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THE COORDINATION AND IRON CHEMISTRY PORPHYRIN OF SOME COBALT COMPLEXES Janine Aron A Dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, for the Degree of Master of Science May 1986 C O R RI N O ID ABSTRACT The r6le of conformationa change in the protein's control of the active metal site in haemoproteins and enzymes is examined with (i) cobalt corrinoid cofacvors for the B -dependent isomerases, (ii) cytochrome c, and (iii) a model for cytochrome c, microperoxidase-8. The half-lives (t/$) of homolytic fission of the cobalt carbon bond ir the B coenzyme, adenosylcobalamin, and in methyl, ethyl and cyclobutylcobalamins have been determined in aqueous solution at 96 °C. The t!£ values increase (rates decrease) in the following ligand order: ethyl (ca. 9 min) < cyclobutyl (ca. 75 min) < adeno^yl (c*». '80 min) < methy) (ca. 80 hrs). These values have been used to provide an estimation of ca. 10 years for the rate in the protein-free coenzyme at room temperature. Comparison with published data on the B -isomer^ses shows that the Co-C bond in the coenzyme is lat^jlisea by ^ 10' when bound by the apoenzymo and by a further ^ 10 in the p r e s e n c e of substrate to achieve the overall labilisation of >,10 (i.e. t’ >} ■ v 2.5 ms) required for the enzymatic reaction. The pK for the alkaline isomerisation of hor?*e-heart cytochrome c (from Type III to Type IV, with cleavage of the iron-methionine (Fe-S) bond) has been confirmed to be 9.05 at 25°C, and extinction coefficients for Type III and Type IV at 695 nm are 867 and 83 M cm respectively. Binding studies with cytochrome c in aqueous solution at 25 °C monitored the 695 nm charge-transfer absorbance band, present when the Fe(III/-S bond is intact. The specific binding of the ^perchlorate anion to cytochrome c occurs with K ( ca. 5 x 10 M (1:1 complex) and coulombic and chaotropic effects are implicated. Donor-acceptor complexes of caffeine and tryptophol with cytochrome c are formed where ^ is ca. 6 x 10 M (1:1 complex) and K is ca. 9 x 10~£ M(?*?2 complex), respectively. Other drugs, including chlorpro m a z i n e, procaine and indole acetic acid, demonstrate thes»* »- * interactions with cytochror c and induce Fe-S bond cleavage. An impro ithod for the preparation and purification of microperoxida^ (MP-8) and a TLC method as a rapid test of purity, have been developed. The binding of thioethers to MP-8 (possible models for the Fe-S bond in cytochrome c) ana of drugs to MP-8 has been studied in 20* Me0H:H,0 (v/v> solution at 25 °C, where 2 x 10 M MP-8 is > ^7% monomeric at pH 7.4. N-acetyl-D, L-m*thionin«* binds to MP-8 with K ca. 1.9 M~ at pH 7.4 and K ca. 2.1 M at pH 6.7 (1:1 compleSPs): the 695 nm band characteristic of th" cytochrome c Fe-S bnnci is exhibited D,L-methionine binds to MP-8 with K ca. 155 M at pH 7.4 (1:1 complex). Doth caffein*- and tryptojSRol form 1:1 donor-acceptor complexes with MP-8: K is ca. 151 M~ at pH 7.4 and ca. 240 M at pH 6.7, for caffeinP? and K is ca. 60 M at pH 7.4, for tryptophol. Other drug molecfi?es, including chlorpromazine, demonstrate these n - ti interactions with MP-8. Donor-acceptor complexes are formed with greater ease with these mode) complexes than with cytochrome c. (iii) Declaration I hereby declare that the work carried out exclusively reported by me and that in this dissertation was the dissertation has not been submitted for a degree at any other university. T & m r u . ,-lrov-L Janine Aron And a H**v«n }n «i Wild Flow#r, Hold Infinity in th* p«]« of yoyr h»nd And in «n hour. tftlllM Blah* ACKNOWLEDGEMENTS I should like to expreaa my gratitude to the following: Dr. D. Baldwin and Professor J. Pratt, my supervisors, for their guidance and encouragement; Mrs. I. Aron, Mr. J. Unterhaltsr and Dr. B. Unterhaltcr for their kindness, generosity and support; Dr. S. Chemaly and Mr. H. Marques for helpful discussion and advice; The technical staff of the Department of Chemistr>, in particular: Mr. J. Zimmermann, Mr. S. van der Schyff, Rev. a . Molefe, Mr. A. Thoane and Mr. B. Fairbrother; Dr. P. Adams for permission to report the HPLC results concerning microperoxidase-B prepared in this study; Mr A. Domleo of Glaxo-Allenbury (S.A.) (Pty) Limited for samples of vitamin , and vitamin Maybaker (S.A.) (Pty) Limited for the gift of chlorpr< ni»xine; Mrs. L. Meredith for typing this dissertation; Mrs. M. Crabb for technical assistance with diagrams; Mrs. S. Dobson, Mr. B. Jersky, Ms. assistance with proofreading. J. Mailer and Mr. G. Yeo for (Vi) TABLE OF CONTENTS CHAPTER 1 - INTRODUCTION 1 1.1 Metallo-enzymes 1 1.2 Cobalt corrinoids used in this work ? 1.3 Iron porphyrins ured in this work 11 1.4 The aims of this dissertation 23 CHAPTER 2 - MATERIALS h NI) METHODS 24 2.1 Materials 24 2.2 Methods 26 CHAPTER 3 - THE PREPARATION AND PURIFICATION OK ORGANOCORRINOIDS 3.1 3V Features of preparation, purification and identification 37 3.2 The preparation of organocobalamins 38 3.3 The preparation of oiganocob*namid*5 39 3.4 Summary 44 CHAPTER 4 - HOMOLYTIC THERMOLYSIS OF ETHYL, METHYL AND CYCLOBDTYLCOBAl.AMINi; AND CORRESPONDING COBINAM IDES, AND ADENOSYLCOBALAMIN 45 4.1 Introduction 45 4.2 i-Ellmtnation and homolytic fission 45 4.3 The rflle of steric strain in cobalt-carbon bond cleavage 46 4.4 Photolysis of organoccbalamins 48 4.5 Adenosy lcoba 1amin-d»*pendent isomerase reactions 50 4.6 The 51 4.7 Experimental procedure 54 4.8 Results 55 4.9 Evidence for homolytic thermolysis 63 4.10 The room rflle of the protein in isomerase reactions order of ligands temperature and half-1 iff decomposition of the coenzyme estimation of the for homolytic 64 (vii ) TABLE OF CONTENTS (contd.) Pace 4.11 4.12 Estimation of the labilising