2-electron mechanism leads to a consideration of the significance of bond-type in determining the reaction paths. In the reactions of catalase and peroxidase kinetic and steric barriers apparently co-operate with thermodynamic factors to establish the rather limited specificity of the enzymes. 2. Reasons are given for favouring a ferryl-type structure in the primary catalase complex, as suggested by George (1952), and the possible nature of the Fe — O bond is discussed, with preference for a form which may be written Fe^^IO. Acknowledgement Dr. N. Ham and Dr. F. P. Dwyer are thanked for helpful advice. REFERENCES Beers, R. F., Jr. (1955). J.phys. Chem. 59, 25. Cahill, a. E. & Taube, H. (1951). /. Amer. chem. Soc. 73, 2847. Chance, B. (1951). The Enzymes, Vol. 2, Pt. 1, pp. 428-53. Ed. J. B. Sumner & K. Myrbiick. Academic Press Inc., New York. Chance, B. & Fergusson, R. R. (1954). The Mechanism of Enzyme Action, pp. 389-98. Ed. W. D. McElroy & B. Glass. Johns Hopkins, Baltimore. Chance, B., Greenstein, D., Higgins, J. & Yang, C. C. (1952). Arch. Biochem. Biophys. yi, 322. Dwyer, F. P., King, N. K. & Winfield, M. E. (1959). Aust. J. Chem. 12, 138. Fergusson, R. R. (1956). /. Amer. chem. Soc. 78, 741. George, P. (1952). Advances in Catalysis, 4, pp. 367-428. Ed. W. G. Frankenburg, V. I. Komarewsky & E. K. Rideal. Academic Press Inc., New York. Gibson, J. F. & Ingram, D. J. E. (1956). Nature, Lond. 178, 871. Gibson, J. F., Ingram, D. J. E. & Nicholls, P. (1958). Nature, Lond. 181, 1398. Keilin, D. & Hartree, E. F. (1951). Biochem. J. 49, 88. King, N. K. & Winfield, M. E. (1959a). Aust. J. Chem. 12, 47. King, N. K. & Winfield, M. E. (1959b). Aust. J. Chem. 12, 147. Theorell, H. & Ehrenberg, a. (1952). Arch. Biochem. 41, 442. Werner, A. (191 1). A^^vi' Ideas on Inorganic Chemistry. London : Longmans, Green & Co. Williams, R. J. P. (1956). Chem. Rev. 56, 299. DISCUSSION Oxidation States of Haemoproteins George: I think there is now a substantial body of evidence to show that the compounds which are formed when ferrimyoglobin, ferrihaemoglobin, ferriperoxidase and ferri- catalase react with strong oxidizing agents are higher oxidation states of the prosthetic group, that can be formally represented as Fe^^' and Fe^' derivatives. Compounds of this type were suggested as the reactive intermediates in systems containing iron salts and hydrogen peroxide at least fifty years ago; but, although Polonovski, Jayle, Glotz and Fraudet proposed their participation in haemoprotein reactions in a series of papers from 1939 to 1941, systematic experimental studies to demonstrate the one and two equivalent oxidation steps, and to distinguish between some of the structures that are possible, have only been carried out over the last ten years. Catalase Oxidation Mechanisms 253 In the physiological pH range these compounds have oxidation-reduction potentials in the neighbourhood of 1 V and are relatively inert, both with regard to mutual disproportionation reactions and to the spontaneous reduction that can occur with oxidizable groups on the protein. This stabilization of higher oxidation states can well be considered as remarkable a property as the reversible oxygenation of the haemoglobins. Only in the case of myoglobin has the complete oxidation-reduction reaction for one of the couples been established. For the single equivalent oxidation it takes the form FcMb"' ^ FcMb"' + 2H+ -f e- On the basis of a hydrate structure for FcMb'" (or an isomer) the appearance of two H+ ions as product is consistent with a ferryl ion type of structure (or an isomer) for FcMb'^, i-e. FeMb+++ (HP) -^ FejibO++ -f 2H+ + e- acidic ferrimyoglobin ferrylmyoglobin (George and Irvine, Symposium on Co-ordination Compounds, Copenhagen, 1953: Danish Chemical Society, p. 135, 1954; Biochem. J. 60, 596, 1955). The aquo ferryl ion was suggested by Bray and Gorin in 1933 as an intermediate in the reactions of iron salts; while higher oxidation states, with structures very similar to that of 'ferrylmyoglobin', are exemplified by the vanadyl porphyrins and the manganyl phthalocyanine pyridine complex referred to by Orgel. As the above reaction and countless other examples in inorganic and organic chemistry show, a knowledge of the way the H+ ion participates to balance the oxidation-reduction equation, as reactant or product or not at all, is an essential piece of evidence for eliminating some of the many structures that otherwise account for the increments in oxidation equivalents, i.e. Fe" -^ Fe"' -^ Fe'^ -> Fe^. This knowledge is clearly important too in deciding the type of reaction by which the reduction of a higher oxidation state is brought about, namely by the net transfer of electrons or hydrogen atoms. However, the structure for a higher oxidation state, or more precisely a family of isomeric structures, can only be specified with certainty if the structure of the lower oxidation state of the couple has already been established. This point is well illustrated by the relationship between the ferric hydrate and ferryl ion structures in the myoglobin reaction as written above. In this case a hydrate structure for the acidic ferrimyoglobin accounts very satisfactorily for all its other reactions, i.e. the reduction to ferromyoglobin, and the formation of cyanide and fluoride complexes, etc. But in the case of ferriperoxidase and ferricatalase, neither the hydrate structure nor the structure with a carboxylate group bonded to the iron in the sixth co-ordination position will account for their combination with the familiar ligands, although such structures are often accepted as being well established. The variation with pH of the equilibrium constants for the formation of their complexes indicates that proton addition accompanies the bonding of anionic ligands — in contrast to ferrimyoglobin and ferrihaemoglobin, where the pH variation is consistent with the simple replacement of the co-ordinated water molecule in the hydrate structure, or the reaction of some equivalent structure. This difference in H+ ion dependence suggests that the parent ferric oxidation states of peroxidase and catalase have another kind of structure entirely, and, in view of the role of H+ ion in oxidation-reduction reactions, it may also be an important clue to difl"erent active types of higher oxidation state. This seems not unlikely, because, as is well known, ferriperoxidase and ferricatalase behave differently with strong oxidizing agents giving two relatively stable higher oxidation states (Fe'^ and Fe^) under conditions where ferrimyoglobin and ferrihaemiglobin give only one (Fe'^ ). Moreover the absorption spectra of the Fe'^' derivatives are not of the same form; the maxima, especially in the visible region, occur at different wavelengths, which is a further indication of important structural differences. Labile crevice structures for ferriperoxidase and ferricatalase, in which the group that is liberated when complex formation occurs has a high proton affinity, e.g. a 254 Discussion phenolic or alcoholic OH group will account for the 'anomalous' pH variation of the equilibrium constants (George and Lyster, Proc. Nat. Acad. Sci. Wash. 44, 1013, 1958). It cannot be a carboxylate group because the pAT is too low. Furthermore, such structures offer the interesting possibility that they remain intact in higher oxidation states, thereby providing an explanation for the different reactivity of peroxidase and catalase toward oxidizing agents. Whatever the true explanation may be, it is clear that any structure suggested for a higher oxidation state that is equally applicable to myoglobin and haemoglobin on the one hand, and to peroxidase and catalase on the other, leaves many questions unanswered. Peroxide Compounds of Catalase and Peroxidase Lemberg : If there is any change in the porphyrin structure it is more likely to be in Cat. H2O2 1 than in Cat. H2O2 II. As has been pointed out by Chance (/. Biol. C/iem. 179, 1331 (1949)), the band in the red part of the spectrum resembling that of verdohaemo- chrome and the low Soret band of the type I compound are indicative of interruption of conjugation in the porphyrin ring; no such evidence is available for the type II compounds of catalase and peroxidase, or for the ferrimyoglobin HjOo compound. Williams: Recently Brill and I have been studying the absorption spectrum of compound I formed from ethyl hydrogen peroxide and bacterial catalase. The spectrum we have obtained is somewhat different from that given by Chance. In particular there is evidence for a new absorption band at about 340-360 m/i not present in the spectrum of free catalase, a weak band at 580-600 m/i very like that of the band found in peroxidase compound I, and a lower Soret band. Brill has shown that there is no evidence for free radicals of catalase. We interpret the spectrum, by using several different lines of other relevant evidence, as indicating that compound I is an equi- librium mixture of two components. One component does not have an intact porphyrin ring. We believe it to have a methene bridge which is oxidized to .CHOH and to have lost an electron from the ring. The second component is a simple ferric complex. In either event the ethyl hydrogen peroxide is a component of the compound I. The two components are also present in peroxidase compound I but the ratio of the two is very different. We will discuss the difference between peroxidase and catalase from the viewpoint of our new evidence. The Nature of Catalase-Peroxide Complex I By B. Chance (Philadelphia) Chance : The nature of catalase complex I is still an enigma in spite of much study. Two views on the structure of this intermediate are possible, viz. that the components of peroxide are: (a) a part of complex I ; (b) are not a part of complex I. Chemical configurations that illustrate these views are: CH3OOH + Fe+++-H20^ Fe+++HOOCH3 + HjO (I) h CH3OOH + Fe+++ • HgO^ Fe+++0 + CH3OH (2) but these are only two of many possibilities, particularly with respect to Eq. (2) where the oxidizing equivalent could alternatively be located in the porphyrin or protein parts of the enzymes. Ogura, working in this laboratory, attempted by kinetic methods to determine whether some intermediate form preceded the given complex I, first by optical studies Catalase Oxidation Mechanisms 255 to times of about 1 millisec and then by studies of the rate laws for peroxide decompo- sition up to about 10 M peroxide (Ogura, Arch. Biochein. Biophys. 57, 288, 1955). No significant deviations occurred for half lives of such complexes of about 5 x 10^* sec or less. This time is long enough, however, to allow intramolecular rearrangements to occur and no definitive conclusions were reached. More recently Schonbaum and I have investigated the types of reactions that could be involved in Eq. (1) and (2) in order to determine whether some difference between the two reactions in their dependence on the concentration of methylhydroperoxide would be expected. If catalase forms a complex of peroxide and the enzyme, the usual form of the equilibrium equation should apply. If, however, instead of forming a complex, the components of peroxide are released as an alcohol, a modified equilibrium equation is required which involves a squared term in the intermediate concentration. We have considered several cases as discussed below. The terminology used here is illustrated for a simple equilibrium: h E + S^ ES (3) {e-p) (x) (p) x(e — p) This represents Eq. (1) above. It is perhaps trivial to indicate that a simple equilibrium followed by an irreversible transformation gives no equilibrium: E + S^ESj (5) ESi-^ESa (6) If the irreversible step is followed by a decomposition of the intermediate to the free enzyme, the system simulates an equilibrium but is actually in a steady state. (7) (8) E + S-^ ► ES (e-p) (x) (P) ES + AH2 — j-E- (P) (a) x(e- -P) ilk. (9) Such a reaction appears to occur in peroxidase where endogenous donor (AHj) participates in the reaction of Eq. (9) and in catalase where endogenous alcohol participates. This equation is valid when such a donor is in excess, i.e. [AHg] = constant. Both these reactions are negligible in pure enzyme preparations which contain no donor, i.e. [AHj] = 0. If the components of peroxide are expended in the initial reaction and a hydrogen donor such as an alcohol is formed, reaction (8) may give rise to a simulated equi- librium: E + S^ES' + AH2 (10) {e-p) (x) (p') (a) This represents Eq. (2) above. Here: x(e — p') 256 Discussion However, in contrast to Eq. (9), a is not a constant, but is formed in amounts equal to/)'. Substituting in Eq. (11) a =/?', Combinations of these reactions may occur and one of some interest is a reversible equilibrium followed by a first order transformation of the intermediate as in Eq. (10). E + Sv^ES (13) (e-v-p') (x) (p) k ES^ES' + AHa (14) k (P) (P') (a) pa = (py = . ^ .^^^^^ , (15) Again a squared dependence is obtained, as in Eq. (12). The derivation is, however incomplete and a term k^ap'jk-j appears in the right hand member. The value of k-, must be sufficient to be consistent with the kinetic data obtained by Ogura, A'7 > 10^ sec-i. This greatly exceeds k^ap (10^ x 10-« x 10-« = IQ-") and the equation has the form ^P ) = TTm7 ^ = -T-T- (16) kj //ca + k^ _ \ kik^ /v 1 ^ Kn I A. 1 /v "7 Under these conditions the p' form would predominate because k^k-, ^ k^^. Lastly we may consider a combination of Eq. (10) and Eq. (13) in which the initial intermediate of Eq. (10) undergoes a transformation to form complex I. E + Sv^ES + AHa (17) K k, ES-^ES' (18) k, ES' + AH2-- E + P (19) k_. P'^ = (py = ^ kAk.a+l)' ^20) Again, for the condition A'7 ^ k^a. These equations suggest that, for all cases examined in which the components of peroxide are released as alcohol and an intermediate having an effective higher oxidation state is formed, a back reaction will be characterized by a squared depen- dency between the intermediate and the usual parameters of the equilibrium constant. On the other hand, a complex which retains the components of peroxide will follow the usual linear dependence of the equilibrium relationship. Catalase Oxidation Mechanisms 257 The analysis just described would appear to have little relevance to catalase since no values for a dissociation constant of the primary complex have been reported; the values given are regarded to be 'apparent' dissociation constants (Eq. 7-9), due to endogenous donor. In bacterial catalase the latter is present in negligible amounts and the possibility of accurate titrations presents itself. Such titrations have been carried out with sufficient spectrophotometric sensitivity that millimicromolar amounts of intermediate compounds can be detected. The experimental details are given elsewhere, but a typical result is included in Fig. 1, which gives both/? and p^ plots for the reaction of catalase and methyl hydrogen peroxide. It is clear that the data P m/ Fe^ (Ferripero.xidase) /Complex II \ /Complex I \ \ Compound 11/ \ Compound 1/ Hydrogen peroxide and alkyl hydroperoxides, both two-equivalent oxidizing agents, are particularly effective in oxidizing Fe'" -> Fe^. With potassium chloriridate, a one-equivalent oxidizing agent, an excess has to be used because of its additional reactions with oxidizable groups on the protein, and the product formed is Fe^. 258 Discussion This presumably occurs through the two single equivalent steps, Fe"' -> Fe'^' followed by Fe'^ -> Fe^'. Evidence that supports this is as follows. First, if the Fe"' derivative is allowed to form by the spontaneous reduction of Fe^ , produced either by peroxides or chloriridate, then the addition of chloriridate very rapidly effects the change Fe"' -^ Fe^ . Secondly, if potassium molybdicyanide is used instead of chloriridate as the one-equivalent oxidizing agent in the original reaction with ferriperoxidase, only the Fe'^ derivative is formed. Then again, if chloriridate is added to the Fe'^ derivative formed in this way, Fe'' results. Apparently chloriridate but not molybdi- cyanide, under the experimental conditions employed, has a sufficiently high £■„' to effect the oxidation of Fe'^ to Fe^ . Furthermore it is an interesting reflection on the ability of peroxides to engage in net two-equivalent oxidations that they are com- pletely ineffective in bringing about this second step, Fe'^ -^ Fe^ (George, Science, 117, 220, 1953; Currents in Biochemical Research, Ed. D. E. Green, 2, p. 338, 1956). Chance: The success of the experiment illustrated by the figure above stems from our finding that the primary intermediate of bacterial catalase and methyl hydrogen peroxide is sufficiently stable to allow its titration with the substrate (Chance and Herbert, Biochem. J. 46, 4, 1950, p. 402). Although chemical depletion of peroxidase of endogenous donor has given preparations in which the primary intermediate is more stable (Chance, Arch. Biochem. Biophys. 41, 416, 1952), it is as yet inadequate for the precision demanded of these titrations. The titrations with one-equivalent oxidants do suggest the mechanism outlined above by George; however, it may be that interaction of the one-equivalent oxidant with portions of the peroxidase protein could produce a two-equivalent oxidant — a possi- bility that could not be disproved on stoichiometric grounds (Chance and Fergusson, in The Mechanism of Enzyme Action, Johns Hopkins Press, Baltimore, 1954, p. 389; Fergusson, /. Amer. chem. Soc. 78, 741, 1956). The experiments described here merely afford a new approach to the problem posed by Eq. (1) and (2) above. It is, however, a "kinetic" approach, and the interpretation of the result surely depends on the reactions postulated. A more elegant test would be aftbrded by chemical determina- tion of alcohol formation in Eq. (2), a topic on which active experimentation is proceeding. DwYER : A simple system recently investigated by Craig, Dwyer and Glaser (Craig, Dwyer and Glaser, /. Amer. chem. Soc, in press) may be useful to this discussion. Trimethyl- amine N-oxide undergoes the rearrangement (CH3)3NO -^ (CH3)2NH -f- H-CHO in H,C I (CHJpN 3'2 Fig. 2. the presence of various metal complexes. The necessary conditions for the catalyst complex containing Fe'", Ru'", or V'^' are (1) a site for the attachment of the N-oxide through the oxygen, (2) an adjacent site containing OH, and (3) the complex must be capable of oxidation. The intermediate complex shown in the figure is self-explanatory. The metal atom, e.g. Fe is either oxidized or polarized to simulate Fe'^ by the oxygen of the N-oxide. This oxygen then breaks the bond to the nitrogen carrying an electron Catalase Oxidation Mechanisms 259 with it, leaving N* and the one-electron oxidation is completed by migration of an electron from carbon. The oxygen finally is protonated to become OH. The metal atom then oxidizes the OH group bound through the hydrogen bond to the a carbon atom. The oxidized OH finally attacks the a carbon atom completing the two- electron oxidation. The resulting carbinolamine spontaneously rearranges to yield the products. It will be appreciated that the original metal complex with the vacant site and adjacent OH group is regenerated in the reaction. M ARGOLiASH '. Some of the observations made during the study of the irreversible inhibition of catalase bear on the points that have just been made. We studied the minimal structural requirements of compounds that showed the aminotriazole type of irre- versible inhibition of catalase. Among other features was an absolute requirement for a free primary amino group. Any substitution on this amino group resulted in a complete disappearance of the inhibitory activity. The second point to consider is that when the haem was separated from the protein and the protein denatured, as occurs with the usual acid-acetone treatment, the irreversible inhibitor remained entirely bound to the protein. The inhibitor must, however, have interferred with the haem in some way since catalase irreversibly inhibited with aminotriazole did not react with the usual ferric ligands such as cyanide or azide. Finally, if one compares the spectrum of irreversibly inhibited catalase with that of catalase-hydrogen peroxide complexes, it seems to be more similar to that of complex I than to any of the others. These various observations led to the idea that the irreversible inhibitors may possibly be covalently bonded to the protein through an amide link to a particular carboxyl group at the active site of the enzyme. This hypothesis is being tested. No direct proof has been obtained as yet, partly because of the obvious experimental difficulties of working with a protein having a molecular weight of 240,000. The similarity of the spectrum of irreversibly inhibited catalase to that of complex I might be due to the binding of the inhibitor to the protein in a manner not entirely dissimilar to the effect of the 'peroxide' in complex I on the active site of the enzyme protein. STUDIES ON PROBLEMS OF CYTOCHROME c OXIDASE ASSAY By LuciLE Smith and Helen Conrad Department of Biochemistry, Dartmouth Medical School and Johnson Research Foundation, University of Pennsylvania INTRODUCTION The best evidence available indicates that the enzyme cytochrome c oxidase is a combination of cytochromes a and a^, and that the cytochrome % reacts directly with oxygen (Keilin and Hartree, 1938, 1939; Chance, 1953; Smith, 1955). The combination can rapidly oxidize ferrocytochrome c, either when the cytochromes a, a^ and c are all attached to insoluble particulate material or when the cytochrome c is in solution and the cytochromes a and a^ are particle-bound. The reaction is usually represented as: cytochrome c -^ cytochrome a -^ cytochrome a.^ -^ O2 The affinity of cytochrome a^ for oxygen is very high (Ludwig and Kuby, 1955; Chance and Williams, 1955). There are several ways of assaying for cytochrome c oxidase: (1) The rate of oxidation of soluble ferrocytochrome c can be measured spectrophoto- metrically, the oxygen in solution being in ample excess. (2) The rate of oxygen uptake can be measured in the presence of a substance which will continuously reduce the cytochrome c nonenzymically; alternatively the rate of oxidation of the reducing agent, which may be a dye which changes colour on oxidation, is measured. reducing agent -^ cytochrome c -^ cytochrome a -> cytochrome % -^ O2 Here the assumption is made that the rate of reduction of the cytochrome c is rapid compared to the oxidation of the ferrocytochrome c by the oxidase. This assumption has been shown in one instance to be incorrect (Conrad, 1951). (3) A direct method of assay would be to measure spectrophoto- metrically the content of cytochromes a and a^ in the preparation. The extinction coefficients for the difference between reduced and oxidized cyto- chrome ^3 and the extinction coefficient for the carbon monoxide compound of cytochrome a^ have been measured (Chance, 1953). In turbid preparations such as tissue homogenates or mitochondria this type of measurement is difficult, except with specialized apparatus (Chance, 1954). 260 Studies on Problems of Cytochrome c Oxidase Assay 261 If all of the above assumptions are correct, a given concentration of cyto- chromes a plus ^3 (which seem to occur in a constant ratio in mammalian tissues) should represent a definite amount of cytochrome c oxidase activity. In a purified preparation of cytochromes a plus a^ this has been shown to be so (Smith, 1955). With this type of preparation the dilution of the enzyme in the assay system must be great enough to eliminate the inhibitory effect of the cholate in the preparation on the enzyme activity. As discussed below, the activity of the oxidase in the purified preparation can be very high under special conditions. On the other hand, when the oxidase activity of tissue homogenates or particulate preparations from cells is assayed by variations of either method (1) or (2) above, the activity often appears to be quite low com- pared to the content of cytochromes a plus ^3. Our studies of the apparently low cytochrome c oxidase activity of many cellular extracts cover several aspects of the problem: (1) It has been shown that soluble cytochrome c actually inhibits cytochrome c oxidase activity (Smith and Conrad, 1956). This work has been pubhshed and will be only briefly summarized. (2) Other basic proteins besides cytochrome c have been found to be inhibitory to cytochrome c oxidase activity, and substances present in some tissue homogenates are also inhibitory. (3) Studies have been made of the activities of fractions separated from homogenates of different kinds in an attempt to determine which type of preparation gives maximal activity. Although many methods have been devised for measuring cytochrome c oxidase activity by varying the type of substance oxidized by the enzyme system or the conditions of the assay, few observations have been made on the effect of the state of the cellular extract. It should be recalled that difficulties are met as a consequence of the attachment of the oxidase to insoluble particulate matter within the cell and that even in purified preparations one is not dealing with a water-soluble enzyme. Our studies have attempted to evaluate the different methods for measuring cytochrome c oxidase activity and particularly the relationship of the oxidase activity to the type of cellular homogenate or fraction. METHODS Preparations The purified preparation of cytochromes a plus ^3 has been described (Smith, 1955). The preparation of heart muscle particles was made by a modification of the method of Keilin and Hartree (Chance, 1952). Mitochondria were isolated from liver (Lardy and Wellman, 1952), kidney (Hollunger, 1955) and heart (Cleland and Slater, 1953). Cytochrome c was removed from liver mitochondria by washing with saline (Estabrook, 1958). Homogenates of rat organs (10% or 20%) were prepared by grinding 262 LuciLE Smith and Helen Conrad the tissues in cold water in a Teflon homogenizer and discarding the material sedimented by centrifugation at 700 rev/min for 5 min in a Servall refrigerated centrifuge. Measurements of the Content of Cytochromes a plus ag in Preparations In the optically clear purified preparations the difference in absorption spectrum between the oxidized preparation (nothing added) and the prepara- tion reduced with sodium dithionite was measured to assay the content of cytochromes a plus a^. These measurements were made either in a Beckman DU spectrophotometer or in the recording spectrophotometer described by Yang and Legallais (1954). The concentration of cytochrome ^3 was measured by recording the absorption spectrum of the carbon monoxide compound formed by gassing the dithionite-reduced preparation with carbon monoxide. The concentration of cytochrome a^ was calculated using the extinction coeflficient reported by Chance (1953). In turbid preparations, such as heart muscle particles, the difference in optical density (A£) at 605 minus 630 m/< between the preparation with the cytochromes reduced (anaerobic preparation containing substrate) and that with the cytochromes oxidized (aerobic preparation) was used as a measure of the cytochromes a plus a^ present. Cytochromes a and a^ were considered to be reduced in anaerobic mitochondria and oxidized in aerobic mitochondria containing substrate and phosphate acceptor (Chance and Williams, 1955). With homogenates, which contained haemoglobin, a diff'erent procedure was followed. Here the diff'erence in absorption spectrum was measured between two samples of aerobic homogenate, one of which contained 10~^ m cyanide. The observed diff'erence in optical density at 605 minus 630 m/t was multiplied by 4/3 to correct for the loss of absorption of the cytochrome a-^ at 605 m// in the presence of cyanide. All measurements were made in the recording spectrophotometer described by Yang and Legallais (1954) or the double- beam spectrophotometer designed by Chance (1951). Both instruments will record small differences in optical density of turbid preparations. Assay of Cytochrome c Oxidase Activity The rate of oxidation of soluble ferrocytochrome c by the oxidase was followed by recording the decrease in optical density at the a, /? or y absorp- tion peak of ferrocytochrome c, as previously described by Smith and Conrad (1956). The cytochrome c was prepared by the method of Keilin and Hartree (1947), purified according to Margoliash (1954) and reduced with hydrogen and palladium (Smith and Conrad, 1956). Chemicals Salmine sulphate, purchased from General Biochemicals, Inc., was dialyzed for several hours, first against 10~^ m ethylenediamine-tetra-acetate (versene), Studies on Problems of Cytochrome c Oxidase Assay 263 then against distilled water. Part of the salmine is lost through the Cellophane membrane during dialysis. The final concentration of protein remaining after dialysis was measured by the biuret method (Gornall, Bardawill and David, 1949) and the molarity of the salmine calculated assuming a molecular weight of 8000. RESULTS Effect of the Concentration of Cytochrome c on Cytochrome c Oxidase Activity Figure 1 shows semilogarithmic plots of the optical density minus the optical density of totally oxidized cytochrome at the wavelengths indicated in 001 Time = ll-5sec Fig. 1. Semilogarithmic plots of the observed changes in optical density as ferro- cytochrome c is oxidized by the oxidase. Ordinate values are E values at given times minus E of the totally oxidized cytochrome c; abscissa represents time. The different experiments were run with different concentrations of cytochrome c in the tests, but with the same concentration of the oxidase. The data of curve A were measured at 415 m/<; those of curves B, C, D, and E at 550 m/i, and those of curve F at 520 m/<. A = 1-07 fiM cytochrome c B = 5-33 /tM cytochrome c C = 80 fiM cytochrome c D = 16 fiM cytochrome c £■ = 32 ^fM cytochrome c F = 128 /j,M cytochrome c the legend against time as ferrocytochrome c is oxidized by the oxidase. The different hues were obtained with different concentrations of cytochrome c in the test mixtures, the concentration of the oxidase being held constant. From the slopes of the plots, the first order velocity constants can be calcu- lated in each case; these are plotted against cytochrome c concentration in 0 5 10 15 20 30 40 50 60 70 80 " IIO 120 130 Cytochrome c cone., /^M 34 r, B-Heo 1 1 rt muscle preparotic n 30 C 1 ^ \ 8 1 2^ \ % \ \, . - \ S 18 i o >« 14 \ \ \ ^"^ \ "^ o O \ ^ ^^^ •S? ^^ ^^^ > 10 - v^ ^ 3 2 1 1 5 10 15 20 30 40 50 60 Cytochrome c cone, //M Fig. 2. The effect of the concentration of cytochrome c on the velocity constant for the oxidase reaction. A. The two curves represent experiments with two different purified oxidase preparations. I. The cytochrome c was the Keilin-Hartree preparation; temperature was 20^C. The final dilution of the oxidase in the test was 3000-fold. II. The cytochrome c was purified according to Margoliash (1954); the tem- perature was 25 C. The final dilution of the oxidase in the test mixture was 6000-fold. The point represented by D was obtained with a mixture of ferri- and ferro- cytochrome c. The other points were obtained with cytochrome c which was more than 95 % reduced. B. Curves III and IV were obtained from experiments with two different heart muscle preparations. III. Final dilution of oxidase preparation in test was 1200-fold; temperature was 25°C. IV. Final dilution of oxidase in test was 600-fold ; temperature was 20°C. Studies on Problems of Cytochrome c Oxidase Assay 265 Figs. 2a and 2b. The different curves were obtained in experiments with two different purified preparations and two preparations of heart muscle particles. Measurements made with mixtures of ferro- and ferricytochrome c showed that the velocity constant is dependent upon the total concentration of cytochrome c (ferro- plus ferri-) in the reaction mixture. This is illustrated by 5-.l^ 20 30 40 50 60 7D 80 90 100 110 120 Cytochrome c cone., //M 800 700 B- Heart muscle preparation X + 600 -^ 500 ' 1 - o^ m^,^- — ,.