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Functional properties of active site variants of yeast cytochrome C Rafferty, Steven Patrick 1992

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FUNCTIONAL PROPERTIES OF ACTIVE SITE VARIANTSOF YEAST CYTOCHROME CbySTEVEN PATRICK RAFFERTYB.Sc., The University of Waterloo, 1986A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIESTHE DEPARTMENT OF BIOCHEMISTRYWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAApril 1992® Steven Patrick Rafferty, 1992In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of_____________________________The University of British ColumbiaVancouver, CanadaDate /‘yr /DE-6 (2/88)ABSTRACTMitochondrial cytochromes c are small, soluble respiratory proteins which exhibit a high degreeof amino acid sequence and tertiary structural homologies across the entire eukaryotic kingdom. Theinfluence of mutations within two conserved regions of yeast-iso-1-cytochrome c on the physicochemicalproperties of this protein were examined. i) Invariant residue Phe82, located at the exposed heme edgeon the protein surface, was replaced by Tyr, Leu, lie, Ala, Ser and Gly. ii) Internal residues AsnS2,Tyr67, Thr78 and 1le75 form the environment about WAT166, a buried water molecule near the heme.This region undergoes relatively large oxidation-state linked conformational changes and these residuesare collectively referred to as ‘water switch’ residues. The mutants Asn52Ala, Tyr67Phe, Ile75Met andThr78Gly were studied. The effects of variation at these conserved positions on i) the oxidation-reductionequilibrium, ii) the electron transfer kinetics with inorganic electron transfer partners, and iii) theequilibrium and kinetics of heme axial ligand Met8O displacement by added azide and alkaline pH of yeastiso-1-cytochrome c, were determined.The reduction potentials (pH 6, 25 °C, 0.1 M) of position 82 mutants decreased by up to45 mV with decreasing size of the side chain. Small differences in the H° and 0 of reduction wereresponsible for the observed reduction potentials. Electron transfer rates (corrected for driving force andelectrostatics) with the inorganic electron transfer partners Fe(edta)2 (as reductant) and Co(phen)3 (asoxidant) increased up to 17-fold as the size of the mutant side chain decreased (pH 6, 25 °C, = 0.1M). Electron transfer reactivity relative to wild type was dependent on the identity of the inorganicelectron transfer partner, indicating that mutations at position 82 change that portion of the surface of theeytochrome that contacts electron transfer partners. Eyring plots of the rate data showed smalldifferences in the activation parameters of the mutants. Mutation decreased the stability of the nativeFe(III)-Met8O bond, as the binding constant of the cytochrome for added azide, K, increased up to5-fold. Kinetic measurements of azide binding (pH 6, 25 °C, = 1.0 M) demonstrated that the higher11affinity for azide was caused by increases in the rate of heme crevice opening, from 74 s_i in wild typeto greater than 160 s_i for Leu, lie, Ser, and Gly variants. Phe82 thus has critical roles in i) maintaininga high reduction potential, ii) maintaining the integrity of the Fe(III)-Met8O bond, and iii) forming partof the contact surface with electron transfer partners.The reduction potentials (pH 6, 25 °C, = 0.1 M) of the water switch mutants were at least 33mV lower than that of the wild type protein. Electron transfer reactivity using the electron transferpartners Fe(edta)2 and Co(phen)3 was up to 10-fold higher than wild type, but unlike the position 82mutants, reactivity relative to the wild type cytochrome was independent of the identity of the electrontransfer partner. This observation indicates that water switch mutations influence the electron transferreactivity of cytochrome c by altering the reorganization energy within the protein rather than changingthe nature of the contact surface of the protein with its electron transfer partners. Measurements of theazide affinity and the alkaline isomerization of the water switch variants indicated that the stability of theFe(III)-Met8O bond of these variants was the same as or greater than wild type, with the exception of theThr78Gly variant. In spite of the improved heme crevice stability and/or electron transfer reactivity ofthe water switch variants Tyr67Phe, Asn52Ala, and Iie75Met, such variants are extremely rare in nature.This observation suggests that the primary role of the native water switch residues is to maintain a highreduction potential.111TABLE OF CONTENTSABSTRACT.. iiTABLE OF CONTENTS . ivLIST OF FIGURES . viiiLIST OF TABLES. xiLIST OF ABBREVIATIONS. xiiACKNOWLEDGEMENTS. xiiiINTRODUCTION 11.1 Overview 11.2 Structural Features of Cytochrome c 31.3 Structural Differences Between Oxidized and Reduced Cytochrome c 121.4 Physiological Roles of Cytochrome c 131.5 Physicochemical Properties of Cytochrome c 161.5.1 UV-Visible Spectra 161.5.2 Electrochemistry 181.5.2.1 Reduction Potential and Its Measurement 181.5.2.2 Factors Affecting The Reduction Potential ofCytochrome c 231.5.3 Electron Transfer Kinetics 251.5.3.1 Electron Transfer Theory 251.5.3.2 Electron Transfer Kinetics of Cytochrome c 311.5.4 The Alkaline Isomerization 331.5.5 Ligand Substitution 37iv1.6 Methods of Structure-Function Analysis of Cytochrome c 381.6.1 Genetic Analysis 381.6.2 Comparative Analyses1.7 Thesis Objectives434343444548494949505050505151525353553941METHODS2.1 Cytochrome c Preparation2.1.1 Fermentation of Yeast2.1.2 Cytochrome c Purification2.2 Electrochemical Experiments2.3 Electron Transfer Kinetics2.3.1 Fe(edta) Reduction Kinetics2.3.2 Co(phen)3Oxidation Kinetics . .2.3.2.1 Synthesis of [Co(phen)3] 12.3.2.2 Ferrocytochrome c Oxidation2.4 Alkaline Isomerization2.4.1 Spectrophotometric pH Titrations .2.4.2 pH Jump Kinetics2.5 Ligand Substitution2.5.1 Azide Titrations2.5.2 Azide Binding Kineticsby Co(phen)3l .RESULTS3.1 Protein Preparation3.2 Electrochemical Experiments . .V3.3 Electron Transfer Kinetics 643.3.1 Fe(edta)2 Reduction Kinetics 643.3.2 Co(phen)3lOxidation Kinetics 703.4 Alkaline Isomerization 773.4.1 pH Titrations 773.4.2 pH Jump Kinetics 783.5 Ligand Substitution 833.5.1 Azide Binding Titrations 833.5.2 Azide Binding Kinetics 87DISCUSSION 924.1 Overview of the Structural Features of the Cytochrome c Variants 924.2 Cytochrome c Oxidation-Reduction Equilibrium 984.3 Electron Transfer Kinetics 1044.3.1 General Comments 1044.3.2 Position-82 Variants 1064.3.3 Water Switch Variants 1074.3.4 Temperature Dependence of Electron Transfer Kinetics 1114.4 Alkaline Isomerization 1124.5 Ligand Substitution 1164.6 Summary 124REFERENCES 125APPENDIX A 136APPENDIX B 142viAPPENDIX C . 147APPENDIX D 158APPENDIX E 160viiLIST OF FIGURESFigure 1 The amino acid sequence of yeast iso-1-cytochrome c 4Figure 2 The heme group of cytochrome c 5Figure 3 The polypeptide fold of yeast iso-1-cytochrome c 7Figure 4 The structure of reduced yeast iso-1-cytochrome c about WAT166 9Figure 5 The environment of the invariant residue Phe82 11Figure 6 The oxidation state-linked conformational changes about WAT 166 14Figure 7 The physiological electron transfer partners of cytochrome c 15Figure 8 The UV-visible spectra of oxidized and reduced yeast iso-icytochrome c 17Figure 9 A cyclic voltammogram obtained for a reversible electrochemicalsystem 21Figure 10 Schematic diagram of the steps involved in outer sphere,intermolecular electron transfer 26Figure 11 A diagram of the reaction coordinate for outer sphere electron transfer . 28Figure 12 The pH dependent ligation states of mitochondrial cytochrome c 35Figure 13 A photograph of the cyclic voltammetry cell used for reductionpotential measurements 46Figure 14 A schematic diagram of the cyclic voltammetry cell 47Figure 15 The dependence of faradaic peak current on the applied potentialsweep rate for cytochrome c 57Figure 16 The temperature dependence of reduction potential for cytochrome cposition-82 variants 58viiiFigure 17 The temperature dependence of reduction potential for cytochrome cwater switch variants 59Figure 18 The pH dependence of reduction potential for several variants ofcytochrome c 62Figure 19 The dependence of pseudo-first-order rate constants forferricytochrome c reduction on Fe(edta)2 concentration for position-82 variants 65Figure 20 The dependence of pseudo-first-order rate constants forferricytochrome c reduction on Fe(edta)2 concentration for waterswitch variants 66Figure 21 Eyring plots for the Fe(edta)2 reduction of position-82 variants offerricytochrome c 67Figure 22 Eyring plots for the Fe(edta)2 reduction of water-switch variants offerricytochrome c 68Figure 23 The dependence of pseudo-first-order rate constants for oxidation ofposition-82 variants of ferrocytochrome c on Co(phen)3concentration 72Figure 24 The dependence of pseudo-first-order rate constants for oxidation ofwater switch variants of ferrocytochrome c on Co(phen)3concentration 73Figure 25 Eyring plots for the oxidation of position-82 variants offerrocytochrome c by Co(phen)3 74Figure 26 Eyring plots for the oxidation of water switch variants offerrocytochrome c by Co(phen)3 75ixFigure 27 pH dependence of the rate of alkaline isomerization from stopped-flowpH jump experiments 79Figure 28 Kinetic trace of the alkaline isomerization of yeast-iso-iferricytochrome c at pH 10.5 monitored at 529 nm 82Figure 29 The visible spectra of yeast iso-i -ferricytochrome c in the presenceand absence of 2.5 M azide 84Figure 30 Azide binding isotherms for position-82 variants of ferricytochrome c . 85Figure 31 Azide binding isotherms for water switch variants offerricytochrome c 86Figure 32 The dependence of observed rate constant of azide binding to position-82 variants of ferricytochrome c on azide concentration 88Figure 33 The dependence of observed rate constant of azide binding to waterswitch variants of ferricytochrome c on azide concentration 89Figure 34 The structures of the position-82 variants of reduced iso-icytochrome c in the vicinity of the mutation site 94Figure 35 The structures of AsnS2Ala and Tyr67Phe variants of reducedcytochrome c about WAT 166 95Figure 36 Comparison of the relative self exchange rates obtained for thereactions of the variant cytochromes with Fe(edta)2 (vertical axis) andCo(phen)3 109Figure 37 A possible minimal mechanism for the alkaline isomerization of theAsnS2Ala variant of ferricytochrome c 114Figure 38 A mechanism proposed by Kihara et al. to account for the alkalineisomerization of horse ferricytochrome c between pH 9 and 12 117Figure 39 Correlation of azide binding with the alkaline plc for cytochromes c. . . 123xLIST OF TABLESTable 1 Thermodynamic parameters associated with the reduction ofcytochrome c variants 60Table 2 Parameters derived from the pH dependence of reduction potential forseveral variants of cytochrome c 63Table 3 Rate parameters for the reduction of variants of ferricytochrome c . . . 69Table 4 Rate parameters for oxidation of variants of ferrocytochrome c 76Table 5 Alkaline isomerization parameters for variants of ferricytochrome c . . . 80Table 6 Azide binding parameters for variants of cytochrome c 90Table 7 Current status of the structure determinations for those Saccharomycescerevisiae iso-1-cytochrome c variants examined in this work 93xiLIST OF ABBREVIATIONSA angstromCAPS 3-cyclohexylamino-1-propanesulfonic acidCD circular dichroismCHES 2-(cyclohexylamino)ethanesulfonic acidCM carboxymethylCN cyanideEapp applied potentialedta’ ethylenediaminetetraacetateE6 midpoint reduction potential at pH 6EPR electron paramagnetic resonanceJR infra-redionic strengthMCD magnetic circular dichroismMES 2-[N-morpholino]ethanesulfonic acidmV millivoltNADH nicotinamide adenine dinucleotide, reducednm nanometerNMR nuclear magnetic resonanceox oxalatephen o-phenanthrolinepy pyridineUV ultravioletxliACKNOWLEDGEMENTSAlthough this thesis bears only a single name, it really consists of the contributions of manysupportive people. It is my pleasure to acknowledge their roles in my career at UBC. I would firstlike to thank my supervisor, Professor Grant Mauk, for providing me with the inspiration, the tools,and, most importantly, the freedom to develop the project. My thanks to Professors Gary Brayer andBrian James for the gift of their time and expertise as members of my advisory committee.For all the members of the Mauk, lab, past and present, I give my warmest regards. Specialthanks go to Dr. Marcia Mauk, who generously offered her help whenever I had a problem. I thankDr. Lindsay Eltis and Dr.2 Tony Lim for their help in the lab in the early days, in addition totrashing buffets and bashing trolls, and to Bhavim Sishta for putting up with us. Thanks go to thefather of fermentation, Dr. Alfred Gartner, and his successors Jackie, Heather, Laura, and Fred, forproviding me with large amounts of cytochrome c. Dr. Paul Barker must be singled out for teachingme the fine art of cyclic voltammetry, and for acting as our tour guide around the pubs of Oxford;I look forward to his Cambridge sequel. Since Dr. Juan Ferrer is an excellent scientist and agenerous friend, he may be forgiven for his less than accurate Spanish translation lessons. The debtof thanks that I owe Dr. Guy Guillemette would require its own chapter; beyond his generosity withhis proteins, his friendship and his wine, I must thank Guy and Sylvie for having their first-born onmy birthday.To those in the Smith lab, especially Jeanette Beatty, who convince yeast cells that it’s coolto carry cytochrome c mutants, I offer my warmest thanks. To those in the Brayer lab (AlbertBerghuis, Mike Murphy, Terry Lo, and the Great Master, Gordon Louie) who persuade cytochrome cthat it’s cool to be a crystal, ditto. To the “new” students in Grant’s lab, Dean, Christie, Alex andFred, good luck; you now see that there is a light at the end of the tunnel and no, it isn’t the lightof an oncoming train.Marital Marshal Peter Durovic, Simon “Mr. Sportsman” Eastman, and beatnik LouisLefebvre have absolutely nothing to do (nor do they want to have anything to do) with biologicalelectron transfer. That’s their loss, but since they’ve been my closest friends and fellow travellerson the Rocky Road to Scientific Success, I thank them anyway.Finally, my love and thanks are offered to mom and dad, who have provided love andconstant support, and who have encouraged my curiosity and desire to learn from the very beginning;this is your thesis as much as it is mine. And for my wife, Janet, I save my greatest appreciation ofall, for sharing in my successes and comforting me in my failures. Coming home with you washesaway all the cares of the day.This thesis is dedicated to my grandfather, Mr. Lloyd Ludgate, and to the memories of mygrandparents, VivianLudgate and Mr. & Mrs. Leo Francis Rafferty.xli’INTRODUCTION1.1 OverviewThe central role of electron transfer reactions in the energy transducing pathways ofphotosynthesis, respiration, and in the catalytic cycles of many enzymes makes biological electron transfera phenomenon that merits the extensive theoretical and experimental effort that has been devoted to thisfield. The study of biological electron transfer systems is a field in which theory, derived frominvestigations of inorganic models, can be applied to biochemical systems. Consequently, our presentunderstanding of biological electron transfer reactions represents the combined efforts of a wide rangeof disciplines, from the highly physical to the highly biological.In such studies, several small, well characterized electron transfer proteins have served as usefulmodels for investigation. Of these proteins, mitochondrial cytochrome c has been perhaps the mostwidely studied example. The popularity of this protein derives in part from its ready availability in largequantities from many species, which provides an abundance of structural and phylogenetic information.Extensive efforts by a large number of investigators have resulted in the description of a great numberof chemically-modified derivatives of cytochrome c that have been employed to address a wide range offunctional questions. Cytochrome c has also been shown to be readily amenable to semi-synthetictechniques that allow substitution of specific amino acid residues with others, including non-naturallyoccurring residues.The functional characteristics that have been studied through investigation of various species ofcytochrome c and various modified or semisynthetic forms of the protein are legion. These includeassessment of the structural factors responsible for the reduction potential of the cytochrome, thestructural elements responsible for the structural integrity of the protein, the nature of binding betweencytochrome c and its physiological electron transfer partner proteins, and the basis for the pH-dependentfunctional properties of the cytochrome.1The application of oligonucleotide-directed site specific mutation of proteins (Zoller & Smith,1983) to the study of cytochrome c was facilitated by the availability of a cloned and sequenced geneencoding the yeast cytochrome (Smith et at., 1979). As a result, cytochrome c was the firstmetalloprotein to be modified by this technique (Pielak et at., 1985). Since this initial work, a multitudeof variant cytochromes has been constructed and characterized in functional and structural studiesdesigned to probe the mechanism by which cytochrome c functions. The balance of this introductionprovides a brief background regarding the structure and function of mitochondrial cytochrome c and themethods used in this thesis to assess the physicochemical properties of structural variants of cytochrome cprepared by site directed mutagenesis. For a comprehensive treatment of all aspects of cytochrome c,the reader is referred to the recent monographs of Moore & Pettigrew (Pettigrew & Moore, 1987; Moore& Pettigrew, 1990).21.2 Structural Features of Cytochrome cCytochrome c is a small protein with a molecular weight of approximately 12 500, and possesses104 to 110 residues depending on the species from which it is isolated. Sequence comparison of morethan ninety species reveals a high degree of conservation, particularly among the residues that define theheme environment. Twenty six residues are invariant among 96 known sequences, and many otherpositions have conservative substitutions (Dayhoff, 1972). Differences in length among different speciesof cytochrome c are due to the presence of extra residues at the N-terminus. In this work, the conventionof numbering residue positions according to the sequence of tuna cytochrome c is followed. Figure 1shows the amino acid sequence of yeast iso-1-cytochrome c (Narita & Titani, 1969).Comparisons of the three-dimensional structures of cytochrome c from several species show thatthe tertiary structure of this protein has been highly conserved over one billion years. There areremarkable similarities in the overall protein fold and consequently in the positions of highly conservedresidues (Bushnell et al., 1990). The structural features of cytochrome c described below are believedto be common to most eukaryotic species, as they occur in the four species of cytochrome c that havebeen characterized by X-ray diffraction analysis: tuna heart (Takano & Dickerson, 1981a,b), rice (Ochiet a!., 1983), horse heart (Bushnell et a!., 1990), and yeast (Louie & Brayer, 1988, 1989, 1990).The primary structural feature of cytochrome c is that it possesses a heme prosthetic group at itsactive site (Figure 2). The oxidation state of the iron of cytochrome c alternates between + 3 (oxidized)and + 2 (reduced). As the dianion of the porphyrin coordinates to the iron, the heme alternates betweena net charge of +1 (oxidized) and 0 (reduced). At neutral pH the heme of cytochrome c remains lowspin and six-coordinate in both oxidation states.Heme is covalently bound to cytochrome c through thioether linkages provided by cysteineresidues 14 and 17, which react with the vinyl groups located on positions 2 and 4 of the porphyrin ringthrough the action of the enzyme heme lyase. The constraints imposed on the heme by the thioether3-5 Thr Glu Phe Lys Ala1 Gly Ser Ala Lys Lys Gly Ala Thr Leu Phe Lys Thr13 Arg Cys Leu Gin Cys His Thr Val Glu Lys Gly Gly25 Pro His Lys Val Gly Pro Asn Leu His Gly lie Phe37 Gly Arg His Ser Gly jn Glu Gly Tyr Ser Tyr49 Thr Asp Ala lie Lys Lys Asn Val Leu Trp Asp61 Glu Asn Asn Met Ser Glu lyr Leu Thr Asn Pro Lys73 Lys Tyr j Pro Gly Thr Lys Met Ala Gly Gly85 Leu Lys Lys Glu Lys Asp Arg Asn Asp Leu lie Thr97 Tyr Leu Lys Lys Ala Cys GluFigure 1 The amino acid sequence of yeast iso-1-cytochrome c, with numbering according to the tunasequence (Narita & Titani, 1969). Residues shown in bold are invariant among 96 known mitochondrialsequences. Underlined residues are the targets of site directed mutagenesis in this work. Lysine 72 istrimethylated, and the N-terminus is not acetylated.4S (Cys 14)COH CO2HS (Cys 17)Figure 2 The heme group of cytochrome c, showing the numbering schemes of the pyrrole rings(Roman numerals), of the pyrrole ring substituents (Arabic numerals), and the thioether bonds from theprotein. Pyrrole rings II and Ill define the more exposed heme edge of cytochrome c.2 318 57 65linkages causes a ‘ruffling’ of the aromatic ring of the heme. Axial coordination to the heme iron isprovided by the E2 nitrogen of His 18 and the sulphur of Met8O.The heme is embedded within the surrounding protein with pyrrole rings II and III closer to theprotein surface. This defines the more exposed heme edge. For horse heart and yeast cytochrome crespectively, 7.5 and 9.6 percent of the surface area of the heme is solvent accessible, representing about0.1 percent of the solvent accessible surface area of cytochrome c (Bushnell eta!., 1990, Louie & Brayer,1989).The heme serves as the template about which the polypeptide chain of cytochrome c folds. Almosthalf of the residues occur within the five a-helical segments of the protein. There are six type II reverseturns in cytochrome c that cause the direction of the polypeptide chain to change abruptly. The tertiarystructure of cytochrome c (Figure 3) has dimensions 25 x 25 x 37 A. Packed against the heme are a setof conserved aromatic residues, which include Trp59, Tyr48, Tyr67 and Phe82.The heme bears two propionates at positions 6 and 7. As the ionization states of the propionates caninfluence the oxidation-reduction equilibrium of the heme through electrostatic interactions, an estimateof their PKa5 has long been sought. The NMR and JR signals of heme propionates of bacterialcytochromes c are sensitive to changes in pH, and pH titrations monitoring these signals have beensuccessful in determining propionate pKs in these proteins (reviewed in Moore & Pettigrew, 1990). Inthis manner heme propionate plçs in the range 6.2 to 8.5 have been measured. The lack of similar pHdependent spectral changes in mitochondrial cytochromes c suggests that the dissociation constants of theirheme propionates are outside the pH interval 4.5 to 9. An experiment comparing the urea induced protonuptake of oxidized and reduced horse heart cytochrome c suggests that one of the plçs is above 9 whilethe other is below 4.5 (Hartshorn & Moore, 1989). IR spectroscopy (Tonge et at., 1989) providesevidence for an unusual carboxylate ionization with a pK of 9.35. Although the results of theseexperiments indicate that the two propionates differ in their pKs by more than 4 pH units, it has not beenpossible to determine which of the two propionates possesses the higher plc. One proposal assigns the6Figure 3 The polypeptide fold of yeast iso-1-cytochrome c, showing the heme, the covalent bondsbetween heme and protein, and the axial ligands (Louie & Brayer, 1990).7pK below 4.5 to heme propionate-7, as this group is in contact with the invariant residue Arg38 whichis proposed to stabilize the propionate anion through electrostatic interactions (Moore et al., 1984).Heme propionate-6 has thus been assigned the abnormally high pK above 9 (Moore & Pettigrew, 1990).However, the proposal that Arg38 is responsible for maintaining the pK of heme propionate-7 below 4.5is not supported by results obtained with position-38 variants of cytochrome c, which show no changein either the pH dependence of their reduction potentials1or their NMR spectra (Cutler et al., 1989).Both propionates are buried within the protein, and the residues forming their environment are highlyconserved, providing for potential electrostatic and hydrogen bonding interactions. These residues includeThr49, Thr78 and AsnS2 about heme propionate-6, and Arg38, Tyr48, AsnS2, and Trp59 about hemepropionate-7. As it is not possible for all of these potential hydrogen bond partners to interactsimultaneously with the propionates, a dynamic equilibrium among hydrogen bonding networks about theheme propionates has been suggested (Moore & Pettigrew, 1990).Of the three solvent molecules buried within the yeast protein, the one that forms hydrogen bonds withthe side chains of the conserved residues Thr78, Tyr67 and AsnS2 is of particular interest (Figure 4).This is the site of the largest conformational differences between oxidized and reduced cytochrome C.The position of this water molecule (WAT 166 by the numbering of Takano & Dickerson, 198 la,b) andits attendant hydrogen bonding partners have been remarkably well conserved in all known structures ofnative cytochromes c (reviewed in Bushnell et al., 1990). Because of the relatively large oxidation-state-linked conformational changes occurring in this hydrogen bond network, the three residues involvedwill be collectively referred to as the ‘water switch’ residues in the balance of this work. This term alsoincludes the conserved residue 11e75, which, although not part of the hydrogen bond network, shields thisregion from exposure to the solvent.