rfile of the protoin 64 Summary 66 CHAPTER 5 - STUD IKb >'?! THE REACTIVITY 0.T THE IRONKKTHIONINK BOND IN CYTOCHROME 68 C 5.1 Introduction 68 5.2 Experimental procedure 72 5.3 The usp of unpurified commercial cytochrome c without further 5.4 5.5 oxidation 74 The pK of the alkaline isomerisation and a calculation of extinction coefficient* at 695 nm 75 The binding of small molecules and drugs to cytochroaie c 81 5.6 Disruption 93 5.7 Summary 99 CHAPTER 6 - PREPARATION OE NICROPEROXIDASE-8 AND ITS ANALYSIS BY THIN-LAYER CHROMATOGRAPHY 101 6.1 Introduction 1C1 6.2 Experimental procedure 1C6 6.3 The preparation of mlcroperoxidase-8 lCd 6.4 The TLC system and R 108 6.5 values “It HPLC results for microperoxidase-8 112 6.6 Discussion 114 6.7 Summary 117 CHAPTER 7 - EQUILIBRIUM STUDIES WITH MICROPEROXIDASE-8 IN AQUEOUS-MKTHANOL SOLUTION 118 7.1 Introduction 118 7.2 Experimental procedure 124 7.3 A monomeric system 125 7.4 Micror r o x idaso-8/thioetht'r complt*x«'s as models for cytochrome c 7.5 7.6 127 Donor-acceptor interactions in microperoxidase8/drug complexes 133 Summary 1 37 (v i ii ) TABLE OF CONTENTS (contd.) Pa8 e CHAPTER 8 - SUMMARY AND CONCLUSIONS APPENDIX 138 - Derivation of equations for kinetic studies (Chapter 4) and equilibrium studies (Chapters 5 and 7) REFERENCES 141 146 (ix) L IST OF TABLES Page Table 1 ,1 E:>zyme reactiona requiring cobalt corrinoids Table 3 .1 UV-visible absorption spectra for 10 organo- corrinoids in this work Table 3 ,2 values of some 43 corrinoid complexes in Solvent I Table 4 ,1 The 44 half-lives of decomposition organocorrinoid complexes oxygen, at pH 6 and 7 under of some nitrogen and (p H - i n d e p e n d e n t ), at 96°C Table 5 ,1 62 Recent determinations of the alkaline isomerisation and and IV of cytoc Table 5 .2 Equilibrium perchlorate pK for the a for Types III one c constants 79 for to cytochrome c the binding of -4 (ca. 1 x 10 M) in alkaline solution Table 5 ,3 Equilibrium constants 86 for the binding of caffeine and tryptophoJ. to cytochrome c (ca. •4 1 x 10 M) in alk* 'ne solution Table 6 1 HPLC analysis of C‘ 'errial MP-8 samples %ni those prepared as „*-• Table 7 1 88 Equilibrium various constants ligands to 2 x 10"' M) in 20* fneOW .bed in the text for the monomeric binding MP-8 ,0 (v/v) solution 1*4 of (ca. 133 (x) LIST OF FIGURES Page Fig. 1.1 Haemin chloride (Fo(III) protoporphyrin IX 3 chloride) Fig. 1.2 The molecular structure of Fig. 1.3 (i) 4 , The structure of the cob^lamin coenzyme (ii) The 5'-deoxyadenosy1 ligand present in coenzyme 6 Fig. 1.4 The structure of cobinamides 7 Fig. 1.5 Organocorrinoid reactions 9 Fig. 1.6 Prosthetic groups of the cytochromes Fig. 1.7. Cytochrome c in Fig. 1.8 The structure of cytochrome c 14 Fig. 1.9 The structure of microperoxidase-8 20 Fig. 3.1 Absorption 1? muitienzyme system spectra of Bj , aquocyanocobinamide ( — Fig. 4.1 12 ( —— ) and ) in wa*er 41 Scheme of possible reaction mechanisms for the isomerase enzymes Fig. 4.2 Scheme of 50 reactions initiated by homolytic fission of th** Co-C bond in or&anoccrrinolds; R may be methyl, ethyl or ad«-ro*;.'l, and Co-R denotes five - and/or six- coordit.ate species Fig. 4.3 Spectral changes deccripos i t ion of under nitrogen: obser d taer.osyl during r>al«tnn at 53 the 9*. °C A ■ 2 mlnu.;'«, B •> 66 minutes, C ■ 145 minutes and D ■ 34* -.M.-nites FI*. 4.4 First-order kinetic plots 56 . ► *< decomposition at 96 °C of a) a d e n o s y l c o t ' '- >> under nitrogen and b) e*hylcobalamln und« » 'hy***n Fig. 4.5 Spectral changes obs decomposition of e thy 1 t 56 during the 1amir. at 96 °C under nitrogen: A ■ 1 minute, K ■ minutes, C *1 18 minutes «»nd D • 37 minutes Fig. 4.6 Spectral changes 58 obf»**rved during decomposition of ethyIcoblnamlde at 96 7, under oxygen: A - 3 minutes, the #C, B pH ■ 46 minutes, C ■ 264 minutes and D ■ 1598 minutes 58 LIST OF FIGURES (contd.) Fig. 4.7 First-order kinetic plot of the decomposition at 96 °C of etnylcobinamide under oxygen Fig. 4.8 First-order kinetic plot of the decomposition at 96 °C of methylcobnlamin under oxygen Fig. 4.9 First-order kinetic plot of at 96 #C Fig. 5.1 the decomposition of cyclobutylcobalamin under nitrogen Relationship of haem to the polypeptide chain, to the bottom of its crevice and to the surface of the molecule Fig. 5.2 The correction method of Kaminsky et a l . (1973) for background absorbance fig. 5.3 The effect of purification and oxidisation (with K^FefCN)^) of cytochrome c on the binding of sodium p«.rchl rate: V purified cytochr me c; • unpurified cytochrome c; o purified and oxidised cytochrome c Fig. 5.4 Acid-base titration of the 695 nm absorption -4 band of 1.13 x 10 M cytocorome c at 25 #C: (a) Af/)S against pH; (b) A^iJt (corr.) against pH (u - 0.05) Fig. 5.5 The data Fig. 5.4 (a) are plotted according to equation 10 (Section 5.4.] Fig. 5.6 Spectra of a titration of the 695 nm absorption ..4 band of ca. 1 x 10 M cytochrome c by sodium p e r c h l o r a t w , pH 9.0: A « 0 M NnC<0.; Fig. 5.7 B • 0.014 M NaC<0.; C - 0.053 M NaC«0„; D • 0.14h M 4 4 NaCIO. 4 The corrected absorbance values at 695 nm as a function of sodium porchloratr concentration: A ■ pH 8.8, B ■ pH 9.0, C ■ pH 9.3 and D ■ pH -4 9.5; cytochrome c concentration ca. 1 x 10 M (tris-hydrofhloric acid buffers, p ■ 0.1) Fig. 5.8 The binding of sodium perchlorate to ca. -4 1 x 10 M cytochrome c: A - pH 8.8, B • pH 9.0, C ■ pH 9.3 and D • pH 9.5 chloric acid buffers, u ■ 0.1) (tris-hydro- (xii ) LIST t FIGURES (contd. ) Pa*e Fig. .9 The plots of «og *^app against pH for perchlorate, caffeine and tryptophol binding to >4 c*. 