^0 a> ^ 2 400 / E 300 iJ Calculated O O O O ^ — m /^^ 1/ 1 1 1 20 30 40 50 60 70 80 Cytochrome c cone., //M B Fig. 3. Variation of the initial rate of oxidation of cytochrome c with the total concentration of cytochrome c in the test mixture. The initial rates were calcu- lated from the data of Fig. 2 by multiplying the rate constants by the concentration of ferrocytochrome c in the test mixture. 266 LuciLE Smith and Helen Conrad the point marked D on curve I of Fig. 2a. When the initial rates of ferro- cytochrome c oxidation are calculated (velocity constant x initial concentra- tion of ferrocytochrome c) and plotted against the total cytochrome c concentration, as in Figs. 3a and 3b, hyperbolic plots are obtained, similar to those seen when measuring cytochrome c oxidase activity in the presence of a reducing substance. The data show that the apparent 'saturating' effect ^uu - 1 1 Liver porticles low in - cytochrome c 320 240 - ) \ \ :\ V 160 - \ \. - \o 80 - - 1 I I 0 20 40 60 Cytochrome c cone, /^M Fig. 4. The effect of the concentration of cytochrome c on the velocity constant for the oxidase reaction of liver particles treated to remove the endogenous cytochrome c. In each test 10 //I. of a 20-fold dilution of the liver particles in 0-05 M phosphate buffer was used. The temperature was 25°C. with increasing concentrations of cytochrome c is actually a reflection of an inhibitory effect of the cytochrome c on the oxidase activity. The above-described experiments on oxidase preparations obtained from heart muscle have been repeated with a preparation derived from rat liver mitochondria treated to remove most of the endogenous cytochrome c. Entirely similar plots were obtained (Fig. 4). Thus in the presence or absence of endogenous cytochrome c the same kinetics are observed. We have interpreted our data to mean that the soluble cytochrome c (either oxidized or reduced) reacts reversibly with the oxidase to form a combination in which the oxidase is inhibited or in which the oxidase is so masked that it cannot react with further cytochrome c in solution. Purifica- tion of cytochrome c in a number of ways did not change the observed kinetics, as illustrated in Fig. 2a. Thus the inhibitory effect of the cytochrome c does not appear to result from an impurity in the cytochrome c solution. Studies on Problems of Cytochrome c Oxidase Assay 267 The inhibitory effect with increasing cytochrome c concentrations on the purified oxidase is greater than with the enzyme attached to tiie heart muscle particles. This could mean that the oxidase of the purified preparation is more exposed to form the unreactive binding with cytochrome c than is the enzyme which is still a part of the respiratory chain particles. As far as the methodology of cytochrome c oxidase assay is concerned, the data indicate that: (1) If comparative studies of the oxidase activity of a given kind of prepara- tion are to be made, the concentration of cytochrome c must be held constant throughout the tests. Then only comparative values will be obtained, since the inhibitory effect of the cytochrome c will be present. If oxidase activities of different tissues or different kinds of preparations are to be studied, the inhibitory effect of cytochrome c on each kind of preparation must also be compared. (2) The high concentrations of cytochrome c usually employed in the manometric method and in most colorimetric methods of cytochrome c oxidase assay will result in a greatly inhibited enzyme. When the velocity constant of the reaction of the purified oxidase is measured at very low concentrations of cytochrome (around 1 /^m), a very active enzyme is apparent. If a second order velocity constant is calculated by dividing the observed first order constant by the concentration of cytochrome ^3, values as high as 10^ m~^ sec~^ are obtained at 25°C (Smith and Conrad, 1956). Ejfect of Proteins Other than Cytochrome c on Cytochrome c Oxidase Activity Since cytochrome c is a protein with an isoelectric point above pH 10, the effect of other basic proteins on the cytochrome c oxidase activity was tested. Table 1 shows the effect of different concentrations of salmine on the oxidase activity of heart muscle particles, all other components being kept constant in the test. The concentration of cytochrome c in the mixture was 7 /tM. Figure 5 plots the oxidase activity with increasing concentrations of cytochrome c in the presence of a constant salmine concentration. The data show that at low concentrations salmine has a greater inhibitory effect than does cytochrome c. The inhibitory effect of cytochrome c is almost entirely ehminated in a preparation inhibited by salmine. Apparently salmine binds at or near the same site as does the cytochrome c. With these effects of basic proteins on the oxidase in mind, we were led into a comparison of the cytochrome c oxidase activity of homogenates of some animal tissues with their content of cytochromes a and O3. This examined the possibility that the low oxidase activity sometimes observed in homogenates results from inhibition of the oxidase activity by substances present in the homogenates. Table 2 shows some results obtained with rat liver homogenates. The oxidase activity of washed particles is about 4-5-fold H.E. — VOL. I — T 268 LuciLE Smith and Helen Conrad greater than that of the whole homogenate when expressed in terms of the content of cytochromes a plus a^. Addition of some of the soluble fraction of the homogenate to the washed particles resulted in an inhibition of the Table 1. Inhibitory effect of salnqne on cytochrome c oxidase activity of heart muscle particles Heart muscle particles were prepared according to a modification of the Keilin-Hartree procedure. The concentration of cytochrome c in the test was 7 fiu. Salmine cone. Cum) Inhibition (%) 0 0 0-67 0 2-66 0 5-32 34 6-65 58 9-31 66 10-64 75 13-3 81 Table 2. Relationship of cytochrome c oxidase activity to content OF cytochromes a + 03 in fractions of liver homogenate The oxidase assay and the method for measuring AE (difference in optical density between reduced and oxidized cytochromes a plus a^) are described in the section on Methods. The concentration of cytochrome c in the test was 15 ixu. Velocity constant X dilution AE 605-630 m/t k|^E Whole liver homogenate Supernatant from centri- fuging homogenate at 900 rev/min for 5 min Washed particles from supernatant 6-1 6-8 7-6 0-034 0-024 0-008 179 281 950 oxidase of the particles, but the inhibition was observed to be variable in extent and to depend upon the concentration of cytochrome c. Again the data indicate the necessity for caution in interpreting data on Studies on Problems of Cytochrome c Oxidase Assay 269 oxidase activity of homogenates. Although we have not carried out experi- ments with plant tissues, some published data show that in homogenates of some plant tissues the oxidase activity is low or absent, but appears on Cytochrome oxidose octivity 70 o of rot heort particles A-withouf solmine B — with solmine c g 50 K - \ C 30 in c o o A .?> ~ >^*-^ o 10 ^''^o-.o...^ 1 1 0 20 Cytochrome C cone • 40 // M Fig. 5. The effect of cytochrome c concentration on the velocity constant of the oxidase reaction in the presence and absence of salmine. The oxidase preparation was a suspension of rat heart sarcosomes treated with 100 vol of distilled water. The concentration of salmine in the test was 1 //m. preparation of washed particles, indicating that something inhibitory has been washed away (James, 1956; Simon, 1957). Studies of Oxidase Activity of Fractions of Tissue Homogenates Some experiments were carried out which attempted to give some idea about the possible localization of the substances in homogenates which are inhibitory to cytochrome c oxidase. The oxidase is a part of the mitochondria, but the oxidase of intact mitochondria suspended in isotonic sucrose does not rapidly oxidize soluble ferrocytochrome c in the suspending medium. The oxidase does react rapidly after the mitochondria have been exposed to water or dilute buffer solutions. We have investigated the oxidase activity of mito- chondria to determine under what conditions maximal oxidase activity can be obtained. When a small volume (5-10 /d.) of a concentrated mitochondrial suspension in isotonic or hypertonic sucrose is added to ferrocytochrome c in 0-05 M phosphate buffer, the changes in optical density during the first 20 sec are not entirely due to the oxidation of cytochrome c, since the usual straight lines on a semilogarithmic plot are not obtained. These early changes in optical density, which presumably result from changes in light scattering as the mitochondria swell (Claude, 1946), are not significant after about 20 sec. However, it was found that if the mitochondria were allowed to stand diluted 270 LuciLE Smith and Helen Conrad Table 3. Guinea pig kidney mitochondria A 4 //I. of mitochondrial suspension in 0-25 M sucrose was added to 2-6 ml of buffer, then cytochrome c added at times indicated to a final concn. of 8 ^m. Time (min) Cytochrome c oxidase 1st order velocity k X dilution 0 15 65 87 28 45-2 89-2 82-7 B 3 ftl. of guinea pig kidney mitochondria was added to test mixture at 0 time, and the optical density readings for the first 20 sec were discarded (see text). 8 /ul. of heart muscle particles was used in the test. The sucrose-KCl medium contained 1 7 1 g sucroses, 0-30 g KCl and 0-121 g MgCl^ in 100 ml 002 m phosphate buffer pH 7-4 Cytochrome c oxidase 1st order velocity k x dilution in sucrose-KCl medium in 0-05 M phosphate buflfer Guinea pig kidney mitochondria Heart muscle particles 60 7-0 30-0 8-3 Table 4. Cytochrome c oxidase of rat heart mitochondria The concentration of cytochrome c in the test mixture was 5-1 ftM. The sucrose-versene mixture was 0-32 M sucrose, 0-001 M versene, 002 m phosphate, 0-01 m KCl, pH 7-4 Cytochrome c oxidase activity 1st order velocity k x dilution (a) 4 /<1. mitochondria added at 0 time to buffered cytochrome c (b) 4 /il. mitochondria incubated in 1-4 ml. water for 34-0 3 min, then buffer + cytochrome c added (c) Mitochondria diluted 100-fold with water 10 min 56-4 before assaying ; 400 fil. of diluted suspension used in test 640 (d) Mitochondria diluted in sucrose-versene and assay run in sucrose-versene 12-3 (e) Same as d + triton (0-13 mg/ml in test) 58-1 Studies on Problems of Cytochrome c Oxidase Assay 271 in buffer for varying lengths of time at room temperature, the cytochrome c oxidase activity increased somewhat. This effect is illustrated in the data of Table 3 obtained with guinea pig kidney mitochondria. As found by others, the oxidase activity is low when the assay is run in isotonic sucrose, as com- pared with the same preparation in 0-05 M phosphate buffer, although the activity of the enzyme in heart muscle particles is nearly the same in the two solutions. Similar observations were made with rat heart sarcosomes; these are summarized in Table 4. About the same maximal cytochrome c oxidase activity (expressed in terms of cytochromes a and ^3) can be obtained with hypotonically treated mito- chondria after standing and with washed particles isolated from a water homogenate. Thus the inhibitory substances do not appear to be present in the mitochondria. A more interesting observation is that the maximal oxidase activity that could be obtained was found to be different for different tissues. Table 5 Table 5. Cytochrome c oxidase activity of several tissues expressed in terms of content of cytochromes a plus o3 The oxidase assay and the method for measuring the difference in optical density between oxidized and reduced cytochromes a plus a^ are described in the text. The concentration of cytochrome c in the test mixture in each case was 15 fiM. Tissue Oxidase activity/Af 605-630 m/z Washed particles from rat liver fiomogenate Rat lieart mitochondria suspended in buffer before 950 assaying Guinea pig kidney mitochondria suspended in buffer before assaying Rat brain mitochondria suspended in buffer before 350 260 assaying 508 summarizes some representative values obtained with preparations from several rat organs, the assays being run at a constant cytochrome c concentration. DISCUSSION Taken all together, the various data show that conclusions based upon measurements of cytochrome c oxidase activity may be quite misleading. In such experimentation the following observations must be kept in mind: (1) The oxidase activity of homogenates, washed particles or purified preparations will depend upon the (total) concentration of cytochrome c in the test system. Thus in a series of experiments comparing activities of a given kind of preparation, the concentration of cytochrome c must be kept constant. The lower the cytochrome c concentration, the higher will be the activity observed, when the activity is expressed as the first order velocity constant. 272 LuciLE Smith and Helen Conrad (2) The extent of the inhibitory effect of cytochrome c is different with different kinds of preparations. The extent of the inhibition must be measured with each kind of preparation. (3) Other proteins besides cytochrome c are also inhibitory to cytochrome c oxidase activity, including some proteins found in tissue homogenates. Sedimentation of the insoluble fraction of the homogenates and washing of this fraction removes inhibitory substances. (4) In several rat organs examined, the same maximal cytochrome c oxidase activity was observed with washed mitochondria treated in hypotonic solution and with washed particles from a water homogenate. However, with mitochondria there is a change in absorption spectrum due to changes in light scattering which lasts for about 20 sec after addition of the mitochondria to the buffer solution. Also the maximal cytochrome c oxidase activity is obtained only after the mitochondria have stood in the hypotonic solution for about 60 min. (5) The maximal cytochrome c oxidase activity that can be obtained using a given cytochrome c concentration in the test mixture appears to be different for different tissues, when expressed in terms of the content of cytochromes a and a^. What can be deduced about the nature of the oxidase from this type of observation? As might be suspected, since the oxidase is a particulate enzyme, the experiments appear to show structural differences related to the oxidase in different kinds of preparations, particularly regarding the extent of 'exposure' of the oxidase to reaction with cytochrome c in solution. (1) In relatively intact mitochondria, the oxidase does not react rapidly with cytochrome c in solution. (2) The oxidase of swollen or ruptured mitochondria or of the insoluble particles derived from the mitochondria is usually in a position to react with cytochrome c in solution. However, in some preparations (Mackler and Green, 1956) the structure of the particulate material is such that rapid reaction with soluble cytochrome c is not observed. Our data indicate that, in addition, cytochrome c or some other proteins can bind to the oxidase particles in such a way that the oxidase becomes inaccessible to reaction with cytochrome c. Some of these inhibitory proteins can be removed by washing. (3) The oxidase of a purified preparation is most susceptible of all to the inhibitory binding of cytochrome c. The simplest explanation of our data seems to be that the inhibitory effect of cytochrome c or salmine on the oxidase results from masking of the enzyme by the binding of these proteins. After addition of salmine, the cytochrome c has httle further inhibitory effect. We have found that the addition of salmine has no effect on the steady-state levels of the cytochromes of heart muscle particles oxidizing succinate with oxygen. This seems to show that the salmine does not react specifically with one member of the oxidase system, but rather Studies on Problems of Cytochrome c Oxidase Assay 273 acts to block interactions physically. The relatively small inhibitory effect of high concentrations of cytochrome c on the oxygen uptake of heart muscle particles indicates that the masking of the oxidase by cytochrome c inhibits the oxidative reaction of the oxidase with cytochrome c in solution, but may not block the reaction between the oxidase and the endogenous cytochrome c of the particles. Preliminary observations on the effect of salmine on the succinate-cyto- chrome c reductase activity of particulate preparations indicate that in this case also the salmine has an inhibitory effect. The postulate might be made that the surface of the particles bearing the electron transport chain has structural parts that can bind basic proteins such as salmine strongly. This binding prevents the interaction of the particulate enzymes with cytochrome c in solution. The poor accessibility of soluble cytochrome c to a catalytic site on heart muscle particles has been suggested by Keilin and Hartree (1955). Thus there appear to be at least two structural effects which can inhibit the reaction of the particulate oxidase with cytochrome c in solution. Some structural barrier inhibits the reaction with cytochrome c of the oxidase of in- tact mitochondria and of some kinds of particulate preparations derived from the breakdown of the mitochondria. With the latter, low concentrations of surface active agents such as cholate or deoxycholate can remove the inhibition (Smith and Stotz, 1954; Mackler and Green, 1956). The present data show that the oxidase can also be masked by binding of some proteins. To what extent the two effects which mask the atjility of the oxidase to react with soluble cytochrome c are interrelated is not yet clear. But the effects are such that different maximal oxidase activities (expressed in terms of content of cytochromes a + flg) are observed with preparations from different rat organs, showing that the extent of the structural inhibitory effects varies in different tissues. Although observations on the factors which may affect the oxidation of soluble cytochrome c by cytochrome c oxidase of tissue homogenates on particles indicate many difficulties in assessing variations in apparent oxidase activity, further experiments of this kind may lead to some insight into the nature of the reaction site of the enzyme. Gamble (1957) has shown that liver mitochondria or mitochondrial frag- ments suspended in media of low ionic strength can definitely bind cytochrome c or salmine and that under these conditions aggregation of the mitochondria or fragments is observed. The binding of cytochrome he describes appears to be irrelevant to the present experiments, since it was not observed in solutions of phosphate buffer comparable to those used in the experiments reported here. SUMMARY Measurements of cytochrome c oxidase activity have been made by obtaining the first order velocity constant for the oxidation of ferrocytochrome c 274 LuciLE Smith and Helen Conrad by the oxidase under different conditions. These studies have shown that: 1. Cytochrome c oxidase is inhibited by soluble cytochrome c in either its reduced or oxidized form. 2. Other proteins besides cytochrome c, notably salmine, are also inhibitory to the oxidase. There is evidence that proteins that occur in water homo- genates of rat tissues are also inhibitory, but some of these can be removed by washing the particles containing the oxidase. 3. With a given tissue the maximum cytochrome c oxidase activity is obtained with either the washed insoluble particles from a homogenate or with washed mitochondria which have been allowed to stand for about 60 min in water or dilute buffer. 4. The maximum oxidase activity obtained with washed particles or water- treated mitochondria is different with different tissues, when expressed in terms of the content of cytochromes a plus a^. 5. The observations on cytochrome c oxidase activity of tissue preparations are most simply explained by assuming that cytochrome c or other proteins can bind to the oxidase in such a manner that its oxidative reaction with cytochrome c in solution is blocked. REFERENCES Chance, B. (1951). Rev. ScL Instrum. 22, 619. Chance, B. (1952). /. biol. Chem. 197, 557. Chance, B. (1953). /. biol. Chem. 202, 397. Chance, B. (1953). J. biol. Chem. 202, 407. Chance, B. (1954). Science 120, 767. Chance, B. & Williams, G. R. (1955). Fed. Pioc. 14, 190. Chance, B. & Williams, G. R. (1955). J. biol. Chem. Ill, 409. Claude, A. (1946). /. exp. Med. 84, 51. Cleland, K. W. & Slater, E. C. (1953). Biochem. J. 53, 547. Conrad, H. (1951). Thesis, University of Rochester. Estabrook, R. W. (1958). /. biol. Chem. 230, 735. Gamble, J. L. (1957). Biochim. biophys. Acta 23, 306. Gornall, a., Bardawill, C. & David, M. (1949). /. biol. Chem. Ill, 751, HoLLUNGER, G. (1955). Acta Pharmacol, toxicol. 11, Suppl. 1. James, W. O. (1956). New Phytol. 55, 269. Keilin, D. & Hartree, E. F. (1938). Pioc. roy. Soc. B125, 171. Keilin, D. & Hartree, E. F. (1939). Proc. roy. Soc. B127, 167. Keilin, D. & Hartree, E. F. (1947). Biochem. J. 41, 500. Keilin, D. & Hartree, E. F. (1955). Nature, Lond. 176, 200. Lardy, H. A. & Wellman, H. (1952). J. biol. Chem. 195, 215. LuDWiG, G. D. & KuBY, S. A. (1955). Fed. Proc. 14, 247. Mackler, B. & Green, D. E. (1956). Biochim. biophys. Acta, 21, 1. Margoliash, E. (1954). Biochem. J. 56, 535. Simon, E. W. (1957). /. exp. Bot. 8, 20. Smith, L. (1955). J. biol. Chem. 215, 833. Smith, L. & Conrad, H. (1956). Arch. Biochem. Biophys. 63, 403. Smith, L. & Stotz, E. (1954). /. biol. Chem. 209, 819. Yang, C. C. & Legallais, V. (1954). Rev. Sci. Instrum. 25, 801. I Studies on Problems of Cytochrome c Oxidase Assay 275 DISCUSSION Assay of Cytochrome c Oxidase Slater: A practical use of cytochrome c oxidase assays is to measure the distribution of mitochondria in various fractions obtained by differential centrifugation. The assumption is made that all the cytochrome oxidase is in the mitochondria, so that the distribution of cytochrome oxidase in the various fractions is the same as the distribution of the mitochondria. We do this by using very high concentrations of cytochrome c, with />-phenylenediamine as reducing agent in 0-05 m phosphate buffer and measure the O2 uptake manometrically. Do you think that the inhibitor which you find in the soluble fraction of tissue homogenates causes serious error in this procedure ? Smith : Yes, I do. Like other workers, we have found that the sum of oxidase activity from different fractions of a tissue homogenate is usually considerably larger than the activity of the unfractionated homogenate. Lemberg : The paper of Smith shows how important it would be to have an analytical method for the estimation of the haem a content of tissues without reference to enzyme activity. I mention only one example. Lahey, Gubler, Chase, Cartwright and Wintrobe {Blood 7, 1053, 1952) had believed that the lack of cytochrome c oxidase activity in organs of copper-deficient swine was due to the lack of copper in the oxidase molecule. However, Gallagher, Judah and Rees {Proc. roy. Soc. 5145, 134, 1956) have shown that haem a was almost completely absent from the liver in copper-deficient rats. Since Lahey et al. {loc. cit.) had found no lack of catalase in copper-deficient swine, it is evident that copper is specifically required for the biosynthesis of haem a from protohaem or its precursors. Three such analytical methods have been worked out by us : (1) The quantitative isolation of porphyrin a in a state of spectroscopic purity or near-purity. This can be satisfactorily applied only to tissues comparatively rich in haem a such as heart and requires at least 5 g of tissue. It is also technically not quite easy. (2) The spectrophotometric determination based on measurements of the optical densities at 587 and 558 m/t of the mixed pyridine haemochromes. This requires less material but still a relatively high haem a content. (3) The separation of haemin a from protohaemin by a modified Rawlinson-Hale procedure, followed by spectrophotometric analysis of the pyridine haemochrome. This method has given satisfactory results in the study of iron incorporation into haem a in rat tissues (Lemberg and Benson, Nature, Lond. 183, 678, 1959). The presence of lipid in the extracts still causes some difficulties which we hope to overcome. Wainio: Suppose a surface active agent was added to the particulates presented in Table 5 (Smith, p. 269), i.e. those that have different oxidase activity/AE 605-630 m/« values, would the results be more uniform? Smith: Addition of low concentrations of cholate to heart muscle particles will increase the oxidase activity/AE 605-630 m/t almost up to that obtained with liver particles. We have not made observations with preparations from kidney or brain. Inhibition of Cytochrome c Oxidase by Cytochrome c Slater: Ever since Smith reported inhibition of the cytochrome c oxidase reaction by oxidized and reduced cytochrome c, we have attempted to incorporate this inhibition in reaction mechanisms, i.e. we thought that the inhibition in some way might be a part of the normal mechanism. Am I correct in concluding that you regard the inhibition as incidental to the enzymic reaction, i.e. that it is a side reaction caused by the fact that cytochrome c is a highly basic protein? If this is so, perhaps we have been wasting our time, and simpler mechanisms will be adequate. Smith: I feel that the observation that the inhibitory effect of cytochrome c can be dupli- cated by another basic protein, salmine, fits better with the assumption that the inhibi- tion is a side reaction. Also in accord with this view is the lack of inhibition of electron 276 Discussion transport down the intact respiratory chain by rather high concentrations of cyto- chrome c; here there is apparently no problem of availability of the oxidase to the reaction site of cytochrome c. Henderson: I wish to ask Lucile Smith whether the result was the same, independent of the iron concentration of the cytochromes ? We have found that both the preparations with 0-34% and 0-43% iron, but particularly that with low iron, combine adequately with copper. This is interesting in view of Wainio's paper. In the low-iron prepar- ation, the cytochrome c is abready combined with non-basic proteins. Did you find any effect of long incubation time as observed by Tsou in 1951? He regained the complete endogenous activity on long incubation. Smith: We used cytochrome c of 0-34% iron, and the same preparation purified according to Margoliash, with identical results. In our hands the period of incubation makes no difference. Interaction of Cytochrome c with Other Compounds The Effect of Cations on the Reactivity of Cytochrome c in Heart Muscle Preparations By R. W. EsTABROOK (Philadelphia) EsTABROOK : I would like to comment on a series of studies I have recently carried out using a cytochrome-c-deficient heart muscle preparation in order to investigate the role of cytochrome c in the succinoxidase system. These studies are complimentary 180 160 120 100 60 • 004 0O8 012 0-16 Phosphote buffer concenfrotion, mole/l Fig. 1. 