‘Replacement of Arg38 by Lys, Ala, Asn, Gin, His, and Leu resulted in reduction potentials as much as50 mV lower than the native protein but did not significantly change the pH dependence of reduction potential inthe pH interval between 5.5 and 8.5 (Cutler et al., 1989).8Figure 4 The structure of reduced yeast iso-1-cytochrome c about WAT166, with the hydrogen bondingpartners to this water molecule indicated by the heavy lines (Louie & Brayer, 1990). Dotted linesindicate hydrogen bonds.Thr78Tyr67His 189Mitochondrial cytochromes c are highly basic proteins owing to an abundance of positively chargedresidues that are located mainly on the protein surface. Yeast iso-i cytochrome c is a typical exampleof these proteins, with three arginines, sixteen lysines and four histidines. The surface distribution ofthese basic groups is asymmetrical, and several of the most conserved such residues encircle the exposedheme edge. Within this ‘ring’ of basic residues and adjacent to the exposed heme edge is the invariantresidue Phe82 (Figure 5). In the crystal structures of cytochrome c, the phenyl ring of Phe82 isapproximately coplanar with the heme. This residue has been the target of several structural (Louie &Brayer, 1988, 1989) and functional (Michel et at., 1989; Pearce et a!., 1989) studies. It is believed tohave roles in maintaining a high reduction potential (Kassner, 1972, 1973) and in facilitating electrontransfer (Poulos & Kraut, 1980). NMR spectroscopy and molecular dynamics simulations have indicatedthat this residue may be highly mobile. The rate at which the phenyl ring rotates about the C-C bondis estimated to be greater than 10 s’ (Williams et a!., 1985). By comparison, aromatic residues whoserotation is restrained have rate constants for ring flips on the order of 10 s1 (Moore & Williams, 1980).In a molecular dynamics simulation of the cytochrome b5 - cytochrome c complex, Wendoloski et at.(1987) observed large movement of the Phe82 side chain about its C-C bond that suggested a role forthis side chain as an aromatic bridge between the two hemes and providing a facile path for electrontransfer. However, this proposal was not supported by an NMR investigation of the cytochrome b5 -cytochrome c complex (Burch et a!., 1990), which found no evidence for significant movement of Phe82about this bond.Cytochrome c undergoes several post-translational modifications to its structure, the most importantof which is the covalent attachment of heme to apocytochrome c. This step is catalyzed by the enzymeheme lyase, and it is coupled to the translocation of cytochrome c across the mitochondrial outermembrane (Hennig & Neupert, 1981, Hennig et at., 1983). Two additional alterations are often observedin mitochondrial cytochromes c. N-terminal acetylation in vertebrate and plant cytochromes c is catalyzedby an acetyl-coenzyme A I N-acetyl transferase (Tsunasawa & Sakujama, 1984). This modification is10Leu85 Argl3Figure 5 The environment of the invariant residue Phe82 (Louie & Brayer, 1990).Phe82MetSOHis 1811believed to increase the resistance of cytochrome c to proteolysis by aminopeptidases (Jörnvall, 1975).Trimethylation of specific lysine residues of plant and fungal cytochromes c is catalyzed by an S-adenosylmethionine-dependent lysine methyl transferase specific for cytochrome c (Dimaria et al., 1979). Thereason for this modification is not clear, although trimethylated cytochrome c binds three times moretightly to the mitochondrial inner membrane than unmodified cytochrome c (Polastro et aL, 1978).1.3 Structural Differences Between Oxidized and Reduced Cytochrome cLong before crystal structures of cytochrome c were available, differences in the physicochemicalproperties between the two oxidation states of cytochrome c suggested that they exhibit structuraldifferences. Oxidized yeast and bovine cytochromes c are readily digested by trypsin and subtilisin, whiletheir reduced counterparts are completely resistant (Nozaki et a!., 1958; Yamanaka et a!., 1959). Thereduced protein is also more resistant to denaturation by heat, guanidine hydrochloride, alcohols, and urea(Myer, 1968; Urry, 1965; Kaminsky et al., 1972). Ulmer and Kagi (1968) used IR spectroscopy tomonitor the hydrogen-deuterium exchange of amide groups in horse heart cytochrome c and detectedoxidation-state dependent differences in the extent of exchange which indicated a more rigid reducedstructure. The temperature dependence of the reduction potential of horse heart cytochrome c has asmall negative entropy of reduction indicating that the reduced state is relatively more ordered than theoxidized state (Taniguchi et al., 1982b). Reichlin and coworkers (1966) observed a small butreproducible difference in the immunological response of reduced and oxidized horse heart cytochrome cto anticytochrome c sera.Direct evidence for structural differences between oxidized and reduced forms of cytochrome c isrevealed by comparison of the crystal structures of the two forms of the protein. Oxidized and reducedstructures are available for tuna (Takano & Dickerson, 1981 a,b) and yeast iso-i cytochromes c (Louie& Brayer, 1990; Berghuis & Brayer, in preparation). There are no significant changes in the positionof the atoms of the polypeptide backbone between the two oxidation states, and side chain movements12are restricted to only a few locations. These include WAT166 and its hydrogen bond network and thehydrogen bond network about heme propionate-7. The largest changes occur in the vicinity of WAT166,which moves 1.7 A towards the heme upon oxidation (Figure 6). Other changes occurring with oxidationinclude breakage of the hydrogen bonds between Met8O and Tyr67 and the hydrogen bonds betweenWAT166 / Asn52 and Asn52 I heme propionate-7.Although the oxidation-state linked conformational changes are similar in tuna and yeastcytochromes c, there are subtle differences between the two species. WAT166 of yeast cytochrome cmoves 1.7 A towards the heme upon oxidation, compared to 1 A in the tuna protein. The largerdisplacement of WAT166 in yeast cytochrome c leads to the loss of its hydrogen bond with Asn52 in theoxidized structure, while this bond is retained in the oxidized tuna protein. In both species ofcytochrome c, the conformational changes are consistent with stabilization of the positive charge on theoxidized heme through charge-dipole interactions.1.4 Physiological Roles of Cytochrome cCytochrome c is a reversible, single electron carrier of the mitochondrial intermembrane space(Figure 7). The major function of this protein is to transfer electrons from ubiquinol-cytochrome coxidoreductase to cytochrome c oxidase without significant loss of free energy.Cytochrome c also channels electrons from two other pathways to cytochrome c oxidase. In yeast,cytochrome c accepts electrons from flavocytochrome b2, which catalyzes the oxidation of L-lactate topyruvate (Bach et a!., 1942a,b). Reducing equivalents from NADH can also be transferred tocytochrome c through a NADH cytochrome 15 oxidoreductase I cytochrome b complex bound to themitochondrial outer membrane. (Ito, 1980a,b; Lederer et a!., 1983).Cytochrome c also takes part in two detoxification pathways. In one of these, cytochrome c acceptselectrons from sulphite oxidase, which is the terminal detoxification enzyme in the catabolism of sulphurcontaining amino acids (McLeod et a!., 1961, Cohen & Fridovich, 1971). In the other, cytochrome c13Asn52Figure 6 The oxidation state-linked conformational changes about WAT166 in yeast iso-1-cytochrome c(Louie & Brayer, 1990; Berghuis & Brayer, in preparation). Thin lines, reduced; thick lines, oxidized.The dashed lines represent hydrogen bonds to WAT166, which is represented by the circles.lie75Thr78 Tyr67eMetSOHis 1814H20+S002+ 4 H”2H0Outer MembraneNADHNAD+HFigure 7 The physiological electron transfer partners of cytochrome c and their location within themitochondrial membranes (Pettigrew & Moore, 1987). Arrowheads denote the direction of electrontransfer. (Abbreviations: CCR, ubiquinol-cytochrome c oxidoreductase; CCO, cytochrome c oxidase;CCP, cytochrome c peroxidase; c, cytochrome C; b5, cytochrome b5; b5R, NADH-cytochrome b5oxidoreductase; Fp-b2, flavocytochrome b2; SO, sulfite oxidase.)S0 +2 H”lactatepyruvate +2 H”‘7H2O÷2HInner Membrane2 H2015donates electrons to cytochrome c peroxidase, which catalyzes the reduction of peroxide to water(Altschul et at., 1940; Abrams et at., 1942).The ability of cytochrome c to interact with partners located on both of the mitochondrial membranesand in the intermembrane space indicates that this protein is mobile and not completely bound to themitochondrial inner membrane, as are other members of the respiratory chain. Cytochrome c formsstable complexes with all of its natural electron transfer partners with dissociation constants on the orderof 10 M at low ionic strength (reviewed in Pettigrew & Moore, 1987).1.5 Physicochemical Properties of Cytochrome c1.5.1 UV-Visible SpectraThe UV-visible spectra of cytochrome c (Figure 8) are largely due to the electronic structure of theheme and its axial ligands. The majority of the observed bands are due to 7r,1r* electronic transitionsin the porphyrin orbitals. Reduced cytochrome c is characterized by three intense absorbance bandsarising from porphyrin 1r_ir* transitions, typically located near 550 nm, 520 nm, and 416 nm. Thesebands are known respectively as the a, 3, and ‘y bands; the last band is the most intense and is also calledthe Soret band. In the oxidized protein, the a and fi bands merge to produce a broad band centred at 528nm, while the Soret band decreases in intensity and shifts to 409.5 nm. Also present in the spectra ofboth oxidation states are bands near 280 nm arising from 1r_ir* transitions of the aromatic amino acidstyrosine and tryptophan.The oxidized cytochrome also exhibits a weak absorption band at 695 nm believed to be caused bya porphyrin ir to iron(III) d transition (reviewed in Moore & Pettigrew, 1990). The presence of this bandis characteristic of methionine as one of the axial ligands (Shechter & Saludjian, 1967). Schejter et at.(1991) demonstrated that a band of similar position and intensity can be reproduced by the addition ofsoft ligands to cytochrome c in which the Met8O sulphur has been chemically modified to prevent itscoordination to the heme.16Reduced —_..jiz1.0• 0.005Oxidized Reduced0000.680 700 720C.0 Wavelength (nm)00.50I I200 400 600Wavelength (nm)Figure 8 The UV-visible spectra of oxidized and reduced yeast iso-1-cytochrome c (25 °C, pH 6, =0.1 M). The reduced protein has absorbance maxima at 550, 521, and 416 nm. The oxidized protein hasmaxima at 695 (inset), 528, and 409.5 nm.171.5.2 Electrochemistry1.5.2.1 Reduction Potential and its MeasurementThe position of the oxidation-reduction equilibrium between two electroactive species (1) is governedby the Nernst equation (2):A0, + Bri + B07, (1)E II = E°-[ATW][BO (2)nFIn the above expression, A and B are the two electroactive species, Eeil is the measured cell potential,R is the gas constant, F is Faraday’s constant, T is the absolute temperature in Kelvin, and n is thestoichiometry of electrons in the reaction. E° is the cell potential when all cell components have unitactivity. The two electroactive species need not be in physical contact as long as there is electricalcontact through a salt bridge (for ionic movement) and a wire (for electronic movement). Thisarrangement allows the complete cell to be subdivided into two ‘half cells’, one of which can be chosenas a convenient reference against which all half cell potentials are measured. Assigning a potential ofO Volts to the reference simplifies the Nernst equation (3):— r’O RT 1 oxüiiz&i] (3)hafcell £ + F FA 1- reducedThe standard hydrogen electrode (SHE), composed of an inert platinum wire in a 1 M solution of HC1in equilibrium with 1 atm of H2 gas, serves as this standard reference. By convention, electrochemicalhalf cell reactions are expressed as reductions. Those half reactions with potentials greater than zero havea greater tendency to be reduced relative to the SHE reference. In practice, the SHE is rarely used;experimentally more convenient reference electrodes, such as the saturated calomel electrode, or SCE[composition Hg/Hg2Cl/KC1 (saturated), E° = 244.4 mV vs SHE] are employed.18By equating the Nernst equation to the expression for the concentration dependence of free energy,the following relation is obtained.AG° = -nFE° (4)Thus reduction potentials are direct measurements of the free energy of the oxidation-reductionequilibrium.Reduction potentials can be measured in several ways. In equilibrium measurements, the cell potentialcan be controlled by a potentiostat and the ratio of the oxidation states of the species of interest measured.Spectroelectrochemistry is an example of this technique, in which species concentrations are determinedspectrophotometrically and the potential is controlled at a semitransparent working electrode (Murray etat., 1967). Electron transfer proteins do not readily transfer electrons with bare metal electrodes evenunder thermodynamically favourable conditions because their active sites are often buried within theprotein. Small inorganic mediators are often necessary to shuttle electrons between the electrode and theprotein. Ideally the mediator should only catalyze the attainment of thermodynamic equilibrium betweenthe electrode and the electron transfer protein. Spectroelectrochemistry has been used to measure thereduction potentials of many electron transfer proteins, including cytochrome b5 (Reid et at., 1982), andcytochromes c from various species (Taniguchi et at., 1982b, Cutler et at., 1987).A second common experiment used to determine reduction potentials is cyclic voltammetry (Bard &Faulkner, 1980; Evans et at., 1983). In this technique, the potential of the working electrode is variedlinearly, and current is monitored as a function of potential. Under conditions where electron transferbetween the electrode and the electroactive species in solution is rapid, the observed current isproportional to the rate of mass transfer of the reactant to the electrode surface, which in turn dependson the concentration gradient of the electroactive species at the electrode. Although electron transferproteins interact poorly with bare metal electrode surfaces, the latter can be modified to promote rapidelectron transfer (vide infra).19Cyclic voltammetry is best understood by describing the events that occur during the experiment, asshown in Figure 9 (Maloy, 1983). Consider an electroactive species that is initially in the oxidized state.The applied potential of the working electrode is initially at a positive value relative to the reductionpotential of the species of interest such that no net electrochemical reaction occurs (1). As the potentialof the working electrode is lowered and approaches the reduction potential of the electroactive species,a cathodic current i begins to flow as electrons are transferred to the oxidized species from the electrode(2). This process causes a depletion of the oxidized species at the working electrode surface and theestablishment of a concentration gradient of the oxidized species between its value at the electrode surfaceand its value in bulk solution far from the electrode surface. The volume between the working electrodesurface and the bulk solution is called the depletion layer, which also corresponds to an accumulationlayer of the reduced species.The observed current will be sensitive to this concentration gradient at the working electrode surface.As the reduction proceeds, the concentration gradient and the current both increase. The concentrationgradient will be steepest when the applied potential is 28.5 mV beyond the reduction potential of theelectroactive species, and at this point the cathodic current reaches a maximum, denoted i,, (3). Thecurrent does not increase beyond this point because the concentration gradient of the oxidized speciesbecomes shallower as the depletion layer expands. As the working electrode is taken to more reducingpotentials, the current begins to fall in a manner independent of the electrode potential (4). When thedirection of the potential sweep is reversed such that the potential of the working electrode is now mademore positive, an anodic current Ia begins to flow in the cell as the reduced species accumulated duringthe first half of the experiment is oxidized. This process gives a current vs potential curve similar to thatobtained during the sweep to low electrode potentials for the reasons already discussed. The result ofthis experiment is the current vs potential plot known as a cyclic voltammogram. The anodic andcathodic peak currents are symmetrically located about the reduction potential of the electroactive species,and their positions are used to calculate the reduction potential.20C0 (bulk)Eapp ,mVvs SHE‘Ixa) b)Figure 9 a) A cyclic voltammogram obtained for a reversible electrochemical system (25 °C). Theterms‘PC and ‘Pa refer to the peak cathodic and anodic currents, respectively. b) The concentrationgradient of the electroactive species at the electrode surface at different points along the voltammogram.The electroactive species is initially in the completely oxidized state, with a concentration of C0 (bulk)far from the electrode surface. The x-axis represents the distance from the electrode surface. Adaptedfrom Maloy, 1983.C0 (bulk)0C0 (bulk)i. :..m57mVE1120C0 (bulk)21For cyclic voltammetry to be useful in determining reduction potentials, electron transfer between theelectrode and the electroactive couple must be reversible. Reversibility requires the heterogeneouselectron transfer rate between the electroactive species in solution and the working electrode surface tobe sufficiently rapid such that the Nernst equation is obeyed at the surface of the electrode. If thiscondition is met, the faradaic current will depend only on the rate of mass transfer of the electroactivespecies to the electrode.There are three means of mass transfer to the working electrode surface: migration of the chargedelectroactive species in response to the electric field at the electrode surface, bulk movement of thesolution (or convection), and diffusion of the electroactive species in response to a concentration gradient.For determination of reduction potentials by cyclic voltammetry it is desirable that the predominant meansof mass transfer is diffusion. Migration is repressed by the presence of a supporting electrolyte insolution, and convection is avoided by performing the experiment in an unstirred solution.The experimental challenge is to make the heterogeneous electron transfer rates of electron transferproteins fast. Electron transfer between metal electrode surfaces and proteins is hampered by lowerheterogeneous rates of electron transfer as well as denaturation of the protein on the electrode surface.However, in the last fifteen years, techniques have been developed to modify electrode surfaces in amaimer that promotes rapid, heterogeneous electron transfer (reviewed by Frew & Hill, 1988). Surfacemodifiers promote reversible association of the protein and the electrode by making the electrode surfacemore hydrophilic and providing for favourable electrostatic and hydrogen bonding interactions. Surfacemodifiers are bifunctional compounds, with one region used for binding to the electrode surface whilethe other region faces the solution and interacts with the electron transfer protein. An example of asurface modifier is the compound 4,4’-bipyridine (Eddowes & Hill, 1977). The electrode-facing regionis frequently a sulphur containing group, while the solution-facing group can be tailored to provide afavourable interaction site for the redox protein to be studied (Taniguchi et a?., 1982a; Allen et al.,1984). Peptides can be used as surface modifiers, with cysteine residues used to anchor the modifier to22the electrode surface while hydrophilic charged residues provide the interaction site for the protein. Inaddition, some electrode surfaces such as pyrolytic edge graphite (Armstrong et a!., 1984) and tin dopedindium oxide (Yeh & Kuwana, 1977) are ‘naturally modified’ and promote reversible electron transferwith proteins.1.5.2.2 Factors Affecting The Reduction Potential of Cytochrome cUnder physiological conditions, the reduction potentials of mitochondrial cytochromes c are in therange + 260 to + 290 mV (Pettigrew & Moore, 1987). These reduction potentials are about halfwaybetween that of the NAD/NADH couple (-320 mV) that is the electron donor to the respiratory chain,and02/H (+ 820 mV), which is the terminal electron acceptor for this system.The reduction potential of a heme containing protein will depend upon the nature of the porphyrin,the axial ligands, and the dielectric environment surrounding the heme (Bottomly et a!., 1982; Moore &Williams, 1977; Moore et a!., 1986). As all mitochondrial cytochromes c possess the same porphyrinand axial ligands, any differences in their reduction potentials are due to differences in the surroundingenvironment. The medium surrounding an electroactive centre can have a considerable influence on itsoxidation-reduction equilibrium. The reduction potential of bipyridine mesoheme methyl ester is 300 mVhigher in benzene than in water (Kassner, 1972, 1973). Heme octapeptid& in the presence of N-acetylmethionine has the same ligands as cytochrome c, yet its reduction potential is 300 mV lower than thatof the intact protein (Harbury et a!., 1965).The charge difference between reduced and oxidized heme is a single positive unit. Thus electrostaticinteractions between the heme charge and the charges and dipoles in the surrounding medium will be thepredominant factors in determining the reduction potential of cytochromes c. The stability of a particularoxidation state will depend on the ability of surrounding dipoles and charges to orient themselves in2 Heme octapeptide is a species prepared from tryptic digestion of cytochrome c. The heme is bound to thepeptide through thioether linkages from Cysl4 and Cysl7 (Tuppy & Paleus, 1955). Hisi 8 provides one of theaxial ligands.23response to the charge at the heme. The most influential groups will be those located nearest the heme.Consequently, Moore and colleagues have directed attention to Arg38 and the heme propionates (Moore,1983; Moore et at., 1984; Cutler et at., 1989). Dipoles from peptide bonds and the side chains of polarresidues, and nearby solvent molecules, including WAT166, will also contribute to the stabilization ofthe heme charge (Churg & Warshel, 1986).The movement of groups adjacent to the heme upon a change in the oxidation state of cytochrome cis consistent with a response to the altered charge on the heme, as previously discussed. However, thestructural changes are comparatively small, with no significant changes in the positions of the main chaindipoles or the surface charges of the protein. The environment about oxidized heme in cytochrome cprovides only partial stabilization of the positive charge, far less stabilization than if the heme wereimmersed in water, where the surrounding solvent dipoles would be free to move in response to thecharge. The inability of the protein to solvate the positive charge on oxidized heme as effectively aswater is the cause of the difference in potential between cytochrome c and N-acetyl methionine I hemeoctapeptide (Churg & Warshel, 1986).Measurements of reduction potentials of cytochrome c variants are useful because they are sensitiveindicators of alterations to the heme environment which influence its ability to stabilize a positive chargeon the heme. In addition, reduction potential measurements can be used to determine the thermodynamicparameters associated with reduction (by measurement of the temperature dependence of reductionpotentials) and the dissociation constants of titratable groups whose ionization states are sensitive to theoxidation state of the protein (by measurement of the pH dependence of the reduction potential).241.5.3 Electron Transfer Kinetics1.5.3.1 Electron Transfer TheoryThe relative simplicity of electron transfer reactions compared to other chemical transformations haslead to extensive experimental and theoretical work aimed at determining the factors contributing toobserved rates and the magnitude of their contributions. Early work in this field emphasized analysis ofreactions between substitutionally inert inorganic complexes and has been useful in identifying the factorsthat contribute to observed biological electron transfer rates (reviewed by Sutin, 1973; Marcus & Sutin,1985)Electron transfer reactions are generally classified as one of two types, according to the involvementof the ligands surrounding the electron transfer centres. ‘Inner sphere’ electron transfer proceeds throughan intermediate in which the reactants share a ligand in their inner coordination shells. This mechanismrequires coordination bond breakage and reformation. In contrast, ‘outer sphere’ electron transfer occurswithout changes in the coordination shells of the reactants. Theories for outer sphere electron transferreactions are easier to consider because such reactions do not involve bond breakage or reformation. Thebalance of this discussion will be concerned with outer sphere mechanisms. Most electron transferreactions involving cytochrome c occur by an outer sphere mechanism (vide infra). Outer sphere electrontransfer is believed to occur in the series of steps shown in Figure 10. In the first step, the electrondonor and acceptor form a noncovalent precursor complex. Formation of the precursor complex maybe influenced by electrostatic interactions between the reactants. The nature of the electrostaticinteractions in a particular reacting pair can be determined from the ionic strength dependence of thereaction rate constant. If the reaction is between species of opposite charge, the observed reaction ratewill diminish with increasing ionic strength.Outer sphere electron transfer theory has been developed from activated complex theory (Eyring,1935), which describes a reaction as proceeding through an intermediate, transitory state of maximumfree energy (the activated