1 x 10 M cytochrome c. The vhJuo ol <?og K is given by the ordinate intercept 85 -4 Fig. .10 The 695 nm absorbance cytochrome c as a of "a. 1 of the finctlon x 10 M increased 86 concentration of sodium nitrate -4 Fig. .1. The binding cytochrome of c: caffeine to A • 8.8, ca. 1 B ■ 9.3 x 10 and M C • 9.5 (tris-hydrochlorlc acid buffers, u ■ 0.1) 89 -4 Fig. .12 The binding of tryptophol to ca. 1 x 10 M cytochrome c: A • pH 9.0, B ■ pH 9.3 and C ■ pH 9.5 Fig. .13 (tris-hydrochloric acid buffers, u » 0.1) Representation of the electric potential generated within human from the left cross-section of the which field ferricytochrome (source: Osneroff et a l . (1980)). protein lies c The view looking approximately 89 is at a along tne dipole axis and contains both the top haem crevice salt bridge between of the lysine 13 and the c-amino group a-carboxyl group glutamic acid 90 and the bottom hydrogen of bond between the c-amino group of lycine 79 and the backbone carbonyl of residue 47. The Italicized numbers Fig. .1 denote the strength of the electric potential lines, in kTD |eD w p The amino acid sequences of the undecapeptlJe of and cytochrome c (MP-11), 96 its tryptic product,the octapeptide (MP-H) Fig. .2 Preparation of MP-8: column 103 chromatography elution profiles. Solvent: 0.1 M NH.HC0*, room 4 3 temperature. Pooled fractions are indicated by arrows. (i) Peptic digestion product of cytochrome c on 4 x 100 cm Siogel P6 at 20 -2 -1 m<cm hr , 2.00 m€ fractions: A ■undigested cytochrome c, B ■ MP-11, C-E » smaller (xii) LIST OF FIGURES (contd.) Page Fig. 5.9 The plots tog of ^app ag a inBt pH f°r perchlorate, caffeine and tryptophol binding to -4 ca. 1 x 10 M cytochrome c. The value of €og is given by the ordinate intercept Fig. 5.10 T.ie 695 nm absorbance cytochrome c as a of ca. 1 of the function 85 x 10 1 M increased 86 concentration of sodium nitrate -4 Fig. 5.11 The binding cytochrome of c: caffeine to A « 8.8, ca. 1 B * 9.3 x 10 and M C ■ 9.5 (tris-hydrochloric acid buffers, u * 0.1) 89 -4 Fig. 5.12 The binding of tryptophol to ca. 1 x 10 M cytochrome c: A « pH 9.0, B » pH 9.3 and C * pH 9.5 Fig. 5.13 (t n s - h y d r o c h l o r i c acid buffers, u * 0.1) Representation of the electric potential generated within human from the left cross-section of the which field ferricytochrome (source: Osheroff et al . (1980)). protein lies c The view looking approximately 89 at is a along the dipole axis and contains both the top haem crevice salt bridge between of lysine 13 and the the f-amino group a-carboxyl group glutamic acid 90 and the bottom hydrogen if bond between the c-amino group of lysine 79 and the backbone carbonyl of residue 47. The <talicized numbers Fig. 6.1 denote the strength of the electric potential lines, in kTD |eD w p The amino acid sequences of the undecapeptide of and cytochrome r 96 (MP-11), its tryptic product, th»* octapeptide (MP-8) Fig. 6.2 Preparation of MP-8: column elution profiles. Solvent: temperature. arrows. 103 chromatography O.i M NHjHCO^, room Pooled fractions arc indicated by (i) Peptic digestion product of cytochrome c on 4 x 100 cm Biogel P6 at 20 -2 -1 m€cm hr , 2.00 m€ fractions: A = undigested cytochrome c, B = MP-11, C-E = smaller (xiii) LIoT OF FIGURES (contd.) Pe.ge unidentified haem-pe p t i d e s . (ii) Tryptic digestion product of MP-11, as in (i) but A with * B =■MP-8, C ■ unidentified haem-o e p t i d e . (iii) Purification of MP-8 on 1.5 x 30 cm Sepnadex - 2 -1 G-50 Superfine, 4 m€cm hr , 2.00 m< fractions: A, B * MP-11 and an unidentified impurity; C * MP-8 Fig. 6.3 109 The separation of a mixture of haem-peptiries on silica gel water/88 0.144 g TLC mt in the rolvent sec-butai.ol/0.2 KCN: A » system m< 0.88 M unidentified impurity found in commercial 88 m< NH,/ haem-peptide MP-8; B * MP-8; C * MP-11; D • cytochrome c Fig. 6.4 HPLC analysis of MP-8 on temperature. 25 - 5C sample gradient 2.25, B = increased Vydac A * TEAP acetoritrile linearly C-18 at injection of eluted; 60/40 111 1 mg/m< buffer, in to 60% B over room A: 20 10% min pH B and then to 95% B over 0.5 min. Detection at 395 nm: purification a) This work, final on Sephadex G-50; b) Commercial MP-8 (Sigma) 113 Fig. 7.1 The structure of microperoxidase-8 119 Fig. 7.2 Beer's Law plot for MP-8 at 25 °C, u * 0.1, pH 7.4 (phosphate) in 1 cm and 10 cm cells in 20% MeOH: H^O (v/v). The ordinate values ore normalised for 1 cm cells Fig. 7.3 128 The binding of N-acetyl-D,L-methionine to ca. 2 x 10"'' M MP-8 in 20% Me0H:H20 (v/v) solution at 25 °C in « 0.1 (phosphate)): A ■ pH 6.7; B * pH 7.4 131 -5 Fig. 7 •4 Absorption spectra of ca. 1.6 M Soret N-acetylregion D ,L-meth i o n i n e , pH A: methionine adduct; A: cytochrome adduct; C: MP-8 2 x 10 c; MP-8; (ii) B: near : M MP-8 7.0: in (i) MP-8/N-acety1infra-red region MP-8/N-acetyl-methionine 1 31 (x i v ) LIST OF FIGURFS (contd). Fig. 7.5 Spectral changes 2 x 10‘6 for M a titration MP-8 in 20% of ca. MeOH: H ,0 solution, pH 7.4 at 25 °C, with D ,L-methionine Fig. 7.6 The binding ot D ,L-methionine to ca. 2 x 10 (v/v) 132 M MP-8 in 20% Me0H:H,0 (v/v) solution at 25 °C (u ■ 0.1 (phosphate)) at pH 7.4 Fig. 7.7 Spectral charges 2 for a 132 titration of ca. x 10~h M MP-8 in 20% Me0H:H,0 (v/v) solution, pH 7.4 at 25 #C, with caffeine Fig. 7.8 135 The binding of caffeine to ca. 2 x I0“6 M MP-8 in 20% M.;0H:H?0 (v/v) solution at 2 5 8C (u « 0.1 (phosphate)) at pH 7.4 Fig. 7.9 The binding of MP-8 in 20% 135 tryptophol M e O H :H ,0 to ca. (v/v) 2 x solution (u ■ 0.1 (phosphate)) at pH 7.4 10~6 at M 25°C 136 CHAPTER 1 - INTRODUCTION 1.1 M e t a llo-enzywes Enzymes are biological catalysts found throughout the plant animal kingdoms which catalyse a diversity of reactions and essential to life. The metabolic rates of hundreds of pathways ire controlled by enz/mes; and they usually steric, for one display high or a substrates. Metallo-enzymes small specificity, number complex cobalt with one corrinoids) cloaely form a sub-group of represent a partnernhip between a metal metal of or more providing the enzymes, (e.g. active iron or complex ( ‘ prosthetic group', the enzyme. metabolism Metallo-enzymes cata *ite, such as the or important enzymatic and a metal. The together w. 