0 20 to those of Smith and bring up a necessary question concerning the interpretation of cytochrome c oxidase assays. During studies to assess the validity of a lipocytochrome c as required for enzymic activity and to determine the accuracy of previous claims that exogenous cytochrome c may be only 1/100 as active as endogenous cytochrome c, it was observed that the K,n for exogenous cytochrome c, in the manometric measurement of succinoxidase activity using the cytochrome-c-deficient heart muscle preparation, was markedly affected by the presence of cations in the medium. This is shown in Fig. 1 where the K^n is plotted as a function of the potassium phosphate concentration employed in the reaction vessel. One sees over a 100-fold difference Studies on Problems of Cytochrome c Oxidase Assay 111 in the determined K,n as low salt concentrations are approached. This lower value for the K,n for cytochrome c, that is 3 x 10"' m, when related to the concentration of endogenous cytochrome c which is normally present in such a heart muscle prepar- ation, shows that exogenous cytochrome c is nearly as effective as endogenous cyto- chrome c thus negating the necessity of assessing the previously low activity values 0060- 1^ o 0O40 0 -0010 r / ...-> N 012m 1 > -i K 002 1 1 bo-< \ 1 Ol 23456789 Time, mjn A 75 / --< r -/' o c '' / o / / ■R 50 ,^ / 1 i/ 4 / t /^onaerobi( O 1 '' / y —>(-Ji—^ 0 — — o 5- 0-06 0 12 018 Phosphate buffer concentration, M/l B Fig. 2. to the requirement for a lipocytochrome c. Other studies have shown that the in- hibitory action of cations is dependent not only on their concentration but on their valency charge. That is, the divalent cations, Ca++, Ba++, Mg+''", are much more inhibitory than the monovalent cations, Na+, K+, and Li+. In turn, the trivalent cation A1+++ is much more inhibitory than the divalent cations. By following spectroscopically the change in steady-state reduction of the exogenous cytochrome c present during succinate oxidation by such a system, it is possible to assess the locus of inhibition as between reduced cytochrome c and cytochrome oxidase. The decrease in oxygen uptake accompanied by the increase in steady-state reduction of the exogenous cytochrome c as the cation concentration increases is summarized in Figs. 2a and 2b. The inhibitory effect of cations as determined from the change in K^ of the system for exogenous cytochrome c is also largely dependent on the enzyme concentration. 278 Discussion Thus the ratio of cation to a hypothetical active site for reduced cytochrome c- cytochrome oxidase interaction appears to be critical, that is, as the enzyme prepar- ation is diluted at a fixed cation concentration more cytochrome c is required to obtain half maximal activity. With cytochrome c oxidase activities as currently determined spectrophotometrically the ratio of cation to hypothetical active site on the protein would be very large indeed introducing a serious source of inhibition of the type described above and would make the interpretation of the results of such assays most difficult. This would also explain in part the inability to saturate the cytochrome oxidase with cytochrome c as determined by such measurements. A second point bearing on the problem of cytochrome a and cytochrome 03 is the unexplained observation concerning the steady-state of these pigments during succinate 0-04 o Ml 370 400 430 460 490 Wavelength, Fig. 3. 550 580 m// oxidation by a cytochrome c deficient heart muscle preparation. Figure 3 shows the difference spectra obtained when succinate is added to such a preparation. The dotted line represents the steady-state obtained in the presence of succinate while the solid hne is that recorded when a sample is anaerobic. As one would expect, the pigments cytochrome b and c^, as determined from the magnitude of the absorption bands at 563 and 553 m/< respectively, are about 80 to 90 % reduced in the steady-state. Un- explained is the large reduction at 605 m/f , a value almost 35 % of that observed on anaerobiosis. If one considers a respiratory chain as represented by the scheme: succinate ->- dehydrogenase -^ b -> c^-^ c -^ a ^i- a^^^i- O2 then the removal of cytochrome c should result in the reduction of cytochromes b and Ci by succinate, but little or no reduction of cytochromes a or 03. This is especially true in the light of the observation that the rate of cytochrome 03-oxygen reaction and that of cytochromes a and 03 interaction (Chance) is extremely rapid. The presence of a small amount of endogenous cytochrome c would be offset by the large velocity of these reactions and would not tend to invalidate the observations presented here. It should be noted, also, that the absorption at 444 m/t is only about 10% of that obtained on anaerobiosis. In agreement with previous measurements on the steady-state reduction of cytochromes a and a^, in a purified preparation (Smith), one must conclude that the 444 and 605 m/t absorption bands are not due to a single pigment, as based on their differences in steady-state values. One must also conclude that the linear chain representation as presented above is not truly representative in this instance and that one may have to place cytochrome a to a side path, much in the manner that Chance has put cytochrome /> in a side path in such modified heart preparations. Studies on Problems of Cytochrome c Oxidase Assay 279 Wainio: In 1951 we demonstrated that the oxidation of ferrocytochrome c by cytochrome c oxidase is maximal at pH 6-0 in 0- 1 m phosphate buffer and that the activity decreases sharply on both sides of the optimum (/. biol. Chem. 192, 349, 1951). More recently (unpublished) we have extended these studies, and although it appears as though the charge on the cation may be the controlling factor, there is still some question as to whether the total ionic strength is not also important. We are investigating the possibility that the increase in activity as the concentration of the buffer is raised from zero may be determined by the total ionic strength and that the decrease after the optimum may be largely an action of the cation charge on the A',,,, as set forth by Estabrook. The extinction coefficient (reduced-oxidized at 605 m/O which we used to calculate the haem content was obtained in our experiments where we used ferrocytochrome c as a reductant {J. biol. Chem. 216, 593, 1955). Under anaerobic equilibrium conditions the moles of ferrocytochrome c oxidized were assumed to equal the gram atoms of iron (or haem) of cytochrome c oxidase that were reduced. From the haem: protein ratio of 7-0 which is given in Table 1 it may be calculated that cytochrome c oxidase has a molecular weight of 140,000. However, this is based on the assumption that the total nitrogen of Fraction 6 (cytochrome c oxidase N + other protein N + lipid N) is cytochrome c oxidase nitrogen. These fractions have at no time been represented as being pure preparations of cytochrome c oxidase. The fractionation technique was applied only for the purpose of obtaining haem:Cu and Cu: activity ratios. The total protein was simply the common denominator before the ratios were taken. HoRio: I should like to make a comment concerning the effects of native and modified cytochrome c on the oxidase activity, about which Henderson asked a question. Of the various modified cytochromes c preparations that we have found, at least one exists in a pure dimer form. Its molecular weight is calculated to be 24,400 based on its sedimentation and its diffusion coefficients. Compared with the native monomer form, this dimer form activates both the purified cytochrome a preparation and the particulate oxidase preparation very little. Margoliash: In view of Smith's findings and those that Estabrook has just referred to in discussion, on the effect of cytochrome c and salmine as well as that of inorganic cations on the cytochrome oxidase reaction, would it not be possible to consider the binding of cytochrome c to the oxidase as due to a polycation-polyanion type of electrostatic interaction ? I think Estabrook's description of the effect of polyvalent cations particularly suggestive in this respect. The extreme basicity of mammalian heart cytochrome c as well as Morrison's finding of an acid isoelectric point for cytochrome oxidase preparations would also fit such an interpretation. Boardman: The interaction between a protein such as cytochrome c and a polyanion appears to be very complex. Some of the factors governing such an interaction have been elucidated by studying the adsorption of cytochrome c on a polymethacrylic acid ion-exchange resin. The adsorption of cytochrome c on the resin is very depen- dent on the cation concentration and the pH of the medium. But it does not seem possible to explain the experimental data by visualizing the interaction as a purely electrostatic one between a polyvalent cation and a polyvalent anion. With substances of high molecular weight, short range forces such as Van der Waals or hydrogen bond forces may play an important role in the adsorption. However, a study of the relationships between cytochrome c concentration, cation concentration and pH on the one hand and oxidase activity on the other does seem to provide an experimental approach to the problem of what is happening in the cytochrome c oxidase complex. Armstrong : There is a cytochrome c in Micrococcus denitrificans which has an isoelectric point of less than pH 7, which might be used to test this theory. Slater : Horio refers in his paper to the fact that his value for the A",,, for cytochrome c in the system reducing agent — cytochrome c-oxidase-oxygen is the same as ours. It is perhaps desirable to point out that, if we accept Smith and Conrad's interpretation of the inhibitory action of cytochrome c on cytochrome oxidase, this Ar„, actually 280 Discussion represents the Ki of the cytochrome-c-cytochrome oxidase inhibited complex. This is rather amusing because this K^ has often been taken to be the dissociation constant of the Michaelis-Menten enzyme-substrate complex. The inhibition by cytochrome c of cytochrome c oxidase can account for the rectangular hyperbolic relationship between cytochrome c concentration and oxidase activity, and the first order kinetics found by Smith on the basis of the following simple mechanism written formally: E + S— ^ "ESi ESi— 5 ^E + P (very fast) E + S ^ ES„ ^ (E)(S) where ESj is the active complex and ESh is the inhibited complex. k^KiC where ^ = concentration of cytochrome c, and e = concentration of cytochrome c oxidase, i.e. Kmax = k^K^e Km — f^i- COMPOSITION OF CYTOCHROME c OXIDASE By W. W. Wainio Bureau of Biological Research and Department of Physiology and Biochemistry, Rutgers, The State University, New Brunswick, N.J. INTRODUCTION When Keilin and Hartree (1938a, 1939) proposed that cytochrome a con- sisted of two components, namely, cytochrome a and cytochrome a^, they introduced a concept that has occupied many of the investigators in this field. In Keilin and Hartree's experiments both carbon monoxide and cyanide caused alterations in the 605 mjti band of reduced cytochrome a, viz. a spreading to 590 mjii. The 448 m/n band disappeared and was replaced by an intensified absorption at 430 m/u when carbon monoxide was used and by a new absorption at 452 m/.i when cyanide was added. It was concluded by them that the component with a strong affinity for carbon monoxide in the reduced state and for cyanide in the oxidized state, and with a weak absorp- tion at 600 m^ (relative to 605 m/u) and a strong absorption at 448 mfi (relative to 452 m/i) was cytochrome a^. The somewhat anomalous component with a strong absorption at 605 m/j, and a weak absorption at 452 m/t was cytochrome a. Cytochrome a^ was tentatively identified with cytochrome oxidase, and the possibility that cytochrome oxidase is a copper-protein was considered in some detail. There was an immediate objection by Stern (1940), who stated that 'If the relationship is actually that described and sketched by the authors [i.e., that the a- and y-bands of cytochrome ^3 are weak and strong, respectively, com- pared with those of cytochrome a], it can hardly be reconciled with the further statement that the a and a^ components are both haem-protein complexes with an identical haem nucleus and that both occur in comparable concentrations in various oxidase preparations'. Stotz (1942) and Lundegardh (1953) raised lesser objections, but since further work from their laboratories has emphasized the separateness of the two enzymes, it may be concluded that these objections are no longer considered valid. We became interested in this problem in 1948 after having successfully prepared a soluble cytochrome oxidase with deoxycholate (Wainio, Cooper- stein, KoUen and Eichel, 1947, 1948). Our view at that time was that cyto- chrome oxidase was a copper-porphyrin-protein and that the anomalous behaviour of the enzyme in the presence of carbon monoxide and cyanide 281 282 W. W. Wainio could be attributed to its copper content. Our evidence in support of this view was that the 605 m/n absorption in the reduced state was related to the copper content in several fractions made from the heart mitochondrial fragment (Eichel, Wainio, Person and Cooperstein, 1950). Because the activity could not be related to the copper content, and because later analysis of the pyridine haemochrome of the prosthetic group of the soluble enzyme revealed the presence of iron (Person, Wainio and Eichel, 1953), we were forced to abandon this view. Our position then became that the enzyme is a tetrapolymer of haemo-protein submolecules, as suggested by the statement of Warburg (1949), 'I almost regret that we cannot . . . assume that the enzyme is a four- fold polymerized haem compound', and by the experiments of Ball, Stritt- matter and Cooper (1951) with carbon monoxide. It was thought that the apparent reactivity of two components could equally well be explained in terms of a differential sensitivity of the parts of a single polymeric molecule. When it became possible by the addition of a lipid activator to relate the activity to the copper content (Wainio, Vander Wende and Shimp, 1959), our hypothesis was altered so that the enzyme is now considered to be a single complex molecule containing both copper and haem. By virtue of its two dissimilar parts the enzyme could exhibit the properties demanded by Keilin and Hartree and others for the cytochromes a and a^ and by Warburg for the oxygen-transporting enzyme. The advocates of two enzymes present the following evidence in support of their position. (1) Morrison and Stotz (1955) and Morrison, Connelly and Stotz (1958) have claimed the existence of two cytochrome fl-haems, an a^ and an a^. They challenge the position of Lemberg (1953), who states that crypto- porphyrin a, which Morrison, Connelly and Stotz (1958) find is closely related to the porphyrin of haem a^, is an artifact derived from the single haem a. (2) Smith (1955) found that in the steady state after the addition of cyto- chrome c to the system ascorbate-cytochromes a + a^-oxygen the 605 m/u peak was 59 % of the totally reduced value, whereas the 445 m/u peak was only 24 % reduced. It was concluded that the two peaks could not be those of one enzyme. (3) Lundegardh (1957) has made a careful study of the effects of carbon monoxide and cyanide on the spectrum of yeast and by optical subtraction has obtained the spectra of the two components. The ratios of the maxima of the absorption peaks were given for cytochrome a, y:y,= 1-0, and for cytochrome a^, y:a = 20-8. The failure by us (Eichel, Wainio, Person and Cooperstein, 1950; Wainio, Eichel and Cooperstein, 1952) and by others to separate the two enzymes by techniques that have separated the other cytochromes may be taken as presumptive evidence in support of the one-enzyme concept. It must be Composition of Cytochrome c Oxidase 283 pointed out that from the very beginning the two enzymes have been viewed as being similar and as occurring in the same proportion wherever they were found (KeiUn and Hartree, 1939). A 1:1-2 ratio has been reported by Chance and Williams (1955) for the cytochromes a and a^ of rat liver mitochondria. The two positions are defined by Slater (1958), who states that 'Those who demand physical separation will not accept the separate identity of the cytochromes a and a^, while those who prefer a functional differentiation will treat the two cytochromes as separate identities'. COMPONENTS OF CYTOCHROME C OXIDASE A. The Haem The view that cellular respiration depends on a haem was proposed by Meyerhof (1924) and Harrison (1924), who believed that the experiments of Warburg and his associates were conclusive enough to show that the inhibi- tory action of cyanide was due to its combination with iron. Warburg (1924) himself was more conservative when proposing his theory for the role of iron as a biological catalyst. He simply characterized the catalyst as a com- plex iron compound. When Fischer and Hilger (1924) isolated a haem from yeast and higher plants the relationship between the iron and the complex iron compound was established. The first separation of the prosthetic group of cytochrome c oxidase was performed by Anson and Mirsky (1925) in Cambridge from material largely supplied by Keilin. They used pyridine to extract the haems and identified two haemochromes: one which corresponded to the pyridine haemochrome of the haem of haemoglobin, and another with its a-band at a higher wave- length. Fink (1932), subsequently, placed one of these bands at 584 m^, while Negelein (1933) found two peaks in the absorption spectrum at 432 m/^ and 587 m/i. More recently, Rawlinson and Hale (1949), Dannenberg and Kiese (1952), Person, Wainio and Eichel (1953), and Morrison and Stotz (1955) have verified that a two-banded pyridine haemochrome, whose peaks are centred at 430 m/< and 587 m^<, can be prepared from mammalian heart muscle or from soluble preparations of cytochrome c oxidase. The past and present evidence points to the nonmetallic portion of the haem as being of the oxorhodo type. Rawlinson and Hale (1949) identified an aldehyde rhodofying group, while Lemberg and Falk (1951) and Dannen- berg and Kiese (1952) agreed that the porphyrin contains a formyl group. Warburg and Gewitz (1951) found two carboxyl groups plus one presumably formyl, and one vinyl group. They also withdrew their earlier suggestion that the haemin might be a phytol ester. Having isolated the haemin from a soluble preparation of cytochrome c oxidase, Kiese (1952) came to the con- clusion that in addition to the formyl group the porphyrin contains a labile H.E. — VOL. I — u 284 W. W. Wainio group, the nature of which was not then known. Lemberg (1953) chose to isolate the porphyrin and identified three characteristic side chains. These were a rhodofying group which is almost certainly a formyl group, a second rhodofying group containing an ethylenic double bond, and a large alkyl or fatty-acid nonesterified side chain. The structure of haem a has also interested Warburg for many years. Warburg, Gewitz and Volker (1955) fused haemin a, obtained from beef heart, with resorcinol and obtained a cytodeuteroporphyrin which differed from deuteroporphyrin in its melting point and in its reaction with bromine. Cytodeuteroporphyrin took up three atoms of bromine which suggested that there were three free positions. The problem has been studied further by Marks, Dougall, Bullock and McDonald (1959), who concluded that cyto- deuteroporphyrin is deuteroporphyrin with hydrogen for methyl in the 8-position. Thus cytodeuteroporphyrin, a derivative of porphyrin a, would have three methyl groups, 1, 3 and 5, two propionic acid side chains, 6 and 7, and three free positions, i.e. hydrogens, 2, 4 and 8. In a very recent report, which is available only in abstract form, Morrison and Stotz (1959) state that haemin a has a molecular weight of 880. According to them it is a dicarboxylic acid having two propionic acid side chains, three methyl groups, a formyl group, a ketone aliphatic side chain containing 14 carbon atoms, and a vinyl group attached to the porphyrin nucleus. The foremost question with regard to the haem is whether there are two haems a or one. Morrison and Stotz (1955) found that they could separate two haemins of the a type on a silicic acid column from the purified cyto- chrome c oxidase preparation of Smith and Stotz (1954). The two haemins were at first labelled a^ and a^, a nomenclature now withdrawn. Their pyridine haemochromes had identical spectra. They subsequently found (Morrison, Connelly and Stotz, 1958) that there was cross-contamination of the two components on the column. With the aid of a new paper chromato- graphic technique they obtained the pure compounds. The reduced pyridine haemochrome of haemin a^ has its a-maximum at 582 m/t and has a small /?-peak at 533 vafx. The corresponding compound of haemin a^, has a maxi- mum at 587 m/i and no /S-peak. They identified haemin a^ with crypto- haemin a of Lemberg (1953), but did not agree with the suggestion that cryptohaemin a and presumably therefore haemin a-^ may be an artifact of the preparative procedure. In the isolations performed by Lemberg (1953), porphyrin a was always accompanied by another porphyrin whose haemin had maxima at 533 mj-i and 582 m/^ in pyridine-dithionite. This component was called cryptopor- phyrin a. Its yield from wet heart muscle was about 0-7 mg/kg which is to be compared with the 16-18 mg/kg of porphyrin a that was obtained. On this basis Lemberg concluded that even if cryptoporphyrin a is derived from haemin a and is not an artifact, it is present in too small a concentration to Composition of Cytochrome c Oxidase 285 be the prosthetic group of either cytochrome a or a^. In their last publication Morrison, Connelly and Stotz (1958) do not record the relative amounts of the two haemins, a^ and a^, which they separated. In the first experiments where there was cross contamination the amounts were about equal (Morrison and Stotz, 1955). The uncertainty regarding the number of haems a has been further compli- cated by the discovery that porphyrin a may exist in two interconvertible forms (Lemberg and Stewart, 1955). The porphyrin which is isolated and purified is identified as «a. On treatment with aqueous hydrochloric acid acf. is partly converted into porphyrin a(i which has the same absorption spectrum, but which differs in its solvent distribution and chromatographic behaviour. B. The Copper The view that copper might be a part of cytochrome c oxidase seems to be contained in the accumulated studies of Warburg (1949) and his associates, who concluded, however, that because of the hght-sensitivity of its carbon monoxide compound, the enzyme could only contain iron. The earliest, as well as the latest nutritional studies have supported an opposite view (Cohen and Elvehjem, 1934; Gubler, Cartwright and Wintrobe, 1957). Keilin and Hartree (1938a, 1939) also briefly considered that cytochrome c oxidase might be a copper enzyme. Hov/ever, when cytochrome a^, an apparent haem-enzyme, satisfied all of the criteria of cytochrome oxidase, they discarded the possibility. Our interest in the copper content of cytochrome c oxidase arose as a consequence of the feeling that, since the response of the enzyme to carbon monoxide was unusual, there must be an unusual component. We prepared fractions from heart muscle mitochondrial fragments with deoxycholate and found a good correlation between the copper content and the height of the 601 mil absorption of the reduced enzyme (Eichel, Wainio, Person and Cooperstein, 1950). Even though in these experiments the correlation between the copper content and the activity was not good (see also : Okunuki, Sekuzu, Yonetani and Takemori, 1958), we concluded that the enzyme was a copper- protein. An analysis of the metal content of the pyridine haemochrome of the prosthetic group prompted us to return to the classical view (Person, Wainio and Eichel, 1953). The view has persisted, however, that cytochrome c oxidase must also be a copper-enzyme. Our position is supported by the recent results of Green, Basford and Mackler (1956) and Mackler and Penn (1957), who find that only those mito- chondrial fragments with a pronounced spectrum for cytochrome c oxidase have considerable amounts of copper. It is also interesting to note that in Mason's (1957) classification of four-electron transfer oxidases, of which cytochrome c oxidase is one, the other three enzymes, laccase, the catechol 286 W. W. Wainio oxidase function of the phenol oxidase complex, and ascorbic acid oxidase, all have copper as the functional group. We have now obtained evidence to show that not only is the copper content of fractions prepared from heart mitochondria related to the haem (as calculated from the 605 m// absorption), but that it is also related to the activity of the enzyme. The latter relationship was established after it was found that surface active agents reduce the activity of cytochrome c oxidase and that certain phosphatides are reactivators of the enzyme (Wainio and Greenlees, 1958; Greenlees and Wainio, 1959). Table 1. RELATIONSHrP OF THE COPPER CONTENT (DETERMINED SPECTROPHOTOMETRICALLY) AND THE HAEM CONTENT OF CYTOCHROME C OXIDASE Fraction Deoxycholate added Protein Copper- protein ratio Haem-* protein ratio Copper-haem ratio Insolublef 1 % 1-0 mg/ml 25-00 12-30 m/t moles/mg 11 0-2 m/i moles/mg 0 m/t moles/ m/i mole 2 0-5 4-86 0-5 0 3 0-5 1-05 2-3 1-2 1-9 4 0-5 0-84 5-3 4-1 1-3 5 0-5 1-21 8-2 5-8 1-4 6 1-0 0-77 8-1 70 1-2 7 1-0 0-78 6-8 4-9 1-4 Residue 4-90 3-3 * Calculated from the AE at 605 m/t with the aid of the factor Ae 7-6 x 10^ cm^ mole~^. t Insoluble heart muscle preparation. Table 2. Relationship