complex). The expression for the rate constant of an outer sphere reaction25(reduced) (oxidized)ketSuccessor Complex0 ÷Donor(oxidized)Acceptor(reduced)Figure 10 Schematic diagram of the steps involved in outer sphere, intermolecular electron transfer(Scott et al., 1985). The reactants form the precursor complex without a change in the coordination stateof the metal centers. is the association constant of the precursor complex; Kd is the dissociationconstant of the successor complex. Shaded and unshaded symbols indicate reduced and oxidizedcomponents, respectively.KaDonor Acceptor Precursor Complex26according to this theory is given byk=xZexp(G) (5)RTIn this equation, Z is the collision frequency (1011 Ms4 for bimolecular reactions, 10’s s’ forunimolecular reactions). K is the probability of electron transfer occurring when the precursor complexis at the transition state and is dependent on the distance between the electron transfer sites as well astheir relative orientation and the nature of the intervening medium. K may have values between zero andunity, representing low to high probability respectively of electron transfer occurring when the precursorcomplex is at the transition state. Reactions for which K is unity are referred to as being adiabatic.IiG is the free energy difference between the ground state and transition state within the precursorcomplex. The free energy of the precursor complex is a function of its nuclear coordinates. Eachpossible conformation has associated with it a free energy, giving rise to a multidimensional free energysurface for all possible conformations. There is a similar surface for the successor complex. Forillustrative purposes, these surfaces are frequently shown as two dimensional curves, with free energyalong the ordinate and the abscissa representing the nuclear coordinate (Figure 11). The parabolicrelation between potential energy and nuclear coordinates assumes that the participating chemical bondscan be described as simple harmonic oscillators that obey Hooke’s law. The wells of the energy curvesare the equilibrium nuclear coordinates about which the nuclei fluctuate in the precursor and successorcomplexes.Libby (1952) proposed that the Franck-Condon principal applied to electron transfer within theprecursor complex, as the positions and momenta of the relatively massive nuclei can be considered tobe unchanged during electron transfer. This premise demands nuclear reorganization of the precursorcomplex to a conformation that is energetically accessible to the successor complex before electrontransfer occurs. This condition is satisfied at the point where the free energy surfaces of the precursorand successor complexes intersect. This point is at the free energy maximum on the reaction coordinate,27Reactants Products Reactants ProductsFigure 11 A diagram of the reaction coordinate for outer sphere electron transfer within the precursorcomplex (Devault, 1984). Symbols: iG°, free energy of the reaction; G*, free energy of activation; X,reorganization energy; R, equilibrium nuclear coordinates of the precursor complex; I, nuclearcoordinates of the transition state; P, equilibrium nuclear coordinates of the successor complex.AG°R I PReaction Coordinate28and the corresponding conformation of the reacting molecules is called the transition state. Nuclearreorganization from the equilibrium coordinates of the precursor complex to the coordinates of thetransition state consists of changes in equilibrium bond lengths and bond angles (inner spherereorganization) and reorientation of surrounding solvent molecules and their dipoles (outer spherereorganization).The activation free energy depends on the reaction free energy, G°, and the reorganization freeenergy X. The reorganization free energy is the energy required to bring the precursor complex fromits equilibrium nuclear coordinates to those of the successor complex without the actual transfer of anelectron. The reorganization free energy can be considered as the sum of two contributions. The innersphere reorganization energy, is associated with changes in the equilibrium bond lengths and bondangles and is determined from the molecular vibrational coordinates. Outer sphere reorganization freeenergy, X0, is the contribution from the changes in the orientation of the surrounding solvent dipoles toadjust to the altered charges on the reaction centres. Its contribution is often calculated using dielectriccontinuum theory (Marcus, 1965). The activation free energy also has contributions from the workrequired to form the precursor complex, wr, and the work required to dissociate the successor complex,-wy. The term AGO’ is the free energy difference between the precursor and successor complexes, whichmay differ from AG°, the free energy difference between the separated products and reactants:AGO’= AG°÷w”—w’ (6)The work terms most often considered are those attributable to electrostatic interactions between theparticipants. The influence of these interactions can be estimated from the size and charge of theparticipants and the application of Coulomb’s Law.The relation between the aforementioned terms is given by the following equation (Marcus, 1964; seealso Devault, 1984, for the derivation of this relationship):AG*= wT + _L(A + AG0’)2 (7)4)29The Marcus expression for activation energy (equation 7) can be substituted into equation 5 and thelatter equation can be used to predict rate constants for electron transfer if the system under study isdefined with respect to the structure and dynamics of the products and reactants. Rarely is a reaction sowell characterized to allow the use of equations 5 and 7 directly. An experimentally more convenientrelationship is the relative Marcus equation, which relates the rate constant of electron transfer betweentwo species (the cross reaction rate constant, k12) to those of the electron transfer self-exchange rateconstants of the individual reactants (k11, k) and the equilibrium constant for the reaction (K1,):k12 = /k11 k22 K12 f12 (8)Wheref2is equal to unity for adiabatic or uniformly nonadiabatic reactions. The key assumption of thisuseful relationship is that the reactants undergo the same activation processes in their cross reaction (k12)as they do in their self-exchange reactions (k11, k2,).Relative Marcus theory has been accurate in predicting cross reaction rate constants from self-exchangerate constants of simple coordination complexes, in which the assumption regarding the similarity of theactivation process is more valid for reactants of similar charge, size, and hydrophobicity. Experimentalvalues for the self-exchange rates of such complexes are obtained from isotope exchange measurements(Baker et at., 1959), NMR saturation transfer experiments (Shporer et at., 1965), or approximated fromcross reaction rates between reactants of similar structure (Wilkins & Yelin, 1968). Self exchange ratesfor several small electron transfer proteins have been measured by NMR saturation transfer experiments,including those of mitochondrial cytochromes c (Gupta, 1973; Concar et a!., 1991). Cross reaction ratesof electron transfer proteins with small nonphysiological complexes are easily measured (reviewed byWherland & Gray, 1976) and the relative Marcus equation is often rearranged to calculate the selfexchange rate constant of the protein. Comparisons of the self exchange rate constants of a given protein30obtained with several different inorganic complexes is a useful way of studying the differences in theactivation processes of these cross reactions (Wherland & Gray, 1976).1.5.3.2 Electron Transfer Kinetics of Cytochrome cBecause of the solubility, stability and commercial availability of horse heart cytochrome c, theelectron transfer kinetics of this protein with simple inorganic complexes was an early target ofexperimentalists using stopped flow spectrophotometry. Electron transfer rates have been measured with(Fe(CN)6, (Morton et at., 1970), Cr (Yandell et at., 1973), Ru(NH62(Ewall & Bennett, 1974),Fe(edta)2 (Hodges et at., 1974), Co(phen)3 (McArdle et at., 1974) and Co(ox) (Holwerda et at.,1980). These experiments demonstrated that in most cases cytochrome c participates in outer sphereelectron transfer. The exception is the reduction of cytochrome c by Cr2, in which the presence of Cr3bound to the reduced protein suggests an inner sphere mechanism.The self-exchange rate for horse heart cytochrome c has been determined directly using NMRspectroscopy by measuring the spin-lattice relaxation time of the Met8O methyl resonance of the reducedprotein in the presence of variable amounts of oxidized protein (Gupta, 1973). The self exchange rate,uncorrected for the effect of electrostatic repulsion, is approximately 1000 M1s at 0.1 M ionic strength,40 °C, pH 7. Self exchange rates for yeast iso-1-cytochrome c wild type and Phe82Gly variant are 200and 350 M1s respectively (Concar et al., 1991).These experiments also demonstrated that the rates of electron transfer involving cytochrome c arecomparable to those of inorganic complexes. This observation indicated that the limited exposure of theheme in cytochrome c does not greatly impede its reactivity. Consequently, attention was focussed onthe conserved basic residues surrounding the exposed heme edge to determine their role in directing theformation of electron transfer active precursor complexes through favourable electrostatic interactionswith electron transfer partners.31Chemical modification of amino acid side chains of cytochrome c has been used to assess thefunctional consequences of altered protein structure at specific locations (reviewed by Brautigan et a!.,1978a). In one particular study, Margoliash and coworkers prepared derivatives of horse heartcytochrome c with singly modified lysine residues at different positions (Brautigan et al., 1978b,c).Lysine residues were modified by carboxydinitrophenylation, which gives these residues negative charges.Those derivatives with singly modified lysines nearest the exposed heme edge had the greatest influenceon the rates of electron transfer of cytochrome c with inorganic complexes (Butler et a!., 1983;Armstrong et a!., 1986a) and the electron transfer proteins cytochrome c oxidase (Ferguson-Miller,1978), azurin, stellacyanin and plastocyanin (Augustin et a!., 1983; Armstrong et a!., 1986b).Chemical modification has been used in a different manner to further establish the role of surfacecharges at the exposed heme edge in cytochrome c electron transfer. In shielding experiments, thesusceptibility of lysine residues of free cytochrome c to chemical modification is compared to theirsusceptibility when cytochrome c is in a complex with an electron transfer partner protein (Bosshard,1979; Rieder & Bosshard, 1980). Those lysines present at the part of the surface of cytochrome c thatinteracts with its electron transfer partner are protected from chemical modification.Both the shielding experiments and the kinetic experiments on chemically modified cytochrome c showthat lysine residues around the exposed heme edge are involved in complexation with other electrontransfer proteins (reviewed in Pettigrew & Moore, 1987). These are lysines 8, 13, 25, 27, 72, 73, 79,86, and 87. Of these, positions 13, 72, 73, 79 and 87 are conserved in yeast iso-1-cytochrome c.Computer graphics models for complexes formed by cytochrome c with bovine microsomalcytochrome b5 (Salemme, 1976) and cytochrome c peroxidase (Poulos & Kraut, 1980) also indicate theinvolvement of the ring of basic residues about the exposed heme edge in complex stabilization. Thesemodels were constructed using the structures of the individual partners determined by X-ray diffractionanalysis and minimizing the electrostatic energy between the two structures. The model complexes are3Position 13 is an arginine residue in yeast-iso-lcytochrome c32stabilized by complementary electrostatic interactions between the charges surrounding the exposed hemeedges of each protein. In addition, the hemes within the complexes are approximately coplanar, withheme edge to edge distances of 18 A and 10 A for the complexes with cytochrome c peroxidase andcytochrome b5 respectively. While bovine microsomal cytochrome b5 is not a natural electron transferpartner of cytochrome c, the complex of these two proteins is relevant because of the sequence homologycytochrome b5 shares with the cytochrome b subunits of flavocytochrome 2, sulphite oxidase andcytochrome b5 of the mitochondrial outer membrane.Because of the relatively long range over which electrostatic forces act, the arrangement ofcomplementary charge arrays on electron transfer partners was proposed to be important in enhancingor promoting rapid reaction by steering the reactants to an optimal docking arrangement. A Browniandynamics simulation of cytochrome c I cytochrome c peroxidase (Northrup et at., 1988) complexformation suggests rather that the electrostatic interactions serve to bring the reactants together in arelatively nonspecific manner. Subsequently there is reorientation of the electron transfer partners withinthe complex to an electrostatically favoured geometry. Further evidence for alternative geometries in theprecursor complexes involving cytochrome c was provided by electrostatics calculations on the interactionof cytochrome c with cytochrome b5 by Mauk et a!. (1986) which suggested the existence of twoisoenergetic, electrostatic complexes at alkaline pH, one of which was the complex proposed by Salemme(1976).1.5.4 The Alkaline IsomerizationUV-visible spectroscopy distinguishes five pH dependent forms of ferricytochrome c, and three suchforms of ferrocytochrome c between pH 0 and 13 (Theorell & Akesson, 1941). These spectraldifferences are attributed to differences in the nature of the axial ligands to the heme iron. A combinationof structural analysis of the native protein and comparison of its pH dependent spectroscopic propertiesto those of model compounds has led to the assigmnent of the pH dependent transitions to the changes33in axial ligation shown in Figure 12. Transitions at the extremes of pH are accompanied by unfoldingof the protein. However, the transition of ferricytochrome c from state III (native) to state IV occurs withthe retention of a folded, globular conformation (Myer, 1968). The transition from state III to IV offerricytochrome c is called the alkaline isomerization, with state IV known as the alkaline isomer.Evidence for an analogous transition in the reduced protein, with a pK of approximately 14.5, has beenreported (Barker & Mauk, in press).The alkaline isomer differs from the native form in several ways. Unlike the native protein, thealkaline isomer is not reducible by ascorbate (Greenwood & Palmer, 1965; Wilson & Greenwood, 1971),and its reduction potential is 450 mV lower than that of the native protein (Barker & Mauk, in press).The UV-visible spectrum of the alkaline isomer indicates that the heme iron remains low spin, but thereare differences in the spectra, particularly the absence of the absorbance band at 695 nm. The 695nmband is associated with the presence of methionine as an axial ligand to ferriheme (Shechter & Saludjian,1967; Schejter et at., 1991) and has been used to monitor the transition from native to alkaline isomer.The native and alkaline isomers also differ in their NMR (Gupta & Koenig, 1971; Wooten et at., 1981),EPR (Brautigan et at., 1977) CD (Myer, 1968), and MCD (Vickery et at., 1976; Gadsby et at., 1987)spectra. There is no crystal structure currently available for the alkaline isomer.The absence of the 695 nm band, the retention of the low spin state, and the lowered reductionpotential of the alkaline isomer suggest that Met8O has been replaced by a strong field ligand at high pH.The ligand replacing methionine 80 is believed to be the deprotonated f-amino group of a lysine residue,based on the similarity of the MCD and EPR spectra of the alkaline isomer to those of heme proteins withknown histidyl/amino axial coordination (Gadsby et at., 1987; Rigby et at., 1988; Simpkin et at., 1989).Additionally, trifluoroacetylation of all lysine residues in cytochrome c prevents the formation of a lowspin alkaline species (Stellwagen et at., 1975). However, which lysine replaces Met8O is unknown. Itis likely that the alkaline isomer consists of several species with different lysines as potential ligands asis suggested from the results of NMR experiments which indicated the presence of at least two alkaline34I I I I IH20—Fe—H H20—Fe—H Met8O—Fe—His18 x—Fe—---HislBI II - — III IV - — VOA 2.5 9 12.8H.S., unfolded H.S., partially folded LS., folded LS., folded LS., unfoldeda)I I IH20—Fe— Hisi 8 Met8O—Fe —Hisi 8 ?—Fe—Hlsl 8I I II— II —__ III<4 >12LS., unfolded LS., folded LS., partially unfoldedb)Figure 12 The pH dependent ligation states of mitochondrial cytochrome c, with the pKs of theassociated transitions, spin states, and state of the protein fold. a) Ferricytochrome c; b)Ferrocytochrome c. Abbreviations: H.S., high spin; L.S., low spin. The ligand ‘X’ of state IVferricytochrome c is likely a lysine residue, but its specific identity is unknown. (Data from Dickerson& Timkovich, 1975).35conformational states of ferricytochrome c in equilibrium with the native structure at alkaline pH(Hong & Dixon, 1989). Additional support for this proposal is provided by the behaviour of single sitemutants of yeast iso-1-cytochrome c in which lysine residues were replaced by alanine residues. Suchmutations at positions 72 and 79, proposed to be the most likely potential ligands in the alkaline isomer,did not alter the alkaline isomerization pK (Inglis et a!., unpublished results).The transition from native to alkaline form involves the net release of one proton from cytochrome c,with a plc that is dependent on the species of cytochrome c (9.0 to 9.5 for horse heart and 8.5 for yeastcytochromes c). Davis and coworkers (1974) measured the pH dependence of the kinetics of the alkalineisomerization of horse heart cytochrome c and proposed a mechanism involving a deprotonation followedby a conformational change that results in the loss of Met8O as the axial ligand:KHNative Ferricytochrome c (protonated) Native Ferricytochrome c (deprotonated)kfNative Ferricytochrome c (deprotonated) Alkaline Ferricytochrome ckbThe measured values were pK = 11, k. = 6 s’ and kb = 0.05 s_i, which were consistent with the overallobserved pK of 9.0. Above pH 10.5, a second, faster kinetic phase is observed (Kthara et al., 1976).The alkaline isomerization is sensitive to the environment about the heme. For example, the influenceof position 82 mutants on this transition was examined by Pearce et a!. (1989) who found thatreplacement of Phe82 by leucine, isoleucine, serine or glycine lowered the alkaline pK, of yeast iso-icytochrome c by 0.8 to 1.3 pH units. Rate measurements from pH-jump experiments revealed that thelower ps of the Leu and lie mutants were caused solely by decreases in the PKH of the deprotonation,with no effect on the subsequent conformational equilibrium. The Ser and Gly variants showed similardecreases in their PKH5, but these were partially offset by changes in the conformational equilibriumwhich stabilized the native isomer.361.5.5 Ligand SubstitutionMitochondrial ferricytochrome c binds added ligands such as imidazole (Schejter & Aviram, 1969;Sutin & Yandell, 1972), azide (Sutin & Yandell, 1972; Saigo, 1986), and cyanide (George & Tsou, 1952)with loss of the 695nm band, indicating displacement of Met8O as one of the axial ligands. At pH 7 andan ionic strength of 1 M, horse heart cytochrome c binds azide and imidazole with equilibrium constantsof 4 M1 and 15 M1, respectively (Sutin & Yandell, 1972; Schejter & Avriam, 1969). The ligand bindingaffmity of ferricytochrome c towards imidazole and azide is low when compared to heme proteins whoserole is reversible ligand binding, but it is consistent with the role of cytochrome c as an electron carrier.However, cyanide binding to ferricytochrome c is an exception, with a binding constant of approximately106 M1 (George & Tsou, 1952).The kinetics of ligand binding to ferricytochrome c were investigated by Sutin & Yandell (1972) usingazide, imidazole and cyanide. In each case, ligand binding was reversible, and at high ligandconcentrations rate saturation was observed. The maximum rate was independent of the identity of theadded ligand, within experimental error. Based on these observations, the following mechanism wasproposed to describe ligand binding to ferricytochrome c where ‘opened’ and ‘closed’ refer to thesusceptibility of the heme to binding of the added ligand:k1Ferricytochrome c (closed) Ferricytochrome c (open)k1k2(10)Ferricytochrome c (open) + ligand Ferricytochrome c-ligandk...2The forward rate constant k1 is rate limiting and has a value from 30 to 60 s1. This rate constant maybe associated with the rate of disruption of the iron(III)-Met8O sulfur bond. Alternatively, it may reflectthe rate at which the heme crevice opens sufficiently to allow access of competing ligands to the heme.The high affinity of ferricytochrome c for cyanide was attributed to a slow rate of dissociation of thecyanide-cytochrome c complex characterized by k2 of less than 1O s’.37Both alkaline isomerization and addition of ligands displace Met8O as one of the axial ligands offerricytochrome c. Saigo (1986) observed that low alkaline pK was correlated to high ligand affinityamong several different species of ferricytochrome c and suggested that both properties were measuresof the stability of the native ligation state of the heme crevice. In addition, Saigo observed a correlationbetween high ligand affinity and increased sensitivity of cytochrome c towards guanidine hydrochloridedenaturation that indicated a link between local (around the heme) and global (protein unfolding)conformational changes of cytochrome c.1.6 Methods of Structure-Function Analysis of Cytochrome c1.6.1 Genetic AnalysisSaccharomyces cerevisiae possesses two forms of cytochrome c known as iso-i and iso-2, that areencoded by genes CYC 1 and CYC7 respectively. Iso-i -cytochrome c accounts for over 95 percent ofthe total mitochondrial protein (Sherman et al., 1966). The expression of iso-i -cytochrome c iscontrolled mainly at the transcriptional level and is induced by the presence of heme, oxygen, and lactateas a non-fermentable carbon source. CYC1 is repressed under anaerobic conditions and in the presenceof glucose as a fermentable carbon source.Genetic studies on the CYC 1 locus by Sherman and coworkers (summarized in Hampsey et al., 1986)have been of great use in examining the influence of changes in the sequence of cytochrome c on itsfunction. In these investigations, randomly mutated yeast strains are screened for their ability to growin a medium containing a nonfermentable carbon source, which requires a functional cytochrome c.Mutation sites which abolish function are located by DNA sequencing of the mutant gene. With thismethod a large number of mutants can be examined rapidly, and critical residues can be identified bytheir tendency to have a high frequency of nonfunctional mutants. Among the positions most resistantto alteration are those hydrophobic residues are in contact with the heme, such as Trp59 (Schweingruberet a!., 1978). An added advantage of this technique is its potential to identify compensatory, second site38revertants, in which mutations at a different residue from the first give a functional gene product. In thismanner the single site mutant 11e52 was prepared and found to exhibit enhanced thermal stability overthe wild type protein (Das et al., 1989; Berroteran & Hampsey, 1991).1.6.2 Comparative AnalysesFour strategies are commonly used to investigate the influence of structure on the function ofcytochrome c. Of these, comparison of the properties of cytochromes c from different species is thesimplest method to perform, but the most ambiguous to interpret. It does not require modification of theproteins to be studied, as nature supplies the structural variants of the protein. However, it is unlikelythat differences in the properties between proteins of different species can be assigned to particularstructural features because proteins from different species generally differ in sequence at several positions.This method is best suited for comparison of the properties of cytochromes c from closely related specieswhere there are fewer sequence differences and for determining the range of variation of a propertyamong diverse species. One example of the latter is the comparison of the susceptibility of mitochondrialcytochromes c to tryptic digestion (Endo et a!., 1985).The remaining techniques are suited to examining the functional consequences of specific structuralchanges to proteins. The use of chemically modified cytochrome c has been discussed previously. Whilethe use of this technique has been beneficial in the study of the structure-function relationships incytochrome c, its limitations include the difficulty of isolating singly modified proteins and the unreactivenature of some residues of interest towards modification.When cytochrome c is cleaved by cyanogen bromide at Met 65, the cleavage products reassociate togive a folded conformation similar to the intact protein (Corradin & Harbury, 1971). Moreover, the twopolypeptide fragments are able to reform the broken peptide bond spontaneously (Corradin & Harbury,1974). This property is the basis of the preparation of semisynthetic cytochromes c, in which the Nterminal heme binding polypeptide derived from cyanogen bromide cleavage is combined with a C39terminal polypeptide prepared by chemical synthesis (Nix et al., 1979). The nature of the C-terminalpolypeptide can thus be specifically altered and covalently reattached to the N-terminal polypeptide (Koulet at., 1979; Wallace et at., 1989). This method is not limited to the use of naturally occurring aminoacids, and proteins that are physiologically nonfunctional can be investigated. However, only