'co-factor* are and porphyrins, protein which controls and enhances the activity protein is termed the 'apoenzyme* and, related ion (e.g. copper(II)) or a ligands the both chemical ie rr,e*al ion 'coenzyme'), in fixation many of forms areas of nitrogen (by nitrogenase enzyme*), energy production or interconversion (e.g. by cytochromes) ana detoxification mechanisms in the body (e.g. by p**roxliases, catalases and cytochrome P-450). Some of the enzymatic reactions do not have analogues among protein-free complexes, as carbon (tkMeton isomerisation b/ the isomeras** enzymes such (which incorporate cobalt corrlnoid co-factors). The enhanced catalytic activity of metallo-^nzymes Is due to a modification by the protein. The prosthetic group is tightly bound by the protein the binding of constant the can properties be as transferrin (Raymond et al., ligands nd stereocnemistry of the high a tetrahedral rather in 1982)). The valency of the metal, the fixf»d protein, usually by than Bterlc 10*1 (e.g. by the protein, (e.g. copper(II) conf igurntior., In p l a s t o c y a n i n ). Some the its usual means, while sometimes is forced to square pathways may introduced by the suitable positioning of groups The protein controls group iron(III) are as inducing strain in the metal complex adopt prosthetic planar be blocked by others may such as thiols. and modifies possible pathways to give be the desired rate enhancement and specificity by varying one or more of: (a) the kinetics of individual steps, (b) the thermodynamics of individual steps or (c) the thermodynamics of the overall reaction. A variation of thermodynamic properties protein conformation, catalytic activity coupled is the predominant mode metallo-enzymes (Pratt, of with for changes in enhancing 1975). the Thus the protein may (i) alter the equilibrium particular constant for the ligand by electronic or coordination steric means of a (e.g. the in the coordination of oxygen by irun(IT ) in haemoglobin); (ii) stabilise s ligand protein-free coordination species five-coordinate by number steric mono-imidazole unusual means iron(II) (e.g. the porphyrin in peroxidases, catalases and cytochromes); (ili) vary the changing redox the potential of hydrophobicity the of metal, its for example environment (e.g. by in cytochrome c); (iv) couple changes in the thermodynamic properties cf the metal Site with the binding of the substrate (e.g. induction of the normally unfavourable homolytic fission of the cobalt-carbon bond in the cobalt corrinoid cofactors of isomerase enzymes); (v) couple changes involving sites distant from the metal with equilibria involving the metal (e.g. in haemoglobin). For a more extensive review of the r81e of iron protoporphyrin IX the protein in metallo-enzymes, see Pratt (1975). The prosthetic group provides an excellent example prosthetic group are altered different proteins. protoporphyrin TX to show how display the propert in consequence of Different great (see Figure haemoproteins versatility the at s of the ichment of containing and 1.1) iron diversity of function. Thus, haemoglobin and myoglobin are respiratory carriers which reversibly bind oxygen for transport o Brunori, 1971); the cytochromes have electron transport in the mitochondrial and Barrett, hydroxylate 1973); mono-oxygenases, hydrocarbons (Hayaishi, 1962); through as storage (Antonint and principal function, respiratory chain (Lemberg such the as cytochrome activation of P-450, oxygen catalases catalyse the decomposition of hydrogen 3 c h 2*c h CH] CHjCM^OOH Fig. 1.1 CH jC H jCOOH Haemin chloride (Fe(III) protoporphyrin IX chloride). peroxide, wnile peroxidases use hydrogen peroxide to oxidise a wide range of substances (catalyses, substrates of peroxidases, may different 1984); specificity (Dixon terminal oxidases and which do not oxidise most of be regarded and Webb, like as peroxidanes 1971)) cytochrome (Frew c the with a and Jones, oxidase, reduce oxygen to water (Caughey et a l ., 1976). In this dissertation, metallo-enzymes has corrinoid-dependent corrinoid models, the rflle of the protein in two types of been studied: isomerase and (ii) enzymes (I) using the haemoprotein the cobalt protein-free cytochrome c, cobalt and a model for cytochrome c, the haem-octapeptide, microperoxidnse-8. 1.2 Cobit It corrinoi<H uned in this work (a ) History Vitamin B 1 , is derivative not of synthesized >y the adenosylcobalamin, cobalt-cofactor for methyl-malonic and methylmalony1-coenzyme the in succinic A mutase vivo acids (the only mammals requiring this B ( , coenzyme) e.g., 0 i'.uman is body. the Yet essential interconversion by known a the of enzyme reaction in A deficiency of vitamin causes pernicious , in the diet, or its maladsorption, anaemia. Minot and Murphy (1926) first reported the alleviation of this condition by feeding patients raw liver; two decades later vitamin B ^ ^ (c y a n o c o b a l a m i n ) (see Figure 1.2) was isolated by Folkers and co-workers (Rickes et a l ., 1948) in the same year as Smith and Parker (1948). Vitamin Bj j is today prepared by bacterial fermentation rather than by isolation from liver. The crystallographic work of Hodgkin (Hodgkin et al., 1956) aided by the chemical studies of Todd end Johnson (see Bonnet et al., 1957) established that B^ , contains 5,6-dimethylben2imidazole and cyanide anion as fifth and sixth ligands to a cobalt(III) ion. The cobalt ion resides corrin ring (which provides four planar nitrogen to the porphyrin ring in haemoproteins (see in donors) a akin tne structure in Figure 1.2). A biologically coenzyme) was discovered crystallographic established . 