of the copper content (determined colorimetrically), THE haem content, AND THE ACTIVITY OF CYTOCHROME C OXIDASE Activity-protein ratio Deoxycholate added Protein Copper- haem* ratio Activity (with fraction I)- Activity (with Fraction fraction 1)- Without With haem ratio copper ratio fraction 1 fraction 1 % mg/ml mn moles/ rail mole kVmg protein kVmg protein kVm/i mole lcVm/< mole Insolublef 25 00 0-660 0-33t 1 10 11-60 10 0-027 2 10 4-54 2-8 0-436 0-690 1-73 0-63 3 10 2-03 11 0-965 2-010 0-78 0-65 4 10 1-57 1-6 0-581 6-900 1-28 0-93 5 10 0-82 3-2 0 3-670 1-27 0-40 6 10 0-34 2-6 0 4-300 1-19 0-46 Residue 4-56 0 0100 0-06 * Calculated from the AE at 605 m/i with the aid of the factor Ae 7-6 X 10' cm^ mole"*. t Insoluble heart muscle preparation. X Activity without fraction 1. Composition of Cytochrome c Oxidase 287 The data showing the relationship of the copper to the haem (Wainio, Vander Wende and Shimp, 1959) for a typical experiment are presented in Table 1. The average ratio of copper : haem is 1-4. It is to be noted that the copper: protein ratio is high in those fractions (5 and 6) which have a marked absorption at 605 m/i in the reduced state (from which the haem content was calculated). In another experiment (Table 2) where the copper was deter- mined colorimetrically, the copper: haem ratios were found to be higher and more variable (average = 2-1). However, since in these experiments the first fraction was employed as an activator, rather constant ratios were obtained for the activity: haem and activity : copper. It is not possible to conclude from these experiments whether 1 or 2 copper atoms are present per haem, although the more hkely figure is 2. The averages, 1-4 and 2-1, are to be compared with the value of 2-3 obtained by recalculating our earlier data (Eichel, Wainio, Person and Cooperstein, 1950). We find that one of our partially purified preparations '2-05-2' has a ratio of 1-6 and our presumed purest preparation '2-4-1-5' (Greenlees and Wainio, 1959) has a ratio of 1-0. According to unpublished data of Mackler (1956) the cytochrome c oxidase moiety of the electron transport particle has a copper : haem ratio of 2-5, Vander Wende (1959) has analysed the state of the copper in cytochrome c oxidase. It has been reported by Okunuki et al. (1958) that the copper is firmly bound to the protein. In our experiments high concentrations (0-1 m) of cyanide, diethyldithiocarbamate, ethylxanthate, etc., and prolonged dialysis (up to 12 hr) were required to remove the copper and to decrease the activity. The copper exists in the cuprous state in the oxidized enzyme as shown by the specific reaction with biquinoline. Cupric ion was ineffective in restoring the activity of preparations from which the copper had been removed by dialysis against diethyldithiocarbamate. Cuprous ion was unexpectedly effective in one experiment, raising the activity to 320 % of the control when 99 % of the copper was restored. These experiments suggest that the metal does not participate in electron transport. It may function, as does the copper in haemocyanin, to complex the oxygen to the enzyme. The spectral changes accompanying the removal of the copper were inconclusive. Some reagents caused no alteration in the spectrum of the reduced enzyme even though they removed much of the copper. However, those that did alter the spectrum, notably cyanide, diethyldithiocarbamate and ethylxanthate, caused a decrease in the absorption at 605 m/i with a shift in the maximum to about 595 m,M and a decrease in absorption at 442 m/i with a shift in the maximum to about 435 m/<. C. The Lipids Cytochrome c oxidase is associated with the insoluble particulate matter of the cell. It is a mitochondrial enzyme which has been shown to be attached 288 W, W. Wainio to the membrane of the particle (Siekevitz and Watson, 1956). The insoluble nature of the particle permitted Battelli and Stern (1912) to separate it from the cell. Keilin and Hartree (1938b) modified the method of preparation so that the grossly impure enzyme could be obtained in a high yield from heart muscle where it occurs in a high concentration. It has been suggested by many investigators that the insolubility of the enzyme may be due to its association with the lipids of the mitochondrion. In fact it has been further suggested that the oxidase may be a lipoprotein complex, because approximately 35% of the mitochondrion is lipid (Bensley, 1937) and because bile salts are needed to solubilize the enzyme (Yakushiji andOkunuki, 1941; Straub, 1941). The soluble preparations that are being studied today are those that are made with either cholate or deoxycholate. It has been clearly shown that the solubility of this insoluble enzyme depends on the continued presence of a solubilizing agent. Smith and Stotz (1954) reported that the solubihty of their preparation is dependent on the presence of both the cholate, and the ammonium sulphate used in the purification. Kremzner and Wainio (1955) found that a complete removal of the deoxycholate from the preparation '2-3' by ion exchange resins led to a precipitation of the enzyme, but not to its denaturation. Among the soluble preparations of cytochrome c oxidase available for study is the preparation of Yakushiji and Okunuki (1941) as modified by Okunuki et al. (1958). However, these authors do not report a lipid analysis. The preparation '2-3' of Eichel, Wainio, Person and Cooperstein (1950) was analysed by Kremzner (1956), who found 45% of total lipid. A modified preparation '2-3', made by first washing the insoluble heart muscle particles with 20% methanol, was still active and contained only 19% of total lipid. The lipids in both preparations were predominantly phosphatidylcholine, and phosphatidylethanolamine. This finding has been verified and extended by Marinetti, Scaramuzzino and Stotz (1957), who reported that the soluble preparation of Smith and Stotz (1954) contains 14-7% of phospholipid (primarily phosphatidylcholine and phosphatidylethanolamine), 12-8% of neutral fat, 1-02% of free cholesterol and 3-12% of unidentified lipids. Green (1958) states that the soluble preparation of Mackler and Penn (1959) contains about 15% of lipid. Marinetti, Erbland, Kochen and Stotz (1958) have more recently extended their chromatographic analysis of the phosphatides and have found again that phosphatidylcholine (33-4 % of the total hpid P) and phosphatidylethanol- amine (19-6 % of the total) were the principal phosphatides. There was 8-2 % of phosphatidylserine, 13-6% of a component tentatively identified as poly- glycerophosphatide, 5-1% of unidentified phosphatides, and traces of lyso- lecithin and lysocephalin. Two compounds distinguished the lipids of the oxidase preparation from those of a purified cytochrome b — c^ preparation Composition of Cytochrome c Oxidase 289 reported elsewhere. There were 13-9% (of the total lipid P) of inositol- phosphatide and 5-9 % of sphingomyelin, both of which occur only in trace amounts in the cytochrome 6 — q preparation. In addition to these differ- ences, the oxidase alone contained an unidentified hpid which had the same mobility as a long chain cholesterol ester, but which gave different colour tests from those given by the ester. The oxidase contained a small amount of a fraction which, after reduction, and passage through florisil, had peaks in its absorption spectrum at 232 m/^ and 272 m/i. The latter peak probably indicates the presence of the coenzyme Q of Crane, Hatefi, Lester and Widmer (1957). Table 3. Reacttvation of cytochrome c oxidase with a deoxycholate extract Preparation Velocity constant 2% deoxycholate extract 4% cholate extract 2-4-1-5* 2-4-1-5 + 2% deoxycholate extract 2-4-1-5 4-4% cholate extract X lO-^sec"^ 2-68 0-54 0-31 7-24 0-31 * 2% deoxycholate to the insoluble heart muscle mito- chondrial fragments, followed by 4% cholate, and then 1-5% deoxycholate. Table 4. REACTrvATioN of cytochrome c oxidase (Preparation 2-4-1-5*) with Phosphatides Additionf Velocity constant None Purified phosphatidylcholine Purified lysolecithin Phosphatidylserine Phosphatidylethanolamine Cephalin plasmalogen Crude phosphatidylcholine sec" Vmg protein per/ml 0-3 0-9 31 6-7 4-0 2-5 46 * 2% deoxycholate to the insoluble heart muscle mitochondrial fragments, followed by 4% cholate, and then 2 % deoxycholate. t 0-012 mg phosphatide P per cuvette, except crude phosphatidylcholine of which 0-25 mg was added. 290 W. W. Wainio Some of these lipids are known to participate in the cytochrome c oxidase system. Wainio and Greenlees (1958) (see also Greenlees and Wainio, 1959) have shown that when a soluble oxidase preparation was made by successively extracting heart muscle mitochondrial fragments with deoxycholate, chelate, and again deoxycholate, the activity was much reduced. Reactivation was accomplished by adding the first deoxycholate extract or one of a number of phosphatides (Tables 3 and 4). The following compounds were ineffective: oleic acid, vitamin K^, cholesterol, DL-a-tocopherol phosphate, choline, and phosphorylcholine. The activation has been verified by Hatefi (1958), who found that the mitochondrial lipoprotein of Basford and Green (1959) increased the oxidase activity of the green particle of Basford, Tisdale, Glenn and Green (1957). The lipid-soluble form of cytochrome c described by Widmer and Crane (1958) was also eff'ective. The role of all of these lipids remains obscure. There are, however, two possibilities to be borne in mind : (1) that they are structural components of the mitochondrion in the sense that they facilitate the optimal arrangement of the reacting enzymes ; (2) that they are actually intermediates in electron transport. D. The Protein Very little is known about the protein of cytochrome c oxidase. Its properties have not been determined because it has not been obtained in a soluble form except with the aid of surface active agents, i.e. it has not been purified free of lipid. Soluble cytochrome c oxidase has the absorption spectrum of a typical protein with a maximum at 279 m/^ (Wainio, Person, Eichel and Cooperstein, 1951). Its molecular weight has been determined to be 75,000 by Warburg (1949) from the molar extinction coefficient of the protein at 283 m/f, as calculated from the photochemical dissociation of the carbon monoxide compound of the reduced enzyme in yeast after correction for the absorption by the carbon monoxide compound of the haem at this wavelength. Wainio, Eichel and Cooperstein (1952) calculated a value of approximately 58,000 by relating the uncorrected sedimentation coefficient of 5-8 x 10~^^ sec to the uncorrected sedimentation coefficient for cytochrome c (1-2 X 10~^^ sec) and to its molecular weight (12,000). REACTIONS OF CYTOCHROME C OXIDASE Since cytochrome c oxidase has been shown to contain copper and to require a lipid for its activity, it becomes necessary to reconsider the reactions of the enzyme in the light of these new data. A. Reaction with Ferrocytochrome c Although it has been known since the early work of Keilin (1930) that ferro- cytochrome c is oxidized by oxidase preparations in the presence of oxygen, Composition of Cytochrome c Oxidase 291 the study of the interactions of the purified preparations has had to await the isolation of a soluble cytochrome c oxidase. Wainio (1955a) has demonstrated that cytochrome c oxidase (as measured at 605 m//) can be partially reduced under anaerobic conditions with the simultaneous partial oxidation of ferro- cytochrome c. The calculated equilibrium constants varied somewhat sug- gesting that perhaps a third component was participating. This may have been residual oxygen. It is also possible to oxidize reduced cytochrome c oxidase partly with ferricytochrome c (Wainio, 1955b), although the equihbrium attained is not the same as when oxidized oxidase and ferrocytochrome c are mixed. With two groups to be considered, the haem and the copper, it becomes necessary to assign tentatively to one of these the position of reacting with ferrocytochrome c. Since the group must accept the electrons from ferro- cytochrome c, and if we assume that the copper is not oxidized and reduced, the role of electron acceptor falls to the haem. The role of the lipid remains more uncertain. It might be asked whether the phosphatide activator serves to link cytochrome c and the oxidase to- gether or even to transfer the electrons from one haem to the other. Further- more, is its role the same or related to the role of the lipid in the lipid-soluble form of cytochrome c ? B. Reaction with Oxygen By definition cytochrome oxidase is the enzyme that reduces oxygen. Again it becomes necessary to ask the question whether it is the copper or the haem that acts as the electron donor. As has already been pointed out, the copper probably does not participate in electron transfer, by reason of its being in the reduced state when the haem is oxidized, but serves merely to attach the oxygen to the enzyme. The role of electron donor, as well as acceptor, would then be assigned to the haem. Although the mechanism of the reduction is unknown, Michaelis (1951) has proposed that, according to the principle of single-electron transfer, e oxygen can only be reduced in the following successive stages : Og -^ O^^ -> 0^~ -^ O^^' -> Oa'*^, which in the presence of water would form the following compounds with four protons : O2 > HO2 > H2O2 > HgO + HO-%2H20. H+ ^ If the copper : haem ratio proves to be 2: 1, as the preliminary data show, the conditions outlined above would be met. There would be two atoms of cuprous copper to attach the oxygen, and one molecule of haem to act as electron donor and to reduce the oxygen in successive one-electron transfers. Sekuzu, Takemori, Yonetani and Okunuki (1959) have postulated the existence of an oxygenated form of cytochrome oxidase which is formed from 292 W. W. Wainio the reduced enzyme by bubbling oxygen through the solution reduced with dithionite. The 444 mju and 605 m/i peaks are diminished in height and the maxima shift to 426-28 mfi and 603 m/<, respectively. The further addition of ferricyanide shifts the maxima to 424 m/n and 600 m/n. These last values are about 5 m/z higher than those reported by other investigators for the oxidized enzyme (see p. 347 of Wainio and Cooperstein, 1956) and seem to suggest that even with ferricyanide the preparation cannot become fully oxidized. As pointed out by Chance in the discussion of the paper of Okunuki, Hagihara, Sekuzu and Horio (1958), it is possible that the oxygenated inter- mediate compound is a mixture of the oxidized and reduced forms of an altered or damaged preparation. The maxima are very reminiscent of the partially reduced solution of our enzyme which we produced with ferrocyanide (Wainio, 1955c). This mixture had maxima at 425 m// and 603 mfi. C. Reactions with Carbon Monoxide and Nitric Oxide Warburg (1926) was the first to suggest that a reduced iron-containing enzyme should be the point of attachment of carbon monoxide. He and his associates went on to determine the photochemical action spectrum of the carbon monoxide compound of cytochrome c oxidase in yeast (Warburg and Negelein, 1928, 1929; Warburg, 1932; Kubowitz and Haas, 1932). Their method was based on the earlier observation that the inhibition by carbon monoxide is light-reversible and that the degree of reversibility is dependent on the wavelength of the incident Hght (Warburg, 1926). The maxima for the absorption by the components which absorbed the energy of the light to cause the reversal were found at 283 m/j, and 430 m/i, at about 520 m/u, and at 540 m^ and 590 m^. Chance (1953) has since shown that the photo- chemical dissociation spectrum of the carbon monoxide compound of the enzyme is the same in mammalian heart muscle. The actual combination of the reduced cytochrome c oxidase with carbon monoxide was demonstrated by Keilin and Hartree (1939), who found a partial shift in the spectrum of a heart muscle preparation with new bands at 432 m^ and 590 m/n. These bands, which correspond to two of the principal peaks found by Warburg for the yeast enzyme, and the partial shift in the spectrum have been confirmed by Ball, Strittmatter and Cooper (1951), Dannenberg and Kiese (1952), Wainio (1955c) and Sekuzu et al. (1959) with soluble preparations of cytochrome c oxidase. The effects noted by Wainio (1955c) are presented in Fig. 1 where it can be seen that the y-peak shifted from 443 m// to 430 m/f, while the a-peak shifted from 605 m/^ to 603 m/« and became asymmetrical on its lower wavelength side. It is to be observed that a considerable absorption still persists at 443 m/< and 605 m/^i. The difference spectrum has maxima for the carbon monoxide compound of the reduced enzyme (downward peaks in Fig. 1) at 428 m/f, 545 m// and 590 m/<. The efi"ect of nitric oxide on the reduced enzyme (Wainio, 1955c) is shown Composition of Cytochrome c Oxidase 293 in Fig. 2 (see also Sekuzu et al., 1959). The existence of two components, one which reacts with nitric oxide and one which does not, is shown even more distinctly here. The difference spectrum has maxima for the nitric oxide compound of the reduced enzyme at 426 m/i, 545 m/< and 597 m/^. .100 - \ T Reduced CO 7 Reduced Difference *.zoo *.I00 -.100 400 450 500 550 600 Wave Lcnqih (m/i) 650 Fig. 1. Effect of carbon monoxide on the spectrum of reduced cytochrome c oxidase. The ordinate on the right is for the curve of the difference spectrum. The effect of carbon monoxide, which prompted Keilin and Hartree (1939) to suggest the presence of two cytochromes a, and the more recently discovered effect of nitric oxide must be reinterpreted in the light of the newer knowledge that the enzyme contains copper. It is our suggestion that, if the site of oxygen-binding is the copper, then it must also be the site of carbon monoxide- and nitric oxide-binding. However, it must be carefully noted 294 W. W. Wainio that Warburg (1949) has not found a carbon monoxide compound of any metal other than iron which is decomposed by light. For example, the carbon monoxide compound of the copper of haemocyanin which has a Cu:CO ratio of 2:1 (Kubowitz, 1938) is not sensitive to light. .800 .700 .600 l\ .500 Densitif III — Rtduczd — NO^Riducid .400 / J •••• Differzncz .500 ; \ .200 Tl I -R • — .100 rr^-^.^ -\ ^ *.zoo *.I00 0 '.100 400 650 450 500 550 600 Vlave Lenqih (m/i) Fig. 2. Effect of nitric oxide on the spectrum of reduced cytochrome c oxidase. Based on a study of the equilibrium between cytochrome c oxidase and carbon monoxide, Wald and Allen (1957) have concluded that the enzyme must contain more than one haem/molecule. They found the curve relating per cent saturation of the enzyme with carbon monoxide to the partial pressure of carbon monoxide to be shghtly inflected and concluded that this was evidence for the interaction of haems. If it is assumed that carbon monoxide combines with the copper and that there are two copper atoms per molecule, the results of Wald and Allen would be explained. Composition of Cytochrome c Oxidase 295 D. Reactions with Cyanide Warburg's (1924) theory of the role of iron in biological oxidations was founded largely on the inhibitory effect of cyanide on respiration. Warburg (1927) later concluded that cyanide combines with the oxidized form of the enzyme. He found that the degree of inhibition by cyanide was independent .700 ' A • \ .600 ;/ 1 ;/ '1 .500 Demitt^ .400 /■ — Oxidized — - ■>■ NaCN 1 I •••• Piftercnce 1 '--'' .. \ .500 /\ \ .200 .100 ,...'''' i. \ _ v. V *.IQ0 - 0 -.100 350 400 450 300 550 Wave Length (mfi) 600 650 Fig. 3. Effect of cyanide on the spectrum of oxidized cytochrome c oxidase. of the oxygen tension and therefore cyanide and oxygen were not competing for the reduced form of the enzyme. The inhibition was amply confirmed by Keilin and Hartree (1939), who used the partial effect of cyanide on the spectrum of the reduced enzyme as support for their concept that there are two cytochromes a. The effects of cyanide on the spectra of both the oxidized and reduced forms of the soluble enzyme (Wainio, 1955c) may be seen in Figs. 3 and 4 (see also Smith, 1951; Ball and Cooper, 1952; Lundegardh, 1953). It is first to be noted that the effects are much more pronounced with the reduced enzyme than with the oxidized enzyme. This would suggest that it is the reduced form of the enzyme that is cyanide-sensitive. Furthermore, a study of the oxidized curves suggests that cyanide, which is a reducing agent, has 296 W. W. Wainio caused the loss of oxidized enzyme and the appearance of reduced enzyme, viz., the 410 m// and 438 m/n peaks on the curve of the difference. We have also observed (Wainio, 1955d) that, whereas oxidized cytochrome c oxidase is readily reduced by ferrocytochrome c in cyanide, the oxidation — *J00 - 0 -.100 450 500 550 600 Wave Lznqfh (mju) Fig. 4. Effect of cyanide on the spectrum of reduced cytochrome c oxidase. of reduced cytochrome c oxidase proceeds only very slowly in air in the presence of cyanide. The slow rate is illustrated in Fig. 5 where the reciprocal of the fraction of cytochrome c oxidase in the reduced state is plotted against the time in minutes. The reaction is second order. These results would seem to support the view that it is not the oxidized, but the reduced form of the enzyme that is inhibited by cyanide. In order to reconcile this conclusion with Warburg's observation that cyanide and oxygen do not compete for the Composition of Cytochrome c Oxidase 297 reduced enzyme, it is suggested that cyanide combines principally with the haem and oxygen with the copper. Not only has Vander Wende (1959) demonstrated that cyanide will remove the copper, but it is known that other copper-containing four-electron transfer oxidases are sensitive to cyanide. The fact that the cyanide will only JO Mimifes Fig. 5. Rate of oxidation of reduced cytochrome c oxidase by air in the presence of cyanide. remove the copper with difficulty in our experiments, suggests that the copper is not too readily available to the cyanide. Whether the copper is a component to be considered in relationship to the phenomenon presented in Fig. 6 (Wainio, 1956), can only be guessed at. If cytochrome c oxidase and a small amount of ferrocytochrome c (to reduce the oxidase) are incubated for 10 min with a range of concentrations of cyanide before the activity is determined spectrophotometrically by adding a substrate amount of ferrocytochrome c, the inhibitions observed in Fig. 6 are recorded. Preincubation of the oxidase alone with the cyanide, or of ferricytochrome c (which is the form of cytochrome c which combines with cyanide) alone with the cyanide, does not give these experimental points which lie on a doubly-inflected curve. The curve is indicative of two binding-sites for cyanide. One complex has a dissociation constant of approximately 3 X 10"^ moles/1, and the other a constant of approximately 5 x 10~^ moles/1. The curve (dashes) is a theoretical curve drawn on the assumption that each 298 W. W. Wainio cyanide is inhibiting the transfer of two electrons, whereas the other (dots) is drawn on the assumption that each cyanide is inhibiting the transfer of one electron. In this experiment the values fit the theoretical indicating that each cyanide is inliibiting the transfer of two electrons, but in other experiments the fit has not been as good, so that the points have fallen between the two curves. ( L^ >: 1 1 1 I 1 ( ^>. 9o' i 60 \ . \ '. \ '. 70 V. >60 V. • ^^ \"- ^ ^•^ '<50 \ - ,^^° O^^ •.\ JO 'A •. \ 20 /O ^ «» •» 1 1 1 1 1 "I-, j; -SO -50 -10 -9 -8 -7 -6 -5 --^ -J Lo<^ M Cone. NaCN Fig. 6. Effect of cyanide on the activity of cytochrome c oxidase. curve based on the assumption that each cyanide is forming a complex with a site transferring two electrons; .... curve based on the assumption that each cyanide is forming a complex with a site transferring one electron. Therefore it can only be concluded that there are two binding sites for cyanide in a cytochrome c — cytochrome c oxidase mixture and that each site may control the transfer of two electrons each. Since this inflected curve is obtained on incubation with the cyanide only when the oxidase and the cytochrome c are together, it cannot yet be concluded that the two cyanide-sensitive sites are the two copper atoms. However, more than a single site must be involved and it must be borne in mind that copper will react with cyanide. SUMMARY The proposition has been explored that cytochrome c oxidase is one enzyme constituted of haem, copper, lipid and protein. It is suggested that the spectral and reaction anomalies which have supported the existence of two components, namely, cytochrome a and cytochrome a^, be reconsidered in Composition of Cytochrome c Oxidase 299 the light of the new evidence. It is particularly emphasized that cyanide may react with both the haem and the copper and that carbon monoxide may react more readily with the copper than with the haem. REFERENCES Anson, M. L. & Mirsky, A, E. (1925). J. Physiol. 60, 161, Ball, E. G. & Cooper, O. (1952). J. biol. Cliein. 198, 629. Ball, E. G., Strittmatter, C. F. & Cooper, O. (1951). /. biol. Chem. 193, 635. Basford, R. E. & Green, D. E. (1958). Quoted by Hatefi, Y. (1958). Biocliim. biopftys. Acta 30, 648. Basford, R. E., Tisdale, H. 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Composition of Cytochrome c Oxidase 301 DISCUSSION Function of Copper in Cytochrome Oxidase Preparations Slater: I was struck by the fact that in Wainio's Table 1 the copper-haem ratio decreased from 1-9 to 1-2, as the haem-protein ratio increased from 1-2 to 7-0. Does this not suggest that the copper concentration is decreasing as the cytochrome c oxidase is further purified ? Wainio: There is only one high value for the copper-haem ratio and that is the first value of 1-9. The others range from 1-2 to 1-4 and are in no particular order. Slater: Is it possible that reactivation by copper after diethyldithiocarbamate treatment is due to removal of inhibitory diethyldithiocarbamate by added copper. Wainio: This kind of reactivation would be possible only if the reintroduced copper were bound in a loose fashion, because diethyldithiocarbamate will not inhibit the intact enzyme even after one hour of contact. Lemberg : We have attempted the addition of copper to various haemoproteins a in the presence or absence of CO, phospholipid, and/or deoxycholate. We have failed to notice any spectroscopic shift by the copper addition which would make the spectra of haemoproteins a more similar to those of cytochrome a and particularly cytochrome Og. We may not have found, of course, the right conditions to reconstitute such a specific copper-haemoprotein as Wainio postulates. Falk: There is a certain amount of evidence that copper is required for cytochrome oxidase synthesis in animal tissues, and it is clear also that this enzyme acts in some kind of lipoprotein complex form. I think it is most important, since some people are trying to postulate chemical reaction mechanisms, to try to determine whether the copper is involved mechanistically in the oxidative activity, or is merely necessary for this activity in a secondary way, perhaps such as holding together the haem- protein-lipid complex. An experiment which might throw further light on this might be to try the effect of a bidentate chelator, which might be able to bind the copper but could not co-ordinate with a haem iron. CYTOCHROME OXIDASES OF PSEUDOMONAS AERUGINOSA AND OX-HEART MUSCLE, AND THEIR RELATED RESPIRATORY COMPONENTS By T. HoRio, I. Sekuzu, T. Higashi* and K. Okunuki Department of Biology, Faculty of Science, University of Osaka, Osaka Keilin and Hartree (1939) observed that their cytochrome oxidase prepara- tion from ox-heart muscle contained a cytochrome component spectro- photometrically similar to cytochrome a, and they reported that the component differed from cytochrome a in the manner in which the component was sensitive to carbon monoxide. They thought that this component might be identical with cytochrome oxidase, calling it cytochrome a^. Thereafter in most reports, cytochrome oxidase preparations were described as con- taining both cytochromes a and ^3. These two components have, however, not yet been separately purified. It is well known that the cytochrome oxidase preparation can oxidize cytochrome c, but not /7-phenylenediamine, cysteine, or ascorbic acid, unless cytochrome c is present. Smith (1956) has found that the purified cytochrome a can be partially reduced by /7-phenylene- diamine. Okunuki et al. (1958) found that their preparation of purified cytochrome a showed the typical activity of cytochrome oxidase if cytochrome c was present, and that the preparation contained no spectrophotometrically detectable cytochrome component other than cytochrome a. Negelein and Gerischer (1934), and Fujita and Kodama (1934) discovered that cytochrome Wg was widely distributed among bacteria, and that it was autoxidizable and could combine with carbon monoxide and with cyanide. These properties led to the assumption that cytochrome a^ had the function of a cytochrome oxidase. There are, however, some reports (Chance, 1953; Smith, 1955) that cytochrome a^ may be the terminal respiratory enzyme in Acetobacter pastewianum ; these argue against the hypothesis that cytochrome ^2 acts as the respiratory enzyme of the bacteria. However, neither cytochrome Qi nor cytochrome a, had been purified; moreover, it was not known in either case which substances could be oxidized by these cytochromes. Horio * Present address: Department of Biochemistry, Medical School, University of Osaka, Osaka, Japan. 