changesin the polypeptide fragment that is not covalently attached to the heme may be studied. Usingsemisynthetic methods, Wallace and coworkers prepared several cytochrome c variants to assess the rolesof residues Tyr67, Thr78, and G1y83 in maintaining the function and stability of the protein (Wallace etat., 1989). In addition, the axial ligand Met8O of cytochrome c has been altered by semisynthesis to giveLeu, His, and Cys variants; these species have reduction potentials 220 to 650 mV lower than the wildtype protein (Raphael & Gray, 1991). In the case of the position-80 variants, the contribution of axialligand heterogeneity, as exemplified by the alkaline transition, to the reduction potentials of these proteinsas measured by equilibrium methods has not been evaluated.The most useful and generally applicable technique for investigation of structure-function relationshipsis to make the desired structural variations at the genetic level by site directed mutagenesis (Zoller &Smith, 1983, Kunkel, 1985). Saccharomyces cerevisiae iso-1-cytochrome c was among the first electrontransfer proteins to be cloned, sequenced (Smith et at., 1979), and modified by site directed mutagenesis(Pielak, et at., 1985). Although a gene for any mutant can be prepared, not all such mutations may beexpressed as the currently used expression system requires a functional cytochrome c. As withsemisynthesis, site directed mutagenesis has the advantage of selectively altering the sequence of theprotein. The two techniques are complementary, and mutagenesis can in principle extend the potentialuse of semisynthesis by introducing methionine residues along the protein sequence (Wallace et a!.,1991). Site directed mutagenesis has been used to generate cytochrome c variants at many positions, asexemplified by studies of the invariant residue Phe-82 (Pielak et at., 1985); the structural (Louie &Brayer, 1988, 1989), functional (Pielak et at., 1985; Michel et at., 1989), and physicochemical (Pearceet at., 1989; Concar et a!., 1991) properties of several position-82 variants have been investigated.401.7 Thesis ObjectivesConserved residues in the beme environment of cytochrome c are proposed to have important rolesin maintaining the heme in a functional state (Kassner, 1972, 1973) and in the oxidation state linkedconformational changes of the protein (Takano & Dickerson, 198 ib). To evaluate this proposal, sitedirected mutants of yeast iso-1-cytochrome c have been prepared at two regions within the heme crevice,and the physicochemical properties of these variants have been measured and compared to the wild typeprotein.Phe82 is an invariant residue located at the exposed heme edge of cytochrome c where interaction withother electron transfer proteins takes place. The identity of the residue at position 82 has significantinfluence on the structure (Louie & Brayer, 1988, 1989), function (Michel et al., 1989; and stability(Pearce et al., 1989) of cytochrome c. Mutants of yeast iso-1-cytochrome c with Tyr, Leu, lie, Ala, Ser,and Gly at position 82 have been supplied by Dr. J.G. Guillemette of the laboratory of Dr. M. Smith.Residues Asn52, Tyr67, and Thr78 form a conserved hydrogen bond network with WAT166 thatundergoes relatively large oxidation state linked conformational changes (Figure 4; Takano & Dickerson,198 la,b; Bushnell et al., 1990). The conserved residue 11e75 protects this region from exposure tosolvent. The yeast iso-1-cytochrome c mutants A1a52, Phe67, Met75, and G1y78 have been supplied byDr. 3. G. Guillemette.Experiments have been performed on these mutants that assess their influence on the function of yeastiso-i-cytochrome c. To determine their influence on the oxidation-reduction equilibrium of cytochrome c,the reduction potentials of the variants have been measured by cyclic voltammetry. The temperaturedependence of reduction potential has been measured for each mutant to determine the thermodynamicparameters of reduction. For several mutants, the pH dependence of reduction potential has beenmeasured. The electron transfer rates of these mutants with the reductant Fe(edta)2 and the oxidantCo(phen) have been measured. Using the results from the electrochemical and kinetic experiments,V 41relative Marcus theory has been applied to assess the influence of mutation in the heme crevice onelectron transfer activity.To determine the influence of heme crevice mutations on the stability of the native heme conformation,the equilibrium and kinetics of alkaline isomerization and ligand binding to cytochrome c mutants wereexamined. The alkaline isomerization of the position-82 variants studied here has been evaluatedpreviously (Pearce et a!., 1989), thus the present work considers the alkaline isomerization of the mutantsin the hydrogen bond network about WAT166 only. Equilibrium and kinetic measurements of azidebinding to both sets of mutants were performed to determine the effects of these mutations on the dynamicproperties of the active site of cytochrome c.In all experiments the results were interpreted with respect to known or possible structural propertiesof the proteins. The likely roles of the conserved residues of the heme crevice are discussed in light ofthe results obtained in this work.42METHODS2.1 Cytochrome c Preparation2.1.1 Fermentation of YeastMutagenesis of yeast iso-1-cytochrome c was performed in the laboratory of Professor Michael Smithby Dr. 3G. Guilemette as described previously (Pielak et aL, 1985; Inglis et aL, 1991). The host yeaststrain, GM3C-2 has no CYC1 gene, and the CYC7 gene has been inactivated by a point mutation. Inaddition GM3C-2 is unable to synthesize leucine due to a point mutation in the leu2 locus. The plasmidthat bears the mutant CYC1 gene also possesses a functional leu2 gene, thus transformed GM3C-2 canbe selected for either on the basis of growth on a nonfermentable carbon source or in the absence ofleucine.Transformed yeast cultures bearing the mutant CYC1 genes were supplied on agar plates in SC leumedium, consisting of yeast nitrogen base, uracil and adenine supplements, amino acids (without leucine),and dextrose as a carbon source. For each mutant cytochrome c, a single isolated colony was used toinoculate three tubes, each containing 5 mL of liquid SC leu medium. The cultures were grown for 24hours at 30 °C in a shaker bath and used to inoculate three flasks each containing 1.5 L of SC leumedium. The large cultures were grown for 48 hours at 30 °C in a shaker bath and were used toinoculate a sterilized 50 L fermenter.The fermenter medium consisted of bactopeptone (2% w/v), yeast extract (1 % wlv), and glycerol asa carbon source (3% v/v). Tetracycline (0.5 g) and streptomycin (2 g) were added to inhibit microbialgrowth, and antifoaming agent (15 mL) was also added. After inoculation the culture was grownaerobically for 48 hours at 30 °C. At this point, neutralized lactic acid was added to a final concentrationof 1% v/v. Fresh antibiotics and antifoaming agent were also added. Growth was continued for another48 hours. A sample of the culture was examined with a microscope to check for microbial contaminationand to calculate the yeast cell density. The culture was harvested using a continuous flow centrifuge(CEPA Model Z41, New Brunswick Scientific).432.1.2 Cytochrome c PurificationAll of the following steps were performed at 4 °C, except as indicated. Buffer A refers to sodiumphosphate buffer, pH 7, = 0.1 M, containing 5 mM mercaptoethanol or 0.5 mM dithiothreitol. Thepurification of cytochrome c described here is based on a method described previously (Cutler et at.,1987), with the addition of a final cation exchange chromatography step using Mono S resin (vide infra).To each kilogram of yeast was added 250 mL of ethyl acetate and 500 mL of 1 M NaC1. The mixturewas combined to a smooth consistency and stirred overnight with a magnetic stirrer. The mixture waspoured into 400 mL polypropylene centrifuge bottles and centrifuged at 7500 rpm in a GSA rotor for 35minutes. The orange-red supernatant fluid bearing the cytochrome c was filtered through cheese cloth.The yeast pellet was resuspended in 1 M NaC1 and centrifuged as before. The supernatant fluids werepooled and diluted with distilled water to give a conductivity less than 8000 2’. Dithiothreitol (2g) wasadded to the solution to reduce the cytochrome c.Cation exchange cellulose resin (Whatman CM-52, 300 mL, equilibrated in buffer A) was added tothe protein solution, and the solution was stirred for 10 minutes by hand using a glass rod. The solutionwas left for 1 hour to let the resin settle. After 1 hour, the solution was decanted, leaving thecytochrome c bound to the settled resin. The resin was transferred to a sintered glass funnel and washedwith several volumes of buffer A, 0.05 M in NaC1. The cytochrome was eluted from the resin withbuffer A that was made 1.0 M in NaC1. The cytochrome c solution was dialysed against 16 L of bufferA using dialysis tubing with an exclusion cutoff of 8000 daltons.The dialysed cytochrome solution was loaded onto a column of cation exchange resin (Pharmacia CMSepharose, 2.5 x 6 cm) and eluted from the column by a salt gradient developed with 400 mL each ofbuffer A, 0.075 M in NaCl and buffer A, 0.25 M in NaC1. The eluent solution was collected in 8 mLfractions with a Gilson fraction collector. Two cytochrome c bands were usually seen. The minor,earlyeluting band was discarded; fractions from the major, later eluting band with absorbance ratios A416 /Agreater than 5 were pooled. The pooled fractions were concentrated by ultrafiltration using progressively44smaller ultrafiltration units: an Amicon stirred cell with a YM-5 membrane (exclusion cutoff 5000daltons) was used to concentrate solutions from volumes greater than 100 mL to less than 20 mL; furtherconcentration steps employed Centriprep and Centricon units (Amicon; both with an exclusion cutoff of10 000 daltons).A solution of concentrated, reduced protein (1 mL, 20-30 mg/mL) was equilibrated with 20 mM MES(pKa 6.1, Perrin & Dempsey, 1974) buffer, pH 6, by passage through a column of gel filtration resin(Bio-Rad P6-DG, 1 x 20 cm). Cytochrome c was simultaneously oxidized on this column with excessNH4[Co(dipicolinato)2](Mauk et aL, 1979) which was applied to the column prior to the protein.Final purification was achieved by ion exchange chromatography at room temperature with an FPLCsystem fitted with a Mono-S HR 10/10 cation exchange column (Pharmacia). A solution of cytochrome c(1 mL, 15-20 mg/mL) was loaded onto the column and eluted with 20 mM MES, pH 6, 1 M in NaCl,using a linear salt gradient from 0.25 to 0.45 M NaC1. The eluate solution was monitored at 280 nm,and chromatograms were acquired with a chart recorder. The eluate fluid was collected in 2 niLfractions, which were placed on ice. Fractions from the main eluting fraction with the highestA5/A2ratios were pooled, concentrated, and the spectrum recorded from 700 to 250 nm. The yield ofcytochrome c was determined spectroscopically using the extinction coefficient€495 = 106.1 mM’ cm’for the Soret band maximum of the oxidized protein (Margoliash & Frohwirt, 1959). Samples wereplaced in cryovials, flash frozen, and stored in liquid nitrogen.2.2 ElectrochemicoJ ExperimentsThe three electrode cell used for cyclic voltammetry is shown in Figure 13. A schematic diagram ofthis cell is shown in Figure 14. The auxiliary electrode is made of platinum mesh, and the reference isa saturated calomel electrode (Radiometer K401) maintained at 25 °C with a water bath. The sample45Figure 13 A photograph of the cyclic voltammetry cell used for reduction potential measurements.Legend: 1, reference electrode; 2, working electrode; 3, auxiliary electrode; 4, sample compartment.46To potentiostatAuxiliary ElectrodeTo potentiostatProtein SolutionFigure 14 A schematic diagram of the cyclic voltammetry cell. The working electrode is a polished goldsurface, treated with 4,4’-dithiodipyridine to promote rapid electron transfer between the electrode andthe protein in solution. The auxiliary electrode is a platinum wire connected to platinum mesh at thebottom of the protein solution. The side arm is filled with buffer and provides contact between thereference electrode and the protein solution by means of a capillary located at the bottom of the proteinsolution. The electrodes are connected to the potentiostat by alligator clips at the indicated positions.Reference ElectrodeSide AnnCapillary47compartment was immersed in ajacketed beaker containing water, and the temperature was regulated witha second water bath.The gold disc electrode (surface area 0.16 cm2) was prepared by polishing with increasingly finegrades of alumina (Beuhier: 1.0, 0.3 and 0.05 m) I water slurry on a polishing cloth (Mastertex). Afterfreeing the polished electrode of alumina by sonication in a water bath and rinsing with deionized,distilled water, the electrode was immersed in a saturated solution of the surface modifier 4,4’-dithiodipyridine (Aldrithiol-4, Aldrich) for several minutes prior to being used.Protein solution (0.5 mL, 0.4 mM) was placed in the sample compartment and deaerated by passageof a stream of water saturated, purified argon over the surface of the solution. The surface modified goldworking electrode was immersed in the sample solution, and the leads from the electrodes were connectedto the potentiostat used to control the cell potential (Ursar Electronics, Oxford U.K.). A potential rangeof -10 to 530 mV vs SHE was scanned at a rate of 5 to 200 mV/s. Cyclic voltammograms were obtainedwith an x-y recorder (Kipp & Zonen Model BD 90). Reduction potentials were measured fromvoltammograms acquired at a sweep rate of 20 mV/s. All experiments were carried out using L = 0.1M buffer (50 mM in KC1 with the balance of the ionic strength provided by sodium phosphate). Thetemperature dependence of reduction potentials was measured between 10 and 40 °C at pH 6.0, with thereference electrode maintained at 25 °C. The pH dependence of reduction potential was measured in thepH interval 5.5 to 8.5 at 25 °C.2.3 Electron Transfer KineticsAll of the kinetic experiments were performed with a Durrum Model D-130 stopped flowspectrophotometer, modified as described previously to improve the anaerobicity of the apparatus (Reid &Mauk, 1982; Reid, 1984). The optical system used was a Durrum Model 120 Rapid KineticsMonochromator with a tungsten source. Data acquisition was controlled by OLIS 4120AT softwarerunning on a Zenith Z-100 microcomputer (On-Line Instrument Systems, Jefferson, Ga.). The reaction48temperature was regulated by a water bath to within 0.2 °C of the desired value. At least four kinetictraces were averaged for each measurement at a given reagent concentration or temperature.2.3.1 Fe(edta)2 Reduction KineticsReactant solutions were prepared on the day of use and were kept under an argon atmosphere.Fe(edta)2 solutions were prepared in phosphate buffer as described by Wherland et a!. (1975). Bufferedferricytochrome c (5 M) solutions were deaerated by a stream of water saturated, purified argon. Thereduction of cytochrome c by’Fe(edta)2 was monitored at 412.5 nm under pseudo-first order conditionswith reductant concentration in at least 20 fold excess (j = 0.1 M, sodium phosphate buffer, pH 6.0,25.0 °C). The temperature dependence of the reaction was measured between 10 and 40 °C.2.3.2 Co(phen) Oxidation Kinetics2.3.2.1 Synthesis of(Co(phen)JC13o-Phenanthroline monohydrate (BDH, 4.0 g, 0.02 moles) and 60 mL water were placed in a modifiedthree necked 300 mL round bottom flask. CoC12.6H0(MCB, 1.6 g, 0.0067 moles) was dissolved in60 mL water; this solution was added in small increments by Pasteur pipette to the phenanthrolinesuspension with swirling of the flask. During the addition of the CoC12, the contents of the flask turnedbrown, and the phenanthroline dissolved completely. The flask contents were heated briefly on a steambath and cooled to room temperature. The CoOl) complex was oxidized by chlorine gas generated bypotassium permanganate oxidation of hydrochloric acid as described by Vogel (1978). The solutioncontaining the oxidized product was heated, with stirring, on a hot plate until the volume had beenreduced to about 20 mL. The product solution was made up to 60 mL with water, and the volumereduced as before; this was repeated twice. The precipitated product was recrystallized from 95%ethanol, washed with diethylether, and air dried.492.3.2.2 Ferrocytochrome c Oxidation by Co(phen)3Cl3Buffered Co(phen)31solutions [,L = 0.1 M (2 mM MES, balance NaC1), pH 6.0] were prepared byweighing and were stored in modified, three necked 100 mL round bottom flasks equipped with Leuerfittings. Buffered ferrocytochrome c solutions were prepared on the day of use by reduction withdithionite followed immediately by passage through a column of gel filtration resin (Bio-Rad P6-DG, 1x 10 cm) equilibrated with deaerated buffer. The eluting ferrocytochrome c was collected in a serumbottle, diluted with deaerated buffer to a concentration of 10-20 M, and kept under a water-saturatedargon atmosphere. Oxidation of cytochrome c by Co(phen)31 was monitored at 550 nm under pseudo-first order conditions, with oxidant in at least 20-fold excess. The temperature dependence of the reactionwas examined between 10 and 40 °C.2.4 Alkaline Isomerization2.4.1 Spectrophotometric pH TitrationsFerricytochrome c was exchanged into 0.1 M NaC1 by ultrafiltration and diluted to a concentrationof 0.15 to 0.2 mM. The cytochrome c solution was placed in a 3 mL quartz cuvette modified to holda pH electrode, a microburette and a magnetic stir bar, as described previously (Pearce et al., 1989).The initial pH of the solution was adjusted with 0.1 M HC1 to pH 5.5. The titrant, 0.1 M NaOH, wasadded in microliter amounts by microburette (Manostat) to the stirred solution. The absorbance changeat 695 nm was monitored after each addition of base. These steps were repeated with increasing pHvalues until no further significant absorbance change occurred.2.4.2 pH Jump KineticsFerricytochrome c solutions (40 M) were prepared in unbuffered 0.1 M NaC1. For experimentsinvolving jumps from low to high pH, the initial pH of the cytochrome c solution was adjusted to pH 650using 0.1 M HC1. Stopped flow experiments were performed as described by Pearce et at. (1989). Theunbuffered ferricytochrome c solution and a buffered solution of known pH were rapidly mixed in thestopped flow spectrophotometer, and absorbance changes were monitored at 695 nm. The final pH ofthe solutions after mixing was recorded. All experiments were done at 25 °C and = 0.1 M. Severalbuffers were used to span the pH region of interest: sodium phosphate, sodium borate, CHES, and CAPS,with pKa’s of 6.8, 9.1, 9.3, and 10.4, respectively (Perrin & Dempsey, 1974). For rapid jumps fromhigh to low pH, the initial pH of the cytochrome c solution was adjusted with 0.1 M NaOH to 1.5 - 2pH units above the expected alkaline pK. For this experiment the final pH was 6 in MES buffer.2.5 Ligand Substitution2.5.1 Azide TitrationsTwo methods were used to measure the stability constants for azide binding to cytochromes c. In thefirst method, ferricytochrome c was exchanged into buffer [pH 6, u = 1.0 M (20 mM MES, balanceNaC1)] and concentrated by ultrafiltration. The concentrated protein solution (200 L) was placed intoeach of two 5 mL volumetric flasks. One flask was diluted to volume with buffer, while the other wasdiluted to volume in 1.0 M NaN, pH 6. The concentration of azide in the latter solution was correctedfor the volume of the protein solution added. The absorbance at 695 nm and spectrum (750-680 nm) ofboth solutions were recorded. Samples of the two solutions were mixed to produce a series of solutionsthat were equimolar in ferricytochrome c but possessing different azide concentrations. The azideconcentration of each solution was calculated, and the absorbance at 695 nm measured. The spectrumof each solution in the range 750-680 nm was also recorded.The second method used involved titration of a ferricytochrome c solution with a 2.5 M solution ofsodium azide. Using the same buffer as above, a solution of ferricytochrome c was prepared in a quartzcuvette to a volume of 2.50 mL and a concentration of 40 to 120 M. The absorbance at 695 nm andspectrum (750-680 nm) of the cytochrome c solution were recorded. A magnetic stir bar was placed in51the cuvette to allow efficient mixing. The absorbance at 695 rim was recorded after each incrementaladdition of azide. Absorbance changes at 695 nm were corrected for dilution resulting from the smallvolumes of titrant solution added.2.5.2 Azide Binding KineticsFerricytochrome c solutions (40 to 120 rM) were prepared in r = 1.0 M buffer, pH 6.0 (20 mMMES, balance NaC1). Azide solutions (0.2 to 1.0 M in ligand) were of the same pH and ionic strength.The ligand exchange was monitored at 695 mn under pseudo-first order conditions, with azide in at least1600 fold excess at 25 °C.52RESULTS3.1 Protein PreparationThe yield of yeast from each 50 L fermentation was 0.8 to 1.4 kg. Protein yields were normallyaround 100 mg, but yields from 10 to 200 mg were obtained. While high yields of proteins areassociated with high yields of yeast, the converse is not true, as the lowest yields of cytochrome c alsogave good yields of yeast. Although cytochrome c is required for growth on a non-fermentable carbonsource, only a relatively small amount of cytochrome c is required. Yeast will grow normally even ifcytochrome c is expressed at only 10 percent of its usual level (Sherman et a!., 1974). Although theplasmid bearing the mutant cytochrome c gene is initially present in multiple copies and is expressed athigh levels, with time the copy number may be lowered as the plasmid is lost from the yeast. Selectionon a non-fermentable carbon source will ensure that only those yeast still bearing the plasmid will grow,but the plasmid copy number may be too low to be useful for large scale cytochrome c isolation. Thisexplanation is consistent with the observation that low levels of expression are associated with mutantbearing yeast cultures prepared over six years ago, while more recently prepared cultures expresscytochrome c at high levels.Fermentations of yeast transformed with plasmids bearing genes encoding the variants Thr78Val andThr78Gln both gave high yields of yeast and cytochrome c, although the protein was not stable in vitro.In both cases, the freshly isolated protein rapidly changed colour from orange to brown as the purificationproceeded. Cation exchange chromatography revealed a mixture of coloured species, which elutedpredominantly at ionic strengths above those normally required for native cytochrome c and mostmutants. As both had sufficient function in vivo to support growth of yeast, their behaviour when isolatedsuggests a conformational change to a nonfunctional state with altered surface properties, and thus aninherent instability in the folded structures of their functional states.53A key step in the purification scheme is the ethyl acetate I salt extraction of the yeast cells. The ethylacetate makes the organelle membranes leaky, and the salt solution dissociates the cytochrome c fromthe mitochondria. As ethyl acetate does not rupture the yeast (extracted yeast appear intact under amicroscope) most of the cellular components remain within the cell, and the supernatant fluid containingthe cytochrome c is relatively free of impurities. The two remaining cation exchange chromatographysteps are greatly simplified by the relative purity of the cytochrome c obtained from the CM-cellulosebatch extraction. The importance of the ethyl acetate / salt extraction cannot be fully appreciated untilone compares it to alternate extraction procedures such as cell disruption using a ball mill. In this methodthe total cell contents are released, which lowers the efficiency of the binding of cytochrome c to the CMcellulose resin and increases the number of impurities that need to be removed.Previously, the final purification step was cation exchange chromatography on CM-Sepharose. Thefurther use of the FPLC Mono S column offers several advantages. The higher resolution of the MonoS column allows one to obtain purer protein through removal of minor components which coelute withcytochrome c on the CM-Sepharose column. In addition, more efficient use is made of the protein, asimpure and used cytochrome c can easily be recycled by passage through the column.Two bands of cytochrome c were often present on the CM-Sepharose ion exchange column. Theminor, rapidly eluting band has been attributed to deamidated cytochrome c (Brautigan et a!., 1978a),or cytochrome c that has not undergone trimethylation of Lys73. However, the relatively low yield thisband precluded confirmation of these suggestions. The rapidly eluting fraction accounted for about 10percent of the total cytochrome c.543.2 Electrochemical ExperimentsThe electrochemistry of the cytochrome c mutants was quasi-reversible as indicated by the peak topeak separations of 54 to 63 mV (25 °C, 20 mV s4 scan rate) obtained from the majority of thevoltammograms. In addition, the dependence of the faradaic current on the square root of the scan ratewas measured for several proteins and was found to be linear up to 50 mV s4. A representative plotis shown in Figure 15.The reduction potential of horse cytochrome c was 270 mV ± 2 mV (pH 7, 25 °C, = 0.1 M, scanrate 20 mV/s), in reasonable agreement with the value of 262 mV obtained by spectroelectrochemistry(Taniguchi et at., 1982b) and the value of 274 mV obtained by cyclic voltammetry (Bond et at., 1990)under similar conditions. For native yeast iso-i -cytochrome c, the reduction potential was 290 mV (pH6’, 25 °C, = 0.1 M, scan rate 20 mV s1). Under these same conditions of temperature, pH and ionicstrength, the reduction potentials of the mutants range from 245 to 286 mV vs SHE (Table 1).The thermodynamic parameters were determined from the temperature dependence of the reductionpotentials by combining the two equations for free energy (Taniguchi et al., 1982b):= flFEm (11)AG° = MI° - TLS° (12)Where the standard conditions are = 0.1 M, pH 6. As the reduction potentials are measured relativeto the standard hydrogen electrode, the thermodynamic parameters of this reference must be taken intoaccount.Fe(III)Cyt c ÷ 1/2 H2 Fe(Il)Cyt c + H (13)= Eli, (products) - Eu, (reactants) (14)By definition, the heats of formation of H and H2 are zero, thus the measured H° is that of thecytochrome c Fe(III) I Fe(1I) couple alone.The entropy change in the cell is treated in a similar manner:55AS° = AS° - AS° (15)cell rc SHEWhere the subscript ‘rc’ (reaction centre) refers to the cytochrome c half cell and the subscript SHErefers to the reference electrode. The expression for the standard free energy of the cell can berewritten asAG° = AR°- (TAS°) + (TAS°)SHEThe partial molar entropies of the proton and of hydrogen gas are 0 and 31.2 entropy units,respectively (Latimer et al., 1938; Latimer, 1952). This gives 15.6 entropy units for the change inentropy of the SHE reference. The reference electrode is maintained at 298 K, while the sampletemperature is varied, thus the term (TzS °)sHE remains constant. Equation 16 can be rewritten to takethis into account:tG° (calories/mol) =- TIiS°,.C + 4649 (17)Finally,E (millivolts) = C T — ‘‘° + 4649) (18)F FFrom a plot of E vs T, Src° can be determined from the slope of the curve, and SH° is obtained fromthe intercept on the ordinate.For horse heart cytochrome c, the reaction centre entropy, determined by cyclic voltammetrywas -9.7 ±0.4 eu and the enthalpy of reduction, tIH°, was -13.8 ±0.1 kcal/mol (j = 0.1 M, sodiumphosphate, pH 7, gold electrode modified with 4,4’-dithiodipyridine). These results are comparable to= -12.9 ± 1.2 eu and }1° = .445 ± 0.4 kcal/mol obtained by spectroelectrochemistry [goldminigrid electrode, [Ru(NH3)5pyJ Cl04mediator, nonisothermal cell, u = 0.1 M sodium phosphate, pH7.0 (1’aniguchi eta!., 1982b)].The piots of reduction potential against temperature for each variant of cytochrome c are shown inFigures 16 and 17, and the calculated thermodynamic parameters are presented in Table 1. All plots are566.0/./////< 4.0 /$.1-: /C /a,/o /w 2.0//1’A//I I I0.0 4.0 8.0 12.0 16.0\i scan rate , (mV/s)1’2Figure 15 The dependence of faradaic peak current on the applied potential sweep rate forcytochrome c [pH 6.0, j = 0.1 M, 25 °C, 20 mV/s].57LU00>>ECDELU315Temperature (Kelvin)Figure 16 The temperature dependence of reduCtiOn potential for cytochrome c positiOfl$2variantsmeasured by cyclic voltammetrY [ph 6.0, = 0.1 M sodium phosphate. Legend: •, Wild type;A, Phe82LeU I, Phe82TY; • , PheS2hle; , Phe82Ala; 0, Phe82Ser D, Phe82GIY. For figures16, 17, and 18, the error in the measured value of each point (±2 mV) is about twice the size of thesymbols. Em,6 is the midpoint reduction potential of the protein measured at ph 6.0 and the specifiedtemperature. Numerical values of the data points in Figures 16 to 18 are tabulated fl Appendix A.30029058300290280270LU2:(1)Cl)>260250_A___A__k__A__A_k___A240—a-230285 290 295 300 305 310 315TemPerat (Keivtn)gUre 17 e temPeta depeflden of reductlofl poteutiat for tocbrome c water switch variantsmeaSUr by cyclic voltaettY 6.0, 0.1 M sodium phosphat Legefld •, Wild type;, Asfl52 a; •, Thr7SGIY S lle75Met.59Protein En6 (mV) AG° (kcal/mol) AS° (eu) ill0 (kcal/mol)Wild Type 290 ± 2 -67 ± 0.2 -9.1 ± 0.3 -14.1 ± 0.1Phe82Tyr 280 ± 2 -6.5 ± 0.2 -8.3 ± 0.3 -13.6 ± 0.1Phe82Leu 286 ± 2 -6.5 ± 0.2 -12.0 ± 0.7 -14.8 ± 0.2Phe82Ile 273 ± 2 -6.3 ± 0.2 -12.3 ± 0.3 -14.6 ± 0.1Phe82Ala 260 ± 2 -6.0 ± 0.2 -8.1 ± 0.5 -13.1 ± 0.1Phe82Ser 255 ± 2 -5.9 ± 0.2 -12 ± 0.9 -14.1 ± 0.3Phe82Gly 247 ± 2 -5.7 ± 0.2 -8.0 ± 0.3 -12.8 ± 0.1a)Protein Em,6 (my) iG° (kcal/mol) AS1.