1.2 active that studies the form by of of vitamin Barker Lenhert macrocyclic et B^? (vitamin B, al. (1958). The and structure Hodgkin and (1961) peripheral The molecular structure of B ( , (source: Pratt (1975)). A deficiency of vitamin causes pernicious , in the diet, or its maladsorption, anaemia. Minot and Murphy (1926) first reported the alleviation of this condition by feeding patients raw liver; two decades later vitamin (c y a n o c o b a l a m i n ) (see Figure 1.2) was isolated by Folkers and co-workers (Rickes et a l ., 1948) in the same year as Smith and Parker 11948). Vitamin Bj ^ today prepared by bacterial fermentation rather than by isolation from liver. The crystallographic work of Hodgkin (Hodgkin et a l ., 1956) aided by the chemical studies of Todd and Johnson (see Bonnet et al., 1957) established that B^., contains 5,6-dimethyIberizinidazole and cyanide anion as fifth and si..ih ligands to a cobalt)III) ion. The cobalt ion resides in corrin ring (wh.4 'h provides fcur -lanar nitrogen donors) to the porphyrin ring >n hacmoproteins (see a akin the structure In Figure 1.2). A biologically coenzyme) was diacovered cryatallographic established g. 1.2 active that studies the form by of of vitamin Barker Lenhert macrocyclic The molecular structure of et »l. and structure (vi‘*min B, , (1959). The Hodgkin and (1961) peripheral , (source: Pratt (1975)). substituents of the coenayme were w hs cyanocohalamin, but that cyanide the same as in replaced by the 5' carbon of an adenosyl group. The resulting Co-C sigma bond represents a unique feature .erivatives of of metals these corrinoids: sigma-bonded alkyl of the first transition series were at the time of the discovery of rather unstab!*?. Coenzyme the ccenzyme considered , (adenosylcobalamin) to be (see Figure 1.3) was the first naturally occurring organometal1ic compound to be discovered, and adenosylcobalamin alkylcobeiamina remain the nly known enzyme!, wat cofartcr Ro s f was recently be • lologically metabolism (as homocysteine later discovered in vivo. determined related orginometal1ic complexes in nature- Methylcobalamin, known to and essential for human 5 N -metrvltetrahydrofolate and active cofactor for methyltransferase The structure of by X-ray this crystallograpny et a l ., 1085). I l?"^, the total synthesis of vitamin , was reported by WondwrtT'i (Woodward, 19"79>. g true-are am -Jbalamin, adenosylcooalamin and methylcobalamin form part a family of compounds known as corrinoids. The naturally o rrurring corrinoid all possess the same conjugated corrin ring «» B , incorporating Corrinoids may differ axial ligands; itself and a central cobalt(III) in the nature of the met*l side ion. chains and rhoee that possess the same side chains as B ^ the nitrogen atom of 5,6-dimethylbenzimidfazole fifth axial ligand, are called cobalamins. propionic acid attached (R)-l-amino-2-propanol , and to C^7 the other In ccbalamins, is amidated side-chains as the with at Cp, C <t C 7 , Cg, C J3 and C^fl terminate in carboxyl groups, converted to amide groups. almost All the propionamide groups point away from planar acetamide macrocycle side-chaina in in the one direction, other and direction. all The the the other nitrogen of 5,6-dimethylbenzimidazole is bound to ribose in an ot-glycosidic linkage and th»- ribose moiety is bound, via a phosphate Cobinamides are linkage, to the aminopropanol in turn, side-chain. corrinoids where the phosphate linkage to the 6 <> ^CMOM < . _~CmOm & *?CH, r (ii) Fig. 1.3 (i) The structure (source: (ii) The moiety of The replaced sixth the benzimif* -ole position ligand present coordinated ligand, in side-chain in often vitamin has been benzimidazole cyanide B ( , t3 cyanide (CN~). Cyunocobalamiri is a diamagnetic, complex containing the d coenzyme (source: Pratt (1975)). by another axial cobalamtn 5'-deoxyadenosy1 hydrolysed (see Figure 1.4). The usually the Lenhert and Hodgkin (1961)). coenzyme ribose of or is water. occupied by six-coordinato cobalt(III) ion. The positive charges on the cobalt ion are offset by the single negative charges on the corrin ring, the cyanide and the phosphate. Adenosylcobalamin (Figure 1.3), methylcobalamin and other cobalamins are also diamagnetic and may formally be regarded as complexes of cobalt(lll) with a carbanion (Abeles and Dolphin, 1976). Reduction of cobalt(lll) corrinoide with thiol under anaerobic conditions results in cobalt(II) complexes (Jaselskis 7 and Diehl, 1954; Dolphin et al., 1964) which are low spin d Co CH,- I N > ^ HOCH . 1.4 The structure of cobinamldon: hydrolysis at — * of Cj7 gives uidechain in cobalamins the the corresponding cobinamide. compounds with one unpaired electron. cobalt(II) complex is probably In neutral solution, five-coordinate with benzimidazole base coordinated to the cobalt (Saveant, Further reduction of or cobalt(III) with the cobalt(II) sodium borohydride, which has been e^tablijhed (Hill et aqueous al., 196?). solution nt complex, to be gives pH to the 1979). reduct-i^n of a green complex a monovalent cobalt complex C o ( I ) corrinoids low the give rapidly Co(II) decompose and in hydrogen, piobably Co(II) via an intermediate cobalt hydride. Both Co(I) complexes are easily oxidised by t»ir to give and Co(III) Q complexes. C o (I > c^rrinoids are spin paired d complexes. The benzimidazole cobalt the base is not coordinated to the in cobalt (I) B ] > complex (Brodie and Poe, 1971). The B,_ (cyanocob a l a m i n ), B,_ 12 12a (c o b ( 1 1 )a l a m i n ) will be used in this abbreviations (aquocobalamin), and B , . 