302 Cytochrome Oxidases of Pseudomonas Aeruginosa 303 (1958a and b), and Horio et al. (1958) succeeded in highly purifying four kinds of respiratory components of Pseudomonas aeruginosa. Pseudomonas (P-) cytochrome-551 and P-blue protein have been crystalHzed (Horio et al, 1958; Horio, 1958b), and P-cytochrome oxidase has recently been obtained in a state of nearly homogeneous purity. The present paper deals with reactions of the animal and bacterial cyto- chrome oxidases with their related respiratory components. Purification of Ox-heart and Pseudomonas Cytochrome Oxidases Ox-heart cytochrome a can be purified with the aid of cholate up to a state spectrophotometrically free of the other cytochromes. Yonetani et al. (1958) found that cholate inhibits the cytochrome oxidase activity of the cytochrome a preparation, and that the inhibitory action can be considerably diminished by the subsequent use of non-ionic detergents such as Emasol 4130 and Tween 80. By this method, the cytochrome oxidase activity of the preparation amounts to as much as one-third of the turnover number (oxygen consumed/cytochrome a) of the original extract. The specific activity of P-cytochrome oxidase was increased approximately 250 times over the first cell-free extract of Pseudomonas aeruginosa, according to the method of Horio (1958a), and Horio et al. (1958), with some modi- fications in which zone-electrophoresis on a vertical starch column was adopted and acetone fractionation was not used. The enzyme could be further purified by dialysing a concentrated sample solution against distilled water, for the enzyme was remarkably less water-soluble in the absence of any salt than in the presence. The purified enzyme has been found to be ultracentrifugally homogeneous. Comparisons between Cytochrome Oxidase Activities of the Purified and Non-purified Oxidase Samples The purified cytochrome a is easily reduced by /7-phenylenediamine, but only slightly by hydroquinone, and ascorbate. Despite this fact, the cyto- chrome a preparation does not consume oxygen with these reductants without the addition of cytochrome c. If a suflftcient amount of cytochrome c is added however, the cytochrome a preparation shows a rapid oxygen uptake by the reductants, as shown in Table 1 . The same fact can be observed with a particulate preparation of cytochrome oxidase which is free of cytochrome c. Moreover, the cytochrome oxidase activity of the cytochrome a preparation is inhibited by the typical inhibitors of cytochrome oxidase, carbon monoxide, cyanide, etc., in a manner similar to that of the cytochrome oxidase activity of the particulate preparation (Green and Brosteaux, 1936). Such similarities are observed with respect to optimal pH of the activity and effect of oxygen 304 T. HoRio, I. Sekuzu, T. Higashi and K. Okunuki tension on the activity between both preparations. These facts indicate that both preparations have the same kind of cytochrome oxidase. Table 1. Influence of addition of cytochrome c on the oxidase-activity of cytochrome a Oxygen uptake was measured with a Warburg manometer at pH 7'4 and at 30°C. Experimental system Substrate (10-^ m) />-Phenylene- diamine Hydroquinone Ascorbic acid (jul. oxygen consumed in 10 min) None Cytochrome a (0-7 x 10-'' m) Cytochrome a + cytochrome c (1-4 X 10-7 ^1) 4 6 104 4 4 126 10 4 80 When Pseudomonas aeruginosa grows anaerobically in the presence of nitrate, or under low oxygen tension (<20%) in the presence or absence of nitrate, the cytochrome content of the cells is greater than for normally grown cells and P-cytochrome oxidase can be purified from these cells as well as the other respiratory components. If grown under higher oxygen tension, the cells produce little or no cytochrome components, the respiration of the cells is not inhibited by the inhibitors, and the oxidase cannot be purified from these cells. Even with the cells grown anaerobically and under low oxygen tension, the oxygen respiration of the cells is not always completely inhibited by cyanide and carbon monoxide. The behaviour of hydroquinone oxidation by the cell-free extract of the cells grown anaerobically in the presence of nitrate towards various inhibitors is very similar to the oxygen respiration of the living cells. From this cell-free extract, two different kinds of enzymes capable of oxidizing hydroquinone can be purified : one is sensitive to cyanide and carbon monoxide (P-cytochrome oxidase), and the other is resistant (P- hydroquinone oxidase: Higashi, 1958). The former oxidase can rapidly oxidize P-cytochrome-551, and P-blue protein, while the latter cannot perform the oxidation. P-hydroquinone oxidase does not show any cytochrome-like absorption spectrum, and its physiological function is not yet known except that it can rapidly oxidize ascorbic acid as well as P-cytochrome oxidase. Properties of the Purified Preparations of Cytochrome a and P-cytochrome Oxidase The cytochrome a contained in the purified preparation is easily reduced by the addition of /j-phenylenediamine, as is indicated by an increase in extinction of a- (605 mp,) and y- (444 m/<) absorption peaks. As shown in Fig. 1, the Cytochrome Oxidases of Pseudomonas Aeruginosa 305 reduction by /j-phenylenediamine occurs even under aerobic condition, though the reduction rate is slow compared with the case under anaerobic conditions. The difference in rate between the two conditions indicates that cytochrome a itself is so slightly autoxidizable that this autoxidation cannot be considered to result from an enzymic activity. At present, it is not known whether the .08 T Aerobe 1 1 1 \ Anaerobe. ■ \ 1 1 1 b y^ o .0 6 \ /^ • CO \ / o .0 4 \ - / c \ ■ 1 1 6 UJ .0 2 .00, a X V c a / ) 10 ; ^0 30 0 10 20 30 TIME IN MINUTES Fig. 1 . Effect of cytochrome c on the oxidation of cytochrome a. Cytochrome a was incubated with /7-phenylenediamine aerobically in 'Aerobe' and anaerobically in 'Anaerobe'. Other additions were made aerobically in both cases. The arrows indicate time of addition of each reagent: a, 10~* m /j-phenylenediamine and a trace of sodium borohydride; b, 5 x 10^* M oxidized cytochrome c; c, a trace of sodium dithionite. The addition of borohydride was made to prevent autoxidation of/7-phenylenediamine. autoxidizability results from the property of cytochrome a itself or from that of its modified form, just like the case of native and modified cytochrome c. If cytochrome c is present, the reduction of cytochrome a by /j-phenylene- diamine rapidly occurs under anaerobic condition. On the other hand, when cytochrome c is aerobically added to the cytochrome a previously reduced by an excess amount of /?-phenylenediamine, cytochrome a cannot maintain its reduced form, and a rapid oxidation immediately occurs. This phenomenon of cytochrome a corresponds to that of the cytochrome oxidase activity of the purified cytochrome a preparation, as well as to that of the particulate preparation. It is, therefore, considered that cytochrome a itself displays an essential role in the cytochrome oxidase activity of its purified preparation. This consideration induces a concept in which the system, 'cytochrome c plus cytochrome a' works as cytochrome oxidase (so-called cytochrome a^^. The physicochemical constants of P-cytochrome oxidase are given in 306 T. HoRio, I. Sekuzu, T. Higashi and K. Okunuki Table 2, The oxidase contains no copper. The frictional ratio (//^) is calcu- lated to be 1-2. Even after purification to a homogeneous state, P-cytochrome oxidase preparation shows a rather complex absorption spectrum, as if it contained so-called cytochrome a^ and c-type cytochrome. The Oa-type haem is easily extracted in acetone containing HCl according to the method of Barrett (1956) but the c-type haem remains in the acetone precipitate in a state bound to the protein moiety. Approximately half of the iron of the oxidase preparation is found in the acidic acetone extract. Therefore, it is sure that each molecule of P-cytochrome oxidase contains two different kinds of haems, a^-Xy^^Q and c-type. Properties of the Respiratory Components Related to the Cytochrome Oxidases Cytochrome c^ can be purified separately from other cytochromes with the aid of cholate from ox-heart muscle. The purified cytochrome c^ does not show enzymic activities such as DPNH-cytochrome c reductase. However, the cytochrome c^ in its reduced form can rapidly transfer an electron to cytochrome c. The general properties of P-cytochrome-551 and P-blue 0.6 E 0.5 0.4 0.3 0.2 0.1 Reduced form 292 oxidized form 630 2 50 300 350 400 450 500 550 WQvelengfh {m>j) 600 650 700 750 Fig. 2. Absorption spectra of crystalline Pseudomonas blue protein. The reduced form was made by adding sodium borohydride. Compared with the reduction of typical cytochrome c's, a much larger amount of the reductant was required for complete reduction. Fig. 3. Crystals of Psendomonas blue protein. P-blue protein was crystallized in its oxidized form. Photograph x SO Cytochrome Oxidases of Pseudomonas Aeruginosa 307 protein crystallized are shown in Table 2. The absorption spectrum of the protein is as shown in Fig. 2. Finely crystallized P-blue protein has a shape as shown in Fig. 3. The copper bound on the protein can be spht off from the Table 2. General properties of P-cytochrome oxidase, crystalline p-cytochrome-551 and p-blue protein 5„„. H' £>.„. w v,„ Mv-/) ^Fe or Cu (at pH 6-40) Isoelectric point P-eytochrome oxidase P-cvtochrome- 551 P-blue protein (Svedberg) 5-8 1-34 1-91 (X 10-' cm- sec ') 5-8 14-8 106 (ml g-i) 0-726 0710 0-749 88,000 7.600 17.400 47,000 8,100 16,600 (Vo) +0-286 +0-328 (pH) 4-70 5 40 protein by dialysing it against cyanide solution at a neutral pH range. The copper-free P-blue protein does not show any absorption peak in the visible wavelengths even in the presence of oxidizing reagents, and the same absorp- tion peak around 630 m/« appears immediately after addition of a small amount of CUSO4 the colour of which can hardly be estimated spectro- photometrically. Reaction of Ox-heart Cytochrome a and Pseudomonas Cytochrome Oxidase with their Related Respiratory Components The hydroquinone oxidation by purified cytochrome a increases in rate up to its upper limit with an increasing concentration of cytochrome c externally added. Just at the concentration of cytochrome c at which the cytochrome oxidase activity reaches its maximal rate, the molar ratio of cytochrome c to cytochrome a is approximately one. This value is indepen- dent of the reactivation of the cytochrome oxidase activity of the cytochrome a preparation by the use of the non-ionic detergents as described above. The K^ for cytochrome c is 3 x 10~^ m, which is similar to that obtained by Slater (1949). Purified cytochrome c^ is only slowly oxidized by cytochrome a, and the rapid oxidation of cytochrome c by the cytochrome a preparation is not influenced by the addition of cytochrome q. On the other hand, the slow oxidation of cytochrome q by the cytochrome a preparation is notably accelerated by the addition of a small amount of cytochrome c, as shown in Fig. 4. P-cytochrome oxidase rapidly oxidizes the reduced P-cytochrome-551, and the reduced P-blue protein, but not the reduced P-cytochrome-554. The typical cytochrome c's crystallized from baker's yeast and animal sources are very slowly oxidized by the oxidase, while the cytochrome a preparation does not oxidize P-cytochrome-551 and P-blue protein. P-cytochrome oxidase can oxidize several reductants such as hydroquinone, /j-phenylenediamine, ascorbate, etc., as shown in Table 3 (Horio, 1958b). The oxidations by 308 T. HoRio, I. Sekuzu, T. Higashi and K. Okunuki P-cytochrome oxidase are similar to those of ox-heart cytochrome oxidase (Okunuki, 1941), except that P-cytochrome oxidase rapidly oxidizes these reagents regardless of the presence or absence of P-cytochrome-551 and P-blue protein, whereas the animal cytochrome oxidase is inactive unless 0.20r o < III o Li_ UJ O CO _) V < LU Ql CL UJ Z o u 1- / Pseudomonas Aeruginosa 309 Table 3. Comparison of specificity between pseudomonas cytochrome oxidase and ox-heart cytochrome oxidase to various electron-donating substances Oxygen consumption was measured by use of a Warburg manometer. Reactions with P-cytochrome oxidase were at 30'C in 004 M phosphate buffer of pH 60, and reactions using ox-heart cytochrome oxidase were carried out in the presence of cytochrome c at 15°C in 001 m phosphate buffer of pH 70 Oxygen consumed Substrate used 1 X 10-2 M P-cytochrome oxidase Ox-heart cytochrome oxidase final concentration O'l.) (%) O'l.) (%) p-Phenylenediamine 60 100 80 100 o-Phenylenediamine 30 50 5 6 w-Phenylenediamine 0 0 0 0 Hydroquinone 65 108 96 120 Pyrocatechol 22 37 25 31 Resorcinol 0 0 0 0 /7-Aminophenol 81 135 41 51 o-Aminophenol 121 202 57 71 /?7-Aminophenol 0 0 0 0 Pyrogallol 86 143 69 86 Phloroglucinol 5 8 0 0 L-Ascorbic acid 144 240 — — containing 2-3 % and 20 % oxygen, respectively. The K^^ values for P-cyto- chrome-551, and P-blue protein are 1-9 x lO^'^M and 3-9 x 10~^m, respec- tively, at 18°C and at pH 5-1, which is the optimal pH for both reactions. The turnover numbers for the oxidations of hydroquinone, P-cytochrome-551, and P-blue protein are 44 moles of oxygen consumed per mole of oxidase per min at pH 6-4 (optimum) and at 30'^C, 87 moles at pH 5-1 and at 18°C, and 100 moles at pH 5-1 and at 18°C, respectively. DISCUSSION The cytochrome a preparation which is free of other cytochromes, shows the same cytochrome oxidase activity as does the Keilin-Hartree particulate preparation, which is free of cytochrome c but contains all other cytochromes and the succinate oxidase system. Moreover, the cytochrome oxidase activity of the cytochrome a preparation is identical in all its properties with the description of the animal cytochrome oxidase (so-called cytochrome ^3), except for the turnover number. Based on the results of the oxidation and reduction of the cytochrome a present in the cytochrome a preparation, it is certain that cytochrome a displays a direct and essential function in the cyto- chrome oxidase activity of the purified a preparation. Because the cytochrome oxidase activity of the purified cytochrome a has a turnover-number one-third that of the original extract, it seems likely that the cytochrome oxidase activity of 'cytochrome a + cytochrome c' is still being partially inhibited 310 T. HoRio, I. Sekuzu, T, Higashi and K. Okunuki by a certain factor, though the inhibitory action of cholate on the oxidase activity has been considerably removed by the use of Emasol 4130 and Tween 80. Independent of this reactivation of the oxidase activity of the system (cytochrome a + cytochrome c) by the use of the non-ionic detergents, the molar ratio of cytochrome c to cytochrome a for the full oxidase activity is approximately one. This may indicate that one mole of cytochrome a binds one mole of cytochrome c to exhibit the oxidase activity. These findings confirm that ox-heart muscle cytochrome oxidase activity is displayed by the system, 'cytochrome a + cytochrome c\ It is therefore considered that the behaviour of 'cytochrome a + cytochrome c' had been incorrectly expressed as so-called cytochrome a^. The P-cytochrome oxidase purified to homogeneity shows a complex absorption spectrum as if it contains c-type cytochrome and so-called cyto- chrome «2- Based upon these facts, P-cytochrome oxidase appears to contain more than one haem moiety. As indicated previously, in spite of the complex absorption spectra, the oxidase preparation was estimated to contain two iron atoms in each mole of the protein (88,000), but no copper, when the experimental value was calculated for correction of a small amount of impurities of the sample. Both mammalian and Pseudomonas cytochrome oxidase are thought to consist of a complex of at least two cytochrome components or to be a protein having at least two haems. Based upon the properties of cytochrome a and P-cytochrome oxidase, and comparing cytochrome oxidase activity in both of them, the cytochrome oxidase activity of the two systems may be visualized in the following way : In [cytochrome a -\- cytochrome c\ electron donor -> cytochrome q t^ ^cytochrome c cytochrome a ■ - oxygen where the brackets indicate the enzymic system (possibly binding of cyto- chrome a and cytochrome c in a complex) capable of displaying cytochrome oxidase activity. In 'P-cytochrome oxidase' succinate -a P-cytochrome-(560)^P-cytochrome-554 l- DPNH I X w P-cytochrome-55 1 ^ P-blue protein P-cytochrome oxidase 'c-type' haem ^ 'gg-type' haem cyanide oxygen carbon monoxide Cytochrome Oxidases o/ Pseudomonas Aeruginosa 311 where the thick arrows indicate an electron-transferring pathway of greater physiological possibihty than the slender ones. The box indicates that the enzymic protein of P-cytochrome oxidase has at least two different kinds of haems, and is capable of displaying cytochrome oxidase activity in the absence of any externally added respiratory components. In spite of the fact that some parts of the absorption spectrum of P-cytochrome oxidase are very similar to the a-, /5- and y- absorption bands of P-cytochrome-551 and P-cytochrome-554, all attempts to split such a c-type cytochrome from native P-cytochrome oxidase have failed. REFERENCES Barrett, J. (1956). Biochem. J. 64, 626. Chance, B. (1953). /. biol. Chem. 202, 383. FujiTA, A. & KoDAMA, T. (1934). Biochem. Z. 273, 186. Green, D. E. & Brosteaux, B. (1936). Biochem. J. 30, 1489. HiGASHi, T. (1958). J. Biocliem. Tokyo 45, 785. Horio, T. (1958a). /. Biocliem. ToliM Fe a_ ' I ■ I ' I ' I ' I 0 10 20 30 40 50 >iMFe a. ® Fig. 1 , An example of a spectrophotometric titration of cytochromes a, a^ and c with 7-8 i-iu oxidizing equivalents. cytochrome oxidase by oxygen, and Yonetani and Nakamura have carried out independent magnetometric titrations. The procedure we have used simulates enzymic activity of the oxidase. Substrate is added to a concentrated solution of the oxidase and after the dissolved oxygen has been exhausted and the oxidase is reduced, a small volume of air-saturated buffer is rapidly mixed by means of a modified Hartridge-Roughton flow apparatus (regener- ative flow apparatus) and the maximal extent of oxidation of the oxidase is recorded in a few tenths of a second by spectrophotometric (double-beam spectrophotometer) or magnetometric (modified Rankine balance) methods. In experiments with the rapid flow apparatus, it has been observed that upon addition of oxygen the absorption bands at 605 m/< and 444 m/< disappear even though cytochrome c is absent. Apparently cytochrome c is needed for the reduction of the oxidase, not for its oxidation as has been inferred above; the rapid oxidation of cytochromes a and ^3 is observable with our technique without the addition of cytochrome c. The added oxygen is slowly expended in enzymic activity and the experiment is repeated with a more dilute solution of the oxidase. The experiment is begun with an excess of oxidase over oxygen and concentrations of the former are diminished until there is considerably more oxygen than oxidase. The utilization of oxygen due Cytochrome Oxidases of Pseudomonas Aeruginosa 317 to 'turnover' of the oxidase is negligible at the time of measurement. Furthermore the time of maximal oxidation of cytochromes a and a^ overlaps sufficiently, so that the time of measurement of the maxima is not critical. When cytochrome c is present, its concentration is measured at the time of the maximum of cytochromes a and a-^ since cytochrome c is not simultaneously maximal. /7-Phenylenediamine plus ascor- bate, ascorbate alone, or cytochrome c plus ascorbate was used as the reductant. The experimental results are of the kind indicated by Fig. 1 which shows the variation of the spectrophotometric effect with the oxidase concentration. It is apparent that the physical effect is maximal at a cytochrome a^ concentration very nearly equal to that of the oxidizing equivalents added. In Fig. 2, the variation of the Z UJ e I d _*' CD T liJ O o »- UJ z o < 2 o X 8 r— 1 1 r 1 1 - 7 ^-o- — o — o.~.___ - 6 / o - 5 / - 4 - 1 - 3 - J - 2 - f - 1 1 \ \ \ 1 1 1 - 0 10 20 30 40 50 60 CONCENTRATION OF O2 (oxidant equivalent) Fig. 2. An example of a magnetometric titration of cytochromes a and O3 with oxygen. Reaction at 100 msec after mixing at 29°C. Concentrations as follows: cytochrome 03, 30 /(m; cytochrome c, 6 / 9 Pyridine (20% v/-/) 13 9 01 NNaOH 0-05 M borate water 587(29-2-31-0) 587(29-9) 587(24-8) none none 530(10-5) 430(117) 430(115) 429(91) 4-6 3-8 3-7 Pyridine Q.Q%\I\) + CO 13 01 NNaOH 592(17-1) none 429(117) 6-9 5 4-Methyl imidazole (2%) 9 005 M borate 594(22-2) 520(10-8) 441(70) 3-3 4-MethyIimidazole (2%) + CO 9 0-05 M borate 605(19-0) none 435(57) 3-0 11 NH3(2%) - - 592-5(21-3) none 435(77) 3-6 Cyanide (0 1 m) 13 01 NNaOH 598(23-0) 534(10-9) 446(101) 4-4 Native human globin (1%) 9 005 M borate 595(24-7) none 442(103) 4-2 Native human globin (1 %) + CO 9 0-05 M borate 602-5(22-0) none 432(126) 5-7 7-5 Apoperoxidase (0-5 %) 9 0-05 M borate 596(15-6) none 441(93) 5-9 Apoperoxidase (0 5 %) + CO 9 0-05 M borate 601(148) 550(10-8) 432(93) 6-3 5 Human serum albumin (0-5 %) 9 005 M borate 590(20-3) none 435(91) 4-5 Human serum Albumin (0-5 "„) + CO 9 7-5 0 05 M borate 0-05 M barbital 596(15-1) 599(14-2) 542(13-2) 575(13-0) 540(12-6) 429(133) 420(101) 8-8 7-1 6 9 Ox serum Albumin (0-5%) 9 7-5 0-05 M borate 0-05 M barbital 590(25-2) 589(19-4) 540(1 11) 510(11-0) 540(11-2) 512(10-8) 435(93) 428(69) 3-7 3-6 Ox serum Albumin (0-5 %) + CO 9 7-5 0-05 M borate 0-05 M barbital 600(20-0) 600(15-0) none 545(11 8) 431(102) 417?(97) 5-1 6-5 10 11 Denatured globin (1 %) 13 0-1 NNaOH 575(19-4) 530(11-7) 505(11-7) 429(101) 5-2 Denatured globin (1%) + CO 13 01 NNaOH 584(15-1) 545(12-2) 424(147) 9-8 9 Denatured ox serum albumin (0-5 "„) 13 0-1 NNaOH 573-5(25-4) 530(14-1) 430(118) 4-6 Denatured ox serum albumin (0-5','„) + CO 13 0-1 NNaOH 583(180) 542-5(16-4) 423-5(155) 8-6 9-5 Denatured human scrum albumin (0-5 °o) 13 01 N NaOH 574 530 505 430 • Concentration calculated as in Table 1 . t Haematin a cf. Table 1 . X A large excess of dithionite must be avoided. Reduction is somewhat slow. § The solution must be saturated with CO (prepared from formic acid-sulphuric acid) before reduction. The Isolation, Purification and Properties of Haemin a 327 when its ethereal solution is washed with neutral sodium chloride solution. In contrast to protohaematin, haematin a does not react, or reacts very little, with cyanide in alkaline solution (0-1 N NaOH). The absorption spectrum is hardly altered by the addition of 0-1 M cyanide. The hydroxyl ion also competes with pyridine for the fifth and sixth co-ordination position of haematin a. Even at pH 9 a solution of haematin a in aqueous pyridine (20% v/v) gives mainly the spectrum of alkaUne haematin a. In aqueous solution, however, without added buffer or alkali, pyridine of this concentra- tion almost completely combines with haematin a to give the ferrihaemo- chrome. With 4-methylimidazole ferrihaemochrome formation is complete with 2% base in weak alkali. Haematin a has thus, like protohaematin, a much greater affinity for the imidazole than for pyridine. Ferrous Compounds The a-bands of the ferrous haem a compounds with bases or proteins can be grouped in three regions: (1) 587 m/t (pyridine haemochrome), (2) 590 vcifi (serum albumins) and (3) 594-596 m/t (4-methylimidazole haemo- chrome and compounds with native globin and apoperoxidase). With the exception of haem a itself, no compound with a-bands similar to those of cytochromes a + a^ (603-605 m,a) has been found. Similarly ferrous chlorocruorin has an absorption band at 604 mj^i, while that of the pyridine haemochrome hes at 582 m// (Fox, 1924). The serum albumin compounds have a-bands similar to that of cytochrome a^. While the absorption spectra of the pyridine and methylimidazole protohaemochromes differ by no more than about 1 mix, those of the corresponding haemochromes a differ by 7 m/<. According to the position of the a-band the haem a iron may be bound to the imidazole of both globin and apoperoxidase, whereas it is assumed that in horseradish peroxidase the protohaem iron is bound to a carboxyl group of the protein. The considerable differences between the spectra of ferrous protohaemoglobin, protohaemperoxidase and protohaeraalbumin are not observed in the series of haem a compounds, nor does the compound with human serum albumin differ in its spectrum from that of ox serum albumin. The absorption maxima of the a-bands of alkali-denatured protein haemo- chromes (but not of urea-denatured globin haemochrome in neutral solution) were found at 573-575 m/<, 12-14 m/< to the blue compared to the band of pyridine haemochrome. This difference is surprising in view of the fact that with protohaem denatured proteins give haemochromes with a-bands within 1-2 m/i of 558 m/^. Possibly the denatured proteins react with the aldehyde group in alkaUne solution; this reaction also appears to occur with chloro- cruorin, whose absorption band of 604 m/< is shifted to 569 m// on denaturation by alkali (Fox, 1924). The millimolar extinctions of the a-bands {e^^ vary between 29-30 for 328 D. B. MoRELL, J. Barrett, P. Clezy and R, Lemberg the pyridine and 25 for the native globin compound to 15-5 for the apoperoxidase compound. The ratio of extinctions y-band/a-band varies from 3-3 for the imidazole compound to 5-9 for the apoperoxidase compound, averaging 4-7 for the haemoproteins. The ratio y-band/a-band for the difference spectra (AFe++ — Fe+++) is similar to this (e.g. 4-4 for the ox serum albumin com- pound); this is in good agreement with the ratio 4-5 observed by Chance and Yonetani (1959) for cytochrome a. It has often been stated that this ratio is abnormally low for haemoproteins. A higher ratio is, however, by no means general for haemoproteins. For cytochrome c, e.g. it is 4-8. Cytochrome a^ rather than cytochrome a appears to have an abnormal y/a ratio. The Soret band of haem a lies at 410^15 m/;, but is shifted towards longer wavelengths by combination with nitrogenous compounds, e.g. to 446 m/^ by cyanide, 442 m// by globin, 441 m^ by methylimidazole and peroxidase apoprotein, 435 m/^ by the serum albumins, and 430 m/f by pyridine or alkah-denatured proteins. The position of the y-band of cytochrome a^ (445 ran) thus resembles those of haem «-methyhmidazole, haem a-globin and haem ^-peroxidase. All the nitrogenous compounds, and also carbon mon- oxide alone, increase the Soret band of haem a. Carbon Monoxide Compounds Carbon monoxide shifts the a-absorption band always towards the red by 5-1 1 m/^ (with the exception of haem a at pH 9, but not at pH 7-5). This also holds for the compounds with denatured proteins, whose CO compounds have absorption maxima at 583-584 m^t. The a-bands of the CO-haemo- proteins and CO-haemochromes lie at 597-605 m/^. Thus so far no CO-haem a compound with an absorption maximum at 590 m/i has been observed, and no compound has as yet been found whose a-absorption band is shifted by 15 m/t towards the blue by combination with CO, as is assumed for cytochrome ^3. In this respect also, cytochrome a^ appears to be an unusual haem a compound. The position of the Soret band of the CO-compounds including CO-haem a has usually been found between 428 and 435 m/f, in agreement with the posi- tion of this band of cytochrome a^. For the denatured protein CO compounds the Soret band was found at 424 m/^. In summarizing; we have obtained no haemoprotein a which resembles cytochromes a and a^ in all properties. In contrast to cytochrome a our haemoproteins combine with carbon monoxide, and their a-bands lie at 590-596 m/( instead of 605 m/<, but their y/a ratio and the position of the Soret band are those of cytochrome a. No haem a compound has been found which like cytochrome a^ forms a carbon monoxide compound with shift of the absorption maximum towards the blue, or a haemoprotein with a y/a ratio of 15 reported for cytochrome a^ by Chance and Yonetani (1959). The Isolation, Purification and Properties of Haemin a 329 SUMMARY 1. A method for the preparation of unaltered porphyrin a and haemin a from heart muscle is given. 