° (eu) fl0 (kcal/mol)Wild Type 290 ± 2 -6.7 ± 0.2 -9.1 ± 0.3 -14.1 ± 0.1Thr78Gly 249 ± 2 -5.7 ± 0.2 -6.3 ± 0.7 -12.3 ± 0.2Ile75Met 245 ± 2 -5.7 ± 0.2 -11.7 ± 0.3 -13.8 ± 0.2Asn52Ala 257 ± 2 -5.9 ± 0.2 -8.0 ± 0.3 -12.9 ± 0.1Tyr67Phe 1 236 ± 2 -5.4 ± 0.2 - -b)1 Guillemette et aL, in preparationTable 1 Thermodynamic parameters associated with the reduction of cytochrome c variants, measuredby cyclic voltammetry [25 °C, = 0.1 M sodium phosphate]. a) position 82 variants; b) water switchvariants. The uncertainties in zS° and 0 were calculated by linear least squares fits of the data setsto equation 18 and are best regarded as a lower limit of the error. More realistic uncertainties in thesefitted parameters are ± 1 eu in and ± 0.5 keal/mol in J060linear with negative slopes, resulting in small negative entropies of reduction ranging from -7 to -13entropy units. The enthalpies of reduction are negative and of similar magnitude for all of thecytochromes studied, ranging from -12.5 to -14.6 kcal mol1. The differences between G° of wild typeand any mutant of cytochrome c are less than 1 kcal rno11.The pH dependence of reduction potential was used to determine the dissociation constants of titratablegroups whose ionization state is sensitive to the oxidation state of the protein. Previous measurementsusing equilibrium techniques have shown that the pH dependence for yeast iso-l-cytochrome c can bedescribed by three dissociation constants, two (Krn, KJ in the oxidized state and one (Kr) in the reducedstate (Moore et al., 1984; Cutler et a!., 1989). The second dissociation constant in the oxidized proteinis generally attributed to the alkaline isomerization (Moore et a!., 1984). Using cyclic voltammetry, theelectrochemical properties of the alkaline isomer do not interfere with observation of the electrochemicalbehaviour of the native conformation, so the pH dependence of reduction potentials determined in thismaimer is described by a single oxidation state-linked pK:E H = E0+ pj’K,+[H9 (19)mpnF K0÷[H]By fitting the measured values to this equation, the oxidation state dependent dissociation constants areobtained, as well as the reduction potential extrapolated to pH 0, (Em0). Figure 18 shows the plots ofreduction potential vs pH for the variants examined. The calculated parameters derived from a nonlinearleast squares fit of the data to equation 19 are presented in Table 2. The values of pK and pK for wildtype cytochrome c are in good agreement with previous measurements (pK0 = 6.6, pKr = 7.2; Cutler eta!., 1989). The data for all mutants studied are consistent with the simple model involving a singleionizing group with a pK near neutrality that shifts by 0.3 to 0.4 pH units upon a change in oxidationstate.61w00>>EE 240Ui2203002802605.5 6.0 6.5 7.0 7.55.0 8.0 85pHFigure 18 The pH dependence of reduction potential for several variants of cytochrome c, measuredby cyclic voltammetry [25 °C, u = 0.1 M sodium phosphate]. Legend: •, Wild type;, Gln42Lys I Ala43His; A, AsnS2Ala; 0, Phe82Ser; •, Ile75Met. The interpolated curves werecalculated by least squares fitting of the data to equation 19.62Protein Em,o (my) PKr pK0Wild Type 292 ± 2 7.1 ± 0.1 6.7 ± 0.1Phe82Ser 259 ± 2 7.0 ± 0.1 6.5 ± 0.1Ile75Met 248 ± 2 7.1 ± 0.1 6.7 ± 0.1Asn52Ala 264 ± 2 6.6 ± 0.1 6.2 ± 0.1Gln42Lys/ 281 ± 2 6.8 ± 0.1 6.4 ± 0.1Ala43HisTable 2 Parameters derived from the pH dependence of reduction potential for several variants ofcytochrome c as measured by cyclic voltarnmetry [25 °C, j = 0.1 M sodium phosphate].633.3 Electron Transfer Kinetics3.3.1 Fe(edta)2Reduction KineticsFor each cytochrome c variant, the dependence of the observed reaction rate on reductantconcentration was linear. The intercept of each plot was within experimental error of zero. The secondorder reduction rate constant (k12) for each cytochrome c variant could be calculated from the equationk01,5 =k12[Fe(edta)29 (20)Plots of k against reductant concentration are presented in Figures 19 and 20, and second order rateconstants calculated are presented in Table 3. For horse heart cytochrome c, k12 = 2.86 x iO M1s,in good agreement with a previously measured value of 2.72 x 10 M’s’ (Hodges et al., 1974) underidentical conditions. The second order rate constants obtained for the yeast cytochromes c variants areall within a two-fold range of the rate constant obtained for the wild type protein [low = 4.18 x 10 M’s’(Ile75Met), high = 1.5 x 10 M1s (Phe82Ser)].Those mutant proteins with an alkaline PKa below 8 (position-82 mutants Leu, lie, Ala, Ser, & Gly,and water switch mutant Thr78Gly) exhibited biphasic kinetics even at pH 6. In these cases, thereduction of the oxidized protein is the principal rate process at this pH and was several orders ofmagnitude faster than the rate of the slower phase. Consequently, the second order rate constants forreduction of the native proteins were readily determined. The slower phase exhibits a similar rateconstant to that observed for the conversion of the alkaline form of these proteins to the nativeconformation in pH-jump experiments (Pearce et al., 1989) and is thus ascribed to this conformationalchange.To account for differences in thermodynamic driving force on the observed reaction rates, apparentself-exchange rate constants calculated for these reactions, k11’°, were determined as described byWherland & Gray (1976). These values are also presented in Table 3. The value ofk11colT for yeast wildtype cytochrome c in this reaction is similar to that of horse heart cytochrome c648060C)a)040Cl).00-2000.00 0.50[Fe(edta)2] (mM)Figure 19 The dependence of pseudo-first-order rate constants for ferricytochrome c reduction onFe(edta)2 concentration for position-82 variants. [25 °C, pH 6.0, t = 0.1 M sodium phosphate].Legend: •, Wild type; A, Phe82Leu; •, Phe82Tyr; •, Phe82Ile; , Phe82Ala; 0, Phe82Ser;, Phe82Gly. For this and subsequent kinetics figures, the error in each point is estimated to be ± 5%.Numerical values of the data points for Figures 19 to 22 are tabulated in Appendix B.0.10 0.20 0.30 0.4065500U)0-0-[Fe(edta)2] (mM)Figure 20 The dependence of pseudo-first-order rate constants for ferricytochrome c reduction onFe(edta)2 concentration for water switch variants. [25 °C, pH 6.0, = 0.1 M sodium phosphate].Legend: •, Wild type; A, AsnS2Ala; •, Ile75Met; •, Thr78Gly.4030201000.00 0.10 0.20 0.30 0.40 0.5066Ic’JTemperature 1 X 10 (Kelvin1 )Figure 21 Eyring plots for the Fe(edta)2 reduction of position-82 variants of ferricytochrome c. [pH6.0, = 0.1 M sodium phosphate]. Legend: •, Wild type; A, Phe82Leu; •, Phe82Tyr;4, Phe82Ile; , Phe82Ala; 0, Phe82Ser; El, Phe82Gly.6.406.005.605204.803.15 3.25 3.35 3.45 3.5567IC’4JCFigure 22 Eyring plots for the Fe(edta)2 reduction of water-switch variants of ferricytochrome c. [pH6.0, JL = 0.1 M sodium phosphate]. Legend: •, Wild type; A, Asn52Ala; I, Ile75Met;•, Thr78Gly.6.005.605.204.804.404.003.15 3.25 3.35 3.45 3.55Temperature1X 10 (Kelvin1)68Protein Rate Constant k11 (M’s) Activation Activation(M1s’) (25°C) (25°C) Entropy (eu) Enthalpy(kcal/mol)Wild Type 7.2 X 10 10.9 -24.7 ± 0.8 3.5 ± 0.2Phe82Tyr 6.2 x 10 11.8 -22.2 ± 0.8 4.3 ± 0.2Phe82Leu 8.8 x 10 19 -18.3 ± 0.9 5.3 ± 0.3Phe82Ile 9.4 X 10 35 -21 ± 1 4.5 ± 0.3Phe82Ala 9.9 x 10 62 -25 ± 1 3.2 ± 0.3Phe82Ser 1.48 x 10 165 -29.6 ± 0.2 1.6 ± 0.1Phe82Gly 1.37 x 10 190 -26.1 ± 0.3 2.7 ± 0.1a)Protein Rate Constant k11 (Ms) Activation Activation(Ms1) (25°C) (25°C) Entropy (eu) Enthalpy(kcal/mol)Wild Type 7.2 x 10 10.9 -24.7 ± 0.8 3.5 ± 0.2Thr78Gly 5.9 X 10 33.5 -14.8 ± 0.8 6.5 ± 0.2Ile75Met 4.2 x 10 19 -13 ± 1 7.3 ± 0.3Asn52Ala 8.9 x 10 56 -16.8 ± 0.9 5.8 ± 0.3Tyr67Phe 1 8.8 x 10 119 - -1 Guillemette et al., in preparation.b)Table 3 Rate parameters for the reduction of variants of ferricytochrome c [iron(II)edta reductant, pH6.0, = 0.1 M sodium phosphate 1. a) position 82 variants; b) water switch variants. The errors inthe cross reaction rate constants are approximately 5 %. Errors in the activation entropies presented hereand in Table 4 are those obtained from least squares fitting of the data points to equation 21 and are bestregarded as a lower limit to the actual error. To calculate k11Ol, the following values were used.Fe(edta)2: Em6 = 104 mV (Reid, 1984), k = 30 000 M’ s’ (Wilkins & Yelin, 1968), radius = 4 A(Wherland & Gray, 1976), charge = -1 (oxidized), -2 (reduced). Cytochrome C: radius = 16.7 A,charge = +7 (oxidized), +6 (reduced).69(Wherland & Gray, 1976), with values of 10.9 and 6 M’s1 respectively. The Phe82Gly and Phe82Servariants possess the highest reactivity, with k11co values of 165 and 190 M1r’ respectively.The temperature dependences of the reduction rates were measured to determine the activationparameters of the reactions using the Eyring equation:= ln-!+___- AH# (21)T h R RTThe activation enthalpies and entropies (Table 3) were calculated from the Eyring plots shown in Figures21 and 22. For most variants, the plots were linear in the interval from 10 to 40 °C. Eyring plots ofvariants with alkaline pK values near 7 were linear over the abbreviated range of 10 to 25 °C. Thesemutants also had a larger contribution of the second, slower kinetic phase as the temperature increased.Activation entropies and enthalpies for all variants studied are similar to those found with horse heartcytochrome c (H* = 6.0 ± 0.3 kcal/mole, AS* = -18 ± 1 entropy units; Hodges et al., 1974).3.3.2 Co(phen)31 Oxidation KineticsThe reduction potential of Co(phen)32 determined by cyclic voltammetry was 370 mV, (goldelectrode, pH 6, = 0.1 M, 25 °C.), in good agreement with previous spectroelectrochemicalmeasurements under similar conditions [377 mV, (Taniguchi et a!., 1982b)]. The extinction coefficientsof Co(phen)31 at 330 and 350 nm were 4700 M1 cm1 and 3730 M1 cm1 respectively, compared to thevalues of 4680 M1cm’ and 3700 M’ cur’ found by Przystas & Sutin (1973).Figures 23 and 24 show the dependence on Co(phen)31 concentration of the pseudo-first-order rateconstants for ferrocytochrome c oxidation. Oxidation of each cytochrome c conformed to the rateequation of the type shown above (Equation 20). The second-order rate constant for each cytochrome cvariant was calculated from the slope of its curve (Figures 23 and 24), and the results are presented inTable 4. For horse heart cytochrome c, this rate constant was 1.5 x 10 M1s’ (JL 0.1 M, pH 6, 2570°C). This value differs from 1.0 x 10 M1s found by McArdle et a!. (1974) under the same conditions.The rate constant was determined several times with different buffer compositions and protein purity, butconsistently gave the higher value.The electrostatics corrected self exchange rates were calculated from the second order rate constantsof the cross reaction (Table 4). For most position 82 mutants, the differences in k11’0 span only a threefold range and show a dependence on the size of the side chain at this position, as was noted in thereduction kinetics measurements previously described. One exception to this general relationship is thePhe82Ile variant, with a k11co almost seven fold greater than wild type. The water switch mutants spanthe same range as they do in the reduction kinetics experiments.The oxidation rate dependence on temperature was treated as described for the reduction kinetics.Eyring plots (Figures 25 and 26) were linear over 10 to 40 °C for all variants. Activation parametersdetermined from these plots are shown in Table 4; they are similar to those previously determined forhorse heart cytochrome c (}{* 11.3 kcal/mol, = -6 entropy units, pH 7; McArdle et a!., 1974).7112.010.00.00 2.00[Co(phen)] (mM)Figure 23 The dependence of pseudo-first-order rate constants for oxidation of position-82 variants offerrocytochrome c on Co(phen)3 concentration [25 °C, pH 6.0, = 0.1 M (2 mM MES, 98 mMNaC1)]. Legend: •, Wild type; A, Phe82Leu; •, Phe82Tyr; •, Phe82Ile; 0, Phe82Ser;[],Phe82Gly. Numerical values of the data points for Figures 23 to 26 are tabulated in Appendix C.0.40 0.80 1.20 1.60720G)U)00-[Co(phen)] (mM)Figure 24 The dependence of pseudo-first-order rate constants for oxidation of water switch variants offerrocytochrome c on Co(phen)3 concentration [25 °C, pH 6.0, = 0.1 M (2 mM MES, 98 mMNaC1)]. Legend: •, Wild type; A, Asn52Ala; •, Ile75Met; •, Thr78Gly; 0, Tyr67Phe.30.025.020.015.010.05.00.00.00 0.40 0.80 1.20 1.60 2.0073I—C’4J14.003.00Temperature1X i03 (KeIvin)Figure 25 Eyring plots for the oxidation of position-82 variants of ferrocytochrome c by Co(phen)3[pH 6.0, = 0.1 M (2 mM MES, 98 mM NaC1)]. Legend: •, Wild type; A, Phe82Leu;•, Phe82Tyr; •, Phe82Ile; 0, Phe82Ser; El, Phe82Gly.2.001.000.003.15 3.25 3.35 3.45 3.55745.0Ic4.J1Temperature1X i03 (KeIvin1)Figure 26 Eyring plots for the oxidation of water switch variants of ferrocytochrome c by Co(phen)3[pH 6.0, JL 0.1 M (2 mM MES, 98 mM NaC1)]. Legend: •, Wild type; A, Asn52Ala;•, Ile75Met; •, Thr78Gly; 0, Tyr67Phe.4.0302.01.0003.15 3.25 3.35 3.45 3.5575Protein Rate Constant k11 (M’s’) Activation Activation(M’s1) (25°C) (25°C) Entropy (eu) Enthalpy(kcal/mol)Wild Type 1.7 x 10 3.1 x 10 -5.5 ± 0.6 11.4 ± 0.2Phe82Tyr 2.4 X 10 4.1 x 10 -5.5 ± 0.5 11.3 ± 0.1Phe82Leu 2.4 X 10 5.0 X 10 -4.4 ± 0.3 11.6 ± 0.1Phe82IIe 6.1 X 10 2.1 x 10 -4.4 ± 0.3 11.0 ± 0.1Phe82Ser 5.4 X 10 8.1 x 10 -6.3 ± 0.4 10.5 ± 0.1Phe82Gly 6.6 X 10 9.2 x 10 -12.2 ± 0.4 8.6 ± 0.1a)Protein Rate Constant k11 (M’s’) Activation Activation(M’s1) (25°C) (25°C) Entropy (eu) Enthalpy(kcal/mol)Wild Type 1.7 x 10 3.1 X 10 -5.5 ± 0.6 11.4 ± 0.2Thr78Gly 5.6 x 10 7.0 X 10 -8.7 ± 0.7 9.9 ± 0.2Ile75Met 4.1 x 10 3.2 x 10 -2.1 ± 0.7 11.9 ± 0.2AsnS2Ala 6.2 X 10 1.1 x 10 -9.0 ± 0.7 9.6 ± 0.2Tyr67Phe 1.5 x 10 3.3 X 10 -5.3 ± 0.5 10.2 ± 0.2b)Table 4 Rate parameters for oxidation of variants of ferrocytochrome c [Co(phen)3oxidant, pH 6.0,= 0.1 M (2 mM MES, 98 mM NaC1) 1. a) position 82 variants; b) water switch variants. The errorsin the cross reaction rate constants are approximately 5 %. To calculatek11C01, the following values wereused. Co(phen)3:Em6 = 370 mV, k = 41.7 M’ s (Baker et al., 1959), radius = 7 A (Wherland& Gray, 1976), charge = +3 (oxidized), +2 (reduced). Cytochrome C: refer to Table 3, page 69.763.4 Alkaline Isomerization3.4.1 pHTitrationsThe pH dependent absorbance changes observed at 695 mu were used to calculate the ratio of alkalineto native isomer according to the relation:naü[alkaline] = A - A695 (22)[native] At - A00hte695 695Where and A5 are respectively the absorbances at 695 rim of the native isomer, thealkaline isomer, and of a mixture of the two at the intermediate pH.The alkaline isomerization pK is calculated from the Henderson-Hasselbach equation:pH= P1a + n log[alkaline] (23)[native]Data from the titration of horse heart cytochrome c plotted in this fashion gave a straight line with slopen = 1, consistent with obedience to the Henderson-Hasselbach equation with a stoichiometry of oneproton. The calculated pK was 9.1, in excellent agreement with the results of other workers (Davis eta!., 1974; Pearce et a!., 1989). Titration of the yeast variant Thr78Gly was also consistent with themodel, with a measured PKa of 7.0; this value is compared to 8.5 obtained for the wild type protein(Pearce et a!., 1989). The pK, values for these proteins are presented in Table 5.773.4.2 pH Jump KineticsFor the five proteins examined (horse heart cytochrome c, and yeast wild type, Thr78Gly, Ile75Met,and AsnS2Ala), the observed rate constants for the loss of absorbance monitored at 695 nm (lç,) werefirst order over the range of pH examined. The model of Davis et al. (1974) and the accompanyingequation were used to determine the pKH of the ionization and the rate constants of the subsequentconformational change:K,,Cyt-H Cyt Cyt (24)kbCyt-H and Cyt are the protonated and deprotonated forms, respectively, of native ferricytochrome c, andCyf is the alkaline isomer.The pH dependences of the first order rate constants were fitted to the following equation, derivedfrom the above mechanism (Davis et a!. 1974):kO=kb+ (25)KH ÷ [H]The reverse rate constant kb was estimated from the observed rate constant of the process at pH 6.The results of the kinetic experiments and their analysis in terms of the model presented above areshown in Figure 27. The rate data for all of the proteins except AsnS2Ala could be fitted to Equation25. The parameters kf, kb, and PKH calculated for each variant are presented in Table 5. There is goodagreement between these results and those of previous investigators for the horse heart and yeast wildtype cytochromes (Davis et a!., 1974; Pearce et a!., 1989). The parameters derived for horse heartcytochrome c, yeast wild type, and yeast variant Thr78Gly also give an estimate of the alkalineisomerization plc (pKH + pK) that is in agreement with the value determined from the titration. Thelowered alkaline p for Thr78Gly is due to a PKH that is 2 units below that of the wild type protein,partially compensated by a conformational equilibrium that is relatively more favourable to the native788.06.000.07.0 11.0pHFigure 27 pH dependence of the rate of alkaline isomerization from stopped-flow pH jump experiments[25 °C, jt 0.1 M]. Legend: •, Wild type; A, Asn52Ala; •, Ile75Met; •, Thr78Gly. Theinterpolated curves were calculated from least squares fitting of the data to equation 25. The data for theAsn52Ala variant could not be fitted to equation 25, thus the line through this data set is presented onlyas a visual aid. Numerical values of the data points are tabulated in Appendix D.8.0 9.0 10.079Protein PKa pKH kr (sec’) kb (sec) K pKH + pKHorse c 1 9.0 11.0 ± 0.1 6 ± 2 0.049 ± 0.003 120 ± 40 8.9Horse c 9.1 11.24 ± 0.05 5.5 ± 0.4 0.035 ± 0.002 160 ± 20 9.0Wild Type 8.52 10.9 ± 0.1 16 ± 2 0.043 ± 0.001 370 ± 60 8.4Ile75Met - 10.7 ± 0.1 7.7 ± 0.9 0.041 ± 0.004 180 ± 30 8.4Thr78Gly 7.0 9.0 ± 0.03 3.8 ± 0.1 0.047 ± 0.004 80 ± 9 7.1Asn52Ala 8.3 - - 0.007 ± 0.001 - -1 Davis et a!., 1974.2 Pearce et a!., 1989.Guillemette et a!., in preparation.Table 5 Alkaline isomerization parameters for variants of ferricytochrome c [25 °C, j = 0.1 M 1.80conformation. The kinetic parameters of Ile75Met are similar to those found for yeast wild type. Thesum pKH + pK for this variant gives a PKa identical to that of the wild type cytochrome.The data for the yeast AsnS2Ala variant could not be fitted to the above equation. As shown inFigure 27, the observed rate constant increases more sharply with pH than observed for the otherproteins. Similar behaviour has been seen in the Tyr67Phe variant (Guillemette, in preparation) and inEuglena cytochrome c, which also has a phenylalanine residue at position 67 (Pettigrew et al., 1975).Above pH 10.5 a second, faster, first-order phase was detected for both yeast wild type and theAsnS2Ala variant. This phase accounted for an increasing amount of the total absorbance change at 695nm as the pH was increased. The presence of this phase was more readily detected by monitoring theabsorbance change at 529 nm (Figure 28). The rate constant for the faster phase was 45 ± 1 s and wasnot dependent on wavelength.810.02CsJU,•10-0.02Time, secondsFigure 28 Kinetic trace of the alkaline isomerization of yeast-iso-1-ferricytochrome c at pH 10.5monitored at 529 tim. The rate constant of the initial phase is 44 ± 2 s1 while that of the second phaseis 4.7 ± 0.4 s_I0 0.5 1823.5 Ligand Substitution3.5.1 Azide Binding ThrationsAzide binding to ferricytochrome c causes several changes to the visible spectrum, most notably a shiftin the position of the Soret maximum from 409.5 nm to 412 nm and a 20 percent increase in the intensityof the Soret absorbance (Figure 29). There is also a shift in the position of the a-band from 530 to 539nm, and a loss of the band at 695 nm (700 nm for Tyr67Phe). In contrast, there were no detectabledifferences between the visible spectra of reduced cytochrome c in the presence and absence of azide.The absorbance changes at 695 nm that were observed upon addition of azide to ferricytochrome cwere fitted to an equation of a binding isotherm for a 1:1 ligand-receptor complex (Connors, 1987):= AA [azide] (26)695 1/K + [azide]In this relationship, is the difference in the absorbance at 695 nm between native ferricytochrome cand ferricytochrome c at a known azide concentration, and is the difference in absorbance at 695rim between native and azido-ferricytochromes c. Figures 30 and 31 show for each cytochrome c thefit of the experimental data to the binding isotherm with normalized to 0.1. The calculated bindingconstants, Kjde, are presented in Table 6 along with the results of the azide binding kinetics experiments.Measurements for all of the variants studied were consistent with 1:1 binding of azide. Ad6 for horseheart cytochrome c was 4.5 ± 0.1 M’, comparable to 4 M1 found by Sutin & Yandell (1972).for the wild type yeast protein was 16.7 ± 0.6 M1 compared to 15 M1 found by Saigo (1986).The affinity of yeast cytochrome c for azide is strongly dependent on the identity of the residue atposition 82. All of the variants at this site possessed greater azide affinities than that of the wild typeprotein. The azide binding constants for the Leu, Ile, Gly and Ser variants were three to five-fold greaterthan that for azide binding to wild type cytochrome c. The azide binding affinity of Phe82Tyr was themost similar to that of the wild type protein but was still two-fold greater.83a)C)c-0Cl)-Wavelength (nm)1.00.50Figure 29 The visible spectra of yeast iso-1-ferricytochrome c in the presence (Az) and absence (N) of2.5 M azide, 25 °C, pH 6.0.400 500 600840.100LO0)CD[N] (M)0.0800.0600.0400.0200.000100Figure 30 Azide binding isotherms for position-82 variants of ferricytochrome c [25 °C, pH 6.0, JL1.0 M]. Legend: •, Wild type; A, Phe82Leu; •, Phe82Tyr; •, Phe82Ile; 0, Phe82Ser;D, Phe82Gly.102 10-1850.100 I I I I I I—0.080//, /10.060I/I0)/1 J0.0404///0.020 _//_s-_—-—0.000 I I Lj — I...—.