1 <?r dissertation. ) Organocorrinolds Both adenosyl and methylc''balamins prepared from B [ , or Bj alkylcobalarrins f: -m ethyl ' 1). All and have been as have many primary and secondary cobinamides, and vinyl coenzymes) to with 1-norbornyl organocorrinolds alkyl ligands (Schrauzer possess a ranging and Holland, cobalt-carbon (Co-C) sigma bond which cleaves homolytically in the presence of light to give cubalt(II) and an organic free radical. ader.osylcobalamin and methylcobalamin are extremely However, stable in the dark both in the solid state and solution (see Chapter 4); this applies to most o r ganocorrinoids, unless the organoligand is particularly sterically hindered (e.g. isop^opylcobalamin). The usual mode of preparation of organocorrinoids the reduction of a cobalt(III) or hydroxocc-balamin) oe cobalamin (e.g. cobalt* III) corrinoid (termed cyanocobalamin cobinamide aquocyanocobinamlde or d i a q u o c obinamide) to cobalt(I) is via (e.g. the corresponding "supernuclecphile" by Grate and Schrauzer (1979)), which is then reacted with compounds such as alkyl halides, expoxides, alkenes or alkynes, to give the desired product via S^2 substitution (see Chapter 3) e.g., NaBH Co( 1 1 1 )-0H , ---- =* Or^nccorrinoida R-Ha< C o d ) ------> engage in a diversity involving th* cobnlt 'oordination sphere; in Figure 1.5. Co( III )-R of reactions rhese are illustrated Co-R Co( I I )«-R‘ - HOMOLYTIC FISSION Co-R C o ( I )+R* - REDUCTION Co-R Co-H-t-R-H - 6 -ELIMINATION Co-R Co(IIIWR“ - HETEROLYTIC FTSSION Co-R Co-R - 0RGAN0L1GAND MODIFICATION Fig. 1.5 Organocorrinoid reactions. (d; Stable yellow corrinoids Stable yellow corrinoids are sometimes formed as by-products of reactions with cobalamins that involve a change in the cobalt oxidation state. They are thought to arise from a fr*tff radical attack on the corrin ring: see for example, Grlining and Gossauer (1979). Stable yellow corrinoids (SYC) display a band at about 460nm in their absorption spectra; they are impervious to the effect of light, oxygen or cyanide, unlike (e ) Enzyme reactions requiring corrinoids as cofactors Three types of reactions in living organisms are catalysed by enzymes containing cobalt corrinoids as c o f a c t o r s 1 1. isomerisation reactions involving the 1,2-shift of a carbon, nitrogen or oxygen atom; 2. methyl transfer reactions, for example in the formation of methionine; 3. the conversion of ribonucleotides to 2'-deoxyribo- nucleotides, by the enzyme ribonucleotide reductase. The enzyme reactions adenosylcobal. min requiring and (ii) cobalt corrinoid methylcobalamin (in cofactors methyl (i) transfer reactions), are classified and tabulated in Table 1.1. The .,-dependent isomerases together with the related ribonucleotide reductases are the first aroup of metalloenzymes (as opposed to reasonably ha e m o p r o t e i n s ) well understood. where The the rfile mechanism of the protein is whereby changes in protein conformation are coupled to cnanges on the metal ion is not known for certain, but several mechanistic proposals have been 10 reported (e.g. Pratt, 1984; Finke and Hay, 1984). In this work only the isomerase reactions are considered. In Chapter 4, mode) studies with protein-free cobalt rorrinoids aim to clarify the r51e of the protein in i&omerase snzymes. For a recent review of model studies concerning isomerase enzymes see Finke et al. (1984). TABLE 1.1 Enzyme reactions requiring cobalt corrinoids Chemaly (1980)). Clot* 1 - Raoction* requiring oatncty icobolo*»in L ____mat;--, ’ tgctior,, Corbo«-»*•Itto" Clutoi*ot* « u t o » * thyl«alony l-co*n*yi«« A mutot* 1 Hijratio" of o" omtne H 1*2 D- Co«v#riio« of d i o l * or amino a l c o h o l * to aid*hyd«i h ' rt^v^ltondti ». ^ 2 ,- d t o « r i b o " v j c l » o ,.i,<'n Bt3C t10" OM OH CM ♦(r s )2 ♦«2o (R5H )2 . dithiol 1 B«aetior, 5 - C H j -THF ♦ H S ( C H 2 )2 C H ( N H 2 ) C OO H ------ ^ THF + CH3S(CH2;2CH(NH;)COOH (source: 10 reported (e.g. Pratt, 1984; Finke and Hay, 1984). In this work only the isomerase reactions are considered. In Chapter 4, model studies with protein-free cobalt corrinoids aim to clarify the r81e of the protein in isomerase enzymes. For a recent review of model studies concerning isomerase enzymes see Finke et al. (1984;. TABLE 1.1 Enzyme reactions requiring cobalt corrinoids (source: Chemaly (1980)). Clot* 1 * Reoctiont requiring — ------ ^ e o r r ^ g e - e - t or oce«ctyicobaio»in I to-- r ° »cc t I 0 " t T«pe of reoctt?" r.~ : II Corb jn-t^eleta" reorrongeaent « I -C. -C..- '* -C.-C.- Glutowote *utat« *«thyl«alonyi-co«ncy*e H R R H A *vtett iutarat* •utote N i g r otio" o* on 0* 1"o grouo NM,M H 1.-?!— »uljii D-e-lvti*e »utote D-or«ithin# sctat* *4 MQ-r, .r,,i 1 ud mO-C C o " v o n l o " of d i o l t o r o«ino L-f-ly»»«e Dioldehydrote *1r?. Glycerol X x dehydrate M o l c o h o l t to i 1 oidohydot * O e C - £ „ - ♦ *H o«*enia-lyot« |2" • M ■* ri00" o cl>-:Ud«t to 2 ' - dec. r 8. c 1»■-». i i t * Re-?c tic* Bote ^ O ^ H ^ Q P P P Bote y O . C H . O P P P Ri bo nueleotid* roductote (RSH)j Q O H OH OH ♦ («S)2 ♦ >y> (* S M ) 2 . dithiol Clott 2 - Reoctiont involving mothylcobalgwjn Reaction 5 - C H j -THF ♦ H S ( C H 2 )2 C H ( N H 2 ) C O O H ^ N^-methy 11 e t r o hydrofoloto: THF ♦ C H . S ( C H ' C H ( N H j C O O H J 2 Z 2 ho«oeyitoino mothyltron*f e ro te 3 - C H 3 - THF ♦ H S ( C H 2 )2 S 0 3 — ) THF ♦ C H 3 $ ( C H 2 "Methone tynth.tate" 5- C H 3.THF ♦ COj ■ THF * t e t r a h ) d r o f o l a t e y THF ♦ C H 3 C O O "Acetate tynthotato" 10 reported (e.g. Pratt, 1984; Finke and Hay, 1984). In this work only the isomerase reactions are considered. In Chapter 4, model studies with protein-free cobalt corrinoids aim to clarify the rflle of the protein in isomerase enzymes. For a recent review of model studies concerning isomerase enzymes see Finke et al. (1984). TABLE 1.1 Enzyme reactions requiring cobalt corrinoids Chemaly (1980)). Clot* 1 - S«actior» requiring o d c n o t y i c o b o l o m i n Ca r b o n . t « « l # t o « rtarrong«*«nt H R * H Gluto*ot« «utoi« thy I m o l o n y i - c o o n Xy«* A Mgtot* «-H*tHylon«glwtarot* ■uttiM C onvortion of d iolt or a«ino o l c o h o l t to oidohydct M -» r i b o n u c ’, 0 ; i j,, »c 2 ' - duo , r ibe-v c > ■ ;♦ .i U % 8 . 