2. Evidence is adduced to show that haem a is the prosthetic group of the cytochromes a, a^ and a^. 3. Determinations of the haem a content of tissues as porphyrin a or pyridine haemochrome a are described and their limitations are discussed. 4. Spectroscopic properties of haem a compounds with nitrogenous bases, cyanide, and native and denatured proteins, as well as their carbon monoxide compounds are investigated. The properties of these proteins and their carbon monoxide compounds are compared with the properties of the cytochromes a, a^ and ^3. ADDENDUM This paper was completed before we became aware of the recent work of Kiese and Kurz (1958). The German authors have also found the shift of the a-absorption bands of the haemoproteins a to longer wavelengths by carbon monoxide, and the peculiar denatured protein haemochromes. There are small discrepancies, e.g. in the position of the oc-bands of ferrohaemoglobin a and ferrohaemalbumin a. Acknowledgement This research has been carried out with grants from the National Health and Medical Research Council of Australia. REFERENCES Chance, B. &. Yonetani, T. (1959). Fed. Proc. 18, 202. Dannenberg, H. & Kiese, M. (1952). Biochem. Z. 322, 395. Fox, H. M. (1924). Proc. Canib. Phil. Sac. {Biol. Sci.) 1, 204. Granick, S. (1952). Fed. Proc. 11, 221. Kiese, M. & Kurz, H. (1954). Biochem. Z. 325, 299. Kiese, M. & Kurz, H. (1958). Biocliem. Z. 330, 177. Lemberg, R., Falk, J. E., Rawlinson, W. A., Hale, J. H. & Rimington, C. (1949). Abstr. Comm. \st int. Congr. Biochem., Cambridge p. 351. Lemberg, R. & Falk, J. E. (1951). Biochem. J. 49, 674. Lemberg, R. (1953). Nature, Lond. 172, 619. Lemberg, R. (1955). Biochemistry of Nitrogen, p. 165. Helsinki: Suomalainen Tiede- akatemia. Lemberg, R., Bloomfield, B., Caiger, P. & Lockwood, W. H. (1955). Aust. J. exp. Biol. med. Sci. 33, 435. Lemberg, R. & Stewart, M. (1955). Aust. J. exp. Biol. med. Sci. 33, 451. Lemberg, R., Morell, D. B., Lockwood, W. H., Stewart, M. & Bloomfield, B. (1956). Chem. Ber. 89, 309. Lemberg, R. & Benson, A. (1959). Nature, Lond. 183, 678. Morell, D. B. & Stewart, M. (1956). Aust. J. exp. Biol. med. Sci. 34, 211, Morrison, M., Connelly, J. & Stotz, E. (1958). Biochim. biophys. Acta Tl, 214. Oliver, L T. &. Rawlinson, W. A. (1951). Biochem. J. 49, 157. Parker, J. (1959). Biochim. biophys. Acta 35, 496. 330 Discussion Warburg, O. & Gewitz, H. S. (1951). Hoppe-Seyl. Z. 288, 1. Warburg, O., Gewitz, H. S. & Volker, W. (1955). Z. Naturforsch. 10b, 541. DISCUSSION Model Systems for Cytochrome Oxidase Absorption Spectra of Ferro- and Ferri- Compounds of Haem a By R. Lemberg (Sydney) Lemberg : With regard to the compounds of haem a and haematin a there appears to be, as in protohaematin compounds, a distinction between ferric compounds with absorp- tion bands at 635 m/i and 500 m/* (probably 'ionic') and ferric compounds with a single band at about 590 m/i similar to but lower than that of the ferrous complex (probably 'co-valent'). As in protohaematin compounds, the (ferric) haematin a-proteins appear spectroscopically to be mixtures of the two types, with the 'co- valent' type prevailing in haematin a-globin and the serum albumin compounds, and 'ionic' compound prevailing in haematin a-apoperoxidase. This seems quite distinct as there appears to be only one single characteristic band for each compound at 635 and 590 m/< respectively. There is, however, a rather poor correlation between A m^ Fe++-Fe+++ for the Soret bands and the ratio £635/e590 m/i in the ferric compounds as shown in Table 1 . Table 1 R635/590 AFe++-Fe+++ m/f Methyl imidazole Pyridine water 0-33 0-38 7 15 Globin-CN pH 7-5 0-60 — Globin-fluoride pH 7-5 0-69-0-74 — Globin pH 7-5 0-76 19 pH9 Human serum albumin pH7-5 0-80 0-91 24 10 pH9 0-91 22-25 Ox serum albumin pH 7-5 0-91 20-23 Pyridine pH 9 Apoperoxidase Pyridine pH 1 3 Haem pH 7-5 0-92 M6 M3 M5 14-20 36 12-15 7 ? pH 13 1-26 15-20 NH3 2% 1-26 - — • The reason that CO always shifted the absorption band of all our models in the visible to longer wavelengths, not to shorter ones as with cytochrome Oj, does not appear to be due to a^ being a more 'ionic' type of compound than any of our model haemoproteins a. Even the compound with apoperoxidase in which the absorption spectrum of the ferric compound as well as A m/t Fe++-Fe+++ indicate a high degree of 'ionic' nature, still gives a shift to the red with CO. Moreover, CO always shifts the Soret band to shorter wavelengths, with our model haemoproteins as well as with cytochrome a^. The haemochromes a formed with alkali-denatured proteins, with bands 13-14 m,« to the blue compared with pyridine haemochrome a are unusual. High pH is necessary for their formation. Urea-denatured globin at pH 9 gives a band quite close to that The Isolation, Purification and Properties of Haemin a 331 of native haemoglobin a, and decreasing the pH to below 9 shifts the absorption band of the alkali-denatured globin-compound from 575 to 596 m/t. The reaction is also given by chlorocruorohaem and monoformyldeuterohaem. The absorption spectrum of porphyrin a is not altered by the addition of denatured globin to its alkaline solution. The reaction is not caused by alkali alone; pyridine haemochrome a is not altered by alkali; nor is the band at 573-5 m/< produced by imidazole, NH3, glycylglycine, aminoacids (histidine, lysine, tyrosine) or SH-compounds (cysteine, GSH, thioglycolate) in 0-1 N NaOH. Possibly a Schiflf's base is formed between the formyl groups of the haems and amino groups in the proteins, resembling alkaline 'indicator yellow', but having the instability to lower pH characteristic of retinyl- idenemethylamine (Morton and Pitt, Biochem. J. 59, 128, 1955). Williams: I wish to show why the model systems of Lemberg and co-workers do not reproduce the physical properties of cytochrome a^. The predicted change in the , High spin type .^ Low spin type X Y Basicity of ligond Fig. 1. Soret band position of ferrous porphyrin complexes with change in the basicity of the ligand in the fifth and sixth positions is illustrated in Fig. 1. For completely ionic high-spin complexes, 0-X, the band position is predicted to move to longer wavelengths with increase in basicity. The same shift is expected for completely covalent complexes, Y-Z. In the region X-Y z. change of magnetic moment occurs and there is a chemical equilibrium involving two spin states. In this region the Amax falls with the increase of basicity of the ligand. Complexes of small molecules such as CO, O2, CN~ cannot be compared with other ligands by their basicity. They shift the absorption bands to longer wavelengths for reasons given in my paper. The point, Q, represents a typical band position for these low-spin com- plexes. Now if we have an ionic complex giving a band at long Amax, then the addition of CO will move the band to shorter wavelengths. A complex giving a band at position Y will show a band shift to the red on addition of CO. The models of Lemberg fall in the general region just before Y. Cytochrome 03 and to a slightly lesser degree cytochrome a fall near to X. Cytochromes Z> and c fall beyond Y with the other haemochromes of protoporphyrin and mesoporphyrin. I consider that nearly all the observations of Lemberg and co-workers are consistent with this interpretation. The argument can also be extended to the visible region of the spectrum. Here the plot of Amax (-x and /3 bands) against ligand basicity is only slightly different (Fig. 3, p. 48 of this volume). In the ionic complexes there is little shift of /Imax with basicity and the greatest change in band position is found in the region X- Y. The work of Falk (this volume, p. 74) defines the region beyond Y. We observe from the two diagrams that in complexes with ligands of very low basicity the Soret band will move in the opposite direction to the a and /J bands on the addition of carbon monoxide. This is observed with FeP(H202). Turning to Lemberg's data on the visible spectra we find that whereas cytochrome a is very H.E. — VOL. I — X 332 Discussion largely ionic, the models are largely covalent. This is consistent with the interpreta- tion of the Soret band shift and to a considerable degree with the changes in intensity of the a-band (see Williams, Chem. Rev. 56, 299, 1956). The same analysis leads us to suggest that cytochrome a^ is more largely in the low- spin form. The interpretation of the spectra of the ferric porphyrin a complexes, both band positions and intensities, needs no elaboration and is as in the text of my paper (this volume, p. 41). Chance: It is highly desirable to emphasize the properties of the CO compound of cytochrome a^, especially in consideration of the models studied by Lemberg and his collaborators. Our data are based upon very precise photochemical action spectra for the relief of CO-inhibited respiration; a compilation of two results from Castor's studies (Castor and Chance, J. biol. Chem. Ill, 453, 1955) is given in Fig. 2 and Table 1. 585 595 '550 = 10 I — I — i — i—r 400 I I I 450 500 I I I — I — r 550 600 650 -Kim) Fig. 2. The absorption bands of the carbon monoxide compounds of cytochrome a^-CO in ascites tumour cell and baker's yeast obtained by photochemical action spectra. The first point to be noted is the great similarity in the position of the peaks for yeast cells and ascites cells, even though the haem may well be bound to different proteins and is probably at a lower pH in yeast than the ascites cells. The second point, in addition to the well recognized properties of the a and y bands, is that there is a clear /S band; the absence of the P band in the CO haemochrome and the multiplicity The Isolation, Purification and Properties of Haemin a Table 1. Comparison of extinction coefficients OF CO compounds 333 Haem a-CO Cytochrome O3-CO muscle yeast a band 19-25 11-4 12 y band 79-105 110 115 of /? bands in some action spectra have both been commented upon (Dixon and Webb, Enzymes, Academic Press Inc., 1958, p. 423). The third point is that the molecular extinction coefficients of the CO compound of cytochrome a^ are known from photo- dissociation kinetics to a reasonable accuracy to be 12cm~^ x mM~^ for the a band and 115 for the Soret band. These data, when compared with haem a-CO data, show reasonable similarities. Cryptohaem a Morell: The origin of cryptohaem is of some interest. In contrast to our earlier belief we do not now think that cryptohaem a, as defined by us, is an artifact. Morrison has suggested in his precirculated paper that cryptohaem a may arise from haem a, in aged cytochrome oxidase preparations. We think that a thorough characterization is necessary before this compound could be identified positively as cryptohaem a. Lemberg: The interesting observation of Morrison of the conversion of haem a into a haem similar to cryptohaem a by a kind of autodestruction of active cytochrome oxidase, requires further studies before it can be concluded that the haem in question is cryptohaem a. The chemistry of our cryptoporphyrin a (see the precirculated paper) and other observations of Parker {Biochim. biophys. Acta 35, 496, 1959), make it appear unlikely that our cryptoporphyrin a is derived from haem a. Porphyrins similar to but distinct from cryptoporphyrin a have been obtained by Miss Parker from haemin a. Some of these porphyrins differed from cryptoporphyrin a in the Rp of their methyl esters; one which had a similar ester- 7?/^ had a distinctly different absorption spectrum. The cryptohaemin a of Morrison and Stotz may be the iron complex of one of these porphyrins. Unless the identity of the porphyrin from their cryptohaemin a with the cryptoporphyrin a now available as a well-crystallized methyl ester has been established, the latter cannot be considered to be a compound derived from one of the cytochromes a. Morrison: It would appear that we are all in agreement on that point, as we do not consider cryptohaemin a to be an artifact either (see Morrison, Connelly and Stotz, Biochim. biopliys. Acta 27, 214, 1958; Connelly, Morrison and Stotz, J. biol. Chem. 233, 743, 1958). Cryptohaemin a is, in our opinion, a naturally-occurring material which may arise as the result of catalytic oxidation of the prosthetic group of the cytochromes a. This may well be a situation comparable to the iron biliverdin complex found in preparations of catalase. In identifying cryptohaemin a, we have used the spectral properties published by Lemberg and Falk (Biochem. J. 49, 674, 1951) as our criteria. Our compound fits these criteria very closely, particularly as our spectra for the porphyrins were taken with pyridine as the solvent, and not with ether as Lemberg assumed. The position of the alpha peak of the pyridine haemochrome is at 582 nyt both in the original work of Lemberg and Falk and in the case of the compound we call cryptohaemin a. In the more recently published work of the Sydney group, the alpha peak is variously located at 579 m/i and 581 m//. It would appear that a clearer definition of what is being called cryptohaemin a is in order. Lemberg : I certainly agree with the last point made by Morrison. It is not justified, e.g., to 334 Discussion identify the non-purified "cryptoporphyrin" of Lemberg and Falk with the pure, crystalline cryptoporphyrin a as isolated by Parker. Mitochrome in Relation to Cryptohaem a Morell: The preparation of cytochrome oxidase which Morrison has aged to obtain the cryptohaem a-like compound is similar to those reported to give 'mitochrome'. According to Elliott, Hiilsmann and Slater {Biochim. biophys. Acta 33, 509, 1959), 'mitochrome' from cytochrome oxidase preparations contains a modified haem a which gives a haemochrome band with pyridine at 575 m/i. Our pyridine crypto- haemochrome a has its a band at 581 mix. Thus aged cytochrome oxidase preparations give at least one modified haem a which is not cryptohaem a. Slater: The difference between the pyridine haemochrome isolated from mitochrome obtained by ageing of cytochrome a + 03, and that obtained by Morrison from his aged preparation is paralleled by a difference in the position of the y-bands of the two preparations. Preparation }'-band (m/<) a-band of pyridine haemochrome (m/i) Cytochrome a + 03 Mitochrome Morrison's aged preparation 444 422 432 587 575 582 It appears, then, either that the transformation of cytochrome (a + 03) has not proceeded all the way to mitochrome in Morrison's aged preparation, or that the latter is a mixture of cytochrome a -\- a^ and mitochrome, yielding a mixture of the two pyridine haemochromes. Morrison: There are two things that are disturbing about the results that Slater cites. The first is the fact that one can obtain mitochrome whether one starts with a cyto- chrome b preparation or a cytochrome c oxidase preparation. These preparations are quite different in both enzymic properties and the nature of their haem groups. The second is that the mitochromes derived from cytochrome b or cytochrome c oxidase appear to be identical spectrally. Yet the haemins derived from the respective mitochrome preparations are quite different, having pyridine haemochromes whose a peaks are 1 7 m/t apart. It is interesting that a haemochrome with an a peak at 575 m/< has been described by Lemberg and termed cryptohaemochrome p. This compound was derived from oxidative procedures applied to protohaemin. Slater's compound, however, appears to have been derived from haemin a or cryptohaemin a. Slater's suggestion that our results might be explained by virtue of a mixed haemo- chrome is not valid since we chromatographed our material. CYTOCHROME OXIDASE COMPONENTS By M. Morrison and E. Stotz Department of Biochemistry, School of Medicine and Dentistry, University of Rochester, Rochester, New York THE PROSTHETIC GROUP OF CYTOCHROME OXIDASE Two of the major unsolved problems in the area of electron transport are: (1) the mechanism by which the electrons are passed on to reduce oxygen and (2) the chemical mechanism by which energy is captured in biological oxida- tion and transformed into a utilizable chemical form. An understanding of the structure of the prosthetic group of the enzyme which catalyzes the reduction of oxygen is of prime importance to the first of these problems. Extensive work by many investigators (see, for example, Dannenberg and Kiese, 1952; Kiese and Kurz, 1954; Lemberg, 1953; Lemberg, Bloomfield, Caiger and Lockwood, 1955; Morrison and Stotz, 1955; Person, Wainio and Eichel, 1953; Warburg, Gewitz and Volker, 1955) has clearly established that the prosthetic group of this enzyme is an iron porphyrin compound which has been labelled 'haemin a" or 'cytohaemin'. One of the problems of estabhshing the structure of the prosthetic group of cytochromes a has been in obtaining adequate quantities of the haemin free of contaminating lipids and other haemins. In the course of developing procedures designed to obtain pure haemin a, several interesting observations were made. A study of the haemins present in a cytochrome oxidase preparation showed that both haemin a and cryptohaemin a were extracted from the preparation (Morrison and Stotz, 1955). Investigation (Morrison, Connelly and Stotz, 1958) of the rate of extraction of haemins from a cytochrome oxidase prepara- tion indicated that cryptohaemin a and protohaemin are extracted more rapidly than haemin a. More recently, the slower extraction rate of haemin a was again demonstrated, using liver mitochondria as a starting material. This differential rate of extraction suggested that cryptohaemin a was not an artifact derived from haemin a by the extraction and isolation procedures, as other workers have implied. This was further strengthened by studies which indicated that haemin a and cryptohaemin a were not interconvertible by any treatment employed in the extraction or subsequent manipulation of the haemins. Thus, cryptohaemin a would appear to be present in the starting haemo- protein preparation, bound to protein. The spectral properties of haemin a 335 336 M. Morrison and E. Stotz and cryptohaemin a are so closely related that either could have been the prosthetic group of either cytochrome a or a^. The cytochromes a and a^ have never been isolated and separated from each other. An attempt (Connelly, Morrison and Stotz, 1959) to achieve such a separation by electrophoresis was made in our laboratory using a purified cytochrome c oxidase preparation as a starting material. This effort was unsuccessful, although an active cytochrome c oxidase was separated from an inactive haemoprotein. This inactive haemoprotein retained the spectral properties of cytochromes a and a^ and even the ability of cytochrome a^ to combine with carbon monoxide. The inactive preparation could not be reactivated and the haemins present in the preparation were identical with those of the active enzyme. In at least one micro-organism, however, nature has already performed the separation of cytochromes a and ^3. Smith (1954, 1955) has shown that Staphylococcus albus contains only cytochrome a, at least insofar as it can be defined by spectrophotometric studies. The haemins from this organism were extracted by procedures that had been developed in our laboratory (Morrison and Stotz, 1955, 1957; Connelly, Morrison and Stotz, 1958). The haemins were separated and studied by chromatographic and spectral techniques with results showing that haemin a was the prosthetic group of the cytochrome a of S. albus. This added a second criterion to the already cited spectrophotometric work which more firmly establishes the cytochrome component in S. albus as cytochrome a, comparable to that of mammalian tissue. It is also an interesting demonstration of the use of a neglected tool for the characterization of cytochromes ; that is, the characteri- zation of the prosthetic groups of cytochromes by chromatographic methods. No cryptohaemin was identified in the haemins extracted from the S. albus. Although there is no comparable source for observing cytochrome ^3 free of cytochrome a, we did turn to Bacillus subtilis, an organism in which the concentration of cytochrome ^3 is 2-3 times (Smith, 1954, 1955) the concentra- tion of cytochrome a. We were able to isolate haemin a from this organism as well as some cryptohaemin a, but the ratio of cryptohaemin a to haemin a was less than that found for the haemins extracted from heart muscle (Morrison and Stotz, 1955; Morrison, Connelly and Stotz, 1958). Providing that the components in the bacteria are strictly comparable to those in heart muscle, these results do not support the suggestion that cryptohaemin is the prosthetic group of cytochrome a^. When a cytochrome oxidase preparation is allowed to stand at 4°C for a prolonged period, it loses its enzymic activity. This loss is shown in Fig. 1, and is paralleled by a loss in haemin, as judged by the extinction at 605 m/<. After standing two weeks at 4°C, the spectrum of the solution is altered markedly. In the reduced form, the aged protein preparation has httle absorp- tion in the visible region and the Soret peak has shifted from 444 m/n to Cytochrome Oxidase Components 337 432 mji. Extraction of the haeniin from this preparation, and a study of the chromatographic properties and spectra, showed that this preparation con- tains both haemin a and cryptohaemin. The total haemin recovered was lower, and the ratio of cryptohaemin to haemin a is greatly increased. These results suggest that cryptohaemin a is a product of oxidative degrada- tion of haemin a. This degradation appears to be the result of an auto- catalytic destruction of the prosthetic group of cytochrome a. It is interesting 100 -I DAYS Fig. 1 . Loss of cytochrome c oxidase activity. The enzyme preparation was allowed to stand at 4°C. to note that at high concentrations of chelate, the cytochrome oxidase preparation is enzymically inactive and that degradation does not take place. On dilution, the preparation is enzymically active and degradation does occur. Since destruction of the enzyme is linked with enzymic activity and hence with activation of oxygen, it is conceivable that this activated oxygen is the cause of the destruction of the enzyme. Sekuzu, Takemori, Yonetani and Okunuki (1959) have already presented spectrophotometric evidence that such an activated form of the cytochrome does exist. The structure of the prosthetic group of the cytochromes has not been neglected. The notable work of Marks, Dougall, Bullock and MacDonald (1959) on the structure of the deuteroporphyrin derived from haemin a, assigns the position and describes the nature of five of the groups substituted on the eight available positions on the porphyrin nucleus. The problems which remain are: first to decide what are the three other groups substituted on the porphyrin nucleus, and second, the arrangement of these groups on the porphyrin nucleus. There is ample evidence to suggest the presence of a formyl group and a 338 M. Morrison and E. Stotz vinyl group as two groups in resonance witli the porphyrin a nucleus (Dannenberg and Kiese, 1952; Lemberg, 1953; Kiese and Kurz, 1953; Warburg, Gewitz and Volker, 1955). Table 1 supplies such evidence. Haemin Table 1. Visible absorption maxima and type of spectrum OF porphyrin a and derivatives Porphyrin IV III II I Spectral type Porphyrin a Porphyrin a oxime Porphyrin a (reduced with HI) Porphyrin a (reduced with NaBH4) 516 512 508 502 558 552 550 540 582 577 576 573 647 645 640 624 O R R A A = actio, R = rhodo, O = oxorhodo. a reacts with dimedon to form a methone derivative; bromine adds to haemin a to form a product of which the haemochrorae band is shifted 5 m/t towards the blue. These observations confirm the presence of a formyl and a vinyl substituent. The third group removed in the resorcinol melt of haemin a may be an a-ketoalkyl group on the following grounds : the infra-red spectrum indicates the presence of two carbonyl groups other than the carboxyl-carbonyls ; one of these is the formyl group, while the other must be present in the third group. The long aliphatic side chain is probably present in the a-ketoalkyl side chain rather than on the vinyl, since oxidation with permanganate yielded no long chain aliphatic acid or aldehyde. From our studies, the molecular weight of chlorohaemin a based on iron determination is 880. By difference, the alkyl group must account for 213 of the molecular weight. The significance of the long aliphatic chain and the aldehyde and ketone groups in the functioning of the cytochromes a and ^3 is not apparent. It is clear that the long chain aliphatic group can help supply a non-polar environ- ment. In view of the findings of Wang (1958) and Corwin and Bruck (1958), it could be suggested that the 'aliphatic' environment makes possible a haem-oxygen combination equivalent to that which takes place in the haemoglobin molecule and that the subsequent oxidation of the iron requires the transfer of an electron from a co-ordinating group on the protein. CYTOCHROMES AND PHOSPHORYLATION The clarification of the mechanism of oxidative phosphorylation in chemical terms is a second outstanding problem in understanding biological oxidation. This problem can be approached in a number of diflFerent ways. It has been investigated by spectrophotometric study (Chance and Williams, 1957) of the kinetics of interaction of the various cytochromes, flavoproteins and Cytochrome Oxidase Components 339 pyridine nucleotides in intact mitochondria. Other approaches (sec, for example, Boyer, 1957; Slater, 1958; Wadkins and Lehninger, 1958) have involved studying the relative rates of incorporation of ^-P or ^^O in an effort to evaluate the steps in the phosphorylation process. The phosphorylation steps have been studied by employing uncouplers and antibiotics (Lardy and McMurray, 1959). Slater (1958) has measured the pH optimum of the various steps involved in this process. More directly, the classical approach of separation and isolation of the enzymes involved in the phosphorylative steps, has only recently shown signs of being fruitful. The process of oxidative phosphorylation has been con- sidered to be catalysed by a complex of enzymes which are very unstable. Until comparatively recently, the intact mitochondria were the smallest units capable of this process. It is now possible, by various techniques (Green and Crane, 1957; Kielley and Bronk, 1958; Lehninger, Wadkins, Cooper, DevUn and Gamble, 1958) to prepare submitochondrial units which are capable of coupling oxidation and phosphorylation. More recently, it has been possible to subfractionate the complex of enzymes even further and isolate the enzymes involved in the terminal steps in the phosphorylation procedure (Remmert and Lehninger, 1959; Wadkins and Lehninger, 1958). Still to be clarified are the enzymic steps in which the electron carriers themselves are involved in the chemical transformations resulting in phos- phorylation. We decided to attack the problem by investigating this phenomenon in the steps involving cytochrome c and cytochromes a and a^. It has been clearly demonstrated that in the oxidative sequence from cytochrome c to oxygen, at least one 'high-energy' phosphate can be derived per passage of two electrons (Judah, 1951 ; Maley and Lardy, 1954; Nielsen and Lehninger, 1954). Initial studies in our laboratories demonstrated that when purified isolated cytochrome c was added to the mitochondria, employing ascorbate as the substrate, the rate of both oxidation and phosphorylation increased. The increase in phosphorylation, however, did not parallel the increase in oxidation, and as a result there was a decrease in the P/O ratio with increasing concentrations of added cytochrome c. Varying the concentration of mitochrondria in the same type of experiment, showed that the rate of phosphorylation was proportional to the amount of mitochondria at any given concentration of added cytochrome c. This was not true of oxidation. At high levels of mitochondria, the oxygen consump- tion does not increase. In the widely used scheme: AH^ + B + I-^A^I -[- BH^ A and B are general symbols for electron carriers and / is a compound which forms a bond with one of the carriers in which the energy available from elec- tron transfer is conserved ; / is the coupler of oxidation and phosphorylation 340 M. Morrison and E. Stotz and the /-electron carrier compound is the inhibited form of the electron transport system. In 'tightly-coupled' mitochondria, oxidation cannot proceed without the presence of phosphate acceptors. This indicates that the component which inhibits oxidation and couples oxidation to phosphorylation, 1, is present in amounts exceeding the electron transport components. Cytochrome c is an electron transport system member which is directly involved in the coupling of oxidation and phosphorylation. The compound, /, which inhibits oxidation is present in amounts exceeding the concentration of cytochrome c initially in the mitochondria. The results of the experiments cited above can then be interpreted simply as a logical consequence of the mass law. On addition of cytochrome c, its concentration will equal and then exceed that of the inhibitor, /. Therefore, at high concentrations, more of the cytochrome c can be oxidized and reduced without reacting with the inhibitor. At low concentrations, where the ratio of 7 to cytochrome c is high, there is a great extent of reaction with the inhibitor. Increasing the concentration of mitochondria results in increased ratios of /to cytochrome c, with a resulting increase in the P/O ratio. Conversely, a lower ratio of / to cytochrome c results in a decreased P/O ratio. From these results, it would appear that cytochrome c does interact with the inhibitor, /, With this information at hand, the following experiments were performed. 