——— I— —t—1 1 ri1 I I I I102 10-1 100 101[Ni] (M)Figure 31 Azide binding isotherms for water switch variants of ferricytochrome c [25 °C, pH 6.0,= 1.0 M]. Legend: •, Wild type; *, Asn52Ala; •, Ile75Met; •, Thr78Gly; 0, Tyr67Phe.86In contrast, the variants at the water switch positions possessed azide affinities similar to or lower thanthat of wild type cytochrome c. This observation was most evident with the Tyr67Phe variant, with azde estimated to be lower than 1 M’. Even in the presence of 2 M azide, this variant could not besaturated with azide, as shown by its incomplete titration curve. The binding constants for the Ile75Metand Asn52Ala variants were closer to that of the wild type protein but both exhibited a lower affinity forazide.3.5.2 Azide Binding KineticsFor all proteins studied, the loss of absorbance at 695 nm followed monophasic, first order kinetics.The relation of observed rate constant to [azidej was fitted to the following equation, which is derivedfrom the mechanism shown in equation 10 on page 37 (Sutin & Yandell, 1972):k01,= k12(1+K[N3i) (27)k1+k2K[N]Here is the observed first-order rate constant for the change in absorbance at 695 nm. Thismechanism assumes a steady state concentration of the active intermediate Cyt-c. The variation inobserved rate constants with the azide concentration is shown in Figures 32 and 33, along with thecalculated fits to equation 27. The resulting rate parameters are presented in Table 6. Equation 27predicts rate saturation at increasing azide concentrations, but this is not always apparent from inspectionof the experimental data. Nevertheless, the data for all variants except Tyr67Phe and Ile75Met areconsistent with this mechanism as judged by their fits to the equation. There is fair agreement betweenthe results for horse heart cytochrome c of this work (k1 = 65 ± 9 s4) and those of Sutin & Yandell(1972), who estimate a forward rate constant k1 of 30 to 60 s under similar conditions.Curvature of the plots is detectable in the Ile, Leu, Ser and Gly variants at position 82. These fourvariants are far more reactive than the wild type protein, with calculated forward rate constants k187160 -Cci---—120-—-7 —C)-7 — A ——0 80-‘—/0 //.D ,/ .V40 - A .•---“,zZZZL0.00 0.10 0.20 0.30 0.40 0.50 0.60[N1 (M)Figure 32 The dependence of observed rate constant of azide binding to position-82 variants offerricytochrome c on azide concentration [25 °C, pH 6.0, = 1.0 Ml. Legend: •, Wild type;A, Phe82Leu; , Phe82Tyr; •, Phe82fle; 0, Phe82Ser; LI, Phe82Gly. Numerical values of thedata points for Figures 32 and 33 are tabulated in Appendix E.8820.016.0C.)a)CoO 80.D0-4.00.00.00 0.60[N] (M)Figure 33 The dependence of observed rate constant of azide binding to water switch variants offerricytochrome c on azide concentration [25 °C, pH 6.0, t = 1.0 M]. Legend: •, Wild type;A, Asn52Ala; •, Ile75Met; 0, Tyr67Phe.0.10 0.20 0.30 0.40 0.5089Ile75MetTyr67PheTable 6 Azide binding parameters for variants of cytochrome c [25 °C, pH 6.0, = 1.0 M j. a)position 82 variants; b) water switch variants.Protein K (M1) k1 (sd) k.2 (s’) k2 1k.Wild Type 16.7 ± 0.6 74 ± 20 1.65 ± 0.06 0.4 ± 0.1Phe82Tyr 31 ± 2 49 ± 3 2.5 ± 0.1 1.6 ± 0.3Phe82Leu 96 ± 11 240 ± 30 2.5 ± 0.1 1.0 ± 0.3Phe82Ile 64 ± 4 160 ± 30 2.1 ± 0.1 1.0 ± 0.3Phe82Ser 53 ± 2 270 ± 20 6.8 ± 0.2 1.3 ± 0.2Phe82Gly 105 ± 1 360 ± 30 4.6 ± 0.2 1.3 ± 0.2a)Protein iç (M1) k1 (s4) k.2 (sd) k2 1k.Wild Type 16.7 ± 0.6 74 ± 20 1.65 ± 0.06 0.4 ± 0.1Asn52Ala 8.2 ± 0.3 58 ± 3 3.71 ± 004 0.5 ± 0.1Keq (P44) kr (M’ s) kb (sd) kr 1kb (1’i’)10.9 ± 0.4 32.4 ± 0.9 3.4 ± 0.3 9.5 ± 10.34 ± 0.03 9.7 ± 0.3 10.8 ± 0.3 0.9 ± 0.1b)90exceeding 160 s. For the Gly and Ser variants, the reverse rate constant k.2 is also increased three tofour fold over the corresponding value observed for the wild type cytochrome.The rate constants that characterize the reaction of the active intermediate, k4 and k2, cannot bemeasured directly; the steady state assumption implies that these steps are not rate limiting.Consequently, only the ratio of these constants can be estimated from the relation= k2(28)The ratios for horse heart cytochrome c and yeast wild type are similar, at 0.4 M1; the other position82 variants have ratios of 1 to 1.6 M1, indicating an increased preference to form the azide bound speciesupon reaching the intermediate complex.The azide binding behaviour of the water switch mutants is variable. The AsnS2Ala variant is similarin most respects to the wild type cytochrome, and the observed decrease in A is caused solely by anincrease in the reverse rate constant k.2. The inertness of Tyr67Phe to azide substitution, previouslyrevealed from the titration experiment, was further exemplified by the small net absorbance changesduring the kinetic experiments and the relatively small dependence of the observed rate constant on azideconcentration. The dependence of observed rate constant on azide concentration obtained from theTyr67Phe variant was fitted to a linear equation. Ile75Met was the most unusual variant, possessing alinear dependence of observed rate constant on azide concentration which suggests a simple two stateequilibrium. The data for the Ile75Met variant was fitted to a linear equation; from the slope andintercept of the line, the forward and reverse rate constants respectively were calculated. The ratio ofthe forward to the reverse rate constant was in good agreement with the value of 1L determined bytitration.91DISCUSSION4.1 Overview of the Structural Features of the Cytochrome c VariantsIn this work, the influence of amino acid changes at several positions within the heme enviromnentof cytochrome c on the electrochemical, kinetic, and ligand binding properties of this protein has beendetermined. Before discussing the results of these experiments, it is appropriate to review briefly theknown structural features of the cytochrome c variants that have been the subject of this work.The three-dimensional structures of several position 82 and water switch variants are available (Table7). This information has been extremely useful, not only for revealing the basis for differences in theproperties of variants of known structure, but also as a departure point for discussion of factors likelyto contribute to the measured properties of variants for which no structural information is available.Figures 34 and 35 present the known structures of the position-82 and water switch variants respectivelywithin the vicinity of the mutation site.The structural features of yeast iso-1-cytochrome c wild type protein that are relevant to this workhave been addressed previously in the Introduction. The structure of the Phe82Tyr variant has beensolved to 1.97 A resolution by Louie (1990). The conformation of the polypeptide backbone is essentiallythe same as that of the wild type structure. The side chain of Tyr82 projects 0.7 A further out of theheme crevice than does the wild type Phe82 side chain. Small alterations in the backbone conformationof residues 81 to 85 compensate for the slightly different position of the tyrosine side chain. Theexposure of the heme to solvent is unchanged from that of the wild type protein. Louie (1990) hasproposed that the hydroxyl oxygen of Tyr82 forms a hydrogen bond with the guanidinium group of thenearby Argl3 side chain.The Phe82Ser mutant possesses a solvent channel at the mutation site extending from the surface ofthe protein to Met8O, thus increasing the solvent accessibility of the heme (Louie & Brayer, 1988).92Protein Oxidation Resolution ReferenceStateWild Type reduced 1.23 A Louie & Brayer, 1990oxidized 1.9 A Berghuis & Brayer, submittedPhe82Tyr reduced 1.97 A Louie, 1990Phe82Ile reduced 2.3 A Louie, 1990Phe82Ser reduced 2.80 A Louie & Brayer, 1988Phe82Gly reduced 2.60 A Louie & Brayer, 1989oxidized 1.76 A Louie, 1990Tyr67Phe reduced 1.95 A Guillemette et a!., in preparationoxidized 2.2 A Guillemette et al., in preparationAsn52Ala reduced 2.0 A Berghuis & Brayer, unpublishedTable 7 Current status of the structure determinations for those Saccharomyces cerevisiae iso-icytochrome c variants examined in this work. At present there is no information for the Phe82Leu,Phe82Ala, Ile75Met, and Thr78Gly variants of this protein.93ArgiS AAr13 ,rgLeu85 Leu85 Leu85/ Leu94“Leu94 “Leu94Phe 2 Tyr 2 11e82MetBO Met8OMet8Oa) b) C)Argl3Argl3‘Leu94Leu85Leu94G1y82 Ser82MetBOMetSOd) e)Figure 34 The structures of the position-82 variants of reduced iso-1-cytochrome c in the vicinity of themutation site (Louie & Brayer, 1988, 1989, 1990; Louie, 1990). a) Wild Type Protein b) Phe82Tyrc) Phe82Ile d) Phe82Gly e) Phe82Ser.9411e75A1a52 (Asn52)Figure 35 The structure of a) AsnS2Ala (Berghuis et al., in preparation) and b) Tyr67Phe (Guillemetteet al., in preparation) variants of reduced cytochrome c about WAT166. Thin lines, native; thick lines,mutants. The heavily shaded circles represent the location of the internal water molecules in the variantstructures; the lightly shaded circles correspond to the position of WAT166 in the wild type cytochrome.lie75Asn5295In addition, WAT166 is 1 A closer to the heme in the reduced structure of this mutant than in the wildtype structure. The presence of a buried water molecule closer to the heme will also contribute to anincrease in the polarity of the heme environment. There is no structure currently available for thePhe82Ala variant. Because of the similar size of a serine and alanine side chain, Phe82Ala might alsopossess a solvent channel, which would account for the similar reduction potentials of the two proteins.The position of WAT166 would also be similar to that of Phe82Ser.In the 2.6 A structure of the reduced Phe82Gly mutant, the polypeptide backbone in the mutation sitefolds to occupy the same space as the phenylalanine side chain in the wild type protein. This is the onlycytochrome c mutant investigated to date with a significantly altered folding of the polypeptide backbone(Louie & Brayer, 1989). This change appears be caused by the flexibility of the sequence of threeconsecutive glycine residues in positions 81 to 83 that is present in this variant. This flexibility isdemonstrated by the observation that the orientations of two peptide bonds (between residues 81 and 82,and 83 and 84) in the oxidized structure of this variant are opposite to those observed in the structure ofthe ferrocytochrome (Louie, 1990). Because of this unique folding of the main chain in the mutation site,the solvent accessibility of the heme in the Phe82Gly variant does not differ significantly from that foundfor the wild type protein. However, the negative ends of several peptide bonds dipoles are directedtowards the heme, which increases the polarity of the heme environment. As seen in the structure of thePhe82Ser variant, the position of WAT166 in reduced Phe82Gly is also 1 A closer to the heme than inthe wild type protein.The structure of Phe82Ile has been solved to 2.3 A resolution (Louie, 1990) and found to exhibit abackbone folding pattern similar to that of the wild type protein. The 3-branching of the Ile side chaincauses unfavourable steric interactions with neighbouring groups, and the j3-methyl group is in van derWaals contact with the methyl and -y-methylene groups of the Met8O side chain. The conformationalstrain within this region may cause flexibility of the side chain, allowing a transient increase in the solventexposure of the heme. Beyond the side chain of the Phe82Ile residue is a void space resembling the96solvent channel of Phe82Ser, which could serve as a transiently exposed solvent channel. The positionof WAT166 in Phe82Ile is approximately 0.5 A closer to the heme than in wild type structure, whichwould also contribute to the lowered reduction potential. There is no three-dimensional structureavailable for the Phe82Leu variant. The -y-branch position for leucine is in the same relative position asthe ‘y atom of phenylalanine, thus the leucine side chain should be more easily accommodated at position82 without as much unfavourable steric interactions with neighbouring atoms as are observed withisoleucine at this position.In both the Tyr67Phe and Asn52Ala variants, the polypeptide backbone configuration is similar tothat of the native protein (Guillemette et at., in preparation; Berghuis et at., unpublished) Thereplacement of the native side chain with smaller, non-hydrogen bonding residues leads to the formationof an internal cavity that is occupied by two water molecules (Figure 35). One of the water moleculescorresponds to the position of WAT 166 in wild type, while the other water molecule occupies the positionof the tyrosine hydroxyl (Tyr67Phe) or the asparagine amide (AsnS2Ala) in the wild type structure. Inthe case of Thr78Gly, for which no structure is available, the lack of a hydrogen bonding side chain couldbe filled by a second internal water molecule, as in the AsnS2AIa and Tyr67Phe variants. However, onemust also consider that in this variant the entire branched side chain of threonine residue would not bepresent. It is possible that the polypeptide backbone in the region of the mutation folds in a mannerdifferent from that of the wild type protein to avoid the formation of a large cavity.Structural information regarding the Ile75Met variant is currently unavailable. If the main chainconformations of the variant and wild type proteins are the same, as appears to be the rule with thecytochrome c variants studied in this work, there are two likely positions that the Met75 side chain couldoccupy with minimal alteration to the protein structure. These positions would place the methionine sidechain on the direction of the longer or shorter 3-branches of the wild type isoleucine. In both cases,unfavourable steric interactions with nearby atoms would be avoided, as the noncovalently bonded atomsnearest to the methionine sulphur atom and methyl group are 3.5 A away. Another possibility, although97one that would cause significant structural changes, is that the methionine side chain is directed inwardand occupies the space of WAT166 to completely alter the nature of the hydrogen bonded network in thisregion of the protein. The results of the electron transfer kinetics experiments and the alkalineisomerization experiments argue against such a drastic alteration to this region of the protein (vide infra).4.2 Cytochrome c Oxidation-Reduction EquilibriumAt pH 6 and 25 °C, all variants investigated had reduction potentials within 45 mV of the valueobserved for the wild type protein. This range corresponds to a difference in the free energy between thetwo oxidation states of 1 kcal/mol or less. The wild type protein possesses the highest reductionpotential, while position 82 mutants have progressively lower potentials in the order leucine > tyrosine> isoleucine > alanine > serine > glycine (Table la, page 60). Qualitatively, smaller residues atposition 82 result in lower potentials, although the presence of a hydroxyl group on the mutant residuewill also contribute to a lower potential. Thus the reduction potential of Phe82Ser is lower than that ofPhe82Ala, and the potential of Phe82Tyr is lower than that of wild type. The contribution of a hydroxylgroup in these variants is to lower the potential by 5 to 10 millivolts. The difference between thereduction potentials of the Phe82Leu (290 mV) and Phe82Ile (273 mV) variants reflects the large effectsthat can result from seemingly small structural differences. In the case of these variants, the differenceis in the position of the branch point of the aliphatic side chain. The reduction potential of the variantPhe82Leu is similar to that of the wild type protein, suggesting that this residue maintains the normaldielectric for the heme environment. The Phe82Gly variant possesses the lowest reduction potential inspite of having a solvent accessibility similar to that of wild type. The lower reduction potential ofPhe82Gly could be caused by transient solvent exposure at the mutation site. Alternatively, the peptidebond dipoles within the mutation site are considerably closer to the heme in this variant, and are orientedin a manner that would be consistent with stabilization of a positive charge on the heme.98Unlike the position 82 mutants, which have reduction potentials distributed evenly across the rangebetween 290 and 245 mV, the water switch mutants all have potentials that are at least 30 mV lower thanthat of the wild type protein (Table lb. page 60). This result cannot be attributed to increased solventexposure of the heme in the case of the two water switch variants whose structures have been solved(Tyr67Phe and AsnS2Ala). A possible reason for the lowered potentials of these two mutants is that thewater molecule that replaces the hydrogen bonding residue of wild type has greater mobility and is betterable to stabilize the oxidized state either by reorientation of its dipole or by moving closer to the hemeupon oxidation. Such a mechanism is consistent with the lower reduction potential of Tyr67Phe (236mV) relative to Asn52Ala (257 mV), as the second water molecule of Tyr67Phe is closer to the hemethan is the second water molecule of AsnS2Ala. New water molecules closer to the heme would beexpected to have a greater influence on the reduction potential than water molecules that are more remote.This proposal would also be consistent with the finding that the reduction potential of Thr78Gly (247 mV)is halfway between those of Tyr67Phe and AsnS2Ala; a proposed second solvent molecule replacing thehydrogen bonding hydroxyl group of the wild type Thr78 residue would be midway between the locationsof the second solvent molecules in these variants.The 45 mV drop in the reduction potential of Ile75Met relative to the wild type protein is likely causedby increased solvent accessibility of the heme as the straight chain methionine side chain would not beas effective as the branched isoleucine at shielding this region from the bulk solvent. After isoleucine,the most commonly occurring residue at position 75 is valine, which also possesses a 3-branched sidechain. Met75 occurs naturally in only two species of cytochrome c whose sequences are known, thoseof Crithidia oncopelti (Pettigrew, 1972) and Crithidiafasciculata (Hill & Pettigrew, 1975). However,the reduction potential of Crithidia oncopelti cytochrome c as determined by an equilibrium method is270 mV (pH 6, Moore et al., 1984). The reduction potential of Crithidia oncopelti cytochrome c mayalso be influenced by other unusual amino acid residues in the heme crevice, the most notable difference99being the lack of one of the thioether bonds from the heme to the protein caused by the occurrence ofan alanine residue instead of cysteine at position 14.None of the mutations studied here altered the net protein charge, nor changed the axial ligands of theheme iron. Potentially the mutations could alter the distribution of the charged residues about the hemeand in this manner alter the electrostatic interactions of these residues with the heme but, with thepossible exception of Phe82Tyr, this seems unlikely. The available crystallographic evidence indicatesthat in most cases the difference in the position of main chain atoms between wild type and mutant aresmall. In the case of Phe82Gly, where a refolding of the main chain occurs, this effect is limited to themutation site and adjacent residues and leaves the rest of the main chain in a conformation similar to wildtype.In the absence of changes in axial ligation and ionic interactions, the source of the differences in thereduction potentials must arise from mutation-induced changes in the dielectric of the heme environment.This environment includes the polypeptide backbone, amino acid side chains, and buried and bulk solventwater molecules. The lower reduction potentials of most of the cytochrome c variants studied indicatesthat the variants are more effective than the wild type protein at stabilizing the oxidized state, or,alternatively, at destabilizing the reduced state.The entropy and enthalpy of reduction for each variant were calculated from the temperaturedependences of the reduction potentials (Table 1, page 60). Small differences in the thermodynamicparameters can have large effects on reduction potentials. For example, the mutants Phe82Leu, Phe82Ile,Phe82Ser and Ile75Met have reduction entropies only 2 to 3 eu more negative than that of the wild typeprotein, but at room temperature this difference contributes to a lowering of the reduction potential by25 to 40 mV. In the cases of Phe82Leu and Phe82Ile, the reduction enthalpy compensates for thisentropy change, and the reduction potentials at room temperature are only 4 to 17 mV lower than thatobserved for the wild type protein, respectively. Phe82Ser and Ile75Met exhibit no enthalpiccompensation. Their lower reduction potentials are due almost entirely to entropic effects that stabilize100the oxidized protein. In the case of these four variants, the relatively larger standard free entropiessuggest slightly larger conformational and/or solvational differences between the fern- andferrocytochromes.In contrast, the mutants Phe82Gly, Phe82Ala, Thr78Gly and Asn52Ala exhibit standard free entropychanges that are similar to or more positive than that of the wild type cytochrome, which indicatesconformational differences between oxidation states may be smaller in these variants than for the wildtype protein. The lowered reduction potentials of these mutants are caused by more positive standard freeenthalpy changes.Despite the differences in thermodynamic parameters, all of the variants investigated exhibitedbehaviour typical of mitochondrial cytochromes c that have been studied previously. The reaction centreentropy changes of -6 to -12.3 eu are small in magnitude and negative, while the standard free enthalpychanges are between -12.8 and -14.8 kcal/mol. In all variants, the entropy change favours the oxidizedstate while the enthalpy change favours the reduced state. Such behaviour data suggest a more orderedand thermodynamically more stable reduced structure for the cytochrome compared to the oxidizedstructure. It seems likely that all of the variants undergo oxidation-state linked conformational changessimilar in magnitude to those observed for tuna and yeast wild type cytochromes c.The small reaction centre entropy of cytochrome c may have a role in promoting rapid electrontransfer. Yee and coworkers (1979) measured the influence of ligands on the reaction entropies oftransition metal complexes. These authors found that Src° for aquo complexes ofM3412 (M = Fe, Cr,Ru, Os, V, Eu) were between 36 and 49 eu, while for ammine complexes ofM3’2 (M = Os, Ru) SrC°was 18 eu, and for Fe32 complexes with the chelating ligands bipyridine and phenanthroline was2 and 3 eu, respectively. Yee et a!. proposed that this relationship between Src° and ligand environmentis due in part to differences in the ability of the ligands to order solvent molecules surrounding the innercoordination shell. Water as a ligand is able to hydrogen bond surrounding water molecules moreeffectively than is ammonia. Large, bulky chelating ligands are effective at shielding the charge of the101metal centre from the surrounding solvent, thus solvent reorientation upon a change in oxidation state isminimized. The authors suggested that the value of AS° is an indicator of the contribution of solventreorganization to the Franck-Condon barrier to electron transfer because those complexes with reactioncentre entropies closer to zero tend to have higher self exchange rates.In cytochrome c, the polypeptide and buried water molecules surrounding the inner coordination shellof the heme iron shield the metal centre from bulk solvent. In addition, the mobility of this environmentin response to a change in oxidation state is limited, as demonstrated by the relatively small oxidationstate linked conformational change. The low reaction centre entropy for cytochrome c presumablyfacilitates rapid electron transfer by minimizing the extent of solvent reorganization, where ‘solvent’includes everything except the heme and the axial ligands. As the observed reaction centre entropies arenot zero, a compromise with other biologically essential requirements, such as the maintenance of abiologically relevant reduction potential, may be a more important evolutionary constraint in thedevelopment of cytochrome c function.The pH dependences of reduction potential for several cytochrome c variants are all consistent withthe presence of a single titratable group, the pK of which is sensitive to the oxidation state of the protein(Table 2, page 63). NMR spectroscopy has been used to identify His39 as the residue whose plc isdependent on the oxidation state (Robinson et a!., 1983). This assignment is consistent with theobservation that horse heart cytochrome c, which has a lysine residue at position 39, has a reductionpotential that is independent of pH between pH 4 and 9, while Candida krusei and Rhodopseudomonasviridis cytochromes c, which possess His39, have oxidation-state dependent pKs similar to that exhibitedby iso-1-cytochrome c.The oxidation-state linked change in the pK, of His39 is small (0.4 pH units), suggesting a weakinteraction between the heme charge and this residue. This finding is consistent with the 14 A distancebetween the a-carbon of this residue and the heme iron. In comparison, the pK, of heme propionate-7of bacterial cytochrome c551 decreases a full pH unit upon oxidation of the heme iron. Moore and102coworkers have proposed that the change in pK of His39 of iso-i -cytochrome c is transmitted from theheme via the small conformational changes that occur in the environment of this residue upon a changein oxidation state (Robinson et al., 1983). His39 is located in a flexible region of cytochrome c thatincludes the residues in contact with the heme propionates.For the wild type cytochrome and the Phe82Ser and Ile75Met variants, the plots of reduction potentialvs pH give similar curves that differ in their vertical displacement by an amount determined by their valueof E0, the reduction potential extrapolated to pH 0. The calculated pKs for these proteins were thusidentical to each other, within experimental error. This finding suggests that the two mutations do notaffect the oxidation state-linked conformational changes in the environment of His39. The mutation sitesin Ue75Met and Phe82Ser are remote from His39, so they are not expected to exert