0 * o c t ion *ibonucl«otid* raductoi* O H OH OH ♦(RS)2 ♦H20 ( R S H )2 . dithiol Clou 2 - W#oction« involving faction THF»t«trohydrofolate leobolowin (source: 11 1.3 Iron porphyrins used in this work 1.3.1 Cytochrome c (a ) History and description The cytochromes are a group of haemoproteins whose principal biological function molecular oxygen is electron and/or hydrogen in reversible valency the respiratory change Union of Biochemistry, of chain, their haem transport by iron virtue of to a (International 1961). Cytochromes also play a role in photosynthesis (Hill, 1954) and in non-photosynthetic anaerobic and chemosynthetic bacterial reactions. Keilin first Identified three different types of cytochrome (cytochromes a, b, and c) (Keilin, 1925; 1926) and showed that the respiratory of cytochromes was related Iron. Ir fact, distinguished four on to a main the valency groups if basis change of on function the cytochromes their prosthetic hsem can be groups: cytohaems a, b, c and d are shown in Figure 1.6. Cytochromes c are unique in covalently that bound to the prosthetic the peptide cysteine residues on typically transfer electrons themselves or the other group, by cytohat-m thioether polypeptide chain. in multienzyme molecules su'-h c, is linkages to Cytochromes systems as the between cytochrome reductases, dehydrogenases or the cytochrome peroxidases. This is illustrated in Figure 1.7 for the mitochondrial chain: the rflle of cytochrome c cytochrome c reductase and in electron cytochrome c respiratory transfer between oxidase is clearly indicated. Cytochrome (1925); its (Mf ^ was wide invertebrates small c first, occurrence and y* ast 12 000) named in has since monohaem and described cells been protein, oxidation chain in the mitochondria, from by Keilin mammals established. It part terminal of the between the ferric and is a which transfers electrons from cytochr >me c reductase to cytochrome c oxidase. The alternates to ferrous states iron with a midpoint potential at pH 7 of 260 ♦ 20 mV (Lemberg and Barrett, 1973). The complete amino acid sequence is known for at least thirty-six species (Lemberg anu Barrett, 1973, p. 13G) ranging 12 C(»M||0 CMj I '0 M CM, CH. i CM. I M, CM, .................... OCM p ^ T 'O ' CO.M CO.M “ CM, CM, CM. I CM, I M. ,CM, *-,cSc#e.c ,c;c,e M M, CM. M M*«m «(CvlohMm «> n c *» m c 'om M CM, Cm . CM, cm / M.C . M.C M .c M>« CO.M CO,M CO.M Pr»«*fc.«NW(C 4). C O lH C<^H«n 4 — CO s NM I M s, 8 ,C0 -NMV M *C ' C m Fig. 1.6 Prosthetic groups of the cytochromes (source: Lemberg und Barrett (1973)). (I) subtlraiui succimio II *Ilit h *|mi »it«tr> <liitm ( ’ ) |I|( I m m i I H % l l l l t l t I l l l t t *1 . l l l l l I . II, III, I V I lie In in M l » ) » H I I* M i l I M l l l t l | « >1 l * V in n i| tli)iv i lli.il tA ii M ill'll H |»llll l it * i h u I i i U i I ( I ) , ( 2 ) . ( lllll' lin t l l i i i t m I i * • »f p l t n t * p l l i n ) l.llH M I. Fig. 1.7 Cytochrome c in a multienzyme and Barrett (1973)). system (source: Lemborg from mammala and birds to reptiles, studies have considerable amphibia and fishes. These evolutionary significance. The tertiary folding of cytochrome c has been maintained at since mammals and fishes diverged, 400 million least years ago yeast was (Dickerson et a l ., 1971). (b ) Structure The preparation of cytochrome c from ox-heart pioneered by Keilin, Hartree and Theorell and in the 1930's (e.g. Keilin, 1930). The tertiary structure of horse-heart cytochrome c was established by Dickerson and Margoliash (e.g. Margoliash 0 et al., 1960), with 4 A resolution. A very close analogue of this cytochrome, with tryptophan 59 substituted phenylalanine has been synthesized (Sano and Kurihara, by 1969). a An X-ray crystallographic structure with 2.8 A resolution was reported by Dickerson (1971) for the same cytochrome specie*.. Recent X-ray crystallographic studies (Takano 1961a; X-ray 1961b) structure) conducted and EXAFS measurements with both (extended (Labhardt ferric and and and Dickerson, absorption have Yuen, ferrous fine cytochrome been c. The structures deduced from X-ray diffraction were confirmed by NMR (nuclear magnetic resonance) studies to be largely conserved in solution (Moore and Williams, 1980). The structure of cytochrome c is shown in Figure 1.8. The X-ray studies confirmed the inclusion of the haem prosthetic group in a crevice of the protein molecule, as postulated (1955a). A polypeptide chain of by Ehrenberg Theorell 104 amino acids wraps around the hatim peptide in two halves, residues 1 and residues 48 - 91 to th«a left of spin haem c, which and to the right the redox centre, a low is recognised as the organising principle (Lemberg and Barrett, 1973; Dickerson et al., 1971). The haem O is contained sideways length), with one bridge 0), over edge, exposed the top of to in the resultant crevice the solvent. and the in residues. Residues 94 - 104 return to the right and are sulphur of the only Four of the ligands to the haem iron are from the porphyrin itself. Histidine ligand, 21 A between pyrrole rings II and I K m e t h i n e the molecule genuinely a-helical (ca. methionine 18 is the fifth 80, the sixth. The Fig. 1.8 The structure of cytochrome c (source: Dickerson et a l . binding of methionine 