'Tightly-coupled' mitochrondria were prepared and incubated with succinate in a nitrogen atmosphere. When the system became sufficiently anaerobic to deoxygenate the haemoglobin present, two volumes of boiling acetone were added. The contents were then placed in a water bath at 60°C for 5 min and the mitochondria were sedimented by centrifuging. The mitochondria were then extracted with salt solutions. The cytochrome c which was extracted was, almost exclusively, in the reduced form. More interesting was the fact that, quantitatively, the amount of cytochrome c extracted under these conditions was a function of the state of oxygenation of the mitochondria. More Table 2. Extraction of cytochrome c Condition of extraction 0-5 M NaCl pH 7-0 0-5 M NaCl pH 7-0 (after pretreatment with dithionite) 0-5 M NaCl pH 7-0 (after pretreatment with ferricyanide) Ratio of cytochrome c extracted from mitochondria anaerobic aerobic 2-0 -1-5 1-75-1-4 1-8 -1-4 Cytochrome Oxidase Components 341 cytochrome c could be extracted from the mitochondria in the anaerobic state than that in the aerobic state. This is not simply a function of the state of oxidation of the cytochrome c. After acetone powders were obtained from aerobic and anaerobic mito- chondria, they were treated in order to oxidize the cytochrome c. The difference in the total amount of cytochrome c extractable from the two types of mitochondrial powders remains the same even when all the cytochrome is oxidized prior to extraction. This difference in extractability, then, can be interpreted as due to the difficult extraction of the cytochrome-coupler compound and not simply due to a difference in the extractability of free oxidized and reduced cytochrome c. When aged mitochondria in which oxidation and phosphorylation are uncoupled were employed, the state of oxidation still affected the amount of cytochrome c which could be extracted. Thus, in agreement with other studies (Remmert and Lehninger, 1959), it would appear that the carrier inhibitor compound is still functioning even after ageing, though the steps involved in the transfer of the high energy bond are disrupted. In addition to the previous evidence for the existence of cytochrome c inhibitor complex, our studies (Crawford and Morrison, 1959) on the succinate-cytochrome c reductase system have also yielded evidence that a cytochrome ^-inhibitor complex may exist. A study of the rate of reduction of cytochrome c with our succinate-cytochrome c reductase preparation has indicated the presence of an inhibitor in the preparation. On extraction of this preparation with /50-octane, the kinetics can be interpreted as an increase in inhibitor. A study of the kinetics after the addition of tocopherol or vitamin K^ indicates that these lipids are able to reverse the effect of the inhibitor. These results are compatible with a scheme in which the cytochrome first reacts with an inhibitor and the cytochrome-inhibitor compound can no longer be alternately reduced and oxidized. Z?++ + / + Ci+++ -^ b+++ ~ / + Cj++ Following this, the effect of the lipid may be to react with the carrier ~/ compound to form a complex of the type I ^^ Xin which Zis the lipid factor. This compound in turn might react with inorganic phosphate (/*,) to form complexes of the X ^^ P type, and preliminary evidence of such lipid- phosphate intermediates has already been obtained (Conover and Witter, 1958; Boyer, Dempsey, Schulz and Andesegy, 1959). ^+++ r^i ^ x-^Ir^ X + b+++ I'^ X + Pi-^I + X ^P Spectrophotometric results are perfectly compatible with these results. On addition of succinate, both the cytochromes b and c^ are reduced. After 342 M. Morrison and E. Stotz wo-octane extraction of the preparation, the addition of succinate results in a complete reduction of cytochrome q and only partial reduction of cyto- chrome b. According to the foregoing scheme, the cytochrome Z?-inhibitor compound cannot be reduced and consequently remains in the oxidized form after having reduced the cytochrome q. Studies are now in progress which are designed to isolate and characterize cytochrome-inhibitor compounds. It is hoped by such studies to clarify the role of the electron carriers in oxidative phosphorylation. A cknowledgemen ts The research in this report was supported by Grant No. H-1322 from the National Heart Institute, National Institute of Health. One of the authors (M.M.) was supported by a Senior Research Fellowship SF-47 from the U.S. Public Health Service. REFERENCES BoYER, P. D. (1957). Proc. Inter. Symp. Enz. Chem. Japan p. 301. BoYER, P. D., Dempsey, M. B., Schulz, a. R. & Andesegy, M. (1959). Fed. Proc. 18. 195. 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Wash. 45, 1. Sekuzu, I., Takemori, S., Yonetani, T. & Okunuki, K. (1959). J. Biochem. Tokyo 46, 43. Slater, E. C. (1958). Aiist. J. exp. Biol. med. Sci. 36, 51. Smith, L. (1954). Bad. Rev. 18, 106. Smith, L. (1955). J. biol. Chem. 215, 847. Stern, A. & Molvig, H. (1936). Hoppe-Seyl. Z. A177, 365. Stern, A. & Wenderlein, H. (1936). Hoppe-Seyl. Z. A176, 81. VVadkins, C. L. & Lehninger, A. L. (1958). J. biol. Chem. 233, 1589. Wang, J. H. (1958). /. Amer. chem. Soc. 80, 3168. Warburg, O., Gewitz, H. S. & Volker, W. Z. (1955). Z. Naturforsch. 10b, 541. THE STRUCTURE OF PORPHYRIN a, CRYPTOPORPHYRIN a AND CHLORIN a^ By R. Lemberg, P. Clezy and J. Barrett Institute of Medical Research, Royal North Shore Hospital, Sydney This paper deals mainly with the chemical structures of the prosthetic groups of the cytochromes of type a. Cytochromes a, a^ and a^ have as their pros- thetic group the iron complex of a formyl-porphyrin (porphyrin a, also called cytoporphyrin by Warburg). The presence in heart muscle of a second formyl- porphyrin, cryptoporphyrin a, suggests that another so far unknown haemoprotein of type a is present in heart muscle in small amounts. The prosthetic group of cytochrome a^ belongs to a different class, being the iron complex of a chlorin (dihydroporphyrin) without formyl side chains. PORPHYRIN A Evidence for the Nature of the Side Chains: The Formyl Group The presence of a formyl side chain in the prosthetic group of the Atmungs- ferment was already postulated in the classical work of Warburg. This assumption, based on the similarity of the photochemical absorption spectrum with the spectra of chlorocruorohaem (also called spirographs haem) com- pounds has been amply confirmed by all later workers. The aldehyde group reacts reversibly with methanolic hydrochloric acid forming a methyl acetal, with hydroxylamine to form an oxime, with hydrazine to form a hydrazone, and with sodium bisulphite. The rapid rate of the reaction of the haemo- chrome with hydroxylamine at room temperature (Lemberg and Falk, 1951), the reaction with bisulphite (Lemberg, cf. Parker, 1959) and the dehydration of the oxime to the nitrile (Dannenberg and Kiese, 1952) prove that the carbonyl side chain is not ketonic. The presence of a ring-ketone group similar to that present in the isocyclic ring of chlorophyll, e.g. in phaeopor- phyrin ctj, is excluded by the failure of phaeohaemochromes to react rapidly with hydroxylamine, by the reversibility of methyl acetal formation of porphyrin a (the conversion of phaeoporphyrins into chloroporphyrins being irreversible), and by the infra-red spectrum of porphyrin a (Lemberg and Willis, unpublished) which does not show the band at 1695 cm~^ characteristic of the ring-ketone group. Recently, three more reactions of the formyl group in porphyrins have been studied in our laboratory, its oxidation by 344 The Structure of Porphyrin a, Cryptoporphyrin a and Chlorin ag 345 chromic acid under mild conditions to carboxyl, its reduction by sodium borohydride to hydroxymethyl, and its condensation with acetone-HCI, probably to -CHiCH-CO-CHg. Porphyrin a undergoes all these reactions. Since acetone-hydrochloric acid is used in the isolation of haemin a from heart muscle, and for its crystallization in the Warburg procedure, the last- mentioned reaction is of interest; unless care is used, this reaction partially converts haemin a into its acetone condensate. The magnitude of the shift of the absorption bands of porphyrin a in the oxime formation (CHO -^ •CH:NOH) shows that only one carbonyl, i.e. the formyl group is present. The average shift for the four bands is 75 A for porphyrin a, 82 for monoformyl deuteroporphyrin, 77 for chlorocruoro- porphyrin and 79 for cryptoporphyrin a. This shift is about the same for acetyl and formyl groups, and two such groups cause a band shift of about 120 A. Porphyrin a does not contain a carboxyl group directly on the nucleus. After treatment of the porphyrin with hydriodic acid or after diazoacetic ester addition to the double bond, hydroxylamine gave aetio-type porphyrins (Rimington, Hale, Rawlinson, Lemberg and Falk, 1949); a carboxyl group would not be altered by these reagents and would retain its rhodofying effect on the spectrum. Two carboxylic acid groups can be demonstrated in porphyrin a by manometry (Morell, unpublished) and both are accounted for as propionic acid side chains, as the conversion of porphyrin a into cyto- deuteroporphyrin shows (Warburg and Gewitz, 1953). In the paper chroma- tography in lutidine-water (Nicholas and Rimington, 1949), porphyrin a had an Rp of 0-84, slightly higher than the 0-81 of other dicarboxylic acids. The Unsaturated Side Chain Conjugated to the Nucleus and Its Position Relative to the Formyl Rimington et al. (1949) believed to have evidence for the presence of two vinyl groups from absorption spectra of the oxime and its presumed diazo- acetic ester adduct. The latter was, however, prepared by diazoacetic ester addition to porphyrin a before oximation. Since the formyl group also reacts with the ester, it is necessary to protect it by oximation first before the diazoacetic ester addition. This experiment was carried out in our laboratory (Parker, 1959) and gave evidence for only one vinyl group (band shift about 2 m/<, as for chlorocruoroporphyrin). Warburg and Gewitz (1951) also found that only two atoms of hydrogen (plus one needed for the reduction of the iron from ferric to ferrous) were added on catalytic hydrogenation of haemin a in borate buffer pH 11. Porphyrin a differs, however, from chlorocruoroporphyrin, which has a formyl and one vinyl group in two aspects. Firstly, its absorption bands are slightly shifted towards the red (cf. Table 1). It may be noted that it is essential to compare absorption spectra in the same solvent. Chloroform, as 346 R. Lemberg, p. Clezy and J. Barrett compared with ether, e.g. shifts all band positions from 2-6 m/t towards the red. Table 1. Absorption spectra of porphyrin a, cryptoporphyrin a AND CHLOROCRUOROPORPHYRIN A in m/i in chloroform Porphyrin a Cryptoporphyrin a Chlorocruoroporphyrin I 646 642-5 644-5 II 584-5 584 585 III 563-5 559 560 IV 520 519 520 Secondly, while chlorocruoroporphyrin has a rhodotype spectrum, with the ratio of bands III/IV 1-3-1-4, porphyrin a has an oxorhodo type spectrum 670 480 670 Fig. 1 . Types of porphyrin spectra. 460 with both bands II and III higher than IV and the ratio III/IV as high as 2-40-2-45 (Fig. 1). The order of strength of the rhodofying influence of side chains is • CHO ^ ring-CO > • CH : CH • COMq > • COMq ^ •COR> -CHrNOHr^ -CHiCHo The Structure of Porphyrin a, Cryptoporphyrin a and Chlorin ag 347 Vinyl alone does not cause a rhodo-type spectrum. This made Lemberg and Falk (1951) postulate the presence of an acrylic acid side chain, which as vinylogue of the carboxyl side chain has a strong rhodofying effect. How- ever, later evidence has not confirmed this assumption. Cytodeuteroporphyrin has two propionic acid side chains, and the infra-red spectrum of porphyrin a ester shows only the 1737 cm~^ band due to the propionic acid side chains, while the acrylic ester should have a band at about 1725 cm"^. It is, in fact possible to account for both the differences between porphyrin a and chlorocruoroporphyrin on the basis of two rhodofying groups, formyl and vinyl (or substituted vinyl) on opposite pyrrole rings, together with the presence of an a-hydroxyalkyl group on the pyrrole ring in between (see below). The average displacement of bands towards the infra-red caused by replacement of an alkyl by an a-hydroxyalkyl is 1 m/<, in agreement with the difference of band positions of porphyrin a and chlorocruoroporphyrin except for band III (3-5 m/<). This band may be shifted further towards the red in porphyrin a owing to overlapping with the high band II, which in turn is shifted towards the blue in porphyrin a. Whereas the position of absorption bands is little influenced by the relative position of the substituents, this is not so for band intensities. A second rhodofying group increases the ratio III/IV very strongly, if it sub- stitutes the pyrrole ring opposite to the first; it decreases it if it substitutes a vicinal pyrrole ring, independent of whether the relative position is 1-4, 2-4 or 2-3 (cf. Table 2, No. 1-5). Table 2. Antirhodofying effects of rhodofytng groups on vicinal pyrroles No. Porphyrin in chloroform Relative position and nature of groups Type of spectrum 1 . Diformyldeutero 2. 3-Desmethyl-3-formylrhodo 3. Rhodinporphyrin gs 4. Neorhodinporphyrin ga but 5. Rhodoporphyrin gg 2F 3 F 3 F 2 V 4F 6 CO.,H 6 CO,H (y—CH,) 3 F (y-CH,) 2 E 3 F (y-CU,) Actio Rhodo F = formyl V = vinyl 2 3 7 6 y = methene bridge. Positions Table 3 shows that the vinyl group has also a rhodofying effect if it substi- tutes an opposite pyrrole (cf. 7 with 6, 10 with 9, Table 3) and an anti-rhodo- fying effect if it substitutes a vicinal pyrrole (cf. 3 with 1, Table 3, or 4 with 5, Table 2). Thus it raises R III/IV from 1-29 to 1-89 opposite to a carboxyl, H.E. — VOL. I — Y 348 R. Lemberg, p. Clezy and J. Barrett and from 1-72 to 2-15 opposite to a ring-ketonyl. Since the rhodofying effect of formyl is even stronger than that of ring-ketonyl (cf. 1 with 9, Table 3), a high R III/IV would be expected for porphyrin a with a vinyl opposite to formyl. The a-hydroxyalkyl group has practically no influence on R III/IV (cf. 2 with 1, Table 3). Porphyrin a carboxylic acid has, indeed R III/IV quite similar to pseudoverdoporphyrin (vinylrhodoporphyrin). Table 3. Effect of substituents on r ra/iv Porphyrin in chloroform Rhodofying Group I Rhodofying opposite Group II vicinal R III/IV 1. Formyldeutero 1 . Monohydroxethyl-formyl deutero 3. Chlorocruoro 4. Porphyrin a CHO CHO CHO CHO alkylvinyl CH(OH)CH3 vinyl 1-77 1-68 1-40 2-40 5. Porphyrin a-carboxylic acid 6. Rhodo 7. Pseudoverdo 8. Oxorhodo COjH CO2H CO2H CO2H alkylvinyl vinyl acetyl — 1-75 1-29 1-89 2-39 9. Phaeo a^ •C0CH(C02R)- — — 1-72 10, Vinylphaeo Og •C0CH(C02R)- vinyl — 2-15 Porphyrin a cannot therefore have the formyl and alkyvinyl groups in the positions at vicinal pyrroles as Warburg and Gewitz (1953) assumed. Lemberg (1953) postulated that the two rhodofying groups must be on opposite pyrroles and therefore one on a ring bearing the propionic acid side chains. There remained the possibility that the two groups substituted one and the same pyrrole ring, no porphyrins of this type being known. However, this possibility was excluded by later work, particularly of MacDonald (see below). By the Schumm resorcinol method, followed by removal of iron, Warburg and Gewitz (1953) obtained from haemin a a crystalline porphyrin ester which they called cytodeuteroporphyrin ester. It differed from deutero- porphyrin ester obtained from protohaemin in its melting point. It has also absorption bands slightly more towards the blue than those of deutero- porphyrin ester (Barrett, unpubHshed). Chromic acid oxidation yielded methylmaleimide and haematinic acid. A formula having two free /5-positions in 2 and 3 was suggested, but bromination revealed the presence of three, not two free ^-positions. Recently Marks, Dougall, Bullock and Mac- Donald (1959) have succeeded in synthesizing cytodeuteroporphyrin. The three free /^-positions are in 2, 4 and 8 (Fig. 2). The Structure of Porphyrin a, Cryptoporphyrin a and Chlorin a^ 349 The two rhodofying groups must therefore occupy the positions 4 and 8 on rings II and IV. The structure of cytodeuteroporphyrin with two propionic acid groups disproves the assumption (Lemberg, 1953; Dixon and Webb, H, H H^ M /2 3\ y/z i\ M/T 4\E M.A 4\H M\^ VM H\8 5/M V ^^ V 6/ P P F P Fig. 2. Cytodeuteroporphyrin. 1958) that the excess carbon and hydrogen in porphyrin a (see below) is present in the form of a long fatty acid side chain. The oi- Hydroxy alky I Side Chain These studies raised the problem of the third substituent removed from position 2 in ring I in the resorcinol melt. As shown above, there was evidence against this being another carbonyl or vinyl side chain. In fact any of these groups in position 2 would have a strong anti-rhodofying effect, not in harmony with the oxorhodotype spectrum of porphyrin a. We have, e.g. recently obtained a porphyrin a derivative having a carbonyl group in this position. This had a rhodotype spectrum with R III/IV 1-21. Of groups known to be removed in the resorcinol melt only a-hydroxyalkyl remained; haematohaemin is known to yield deuterohaemin. The low Ry of porphyrin a ester in chloroform-kerosene (0-10) or propanol-kerosene (0-26) was in agreement with such an assumption (Barrett, 1959) and also the analyses of haemin a (Table IV) which indicated the presence of at least 6 atoms of oxygen. Barrett (1959) has been able to acetylate the hydroxyl groups by acetic anhydride in pyridine with the increase of Rp from OTO to 0-56 and from 0-26 to 0-62 respectively. This hydroxyl group is present in the form of an a-hydroxyalkyl group (Clezy and Barrett, 1959). In this work the use of the porphyrin a carboxylic acid (CO2H replacing CHO) was found useful, since it abolished by-reactions due to the sensitivity of the formyl group to oxidation. This compound had been obtained by mild chromic acid oxidation of the acetate of porphyrin a, followed by hydrolysis of the acetate of the oxidation product. As expected, oxidation of the a-hydroxyalkyl to ketonyl by chromic acid-H2S04 in acetone decreased R III/IV (see above) from 1-75 to 0-78. If the reaction was carried out with porphyrin a itself (R III/IV 2-3), a compound of R III/IV 1-27 with intact formyl group was obtained in addition to the carboxylic acid compound with R III/IV 0-78. The resulting porphyrins resembled acetylporphyrins spectroscopically. Confirmatory evidence was obtained by dehydration. Using either porphyrin a carboxylic acid or porphyrin a alcohol (CH.jOH replacing CHO), the a-hydroxyalkyl side chain could be demonstrated by the band shift (2-3 m.[i towards the red) 350 R. Lemberg, p. Clezy and J. Barrett accompanying dehydration to vinyl or substituted vinyl, which was achieved by heating in acetic acid or in pyridine plus toluene-sulphonylchloride. The introduction of the vinyl at position 2 lowered, as expected, R III/IV of porphyrin a acid from 1-75 to 1-50. The a-hydroxyalkyl group is therefore present on ring I (in position 2) between the two rhodofying groups at rings II and IV. Possible Formulae of Porphyrin a These findings can be summarized as establishing one of the two formulae of Fig. 3 for porphyrin a. CHOH CHO H,C OHC CO2H COgH I n Fig. 3. Alternative formulae for porphyrin a Nicolaus (private information) has been unable to obtain the pyrrole carboxylic acids CH HO,C XN c^^3 CO2H HOpC C02H OgH from the nitrile of porphyrin a oxidized by his alkahne permanganate method. This would seem to exclude formula I and support formula II of Fig. 3. However, the value of such negative evidence is uncertain. Dr. Clezy is attempting to solve this problem by Wolflf-Kishner reduction of the formyl to methyl. A resorcinol melt of the product should then give deuteroporphyrin The Structure of Porphyrin a, Cryptoporphyrin a and Chlorin ag 351 IX if formula II is correct, but another porphyrin with two methyl groups on one pyrrole from a porphyrin of formula I. The results are as yet inconclusive. The dimethyl porphyrin ester obtained greatly resembled deuteroporphyrin IX ester, and gave no melting point depression with it, but had a lower melting point (203-206° compared with 220-222° of the deuteroporphyrin ester). Large A Iky I Group (or Groups) and Molecular Weight of Porphyrin The molecular weight of porphyrin a can be calculated from the analyses of haemin a, in particular its iron content (Table 4), and from the ratio of molar extinction to specific extinction of pyridine haemochrome a and porphyrin a. While these results are as yet not fully concordant (Table 5), they show that the molecular weight is considerably greater than that of protoporphyrin and that R^ + R2 of the formulae in Fig. 3 is between Table 4. Analyses of haemin a Warburg and Gewitz (1951) C 64-47 H 6-79 N 6-50 Fe 642 CI 4-20 Lemberg (unpublished) (haemin reconstituted from porphyrin a) C 65-41 H 6-56 N— Fe 5-90 CI 3-75 Possible formulae C46H56N406FeCl CaiHesN.OvFeCl Table 5. Molecular weight of porphyrin a and size of R groups {R, + R,) M.W. of porphyrin Additional C atoms Warburg haemin analyses Lemberg haemin analyses Pyridine haemochrome a, fji/^s Porphyrin a, fji/^sp 760-790 835-860 740-810 < 875 12-14 16-18 11-16 <20 C12H25 and C20H41. These alkyl groups or group can only be attached to the vinyl and the a-hydroxylalkyl, since they are removed in the resorcinol melt. Some of it at least, must be attached to the vinyl since no crystalline porphyrin was obtained in the resorcinol melt after catalytic hydrogenation of the unsaturated group. Since we have observed that an acetyl group is not removed by milder conditions in the resorcinol melt, we hope to solve the problem whether the group resulting from oxidation of the a-hydroxyalkyl group is acetyl or a substituted acetyl group. We have so far refrained from an attempt to obtain information by an oxidation of porphyrin a and the attempt at isolation of the fatty acid or acids, 352 R. Lemberg, P. Clezy and J. Barrett because most of our porphyrin a preparations still contain lipid impurities in amounts of the same order as the large alkyl side chain. The position of these alkyl groups (or group) and their structure is of interest for the problem of the biosynthesis of haem a. The formyl group would not appear to present a problem; it can arise by oxidation of a methyl, as in chlorophyll b, leading to a porphyrin of formula II (Fig. 3); or, leading to a porphyrin of formula I, by oxidation of a vinyl, since in chlorocruorin formyl is found instead of a vinyl in haemoglobin. Formation of an a-hydroxy- ethyl side chain (in position 2) from vinyl also can easily be understood, since Granick, Bogorad and Jaffe (1953) have found this group in porphyrins from Chlorella mutants. The formation of an a-hydroxyalkyl group, other than a-hydroxyethyl, would, however, require a different explanation. The • CH : CH • R group can arise either by condensation of • CH3 (or an original • CH2CO2H side chain) with a fatty aldehyde, which would explain the double bond joining it to the porphyrin ring; or by one molecule of succinyl co- enzyme A being replaced in the synthesis by the coenzyme A compound of an a,/3-unsaturated fatty acid. CRYPTOPORPHYRIN A The fractionation with hydrochloric acid of porphyrins from heart muscle by the Willstatter procedure yielded in the 8% HCl fraction a porphyrin which could not be a mixture of protoporphyrin with /h porphyrin a, since the ratio £'580m;) in ether was 0-65, identical with that of protoporphyrin and different from that of porphyrin a (1*2), whereas a \^ X distinct absorption band at 555 n\fi could be observed P P (Lemberg, 1953; Lemberg and Parker, 1955). By alumina Fig. 4. chromatography and crystallization from ether at low Cryptoporphvrin a temperature, the well-crystallized methyl ester, m.p. 259- 260°C was obtained (Parker, 1959). The absorption spectrum of cryptoporphyrin a is very similar to that of chlorocruoroporphyrin (Table 1) and R III/IV of the two porphyrins is also similar. Cryptoporphyrin a contains one formyl group and one vinyl (or alkylvinyl) side chain in the same relative position to one another as in chlorocruoroporphyrin. However, the specific extinctions of cryptoporphyrin a show that the molecular weight is similar to that of porphyrin a and larger than that of chlorocruoroporphyrin, and indicate that cryptoporphyrin a contains a large alkyl group. The formula given in Fig. 4 which differs from that of chlorocruoroporphyrin only by its R group on the vinyl group has been suggested (Parker, 1959). Barrett (unpublished) has found that the resorcinol melt yielded a porphyrin of deuteroporphyrin type, but insufficient material has been available for exact identification of this porphyrin with deuteroporphyrin which would result from a porphyrin of the formula The Structure of Porphyrin a, Cryptoporphyrin a and Chlorin 82 353 suggested. In spite of its larger molecular weight, cryptohaemin remains together with protohaemin in the aqueous phase of the Rawlinson distribution between ether-pyridine and aqueous pyridine-HCl (Rawlinson and Hale, 1949), and also in the aqueous phase in the countercurrent distribution phase between light petroleum-acetone and aqueous HCl-acetone of Kiese and Kurz (1954), thus differing from porphyrin a of similar molecular weight. Cryptoporphyrin a is not an artifact derived from protohaem. Removal of myoglobin from heart muscle before processing greatly decreases the protoporphyrin yield, but does not decrease the cryptoporphyrin yield. Porphyrins of similar spectroscopic properties can be obtained from red cells (cryptoporphyrins/?) but these are chemically quite different compounds; they contain a ketonyl side chain and chlorine, not a formyl side chain (Lem- berg, 1953; Clezy and Parker, unpubhshed). At first it was assumed (Lemberg, 1953; Lemberg and Parker, 1955) that cryptoporphyrin a was an artifact derived from porphyrin a by alteration during the isolation process. Its yield, though always much smaller than that of porphyrin a varied considerably, and porphyrin a gave rise to a porphyrin resembling cryptoporphyrin a during treatment with acetone-HCl and reintroduction and removal of iron. It could be shown, however (Parker, 1959), that the altered porphyrin a thus formed was not identical with cryptoporphyrin a, while its formation in variable amounts can explain the apparent variation in yield. The variation was also less in later experiments. The yield of cryptoporphyrin a from heart muscle is between 5% and 10% that of porphyrin a. These results make it likely that cryptoporphyrin a is derived from a so far unknown haemoprotein of heart muscle which has cryptohaem a as its prosthetic group. Connelly, Morrison and Stotz (1958) have independently come to the same conclusion by chromatographic separation of heart muscle haemins on silica gel. A reservation must be made, however. The spectrum of the porphyrin obtained from their "haemin a^' (Morrison, Connelly and Stotz, 1958) is not identical with that of cryptoporphyrin a, since its spectrum in ether is compared with that given by Lemberg (1953) for a solution of cryptoporphyrin a in chloroform. We have also found cryptoporphyrin a in chicken heart and liver. Pyridine cryptohaemochrome a differs from pyridine haemochrome a in the position of its a-band by 6 m//, and in having a second absorption band in the green. Owing to these relatively small differences and the low concen- tration of cryptohaem a, it will be very difficult to discover the cryptohaemo- protein a in cytochrome oxidase preparations or in tissues. Its low concentra- tion would not appear to suggest for it a role in the main respiratory chain, nor can it be the prosthetic group of cytochrome a or ^3. However, its dis- covery shows that careful chemical isolation can still give results unobtainable by spectroscopic methods alone. 