an influence on theionization of this residue.The difference in pK0 and pK for the AsnS2Ala variant is 0.4 as for the other yeast cytochromesstudied, but both values are 0.5 pH units lower than those determined for the wild type protein. Incontrast to the lle75Met and Phe82Ser variants, residue 52 is located near the heme, between the hemepropionates and His39. The lower values for pK0 and PKr of this mutant suggest a more hydrophobicenvironment for His39. Such a change could be caused by closer contact with neighbouring residueLeu58.The double mutant Asn42Lys I Ala43His was constructed to increase the pH dependence of thereduction potential in the physiological range. In certain bacterial cytoehromes c, an oxidation-statedependent ApK of 1 pH unit is attributed to the ionization of a heme propionate in the physiologicalrange between pH 5 and 8. The presence of additional, positively charged side chains near the hemepropionates is expected to lower the pK of these groups. As one of the heme propionates is believed toionize above pH 9 in the wild type protein (vide supra), the double mutation Asn42Lys I Ala43His wouldbe expected to lower the pK of this heme propionate into the physiological range. This was notobserved. Although the reduction potential of this mutant was lowered by 10 mV, the pH dependence103of the reduction potential was not altered. Examination of the structure of the protein suggests that thealtered residues would be directed towards the solvent, thus having minimal influence on the pHdependence of the oxidation-reduction potential. The lower reduction potential of this mutant isinteresting, as the addition of positively charged residues to cytochrome c would be expected todestabilize the oxidized state. This result illustrates the complexity of determining the influence amutation can have on reduction potential, as a single mutation can have several and often opposing effectson the observed property. While the electrostatic interaction between the mutated residues and the hememay favour a higher reduction potential, the influence of these residues on the local polypeptide structureand other structural characteristics may have a stronger tendency to lower the reduction potential.4.3 Electron Transfer Kinetics4.3.1 General CommentsPrevious workers (Augustin et a!., 1983; Armstrong et a!., 1986a) have investigated the influence ofmodifying the charge properties of cytochrome c on its reactivity toward small inorganic electron transferagents and other electron transfer proteins. The present work demonstrates that the electron transferreactivity of cytochrome c is equally sensitive to mutations that do not alter its electrostatic properties.Rather than comparing directly the cross reaction rates of the cytochrome c variants with the inorganiccomplexes used in this study, each experimentally determined cross reaction rate constant is used withthe known self exchange rate constant of the inorganic complex and the driving force of the reaction tocalculate the self exchange rate constant exhibited for the protein in each reaction according to the relativeMarcus equation. These calculated protein self-exchange rate constants are the basis for comparison ofthe influence of mutation on electron transfer reactivity. Thermodynamic driving forces are calculatedfrom the measured reduction potentials of the cytochrome c variants and their electron transfer partners.The calculated protein self exchange rates, k110, have also been corrected for the influence that104electrostatic interactions between the reactants has on the reaction rate by the inclusion of appropriatework terms for precursor complex formation and successor complex dissociation.There is an apparent contradiction if the value of the self exchange rate constant of cytochrome cdepends on the inorganic complex used. Thus the self exchange rate for wild type iso-1-cytochrome cis 11 and 3100 M’stwhen calculated with cross reaction measurements using Fe(edta)2 and Co(phen)3respectively. This apparent fault in the relative Marcus equation is resolved by examining the assumptionthat the activation free energy of the cross reaction is the average of the activation free energies of eachreactant in its self exchange. Except for electrostatic work terms, no provisions are made for specificinteractions between the two reagents in the cross reaction that do not occur in the component selfexchange reactions. The dependence of the calculated self exchange rate constant of cytochrome c onthe electron transfer partner employed in the cross reaction is not a drawback, as disparity in selfexchange rate constants demonstrates differences in reaction mechanisms. This reasoning has beenutilized by Wherland and Gray (1976) who considered the five orders of magnitude difference in selfexchange rate constants of horse heart cytochrome c with various electron transfer partners. Differencesin the hydrophobicity of the electron transfer partners of cytochrome c and differences in the nature ofthe molecular orbital types employed by these partners in electron transfer with the protein were proposedto explain the observed variation in the calculated self exchange rate of cytochrome cMore important than the absolute value of k11co for a mutant protein is this value relative to that ofthe wild type species. Describing the electron transfer reactivity of each variant in terms of its relativek11COI simplifies discussion of the results and facilitates comparisons between the results obtained usingdifferent electron transfer partners. In the following discussion the terms ‘reactivity’ and ‘reactive’ willrefer to relative values of the calculated self exchange rates.1054.3.2 Position-82 VariantsThe results obtained with the position 82 mutants show that while electron transfer occurs via theexposed heme edge, Phe82 itself is not essential for electron transfer with inorganic complexes. This hasalso been shown to be true for the reaction of position 82 variants with cytochrome b5 (Barker et a!.,unpublished), cytochrome c peroxidase (Pielak et a!., 1985), and cytochrome c oxidase (Michel et a!.,1989).The main influence of mutations at position 82 on the reactivity of cytochrome c is on the nature ofthe precursor complex formed between cytochrome c and its electron transfer partners. As the size ofthe residue at position 82 decreases, reactivity increases. This affect is probably attributable to shorterdistances between electron transfer centres, with position 82 mutants having small side chains allowingthe electron transfer partner to approach the heme more closely. This trend is seen clearly with theresults of kinetics experiments using Fe(edta)2,where k11col varies over a seventeen-fold range (Table3a, page 69). With Co(phen)3 oxidation of ferrocytochrome c the trend is the same, but it is lesspronounced, as the reactivity varies only over a seven-fold range (Table 4a, page 76). For example,Phe82Gly and Phe82Ser are 15 to 17 times more reactive than wild type cytochrome c towards Fe(edta)2but only 2.6 to 3 times more reactive towards Co(phen)3. The difference in results obtained forcytochrome oxidation and reduction is likely a consequence of the difference in the sizes of the twoinorganic complexes used. The smaller radius of Fe(edta)2 (4 A) compared to Co(phen)3 (7 A)(Wherland & Gray, 1976) allows the former reagent to probe the surface of cytochrome c moreintimately, allowing shorter distances between the metal centres.The higher reactivities of Phe82Ser and Phe82Gly toward Fe(edta)2 may also be caused by adifference in the orientation of this electron transfer partner with respect to the surface of cytochrome c.As noted by Wherland & Gray (1976), Fe(edta)2 can present two faces to cytochrome C: a polar facebearing the carboxyls of the edta ligand, or a more hydrophobic face consisting of the ethylene backboneof the ligand. The hydrophilic face would be more favourable for electron transfer because of the106opportunity for ir-ir overlap of the carboxyl orbitals with the corresponding heme orbitals. However, thisorientation of the hydrophilic face of Fe(edta)2 with the hydrophobic Phe82 residue would not befavourable. Instead, Fe(edta)2 would present its more hydrophobic face to the surface of wild typecytochrome c. In both Phe82GIy and Phe82Ser the surface of the protein in the mutation site would bemore hydrophilic, allowing Fe(edta)2 to present its more reactive face to the protein.In addition to altering the nature of the precursor complex by changes in the relative orientation ofthe reactants and shortening of the distance between electron transfer sites, Phe82Gly and Phe82Ser mayincrease reactivity by lowering the reorganization energy of cytochrome c. In the reduced structures ofboth proteins, WAT166 is 1 A closer to the heme than it is in the wild type protein, giving the reducedstructure some characteristics of the oxidized structure.Although most position 82 mutants are more reactive toward Fe(edta)2 than Co(phen)3,oneexception is Phe82Ile. The heme crevice of this mutant is structurally unstable, as described previously,and may allow the hydrophobic Co(phen)3 to penetrate the protein surface. This interaction could bedescribed as an encounter between a hard sphere (Co(phen)3)and a soft sphere (Phe82Ile). In contrastto the behaviour of Phe82Ile, the electron transfer reactivity of its Phe82Leu isomer is closer to that ofthe wild type protein.4.3.3 Water Switch VariantsThe reactivity exhibited by the water switch mutants towards Fe(edta)2 and Co(phen)3demonstratesthat the specific arrangement of the hydrogen bond network in the region of the mutated residues is nota prerequisite for rapid electron transfer. Variants in which critical residues of this network have beenremoved all have similar or even greater reactivity than the wild type cytochrome. In at least two of themutants studied, replacement of a hydrogen bonding side chain by a second buried water molecule allowselectron transfer reactivity to be increased.107The case of Tyr67Phe clearly illustrates the need to take into account driving force differences throughthe application of the relative Marcus theory when comparing the electron transfer rates. The crossreaction rate of Tyr67Phe with Fe(edta)2 is similar to that of wild type. Without consideration of the 55mV difference in reduction potential between these two proteins, the conclusion would be drawn that themutation has no significant effect on the electron transfer properties of cytochrome c. One would thenbe hard pressed to explain the reason why the cross reaction rate of Tyr67Phe with Co(phen)3 is tentimes that observed for the wild type protein. When the cross reaction rates and the reduction potentialsare used with the relative Marcus equation to calculate the self exchange rate for each protein, one findsthat Tyr67Phe is eleven times more reactive than the wild type cytochrome towards both electron transferpartners.The observation that the electron transfer reactivity of Tyr67Phe relative to the wild type protein isindependent of the identity of the electron transfer partner is true for all of the water switch variantsexamined (Figure 36). This finding indicates that these substitutions do not influence the nature of theprecursor complex as do the position 82 replacements. Rather, the differences in reactivity that areobserved with the water switch variants are caused by adjustments in the reorganization energy of theprotein.The Ile75Met variant has the same reactivity in both reactions as the wild type cytochrome. Its lowercross reaction rate with Fe(edta)2 and higher cross reaction rate with Co(phen)3 can be accounted foralmost completely by the relatively low reduction potential of this variant. Thus the reorganizationenergy of Ile75Met is the same as that of the wild type protein. As this mutation likely does not removeany hydrogen bonding partners to WAT 166, it is likely that the hydrogen bond network is intact inIle75Met and that this protein undergoes oxidation state-linked conformational changes similar to thoseobserved for the wild type protein. In contrast, the mutants Thr78Gly, AsnS2Ala, and Tyr67Phe, eachof which removes one of the hydrogen bond partners to WAT166, are all more reactive than the wildtype protein. For AsnS2Ala and Tyr67Phe, it is known that a second buried water molecule replaces the10820.0.. Phe82GIy0o • Phe82SerD12.0. Tyr67PheI8.0Asn52AIa. 4.0 Thr7SGlylie75Met •‘ • PheS2iie—• .Phe82Leu-. Phe82TyrLL 0.0 I I I I0.0 2.0 4.0 6.0 8.0 10.0 12.0Relative k1°’T (Oxidation)Figure 36 Comparison of the relative self exchange rates obtained for the reactions of the variantcytochromes with Fe(edta)2 (vertical axis) and Co(phen)3 (horizontal axis). The line corresponds toequal values for the two relative self exchange rates.109lost hydrogen bonding group (Figure 35; Guillemette et a!., submitted; Berghuis et at., unpublishedresults). Although there is no structural evidence that a similar compensatory change in solvation occursin the Thr78Gly variant, it is reasonable to suggest that a similar replacement does takes place. Notably,the Tyr67Phe mutant, with the second water molecule closest to the heme, has the highest reactivity,while the mutants Asn52Ala and Thr78Gly are less reactive than Tyr67Phe. The new water moleculein the Asn52Ala variant is further removed from the heme than that of the Tyr67Phe variant, and theAsn52Ala variant has a reactivity more similar to the wild type protein. The reactivity of the Thr78Glyvariant is between those of Tyr67Phe and AsnS2Ala variants. A proposed second water molecule thatreplaces the lost Thr78 side chain in Thr78Gly would be closer to the heme than the additional watermolecule in the AsnS2Ala variant, but it would be further from the heme than the new water moleculein the Tyr67Phe variant.The lower reorganization energies of the Tyr67Phe, AsnS2Ala, and possibly the Thr78Gly variantsmay be caused by smaller movements in the protein surrounding the heme and greater movement of theinternal water molecules. In wild type cytochrome c, reorganization in the hydrogen bond network aboutWAT166 requires movement of hydrogen bonding side chains that are held in place to the proteinbackbone. In the three water switch variants, the second internal water molecule should have morefreedom to move than the hydrogen bonding side chain that it replaces. Reorganization may simplyinvolve facile movement of internal water molecules rather than movement of somewhat constrained sidechain residues. Internal water molecules closest to the heme would be expected to have the largest effect,which would diminish as their distance from the heme increases. This possibility would explain the orderof reactivity of the three internal site mutants.The electron transfer properties of the water switch variants suggest that one of the roles of thehydrogen bond network of WAT166 is to lower the reorganization energy of cytochrome c by promotingmovement of one or more internal solvent molecules so that movement of the surrounding polypeptideis minimized. Recently the structure of another water switch mutant, Asn52lle of yeast iso-i-110cytochrome c, has been reported (Hickey et a!., 1991). This mutant completely lacks internal solventmolecules, including WAT166. Despite the absence of internal water molecules, this mutant appears tobe fully functional and thermodynamically more stable than the wild type protein. Electron transferkinetics of AsnS2Ile with inorganic complexes have not yet been reported. The absence of internal watermolecules requires that the reorganization energy of 1le52 prior to electron transfer is due solely tomovement of the polypeptide chain in the vicinity of the heme. The results obtained in this work suggestthat the reactivity of 11e52 will be lower than those of Tyr67Phe, Asn52Ala, and Thr78Gly.4.3.4 Temperature Dependence of Electron Transfer KineticsTransition state theory has been applied to the analysis of the temperature dependences of the electrontransfer reactions of the cytochrome c variants studied here to calculate their activation parameters, whichare presented on pages 69 and 76. Results of this type must be interpreted cautiously for two reasons.First, the error inherent in collecting data over a short temperature interval and obtaining activationparameters by extrapolation to a point far removed from the data points is significant. A difference infree energy of activation of less than 1 kcal/mol can have a dramatic effect on the observed rates. Themost striking example of this is the similarity of the activation parameters for reduction by Fe(edta)2 ofthe Phe82Gly variant and of the wild type protein. The difference in activation entropies and enthalpiesfor the two proteins are 2 eu and 0.8 kcal/mol respectively, yet the cross reaction rate constant of thePhe82Gly variant is almost twice that of the wild type protein at 25 °C. A second consideration is thatthe activation parameters are derived from the cross reaction and are not corrected for differences indriving force and the nature of the reactants. Such factors tend to dominate the activation parameters andmay obscure the contributions of the mutations. For example, the lower activation enthalpy of thereaction of wild type cytochrome c with Fe(edta)2 compared to Co(phen)3 is due mainly to the higherdriving force and electrostatic attraction in the former reaction. By comparison, the higher activationentropy associated with the oxidation of wild type by Co(phen)3compared to Fe(edta)2 is a consequence111of the more symmetrical nature of the structure of the oxidant, which increases the number of ways thatthe specific geometry of the precursor complex can be reached. For these reasons, the differencesbetween the activation parameters of mutant and wild type cytochromes c with a particular electrontransfer partner tend to be small, even when there are clear differences in the cross reaction rates.Comparison of the activation parameters for the reduction of Phe82Ala and Phe82Ser variants byFe(edta)2 supports the proposal that the enhanced reduction rate of Phe82Ser is due in part toreorientation of the inorganic complex with respect to the protein surface. As discussed previously, thetwo variants should have a similar indentation in the protein surface. However, the activation parametersof Phe82Ala are within experimental error of those determined for the wild type protein. The loweractivation enthalpy and more negative activation entropy of Phe82Ser are what would be expected forformation of a precursor complex with more effective orbital overlap with fewer ways of producingelectron transfer active precursor complexes.4.4 Alkaline IsomerizationStopped flow pH-jump experiments were used by Davis et al. (1974) to characterize the mechanismof the alkaline isomerization of ferricytochrome c, as described in the Introduction. The results of theseexperiments were consistent with a two-step process consisting of an initial deprotonation characterizedby PKH, followed by a conformational change in which the axial Met8O is replaced by another ligandsupplied by the protein. The second step is characterized by the equilibrium constant R = kf/kb. Thealkaline isomerization pK measured by spectrophotometric titration is equal to pKH + pK. Spectroscopicmeasurements on the alkaline isomer and comparisons with heme complexes of known ligation suggestthat the alkaline isomer possesses His-Lys axial ligation, as previously discussed. It should be noted thatthe titrating group responsible for the observed pKH need not be the group that replaces methionine asone of the axial ligands to the heme, although this possibility represents the simplest mechanism. Thus,112deprotonation of a heme propionate (Tonge et at., 1989), WAT166 (Takano & Dickerson, 198 ib) andHis 18 (Gadsby et at., 1987) have each been proposed as the group responsible for the observed pKH.The equilibrium and kinetics of the alkaline isomerization of several position 82 variants of yeast iso1-cytochrome c have been studied previously (Pearce et a!., 1989). Although position 82 variants showedlarge differences in their alkaline pKs and rate parameters, all behaved in a manner consistent with themechanism described above. Replacement of Phe82 by a nonaromatic side chain destabilizes the nativeconformation by lowering pKH by 1 to 1.4 pK units. The equilibrium constant K0 is either unaffected(Phe82Leu and Phe82Ile) or slightly lowered (Phe82Ser and Phe82Gly). A lower pK0 partially offsets theeffect of the lower pKH on the pK for the overall process.The alkaline isomerization is also sensitive to mutations at residues that hydrogen bond to WAT166(Table 5, page 80). The Thr78Gly variant destabilizes the native ligation state and has the lowest pKmeasured for any water switch or position 82 mutant. This behaviour results entirely from a loweringof the pKH of the titrating group by 2 pH units. The equilibrium constant K0 is also lower for Thr78Glythan in the case of the wild type protein. This lower value for pK3 partially compensates the effect ofthe lower pKH, as previously reported for the Phe82Gly and Phe82Ser variants.Although the Asn52Ala variant has an alkaline PKa within experimental error of that determined forwild type cytochrome c (Guillemette, unpublished), the kinetic data for the alkaline isomerization of thisvariant are inconsistent with the mechanism described above. Similar kinetic behaviour has been recordedfor the alkaline isomerization of Tyr67Phe (Guillemette et at., in preparation). The spectrophotometricpH titration curve of Asn52Ala indicates the release of one proton upon isomerization to the alkalineform. To be consistent with both the kinetic and equilibrium experiments, the mechanism of the alkalineisomerization of this variant would require at least four steps: two deprotonations, a protonation, and aconformational change. A possible mechanism is shown in Figure 37. The following equation for theobserved rate constant is derived from this mechanism:k = kbKfl3 + k KH, Kfi2 (29)KR3 + [H] KRJ KR2 + KHj[H9 + [H]2113XH NH2 XHS (Met8O) S (Met8O)I pKH1Fe FeN(Hisl8) N(Hisl8)NH2 XH NH2S (Met8O) S (Met8O)I pKH2 IFe FeN (Hisl8) N (Hisl8)NH2S (MetBO) NH2____I____k____I____Fe FeI kb IN (Hisl8) N (Hisl8)I pKH3 IFe FeN (Hisl8) N (Hisl8)Figure 37 A possible minimal mechanism for the alkaline isomerization of the AsnS2Ala variant offerricytochrome c. For simplicity, this mechanism is presented with a lysine as one of the titratinggroups as well as being the replacing ligand. Among groups proposed as the titrating residue responsiblefor pKH1 are a heme propionate (Tonge et at., 1989) and WAT166 (Takano & Dickerson, 1981b). Theporphyrin plane is symbolized by the horizontal line through the symbol for iron.114The initial deprotonation may be similar to that of the wild type protein. The second deprotonation mayinvolve the disruption of a stabilizing interaction, such as a hydrogen bond. After the seconddeprotonation, the protein becomes sufficiently flexible to undergo the conformational change to thealkaline form. A subsequent protonation event may then stabilize the alkaline isomer.The presence of a protonation following the conformational change to the alkaline isomer is consistentwith the observed rate constant for the pH jump from high to low pH, which in the AsnS2Ala is an orderof magnitude smaller than observed for wild type. This behaviour suggests a pH dependent reverse rate.The rate of conversion from alkaline to native ligation state upon lowering the pH would depend on theconcentration of alkaline isomer present without the proposed stabilizing hydrogen bond. At low pH,this interaction will be strengthened, resulting in only a small concentration of alkaline isomer withoutthis stabilizing interaction that is capable of forming the native species. The kinetic data can be fittedwell to an equation that describes such a mechanism. However, attempts to determine these parametersby use of a non-linear least squares analysis were not successful, as the calculated parameters were highlydependent on the initial estimates used in the calculation.The strong influence on the alkaline isomerization of ferricytochrome c by mutations at positions 52,67 and 78 suggests that the trigger for this process resides within the protein, rather than on a surfacegroup such as the N- amino group of a lysine residue. It is unlikely that mutations at these internalpositions influence the ionization of the replacing lysine residue(s) on the protein surface. This reasoningsupports the proposal that a heme propionate, Tyr67, or WAT 166 is the titrating group responsible fortriggering the alkaline isomerization. Which one of these three possibilities is ultimately responsiblecannot be determined from the results of this work, as the mutation sites occur at or close to all threepotential candidates.The mutation of 11e75 to Met has no effect on the alkaline isomerization. This finding suggests nosignificant structural changes within the hydrogen bonding network about WAT166 result from this115substitution. This observation argues against the possibility that the Ile75Met side chain is directed intothe space occupied by WAT166 as it is unlikely that such a large change in the heme environment wouldhave no effect on the alkaline isomerization.In stopped-flow experiments performed above pH 10.5, biphasic kinetics were observed with theAsn52Ala variant and the wild type protein. The observed rate constant for this second kinetic phase was37 to 45 s_i for both proteins, and its contribution to the total absorbance change increased with pH.Biphasic kinetics of the alkaline isomerization of horse heart ferricytochrome c above pH 10 wereobserved using stopped flow pH jump experiments by Kihara and coworkers (1976), who attributed thefaster kinetic phase to the formation of a transient intermediate species on an alternate pathway to thealkaline isomer. This pathway represents a ‘short-cut’ to the alkaline form, but only becomes availableafter a second deprotonation (Figure 38). An alternative explanation for the biphasic kinetics is that thefaster phase represents the rate of conversion of the native protein to a second alkaline isomer with adifferent lysine residue as the axial ligand than the alkaline isomer observed below pH 10. Thismechanism is consistent with a 2D NMR investigation that provides evidence for two distinct alkalinespecies of horse heart ferricytochrome c (Hong & Dixon, 1989). The second lysine residue may havea higher pKH, but once it is deprotonated it may be able to displace Met8O more rapidly than the lysineresidue that forms an alkaline isomer below pH 10.5.4.5 Ligand SubstitutionAzide binds to cytochrome c by displacing Met8O sulphur as one of the axial ligands, a process whichcan be monitored by the loss of absorbance at 695 nm with increasing azide concentration (Sutin &Yandell, 1972; Saigo, 1986). The binding isotherms determined here are consistent with one ligandmolecule binding per molecule of cytochrome c, except for the Tyr67Phe mutant, where binding wasincomplete even at ligand concentrations greater than 2 M.116Native-H2pKH1 = 11.4k1 =31 s1Native-H— Transient-H1ç=58sk2 =6.7s1 k..2=Q.05s4Alkallne-H pKH2 = 10.4pKHS = 11.41(3 = 0.25 s1Alkailne Transientk3=78s-1Figure 38 A mechanism proposed by Kthara and coworkers to account for the alkaline isomerization ofhorse heart ferricytochrome c between pH 9 and 12 (Kihara et al., 1976). ‘Native’ refers toferricytochrome c with histidine-methionine axial ligation to the iron; ‘Alkaline’ refers to the alkalineisomer of ferricytochrome c in which methionine has been replaced as one of the axial ligands.