80, postulated by Harbury et a l . (1965) and supported by a model study (Shechter and Saludjian, and NMR studies (e.g. WUtr ich (1969)), was confirmed 1967) by the bind via X-ray study. Histidine 18 and cysteines thioether linkages to 2) and II (position the 4), 14 porphyrin and 17, which (pyrrole rings respectively) extend I (position from the right wall of the crevice. Methionine 80, which extends from the left wall, forms part of a (67 - 82) which largely includes invariant sequence of lysine residues 72, channels filled wit.h hydrophobic side-chairm 73 and residues 79. Two le.id to the right and left from the haem to the surface, and each is surrounded by a cluster of positively charged c-amino groups residuee, where it meets the surface. molecule between the positive regions, negatively charged acidic groups. been implicated in the At the rear lysine i>/ the lies a cluster of nine These surface of cytochrome linkages of features have c to other enzymes. ( c ) Electron transfer and conformational change Cytochrome c interacts cytochrome oxidase with both complexes in cytochrome the reductase terminal and mitochondrial oxidation chain, in complex reactions (Moora et a l ., 1982). The reductase and oxidase molecules are additionally coupled to ATP synthesis and the oxidase also redices oxygen to water. The Interaction site on cytochrome c for the oxidase and reductase complexes appears to be largely the same for both complexes and includes the exposed edge of the haem (Rieder tind flosshard, 1980). The electron transfer site is probably the exposed edge of the haem (Moore et al., 1962) where protein residues are relatively mobile. Poulos and Kraut (1980) have suggested that ph-nylalanine 82 forms part of the electron The electron transfer function has been transfer pathway. modelled with iron, cobalt and ruthenium hexacyanides (Ragg and Moore, 1984) where the and interaction electron site transfer is the probably exposed occurs. haem An edge oxidation direct linked conformational change of the cytochrome c protein on the basis of differences in the ferric and ferrous species was suggested as early a* 1913 (Zelle and feuter, 1933). Reduced cytochrome c appears to be more compact and rigid than the oxidised species. It is less susceptible to digestion by trypsin (Okunuki et al., 1965) and shows a greater thermal stability than the oxidised molecule (Butt and Keilin, 1962). The methionine bond to haen iron, which in ferric cytochrome c is relatively easily broken 0 by pH or increasing temperature (Angstrom et a l ., 1982), is particularly stable in the ferrous species., even at extremes of temperature and pH (Moore et a l ., 1982). No denatured protein where the methionine-iron lyophilised ferrous bond cytochrome has c, been as it brokan is for is fo^nd the in farric spocies (Avirtn and Schejter, 1972). Hefined X-rav structures of bonito cytochrome c have confirmed between the two redox states that dtfferenceB exist (Takano ind Dickerson, 198:a; 1981b). Thus, cytochrorm the oxidised c is far more ste-.t e in the state; the environment also serves the ferric r»tate. hydrophobic to stabilise In consequence, relatively high (260 ♦ 20 mV) lured than nature th* tr - ferrous the re >x (Lemberg and nature of the conformational change is reli<-ive to : otential ot k own for protein are likely (Moore et a l ., 1 9 8 2 1 X-raj studies has been tain, movement o'.' the haem group the 1981a; and in structure is at the axial mcludn g&nds, th- of th- isoleucine 57. This is supported ty *n«* some -he gr ups tyrosine 67). 1980) agr* back conformatio-.al “ -:ri : it moving by up to 0.07 nm (asparagine 52 ar and NMP data (Moore et al., jf the ietailed model based proposed change ^Takf;no and Dickerson, is 19"’ ’ J . The Barrett, but minor changes in structure extending -o tne surface on haem -ray major •» Ue, - U nge clo**- modif: to -ion studies of Bosahard and ZLirrer (19*<’ . The role of the methionine-iron linked conformational change remains (Moore et al., cytochrome c 1980) of reported b the o n — --- - .a l . NMP studies cobalt, yr- n r — - a smal oxidation- incrwta* c ir and metal-sulphur interaction, on reduction, interpvafead aa a b m d length But X-ray crystallographic atucj es 1981a; 1981b) and EXAFS m e a s u r e ^ and Yuen, 1979) and model contradict the properties" (Moore et -s on complexes assertion. al., (Takar -yt 19*.') and 1 ■ Dickerson, nr- me c iLjbhardt (Mash * H wever, native et al., inomaloua d i s p l a y - 1 by the 198; ', dynamic bond in ferricytochrome merit its further investis»-ion. (d ) The haem ir«m-methion in»» bond The methionine ligand of cytochrome c is displaced by cyanide, azide and imidazole at pH 7 (Stellwagen, Redfield, complexes, 1970; Sutin and Yandell, and by a residue at ca. 1966; 1972) 1968; Gupta and producing pH 9.0, the low-spin identity of which has not definitely been established (Bosshard, 1981). The Author Aron Janine Name of thesis The Coordination Chemistry Of Some Cobalt Corrinoid And Iron Porphyrin Complexes. 1986 PUBLISHER: University of the Witwatersrand, Johannesburg ©2013 LEGAL NOTICES: Copyright Notice: All materials on the U n i v e r s i t y o f t h e W i t w a t e r s r a n d , J o h a n n e s b u r g L i b r a r y website are protected by South African copyright law and may not be distributed, transmitted, displayed, or otherwise published in any format, without the prior written permission of the copyright owner. Disclaimer and Terms of Use: Provided that you maintain all copyright and other notices contained therein, you may download material (one machine readable copy and one print copy per page) for your personal and/or educational non-commercial use only. 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