354 R. Lemberg, P, Clezy and J. Barrett HAEM A2 AND CHLORIN A^ Haemin a., (Barrett and Lemberg, 1954; Barrett, 1956) has been isolated from all micro-organisms in which the cytochrome a^ band at 630 m/i could be observed, and in small concentration in a few in which it had previously not been observed, e.g. Bacillus subtilis. Its amount paralleled the strength of the cytochrome Ao band. Further, anaerobically cultured bacteria con- taining no cytochrome a^ also failed to yield haemin a^- Aerobacter aerogenes was our main source. The bacterial haemins in acid ethereal solution showed an absorption band at 630 m/f not found in the spectrum of protohaemin. This was used as guide in the isolation procedure as ^603m///'£^635m/i- This Varied from 1-3 in cells with much cytochrome a^ to 1-0 in those with little, corresponding to a protohaem/haem a^ ratio of from 4-10. The separation of the green haemin from protohaemin was achieved by silica gel chromatography and freezing out of protohaemin together with phospholipids at — 70°C from acetone solutions. Purified haemin a^ had an ^603m;./^635m,. ratio of 3-18. Iron is removed more readily from haem a^ than from protohaem and even from haem a. This process could therefore be carried out with relatively low HCl concentration (0-4 % w/v) at room temperature. This yielded a chlorin (dihydroporphyrin). It is known that metals are less strongly bound by dihydroporphyrins than by porphyrins. Re-introduction of the iron was correspondingly more difficult, but restored haemin a.^- Further purification was achieved by silica gel chromatography, counter- current distribution in aqueous methanol-1 % HCl and fight petroleum, using as a guide the ratio £'653m///-^503m// which is 3-3 in the purest chlorin, and by HCl-fractionation. The last traces of lipid are held very fast, as by porphyrin a, and haemin and chlorin a^ remained oily. It is therefore likely that haemin ^3 contains a large alkyl side chain. Unfike haemin a, but fike cryptohaemin a, it accompanies protohaemin in the Rawlinson separation. Paper chromatography by the lutidine- water method of Nicholas and Rimington (1949) shows that chlorin a^ is a dicarboxylic acid, but fike porphyrin a its Rp (0-87) is somewhat higher than those of usual dicarboxyfic porphyrins (0-80). Haem a^ does not contain a carbonyl side chain ; neither the haemochrome nor the chlorin reacted with hydroxylamine. Like other chlorins, chlorin a.^ was converted into porphyrins by catalytic hydrogenation and reoxidation, and by hydriodic acid. Neither of these reactions proceeds, however, without alteration of side chains. Preliminary experiments of dehydrogenation of vinylchlorins by photo-oxidation in the presence of quinones have been carried out and this method is being studied with chlorin ^2. The product of the catalytic hydrogenation closely resembled The Structure of Porphyrin a, Cryptoporphyrin a and Chlorin di^ 355 mesoporphyrin, that of the hydriodic acid reaction monoethyl-monohydroxy- ethyl-deuteroporphyrin, porphyrins which are also obtained from proto- porphyrin under these conditions. This evidence would not exclude that chlorin a, is protochlorin with two vinyl side chains, perhaps with an additional large alkyl group. The presence of one vinyl group, possibly two, can be demonstrated by diazoacetic ester addition; the shift of the main absorption band in the red to the blue was like that of phaeophorbide a (with one vinyl), but also like that of protoporphyrin with two. The position of the absorption bands of chlorin a, resemble those of pyrrochlorin (with one vinyl) rather than those of mesochlorin or mesopyrrochlorin (with none), except that the whole absorption spectrum is somewhat contracted. However, the presence of an a-hydroxyethyl (or a-hydroxyalkyl) group is indicated by the low Rp values of chlorin a^ in kerosene-chloroform (0-32) and kerosene-propanol (0-55) compared with those of mesochlorin (0-89 and 0-88) and by the fact that acetylation in pyridine-acetic anhydride increased the Rp. It is therefore most hkely that chlorin a^ is a monovinyl-mono- hydroxyethyl-deuterochlorin, differing from protochlorin by the hydration of one vinyl group as well as by addition of a large alkyl group to one or the other (or both) of these side chains. Table 6. Absorption bands of haem a^ compounds and of chlorin oj (Barrett, 1956) A in m/f X in m/< Fe++ cytochrome a^ 628-630 Chlorin a^ 653, 598, 573, 534, 503, 405 in ether Fe++ CO-cytochrome a^ 635 Acid haematin (750) 603 Alkaline haematin 663 in20%HCl 647 Haem 618 in 10%HC1 630 CO-haem 619 Cu complex 613, 562, 526,401 Pyridine haemochrome 614 Zn complex 615. 564, 529,408 CN-haematin 604 CN-haem 618 The absorption maxima of haem a.^, chlorin a.^ and some of their compounds are given in Table 6. The ferrous cyanide compound of haem a.^ is more stable than its very unstable pyridine haemochrome. Since the position of the band of cytochrome a^ from Acetohacter suboxidans (Chin, 1952) coincides with that of pyridine haemochrome a^, cytochrome a^ may stand to cytochrome flg in the same relation as cytochrome a^ to cytochrome a, but the haemochrome of the Acetobacter peroxidans cytochrome has not yet been studied. Being an iron-dihydroporphyrin complex, haem a^ is an interesting intermediate between chlorophylls and haem compounds, a new type of 356 R. Lemberg, P. Clezy and J. Barrett intermediate, different from the magnesium porphyrins isolated by Granick and Bogorad (Granick, 1950; Bogorad and Granick, 1953) from Chlorella mutants. Chlorophyll in the chloroplasts, haem a in the mitochondria, and haem a^ in much smaller bacterial particles perhaps derived from the protoplasmic membrane (Moss, 1954; Tissieres, 1954) are all closely associated with phospholipids. While the lipid character of chlorophyll is due to its phytol ester group, neither haem a nor haem a^, are esters, but have lipophilic character owing to their large alkyl side chains. Nothing is as yet known about the oxidation-reduction potential of cyto- chrome fiTa- Nor is it as yet ascertained that cytochrome flg acts as oxidase in the cytochrome system of Aerobacter, although the balance of the evidence appears to be in favour of this hypothesis (Tissieres, 1952; Moss, 1952; Chance, 1953). The concentration of haemin a^, in Aerobacter is variable, but can be almost half as great as the concentration of haem a in ox heart muscle (14-43 mg/kg of dry weight). It is an induced enzyme, requiring oxygen for its formation, but in contrast to cytochrome oxidase is sensitive to iron-deficiency. While the recent studies of Layne and Nason (1958) and Horio (1958) do not yet exclude the possibihty that their oxidase preparations from Pseudomonas contain mixtures of cytochrome a.^ with cytochrome of type c, a double-headed enzyme containing haem Og as well as cytochrome c type prosthetic groups is another possibility. SUMMARY 1 . Porphyrin a, the iron-free prosthetic group of cytochrome oxidase, is a dicarboxylic porphyrin substituted with formyl, with alkylvinyl (on the pyrrole ring opposite to that bearing the formyl) and with an a-hydroxyalkyl on the pyrrole ring in between (pyrrole I). The alkylvinyl group and probably the a-hydroxyalkyl group contain large alkyl groups. Two partial structural formulae are suggested which are in harmony with the available evidence. 2. Cryptoporphyrin a is another formyl-porphyrin which occurs in heart muscle in the form of a haematin compound, though only in small amounts. It greatly resembles chlorocruoroporphyrin from which it appears to differ only by the presence of a large alkyl group on the vinyl side chain. 3. Chlorin a^ is the iron-free prosthetic group of cytochrome a^. It is a dihydroporphyrin without formyl groups and with one vinyl (or alkylvinyl) and one a-hydroxyalkyl side chain instead of the two vinyl side chains of protoporphyrin. Acknowledgement This research has been carried out with grants from the Australian National Health and Medical Research Council. The Structure of Porphyrin a, Cryptoporphyrin a and Chlorin a^ 357 ADDENDUM Piattelli in the laboratory of Nicolaus has recently obtained a 2 : 5-pyrrole dicarboxylic acid with propionic and nitrile side chains in the /5-positions by permanaganate oxidation of porphyrin a nitrile (private information). This estabhshes the formyl group in position 8 on ring IV (formula II of Fig. 3). Porphyrin a is thus 1:3: 5-trimethyl-2-a-hydroxyalkyl-4-/3-alkylvinyl-8- formyl-6 : 7-di(/^-carboxyethyl)-porphyrin. No ethyl-methyl-2 : 5-pyrrole-dicar- boxylic acid could be discovered among the oxidation products of hydro- genated porphyrin a. This lends further support to the vinyl group of porphyrin a in position 4 carrying an alkyl substituent. Piattelli and Nicolaus {R.C. Accad., Napoli, Ser. 4a, 26, p. 44, 1959) have now obtained the 2:5- pyrrole-dicarboxylic acid with methyl and carboxylic acid groups in the /^-positions; the carboxyl is here derived by oxidation of the unsaturated side chain in 4 and probably also the a-hydroxyalkyl side chain in 2 of porphyrin a (cf. also R. A. Nicolaus, Rassegna di Medicina sperimentale, 7, suppl. 2 (I960)). REFERENCES Barrett, J. & Lemberg, R. (1954). Nature, Land. 173, 213. Barrett, J. (1956). Biochem. J. 64, 626. Barrett, J. (1959). Nature, Loud. 183, 1 185. BoGORAD, L. & Granick, S. (1953). J. biol. Chem. 202, 793. Chance, B. (1953). J. biol. Chem. 202, 383. Chin, C. H. (1952). Abstr. Comm. 2nd int. Congr. Biochem. Paris p. 277. Clezy, p. & Barrett, J. (1959). Biochim. biophys. Acta 33, 584. Connelly, J., Morrison, M. & Stotz, E. (1958). J. biol. Chem. 233, 743. Dannenberg, H. & KiESE, M. (1952). Biochem. Z. 322, 395. Dixon, M. & Webb, E. C. (1958). Enzymes, p. 414, London: Longmans, Green & Co. Granick, S. (1950). J. biol. Chem. 183, 713. Granick, S., Bogorad, L. & Jaffe, H. (1953). J. biol. Chem. 202, 801. HoRio, T. (1958). J. Biochem. Tokyo 45, 195, 267. KiESE, M. & KuRZ, H. (1954). Biochem. J. 325, 299. Layne, E. C. & Nason, a. (1958). J. biol. Chem. 231, 889. Lemberg, R. & Falk, J. E. (1951). Biochem. J. 49, 674. Lemberg, R. (1953). Nature, Lond. 112, 619. Lemberg, R. & Stewart, M. (1955). Aust. J. exp. Biol. med. Sci. 33, 451. Lemberg, R. & Parker, J. (1955). Aust. J. exp. Biol. med. Sci. 33, 483. Marks, G. S., Dougall, D. K., Bullock, E. & MacDonald, S. F. (1959). J. Amer. chem. Soc. 81, 250. Morrison, M., Connelly, J. & Stotz, E. (1958). Biochim. biophys. Acta 27, 214. Moss, F. (1952). Aust. J. exp. Biol. med. Sci. 30, 531. Moss, F. (1954). Aust. J. exp. Biol. med. Sci. 32, 571. Nicholas, R. E. & Rimington, C. (1949). Scand. J. din. Lab. Invest. 4, 12. Parker, J. (1959). Biochim. biophys. Acta 35, 496. Rawlinson, W. a. & Hale, J. H. (1949). Biochem. J. 45, 247. Rimington, C, Hale, J. H., Rawlinson, W. A., Lemberg, R. & Falk, J. E. (1949). Abstr. Comm. \st int. Congr. Biochem. Cambridge p. 379. Tissieres, a. (1952). Biochem. J. 50, 279; Nature, Lond. 169, 880. Tissieres, a. (1954). Nature, Lond. 174, 183. Warburg, O. & Gewitz, H. S. (1951). Hoppe-Seyl. Z. 288, 1. Warburg, O. & Gewitz, H. S. (1953). Hoppe-Seyl. Z. 292, 174. 358 Discussion DISCUSSION The Structure of Haem a and Haem a. The Structure of Porphyrin a By M. Morrison (Rochester) Morrison: Barrett {Nature, Lond. 183, 1185, 1959) and Clezy and Barrett (Biochim. biophys. Acta 33, 584, 1959) recently reported that porphyrin a contains a hydroxy! group that can be acetylated and that this group is present as — CHOH — group adjoining the porphyrin ring. Haemin a also can be acetylated by the procedure of Barrett. It is converted to two products, one with increased chromatographic mobility and the other with decreased chromatographic mobility in non-polar solvents. This acetylation causes a spectral shift of the a-band of the pyridine haemochrome to shorter wavelengths (cf. Table 1). This indicates that an electron-withdrawing group has been removed from resonance with the porphyrin nucleus. The position of the a-peak of the pyridine haemochrome is an excellent index of the number and type of electron-withdrawing groups in resonance with the porphyrin nucleus. Two of these groups are known to be formyl and vinyl. A comparison of haemochrome a (a-peak 587 m/0 with Spirographis haemochrome (a-peak 583 m//) and of the haemochromes of their respective oximes (571 m/f and 561 m//) also indicates the presence of a third electron-withdrawing group in addition to formyl and vinyl. It should be noted that the effects of the electron-withdrawing groups except vinyl are lost on reduction with borohydride (cf. Table 1). Table 1 Compound Position of the a-peak of pyridine haemochrome Haem a 587 Oxime 571 Br;, addition 582 BH4- reduction 552 Dimedon 558 Acetylation Spirographis Haem Oxime 582 583 561 Since the infra-red spectrum of haemin a suggested the presence of another carbonyl group in addition to formyl, one can speculate that the third group is — CH(OH) — CO — R. The effect of this group is abolished by borohydride reduction and by acetylation. Since the formyl and vinyl groups must occupy opposite pyrroles, this group must occupy position 2, and if the formyl group occupies position 8 (according to the evidence of Piattelli quoted by Lemberg, p. 357), the formula of Lemberg et a/., p. 350, Fig. 3 (11), but replacing -CHOH— CHj-R, in position 2 by — CHOH-CO — R, can be assumed for porphyrin a. From the molecular weight of chlorohaemin a (880) it can be calculated that the R group has a molecular weight of 182 and might represent a 13 carbon saturated alkyl: — CH(OH) — CO — (CH2)i2CH3. Clezy: Morrison's formula shows an a ketol conjugated with the porphyrin ring system. It is difficult to reconcile this structure with the dehydration experiments reported in the paper by Lemberg, Clezy and Barrett (this volume, p. 349). Porphyrin a and its derivatives can be dehydrated with />-toluene sulphonyl chloride to a compound showing spectroscopic properties in accord with the introduction of a carbon-carbon double bond conjugated with the porphyrin ring system. This reaction requires at least one hydrogen atom on the carbon atom ^ to the porphyrin ring system. Lemberg : The conclusions of the Rochester school on the structure of porphyrin a as reported by Morrison are now in close agreement with ours, with the exception of The Structure of Porphyrin a, Cryptoporphyrin a and Chlorin ao 359 two points, namely, (a) the formulation of the side chain in position 2 as — CH(OH)— CO — R, and (b) the distribution of the extra alkyl groups between the positions 2 and 4. The analyses of haemin a would appear to allow the presence of a seventh oxygen atom, assumed by Morrison in the — CH(OH) — CO — R side chain. We have observed a shift of 2 m/< of bands III and IV of porphyrin a on acetylation; on hydrolysis the spectrum of porphyrin a is restored unaltered. Morrison observed a similar shift of 5 m/t in the position of the a-band of the pyridine haemochrome. These observations may indicate that the group formulated by us as a-hydroxyalkyl group is more complicated and bears another oxygen atom. However, the presence of a — CH(OH) — CO — grouping appears unlikely since it is not in harmony with our dehydration of a-hydroxyalkyl to )?-alkylvinyl by /7-toluenesuIphonyl-chloride. We have evidence indicating that the a-hydroxyalkyl group is not a-hydroxyethyl. The ketonylporphyrins obtained by the resorcinol melt of haemins after cautious oxidation of a-hydroxyalkyl to a-ketonyl by chromic acid differed from acetylpor- phyrins by higher HCl-numbers and higher /?/• in chloroform-kerosene. In particular the ketonylporphyrin thus obtained after conversion of formyl to methyl by the Wolff-Kishner reaction was not monoacetyl-deuteroporphyrin. Again, the porphyrin R HCOH M m/ \cho HOHjCx^ P 'M (obtained by a succession of reactions from porphyrin a) which differs from hydrated isochlorocruoroporphyrin only by the replacement of methyl by hydroxymethyl, should have a low HCl-number, if R is CH3. In fact, it has a high HCl-number and still shows the a -> /? conversion typical for porphyrin a. There is some evidence that the vinyl group in 4 is also substituted. More important than Warburg's inability to obtain cytodeuteroporphyrin from hydrogenated haemin a in the resorcinol melt, is Piattelli's recent finding (see p. 357, this volume), that no /3-ethyl-/J-methyl-pyrrole-2 : 5-dicarboxylic acid can be obtained by permanganate oxidation of hydrogenated porphyrin a. The fact that the alkyl group substitutes the vinyl group in /J, not in a is shown by the possibility of oxidizing it to formyl. Either the additional carbon atoms are distributed between a-hydroxyalkyl in position 2 and /S-alkylvinyl in position 4, or these two groups are interconnected by an alicyclic polymembered ring. As we can now assume that these two groups replace vinyl, not methyl, of proto- porphyrin, a mode of biosynthesis of haem a different from those discussed in the pre- circulated paper must be postulated, i.e. oxidation of the two vinyls to formyl and coupling with a saturated fatty acid whose a-CHj is activated, perhaps by coenzyme A, followed by hydration of one of the two groups; or if an alicyclic ring is formed between 2 and 4, by condensation of the two formyls with a similar compound of a saturated dicarboxylic fatty acid. Winfield: I should like to ask Lemberg by which method the molecular weight of his porphyrin was determined. If it was not by Brintzinger's diffusion method, I suggest that this might be used to advantage. For some compounds it is possible to determine the molecular weight with considerable accuracy, and with no necessity for pure preparations. It might well be possible to determine from the molecular weight the number of oxygen atoms present in the porphyrin. Lemberg : For the determination of the molecular weight of porphyrin a or haemin a we have relied either on the iron content of the haemin, or on the ratios of the specific extinctions of porphyrin a and its molar extinction as established by copper titration, or on the specific extinction of pyridine haemochrome a and its molar extinction as established on the basis of its iron content. These methods all depend on the purity of the preparation, but give concordant results for the purest preparations which agree with those of Warburg and of Morrison. As Winfield has pointed out, the diffusion method has the advantage of being not 360 Discussion dependent on purity. However, it may not overcome the difficulty of the formation of rather firm linkages between the porphyrin and lipids which we have found. Estabrook: I would like to ask Lemberg about the 2-a hydroxy in the structure of haemin a. The presence of such a hydroxy group confers assymmetry upon the associated carbon and should introduce optical activity to haemin a. Is haemin a optically active ? Lemberg: We have no evidence on optical activity. Morrison: We have looked for optical activity in many ways, but have been unable to detect it. Williams : Since the — CHOH group sits on a formylporphyrin ring, racemization might easily occur. Estabrook: What is the evidence for the presence of the 2-a hydroxy group? Lemberg : The evidence is as follows : (a) Acetylation with alterations of Rp. (b) Dehydration with /7-toluene-sulphonylchloride in benzene, shifting absorption to longer wavelengths. (c) Oxidation (by chromic acid) of CHOH — R to CO — R with the formation of an a-ketonylporphyrin. The Properties of Haem a^ and Cytochrome a<^ By J. Barrett (Sydney) and J. P. Williams (Oxford) Williams: We should like to make certain observations about cytochrome a^. The prosthetic group is a dihydro-porphyrin and therefore has one p/C value much lower than either of those of simple porphyrins (Conant and Dietz, /. Amer. chem. Soc. 56, 2185, 1934). Reduction in p/r of the ligand reduces the stability of the iron com- plexes, ferric much more than ferrous (Williams, Chem. Rev. 1956; Falk and Perrin, this volume, p. 56). Thus where the further co-ordination of an iron-protoporphyrin and an iron-dihydroporphyrin to a protein are the same, equilibration between the ferrous and the ferric form of the chlorin should favour the ferrous. The addition of two hydrogen atoms to the beta position of one of the pyrrole rings results in an altered resonance pathway which is now markedly asymmetrical. As a consequence the stability of the low-spin forms relative to the high-spin states will be reduced. These changes from a simple porphyrin make the removal of iron from haem a^ relatively easy, as is observed. The model compounds of the iron-dihydroporphyrin are also of interest. Using Barrett's data (this volume, p. 355) we note the following: (a) CO and CN~ move the peaks to longer wavelengths relative to the pyridine haemochrome but less than expected from comparison with protoporphyrin and mesoporphyrin haemochromes. (b) The pyridine haemochrome is rather unstable. Both (a) and (b) are in keeping with the suggestion that the pyridine haemochromes are not 100% low-spin complexes. (c) The acid haematin gives a band at 740 m/< which could be the charge-transfer band in these complexes. If this is so then the anomalous band-position of the hydroxide at 662 m/i would be a charge-transfer band too. We now consider cytochrome a^ itself. The following points suggest that it is at least partly in the high-spin state in both the reduced and oxidized forms : In the reduced form: (1) the band max. 628-630 m/< is at a considerably longer wavelength than the pyridine haemochrome; (2) a CO complex is readily formed; (3) an O2 complex is obtained (Horio, this volume, p. 315). In the oxidized form: there is a band at 750 m/< similar to that in the acid haematin compound. This is the charge-transfer band typical of ionic ferric species. Following the remarks made about model complexes which pick up oxygen rever- sibly (Williams, this volume, p. 49) we conclude that the protein binds iron through an imidazole group. If this conjecture is correct the redox potential of cytochrome a^ will be about -f 0-30 V. We predict that (1) the value of A m// (Soret band) between the Fe++ and Fe+"'""'' The Structure of Porphyrin a, Cryptoporphyrin a and Chlorin a, 361 forms will be large; (2) the ratio — will be larger for cytochrome a.^^ than for pyridine haemochrome a^. In some bacteria where cytochrome a^ occurs, cytochromes a^^ and b^ are also found. In others, cytochrome 02 occurs with c type cytochromes (as well as cytochromes aj and 61). Castor and Chance {J. biol. Chem. 234, 1587, 1959) have demonstrated that in some bacteria cytochrome a^ can function as an oxidase and that it also combines with carbon monoxide. Nevertheless it appears to us that in other organisms it may function similarly to cytochrome c in other electron transporting sequences. Like cytochrome c, cytochrome a^ appears to be a di-imidazole complex from consideration of its spectroscopic properties, which resemble those of pyridine haemochrome a. The redox potential of cytochrome Oj we would expect to have a value around 0-20 V. We then speculate that in bacteria there exist the transport systems: O2 02 ^1 ^i substrate O2 02 (^ b substrate E° = 0-30 0-20 005 These systems are very like the graded redox systems of animal cells. Terminal oxidase systems with a redox potential around 0-30 V can utilize oxygen with an almost optimal efficiency. There are now two ways of raising the redox potential and destabilizing the iron-protein complexes sufficiently for such efficient reactions to occur with oxygen (1) by introducing — CHO groups into the porphyrin ring, e.g. porphyrin a; (2) by addition of hydrogen to one of the pyrrole rings, e.g. chlorin 02- In these proceedings it has been indicated why a chain of catalysts is required. In Pseudomonas oxidase, Horio (this volume, p. 302) has found that the oxygen-combining haem a^ is accompanied by a type c haem. Okunuki and his school in their studies on "cytochrome a" have brought forward evidence that the initial oxidase combines with oxygen reversibly, and that it is only the presence of a second cytochrome which enables it to act as a cytochrome oxidase and not as an oxygen transporting haem- protein. Such may be the case with cytochrome a^. Evidently more model experiments are required to establish this argument. Extractability of Ferro- and Ferricytochrome c Slater: In Morrison's experiments, is the degree of extractability of cytochrome c from mitochondria a function of the degree of swelling of the mitochondria? Morrison: I do not believe that the degree of swelling of the mitochondria has much effect on the extractability of the cytochrome c since the structure of the mitochondria is destroyed by the acetone and salt treatment. Margoliash: Your treatment with boiling acetone would be expected to denature cyto- chrome c, and the degree of this denaturation might depend on its oxidation state. Legge: Theorell's earliest preparation of cytochrome c made use of the differential adsorption of oxidized and reduced cytochrome c on cellophane. If there is doubt as to whether cytochrome c is denatured by hot acetone, there would be no doubt that the other proteins would be, and therefore perhaps be able to adsorb the oxidized and reduced cytochrome differentially. Henderson : In connexion with the extraction of cytochrome c from mitochondria treated with boiling acetone and then at 60'C, your experiments indicate that the well-known difference between the adsorbability of Fe+++- and Fe++-cytochrome c is not involved. Have you considered, however, the difference in the degree of modification brought about by this treatment on Fe+++ as compared with Fe++ cytochrome c? Okunuki's group (see, e.g. Hagihara et al. Nature, Land. 181, 1588, 1958) have reported that Fe+++-cytochrome c is considerably more susceptible to modification than Fe++- cytochrome c and that this is reflected largely in the increased adsorbability of the former. It would seem that this could explain the observed results without implicating the extraction of a 'cytochrome coupler compound'. ■wii^Bi Miff m 'M-M:M »!;W'l'y 'f/ji