‘Transient’ refers to a proposed intermediate of unknown ligation. The protonation state of each speciesis indicated by the number of H’s which follow the name of each component. Note that the portion ofthe mechanism defined by pKH1,k2, and k2 is identical to the mechansim proposed by Davis et al. (1974).117Although the stereochemistry of azidoferricytochrome c is not precisely known, structural studies ofcoordinated azides and knowledge of the electronic structure of the ligand predict several features thatwill be exhibited by this complex (Don & Ziolo, 1973). The lone pair of electrons that a.zide donatesto iron(III) are from an sp2 hybridized orbital, giving an Fe-N1-N2 angle of 1250 and a coordinate bondlength of 1.93 A in the complex of azido(pyridine)tetraphenylporphyrinatoiron(III) (Adams et al., 1976).Azide methemoglobin possesses similar geometry, but the low resolution of the crystal structure of thiscomplex did not allow precise calculation of the bond lengths and angles (Deatherage et a!., 1979). Itis highly likely that azide will bind to fernicytochrome c in a similar fashion. The short azide-iron bondlength (compared to the 2.43 A iron-sulphur bond of the native protein (Louie & Brayer, 1990)) and thepreference of azide for a bent coordinate bond are factors that favour minimal structural perturbation uponazide binding to cytochrome c.The orientation of coordinated azide with respect to the plane of the heme in ferricytochrome c isunknown. Steric considerations suggest an orientation that places bound azide along the line defined bypyrrole rings II and IV. The side chain of Met8O lies over pyrrole rings I and III in nativeferricytochrome c and, barring movement of the polypeptide backbone, would continue to occupy asimilar position after disruption of the iron-sulphur bond. Additionally, the azide nitrogen atom that iscoordinated to the iron bears a second lone pair in an sp hybridized orbital that could potentiallyhydrogen bond to the hydroxyl group of Tyr67, as does the Met8O sulphur atom in ferrocytochrome c.This would place the azide mainly over pyrrole ring II. Formation of a Tyr67-OH / azide hydrogen bondwould require a shortening of the distance between the potential hydrogen bonding partners.Ligand substitution at the heme of ferricytochrome c is sensitive to the structure of the hemeenvironment. If Phe82 is replaced by nonaromatic residues, K., increases three to six fold. The resultsof the stopped-flow kinetics experiments show that the increase in azide binding affinity is caused by a2.5 to 5-fold increase in k1, the forward rate constant, and by an increase in the tendency of the118intermediate state to proceed to the azide bound form, characterized by an increase in the ratio ofk2/k1.These kinetic parameters are similar for Leu, Ser, and Gly mutants at position 82. The increase in k1for Phe82Ser and Phe82Gly is consistent with greater exposure of the heme to solvent, the former becauseof a solvent channel extending from the surface to Met8O, the latter because of flexibility of thepolypeptide chain in residues 81 through 84 (Louie & Brayer, 1988, 1989). The causes of the elevatedk1 for the Phe82Leu and Phe82Ile variants are not obvious, but the increased affinity of these species forazide is. consistent with their lower alkaline pKs, which also indicates a weakened heme crevice.Instability of the heme crevice in the Phe82Leu and Phe82Ile variants may be caused by an absence ofhydrophobic contacts that the wild type Phe82 side chain normally makes with nearby residues or bydisruption in packing against neighbouring side chains caused by replacing the planar aromatic ring ofphenylalanine with branched aliphatic side chains.Differences in the rate constant k2 contribute to the difference in the observed azide binding constantsof the Ser and Gly variants. The variation in k2 observed here implies structural differences in the azidocytochrome c complexes of these species. Both variants have similar forward rate constants that are atleast three-fold greater than that of the wild type protein. The solvent channel of Phe82Ser allows readyaccess of the ligand to the heme, but it also offers no hindrance to the release of azide. Thus this varianthas the largest rate constant for dissociation of azide from ferricytochrome c. In contrast, the Phe82Glymutant has a greater forward rate constant and a smaller reverse rate constant that of the Ser variant,giving Phe82Gly a greater overall affinity for azide. Because azide binding to ferricytochrome cneutralizes the positive heme charge, it is possible that the protein undergoes conformational changesupon azide binding similar to those that occur upon reduction. Such a change would explain the lowerk2 for the Phe82Gly variant relative to Phe82Ser variant as in the former protein a conformational changein the reduced protein reduces heme solvent accessibility by movement of the polypeptide backbonetoward the heme. This movement would sterically hinder the release of bound azide.119The mechanism by which azide association occurs is not known with certainty. This process may bedominated by the inherent strength of the iron(III)-sulphur bond, by the flexibility of the polypeptidearound the heme, or by a combination of the two. The ratio of the rate constants leading away from theactive intermediate is calculated indirectly from the value of the binding constant (KJ and the forward(k1) and reverse (k2) rate constants. The magnitude of this ratio will depend on the nature of this activeintermediate and on the position of the incoming/outbound azide molecule. The accuracy with which thisratio can be determined will thus be dependent on the accuracy of the parameters from which it isderived. The lower ratio of k2 /1c determined for the wild type protein relative to the position-82variants reflects the greater tendency of the former to re-form the native ligation state and suggests thateven when the heme crevice opens that the azide still has only limited access to the heme. The higherratios of the Gly, Ser, Leu, and Ile variants indicate an increased tendency of the active intermediate toform the azido-cytochrome c complex.In contrast to the position 82 mutants, the water switch mutants Tyr67Phe, Asn52Ala and Ile75Metall have lower affinities for azide than does wild type yeast cytochrome c. Tyr67Phe is highly resistantto ligand substitution, with a binding constant for azide of 1 M1 or less. Consequently, it was notpossible to extrapolate a rate of heme crevice opening from the rate data as there was no evidence of ratesaturation up to azide concentrations of 0.5 M. However, the rate data indicate that low azide affinityresults from a combination of a high reverse rate constant k2 and a low ratio ofk21k. This suggests thatthe rate of heme crevice opening (k1) may be little changed from the native protein. Unfavourable stericinteractions in the azide bound species may increase k.2. Additionally, the Met8O sulphur may be in amore favourable position to compete with azide for the axial coordination site, causing a decrease in theratio ofk2/k1.The lower affinity of Asn52Ala for azide is entirely caused by an increase in k2, indicatingdestabilization of the azide bound complex. There are no significant differences in the other rateparameters between Asn52Ala and the wild type protein. In all experiments where the properties of120Tyr67Phe and AsnS2Ala mutants are compared to those of the wild type protein, the mutation closer tothe heme always has the larger affect. Thus both mutants have lower reduction potentials, but that ofTyr67Phe is lower; both undergo electron transfer with inorganic complexes more readily, but Tyr67Pheis more reactive; both have altered alkaline isomerization kinetics, but Tyr67Phe has the higher alkalineplc. The azide binding affinities of these two mutants also follow this trend.The rate data for the Ile75Met variant best fit a simple two state equilibrium model characterized bya forward and reverse rate constants kf and kb, respectively. Although this variant exhibits a strongdependence of the observed rate on azide concentration, it shows no indication of rate saturation. Theequilibrium constant determined from the ratio kflkb is in excellent agreement with that found by titration(Table 6). The means by which this substitution changes the mechanism of azide binding to cytochromec is unknown. A similar mechanism has been proposed for the binding of imidazole to Candida kruseiferricytochrome c (Creutz & Sutin, 1974), which possesses isoleucine at position 75. Both S. cerevisiaeiso-i and C. krusei cytochromes c have high sequence homology among the residues defining the hemeenvironment and have identical sequences between residues 7 i and 87. Residue 1le75 shields thehydrogen bond network about WAT166 from exposure to solvent. The lower reduction potential of theIle75Met variant may suggest that the heme is more solvent accessible in this variant, though other originsfor this behaviour are also possible. A new route for azide to the active site through the mutation sitemight explain the kinetic properties of ligand substitution in this mutant.Comparison of various species of cytochrome c led Saigo (1986) to note a correlation between thealkaline isomerization PKa of a cytochrome c and its affinity for exogenous ligands such as imidazole andazide. A similar correlation has been observed in this work (Figure 39). The common feature of bothprocesses is the disruption of the iron(1II)-sulphur bond and the native heme crevice structure. Theobservation that the rates of substitution by exogenous ligands are faster than the rates of conformationalchange in the alkaline isomerization reflects the difference in the degree of freedom of the free azideligand compared to the lysyi residue, which is constrained by its attachment to the rest of the protein.121The correlation does point out the inherent weakness of the ironlll)-suIphur coordination. This weaknessis demonstrated by the observation that oxidized heme octapeptide requires 2 M N-acetylmethionine toform the iron(1lI)-Met complex arhury. 1965). Thus one of the functions of the protein environmentaround the heme of cytochrorne C IS to stabilize the intrinsically unstable axial ligation of the heme ironby Met8O in the ferricytochrome.12210.5 I I I I IY67F9.5..Horse cN 175M8.5 .WTN52A. • F82YF82 • FS2G75 NNF82LFad. .‘ I I I I I-1.00 0.00 1.00 2.00log KFigure 39 Correlation of azide binding with the alkaline plc for cytochromes c.1234.6 SummaryIn this work, the effect of mutation at residues within the heme environment of yeast iso-icytochrome c on reduction potential, electron transfer reactivity, and stability of the native coordinationstate has been examined. From the results obtained in these experiments, several conclusions can bedrawn regarding the roles of the residues occurring at these positions in the wild type protein.The invariant residue Phe82 serves two major roles. First, it is required to maintain a high reductionpotential. Except for the Phe82Leu variant, the reduction potentials of all other mutants at this positionwere 10 to 45 mV lower than wild type. Second, Phe82 stabilizes the iron(III)-Met8O sulphur bond bylimiting the access of solvent to the heme. Phe82 is not essential for electron transfer activity. All ofthe position 82 mutants studied were capable of transferring electrons with inorganic complexes at ratessimilar to or greater than those exhibited by the wild type protein.A major role of the water switch residues in the environment of WAT166 is to maintain a highreduction potential. All of the substitutions in this region lowered the reduction potential by at least 33mV. The wild type residues are not required for efficient electron transfer, as several variants are morereactive than wild type. 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(1983) Oligonucleotide-Directed Mutagenesis, Methods Enzymol. 100, 468.135APPENDIX A: Reduction Potential MeasurementsProtein pH Temperature E°(Kelvin) (mV vs SHE) (mV)Horse heart cytochrome c 7.00 283.2 275.9 65276.9 65288.0 275.9 63274.9 63292.8 272.9 61272.9 61297.7 270.4 61269.9 61303.1 268.9 61268.9 61308.0 266.4 62267.4 62314.0 263.9 65262.9 63Yeast iso-1-cytochrome c 5.50 298.0 290.9 63292.4 606.00 283.0 295.9 59287.8 294.4 56293.0 292.4 58297.8 290.9 55297.8 287.9 58303.1 287.9 54307.9 285.9 55313.4 284.4 547.00 298.0 277.0 56277.5 577.00 298.0 281.5 61136Protein pH Temperature E°(Kelvin) (mV vs SHE) (my)Yeast iso-i cytochrome c 7.41 298.0 272.4 687.43 274.9 577.78 271.9 557.86 270.4 547.89 269.9 55Asn42Lys/Ala43His 5.46 298.0 279.4 566.01 276.4 566.47 272.4 60700 2669 677.49 263.4 618.00 262.4 688.46 261.4 70AsnS2Ala 5.50 298.0 262.4 60261.9 546.00 282.9 262.4 54262.4 54287.9 260.9 55292.8 258.4 54297.9 257.4 54257.4 54257.4 56303.2 254.9 54307.9 253.4 54313.2 252.4 566.48 298.0 253.4 546.97 249.9 557.36 246.4 567.90 243.9 54137Protein p11 Temperature E° iE(Kelvin) (mV vs SHE) (my)Ile75Met 5.57 298.0 249.4 606.00 282.3 255.4 64254.4 62287.6 2499 57250.4 58292.8 247.4 56247.9 55298.0 245.4 54244.9 54244.9 83303.0 243.4 58244.9 57308.1 241.4 56240.9 57313.7 237.4 60237.4 606.52 298.0 242.9 637.02 235.9 717.04 235.9 637.47 231.4 667.84 229.4 708.16 228.4 80Thr78Gly 6.00 281.5 254.9 59281.2 252.4 60288.0 252.4 62253.9 61293.0 251.4 62298.0 249.4 62302.9 248.9 63138Protein pH Temperature E°(Kelvin) (mY vs SHE) (mV)Thr78GIy 307.9 247.4 62314.4 244.4 76Phe82Ala 6.00 283.1 265.9 63266.9 61288.2 264.4 60263.9 61293.1 261.9 61261.9 61298.0 260.4 60260.4 60307.9 257.4 72257.9 73Phe82Gly 6.00 283.2 252.4 54253.4 54288.0 251.4 54251.4 54293.2 249.4 54249.4 54298.1 247.9 54248.4 54303.0 245.4 54303.0 246.9 54307.8 243.9 55244.9 54313.4 242.9 59241.9 57139Protein pH Temperature E°(Kelvin) (mV vs SHE) (mV)Phe82Ile 6.00 283.0 282.9 59282.4 64282.4 60288.0 278.9 55287.9 57293.0 276.4 54276.4 56298.0 273.4 54273.4 56303.0 271.4 54271.9 55307.9 268.9 55269.4 56313.3 265.4 54266.4 56Phe82Leu 6.00 283.0 292.9 61287.9 290.9 61290.6 290.4 60293.0 289.4 60295.5 287.4 60298.1 286.9 59303.1 284.4 60308.1 280.4 62313.2 277.4 69Phe82Ser 6.00 283.1 263.4 58287.9 260.9 57292.9 257.9 53297.7 255.9 57140Protein pH Temperature E°(Kelvin) (mV vs SHE) (my)Phe82Ser 298.0 254.4 56303.5 252.9 546.00 307.8 252.4 76313.2 246.4 566.48 298.0 248.4 546.99 239.9 55241.4 567.42 298.0 236.4 60237.4 607.88 234.4 54231.4 548.17 231.9 55Phe82Tyr 6.00 283.0 286.4 54287.9 284.4 56292.9 292.9 57297.8 280.4 54303.0 279.4 54307.9 277.4 54313.2 275.4 62141APPENDIX B Fe(edta)2Reduction Kinetic DataProtein Temperature [Fe(edta)2-](Kelvin) (mM) (s1)Horse Heart cytochrome c 298.0 0.05 1.36 ± 0.010.10 2.93 ± 0.010.15 4.43 ± 0.010.20 6.05 ± 0.020.25 7.53 ± 0.040.50 13.6 ± 0.1Yeast iso-1-cytochrome c 283.0 0.20 10.12 ± 0.04290.2 11.11 ± 0.04298.0 0.05 2.72 ± 0.010.10 7.08 ± 0.030.15 10.25 ± 0.07298.0 0.20 13.49 ± 0.050.20 13.72 ± 0.040.25 18.68 ± 0.080.50 32.6 ± 0.4303.2 0.20 15.94 ± 0.06308.6 17.83 ± 0.06313.8 19.96 ± 0.05Asn52Ala 282.8 0.05 2.18 ± 0.022.32 ± 0.02287.8 2.46 ± 0.022.72 ± 0.03293.2 3.10 ± 0.023.23 ± 0.02298.0 0.05 3.69 ± 0.033.53 ± 0.03142Protein Temperature [Fe(edta)2-] kthS(Kelvin) (mM) (srn’)AsnS2Ala 298 0.05 4.06 ± 0.010.10 8.72 ± 0.050.15 14.04 ± 0.070.20 18.6 ± 0.10.25 22.7 ± 0.20.50 42.9 ± 0.4303.2 0.05 4.77 ± 0.034.81 ± 0.04308.8 5.63 ± 0.045.76 ± 0.04Ile75Met 283.5 0.10 1.65 ± 0.01287.7 2.07 ± 0.01292.7 2.57 ± 0.02298.0 0.05 2.07 ± 0.010.10 3.77 ± 0.023.50 ± 0.020.15 6.23 ± 0.020.20 8.94 ± 0.040.25 11.83 ± 0.070.50 19.7 ± 0.1302.8 0.10 4.17 ± 0.04308.0 0.10 4.91 ± 0.03Thr78Gly 283.8 0.20 6.58 ± 0.05288.4 8.2 ± 0.1294.0 10.1 ± 0.2298.0 0.05 2.36 ± 0.020.10 5.2 ± 0.1143Protein Temperature [Fe(edta)2-] k8(Kelvin) (mM) (s1)Thr78Gly 298.0 0.15 9.1 ± 0.10.20 13.1 ± 0.20.20 12.2 ± 0.20.25 15.2 ± 0.10.50 29.3 ± 0.3303.4 0.20 13.1 ± 0.2308.4 14.6 ± 0.2316.4 15.6 ± 0.2Phe82Ala 283.0 0.20 13.99 ± 0.05288.0 15.30 ± 0.04293.0 16.97 ± 0.06298.0 0.05 4.84 ± 0.020.10 9.84 ± 0.050.15 14.83 ± 0.070.20 19.75 ± 0.0521.06 ± 0.080.25 25.1 ± 0.10.50 47.5 ± 0.1303.4 0.20 20.8 ± 0.1309.4 21.4 ± 0.1Phe82Gly 283.4 0.20 19.76 ± 0.04288.2 21.67 ± 0.08293.0 23.76 ± 0.05298.0 0.05 6.46 ± 0.040.10 13.71 ± 0.050.15 21.41 ± 0.060.20. 28.24 ± 0.050.20 26.72 ± 0.06144Protein Temperature [Fe(edta)2-] kth(Kelvin) (mM) (s’)Phe82Gly 298.0 0.25 32.86 ± 0.070.50 65.9 ± 0.2303.8 0.20 29.19 ± 0.07308.6 31.6 ± 0.1Phe82Ile 283.6 0.20 12.39 ± 0.04288.7 14.09 ± 0.04293.0 16.60 ± 0.04298.0 0.05 4.28 ± 0.020.10 8.87 ± 0.040.15 14.62 ± 0.040.20 18.58 ± 0.0618.99 ± 0.050.25 24.68 ± 0.040.50 45.33 ± 0.07304.0 0.20 19.1 ± 0.3308.2 19.3 ± 0.2Phe82Leu 283.0 0.20 10.31 ± 0.04289.2 12.48 ± 0.03294.6 15.55 ± 0.07298.0 0.05 3.71 ± 0.070.10 7.5 ± 0.10.15 14.5 ± 0.10.20 18.9 ± 0.10.25 23.8 ± 0.30.50 44.4 ± 0.4299.8 0.20 18.26 ± 0.09305.0 19.5 ± 0.220.2 ± 0.2145Protein Temperature [Fe(edta)2-] k(Kelvin) (mM) (s1)Phe82Leu 311.2 0.20 22.2 ± 0.2Phe82Ser 283.2 0.20 24.1 ± 0.2289.8 25.9 ± 0.3293.2 27.4 ± 0.2298.0 0.05 7.51 ± 0.090.10 14.8 ± 0.10.20 30.0 ± 0.328.9 ± 0.30.25 37.5 ± 0.40.50 73.9 ± 0.6304.4 0.20 31.5 ± 0.3308.8 32.9 ± 0.2Phe82Tyr 283.6 0.20 7.39 ± 0.04289.6 9.53 ± 0.04298.0 0.05 2.84 ± 0.010.10 6.07 ± 0.030.15 8.96 ± 0.050.20 12.21 ± 0.060.25 15.31 ± 0.080.50 30.0 ± 0.2298.6 0.20 11.79 ± 0.0512.53 ± 0.04303.8 13.67 ± 0.0514.05 ± 0.06309.6 15.64 ± 0.0615.83 ± 0.06146APPENDIX C Co(phen)31Oxidation Kinetic DataProtein Temperature [Co(phen)31] k(Kelvin) (mM) (s’)Horse Heart cytochrome c 298.0 0.20 0.299 ± 0.00030.301 ± 0.00030.40 0.599 ± 0.00030.599 ± 0.00030.80 1.197 ± 0.0011.204 ± 0.0021.00 1.487 ± 0.0021.20 1.771 ± 0.0011.771 ± 0.0021.40 2.064 ± 0.0022.089 ± 0.0021.60 2.350 ± 0.0032.390 ± 0.003147Protein Temperature [Co(phen)31](Kelvin) (mM)k(s1)Yeast iso-1-cytochrome c 283.5 1.00 0.597 ± 0.00030.593 ± 00003288.3 0.775 ± 0.00040.788 ± 0.0005293.0 1.00 1.073 ± 0.0011.088 ± 0.001298.0 0.20 0.310 ± 0.00030.317 ± 0.00030.40 0.695 ± 0.0010.705 ± 0.0010.80 1.350 ± 00021.377 ± 0.0021.00 1.702 ± 0.0021.741 ± 0.0021.523 ± 0.0011.529 ± 0.0011.20 2.027 ± 0.0032.061 ± 0.0032.024 ± 0.0041.40 2.404 ± 0.0032.417 ± 0.0031.60 2.729 ± 0.0042.762 ± 0.004303.3 1.00 2.206 ± 0.0022.250 ± 0.002308.3 2.910 ± 0.0093.160 ± 0.0053.178 ± 0.005313.3 4.21 ± 0.014.415 ± 0.005148Protein Temperature [Co(phen)31](Kelvin) (mM) (s1)Asn52AIa 283.0 1.00 2.483 ± 0.003288.0 3.308 ± 0.0043.314 ± 0.004293.0 4.370 ± 0.0034.365 ± 0.004298.0 0.20 1.233 ± 0.0011.221 ± 0.0010.40 2.505 ± 0.0022.516 ± 0.0020.80 5.093 ± 0.0064.987 ± 0.0051.00 6.031 ± 0.0055.985 ± 0.009298.0 1.00 5.663 ± 0.0055.707 ± 0.0061.20 7.49 ± 0.017.45 ± 0.011.40 8.52 ± 0.018.54 ± 0.011.60 9.87 ± 0.029.81 ± 0.02303.0 1.00 7.75 ± 0.017.73 ± 0.01308.2 9.96 ± 0.0310.94 ± 0.02313.0 14.46 ± 0.0413.74 ± 0.04149Protein Temperature [Co(phen)31] kth$(Kelvin) (mM) (s’)Tyr67Phe 283.5 1.00 6.139 ± 0.005288.4 8.384 ± 0.018.159 ± 0.01293.1 10.84 ± 0.0110.89 ± 0.01298.0 0.20 2.952 ± 0.005V 3.006 ± 0.0040.40 5.716 ± 0.016.156 ± 0.020.80 11.92 ± 0.0512.38 ± 0.041.00 14.79 ± 0.0515.10 ± 0.0514.42 ± 0.0314.18 ± 0.03298.0 1.00 14.75 ± 0.051.20 18.47 ± 0.0618.55 ± 0.061.40 20.55 ± 0.0722.04 ± 0.091.60 25.23 ± 0.0925.1 ± 0.1303.2 1.00 20.3 ± 0.1308.2 27.7 ± 0.127.6 ± 0.1313.2 36.8 ± 0.136.8 ± 0.1150Protein Temperature [Co(phen)31] k(Kelvin) (mM) (r’)Ile75Met 283.0 1.00 1.374 ± 0.002288.0 1.876 ± 0.0021.871 ± 0.002293.0 2.646 ± 0.0032.605 ± 0.0042.725 ± 0.002298.0 0.20 0.767 ± 0.0010.772 ± 0.0010.40 1.558 ± 0.0031.576 ± 0.0040.80 3.237 ± 0.0053.257 ± 0.0051.00 3.997 ± 0.0073.985 ± 0.0093.882 ± 0.0043.887 ± 0.0061.20 4.72 ± 0.014.77 ± 0.011.40 5.60 ± 0.015.65 ± 0.011.60 6.21 ± 0.026.62 ± 0.02303.0 1.00 5.45 ± 0.015.43 ± 0.01308.0 7.37 ± 0.027.78 ± 0.02313.0 10.31 ± 0.0511.48 ± 0.0411.61 ± 0.04151Protein Temperature [Co(phen)31] kth,,(Kelvin) (mM) (s’)Thr78Gly 283.0 1.00 1.936 ± 0.005288.5 2.708 ± 0.0032.820 ± 0.005293.2 3.556 ± 0.0083.572 ± 0.009298.0 0.20 1.099 ± 0.0031.105 ± 0.0020.40 2.156 ± 0.0072.189 ± 0.0050.80 4.30± 0.014.56 ± 0.011.00 5.67 ± 0.025.55 ± 0.024.99 ± 0.024.96 ± 0.021.20 6.49 ± 0.026.75 ± 0.031.40 7.96 ± 0.037.87 ± 0.031.60 8.65 ± 0.049.20 ± 0.04303.0 1.00 6.40 ± 0.026.63 ± 0.026.53 ± 0.03152Protein Temperature [Co(phen)31](Kelvin) (mM) (srn’)Phe82GIy 283.5 1.00 2.729 ± 0.0042.815 ± 0.006288.5 3.529 ± 0.0063.526 ± 0.006293.1 4.64 ± 0.014.623 ± 0.008298.0 0.20 1.141 ± 0.0041.173 ± 0.0030.40 2.596 ± 0.0062.620 ± 0.0070.80 5.05 ± 0.015.20 ± 0.011.00 6.60 ± 0.026.35 ± 0.026.08 ± 0.016.16.± 0.011.20 7.97 ± 0.027.61 ± 0.031.40 8.60±0.049.19 ± 0.031.60 9.76 ± 0.0410.20 ± 0.04303.5 1.00 7.96 ± 0.027.80 ± 0.03308.2 9.79 ± 0.059.79 ± 0.069.79 ± 0.06313.3 13.54 ± 0.0512.96 ± 0.06153Protein Temperature [Co(phen)31] k8(Kelvin) (mM) (r’)Phe82Ile 283.5 1.00 2.137 ± 0.0032.184 ± 0.004288.0 2.941 ± 0.0072.956 ± 0.007293.0 4.032 ± 0.0074.068 ± 0.007298.0 0.20 1.050 ± 0.0021.079 ± 0.0020.40 2.335 ± 0.0062.361 ± 0.0070.80 4.68 ± 0.014.87 ± 0.011.00 5.72 ± 0.025.87 ± 0.015.70 ± 0.015.78 ± 0.011.20 7.12 ± 0.027.09 ± 0.021.40 8.38 ± 0.028.41 ± 0.021.60 9.59 ± 0.029.78 ± 0.02303.3 1.00 7.94 ± 0.017.95 ± 0.02308.4 11.17 ± 0.0211.53 ± 0.0311.37 ± 0.03313.1 15.21 ± 0.0514.75 ± 0.05154Protein Temperature [Co(phen)31] kth8(Kelvin) (mM) (s’)Phe82Leu 283.5 1.00 0.784 ± 0.0020.761 ± 0.002288.2 1.089 ± 0.0021.059 ± 0.002293.0 1.472 ± 0.0021.491 ± 0.002298.0 0.20 0.425 ± 0.0010.438 ± 0.0010.40 0.933 ± 0.0050.973 ± 0.0060.80 1.895 ± 0.0081.895 ± 0.0081.00 2.23 ± 0.012.30 ± 0.012.09 ± 0.0042.153 ± 0.0041.20 2.84 ± 0.022.77 ± 0.021.40 3.19 ± 0.032.93 ± 0.03303.0 1.00 3.130 ± 0.0073.014 ± 0.0063.108 ± 0.007308.0 4.11 ± 0.014.24 ± 0.014.16 ± 0.01313.3 5.87 ± 0.025.83 ± 0.026.15 ± 0.02155Protein Temperature [Co(phen)31] k(Kelvin) (mM) (r’)Phe82Ser 283.5 1.00 2.048 ± 0.0022.045 ± 0.003288.0 2.673 ± 0.0052.684 ± 0.005293.2 3.947 ± 0.0094.010 ± 0.0063.917 ± 0.007298.0 0.20 0.951 ± 0.0020.938 ± 0.0020.40 2.184 ± 0.0042.143 ± 0.0040.80 4.151 ± 0.0094.224 ± 0.0091.00 4.95 ± 0.015.01 ± 0.015.35 ± 0.015.36 ± 0.011.20 5.93 ± 0.026.20 ± 0.021.40 7.30 ± 0.027.54 ± 0.011.60 8.54 ± 0.028.61 ± 0.02303.0 1.00 7.281 ± 0.027.307 ± 0.009308.2 9.63 ± 0.029.68 ± 0.029.94 ± 0.02313.3 13.18 ± 0.0413.82 ± 0.03156Protein Temperature [Co(phen)31] k(Kelvin) (mM) (c’)Phe82Tyr 283.4 1.00 0.866 ± 0.0010.836 ± 0.001288.3 1.097 ± 0.0011.105 ± 0.001293.0 1.512 ± 0.0011.539 ± 0.001298.0 0.20 0.423 ± 0.0010.432 ± 0.0010.40 0.963 ± 0.0020.954 ± 0.0020.80 1.898 ± 0.0021.900 ± 0.0021.00 2.313 ± 0.0032.379 ± 0.0032.168 ± 0.0022.193 ± 0.0021.20 2.788 ± 0.0062.822 ± 0.0031.40 3.215 ± 0.0063.245 ± 0.0051.60 3.772 ± 0.0073.747 ± 0.005303.1 1.00 3.031 ± 0.0063.07 ± 0.01308.0 4.269 ± 0.0044.278 ± 0.0074.280 ± 0.006313.4 5.88 ± 0.02157APPENDIX D pH Jump Kinetic DataProtein p11 k (r’)Horse Heart cytochrome c 6.1 0.035 ± 0.0018.99 0.071 ± 0.0019.18 0.083 ± 0.0010.086 ± 0.0019.50 0.136 ± 0.0029.70 0.192 ± 00039.99 0.328 ± 0.00510.5 0.89 ± 0.01Yeast iso-1-cytochrome c 5.9 0.043 ± 0.0018.99 0.150 ± 0.0019.53 0.445 ± 0.00210.01 1.47 ± 0.0110.51 4.7 ± 0.03 (61 %)45 ± 1 (39 %)10.88 7.2 ± 0.2 (32 %)37 ± 1 (68 %)Thr78Gly 6.00 0.047 ± 0.0017.45 0.148 ± 0.0037.65 0.199 ± 0.0037.95 0.333 ± 0.0038.14 0.508 ± 0.0048.53 1.03 ± 0.018.72 1.345 ± 0.0068.98 1.90 ± 0.02158Protein pH k (s1)AsnS2Ala 6.00 0.007 ± 0.0019.55 0.204 ± 0.0029.72 0.387 ± 0.002.9.74 0.364 ± 0.0029.82 0.435 ± 0.0039.90 0.633 ± 0.00310.03 0.761 ± 0.00410.10 0.989 ± 0.00510.23 1.586 ± 0.00910.32 1.817 ± 0.00610.46 3.27 ± 0.0110.64 5.25 ± 0.0350 ± 210.83 7.35 ± 0.0445 ± 1fle75Met 6.08 0.041 ± 0.0018.48 0.074 ± 0.0010.086 ± 0.0018.98 0.177 ± 0.0010.167 ± 0.0019.43 0.392 ± 0.0020.396 ± 0.0029.88 1.080 ± 0.0071.114 ± 000810.27 2.18 ± 0.022.10 ± 0.02159APPENDIX E Azide Binding Kinetic DataProtein [Azide] (M) (s1)Horse Heart cytochrome c 0.05 7.31 ± 0.087.42 ± 0.080.10 9.23 ± 0.068.83 ± 0.060.20 10.23 ± 0.0911.15 ± 0.070.25 12.27 ± 0.0812.22 ± 0.060.30 12.82 ± 0.0813.03 ± 0.060.40 14.82 ± 0.0514.77 ± 0.060.50 16.76 ± 0.0717.37 ± 0.06Yeast iso-1-cytochrome c 0.05 3.20 ±. 0.020.10 4.31 ± 0.020.20 6.77 ± 0.030.25 7.85 ± 0.040.30 8.48 ± 0.050.40 11.00 ± 0.040.50 13.13 ± 0.07160Protein [Azide] (M) kthS (s’)Asn52Ala 0.05 4.94 ± 0.025.22 ± 0.020.10 6.63 ± 0.026.53 ± 0.030.20 8.94 ± 0.038.83 ± 0.030.25 9.95 ± 0.049.81 ± 0.040.30 10.98 ± 0.0411.20 ± 0.040.40 13.03 ± 0.0413.18 ± 0.040.50 15.09 ± 0.0415.04 ± 0.04Tyr67Phe 0.10 11.7 ± 0.211.9 ± 0.20.20 12.7 ± 0.10.25 13.1 ± 0.10.30 13.6 ± 0.10.40 14.7 ± 0.10.50 15.7 ± 0.1Ile75Met 0.05 5.12 ± 0.070.10 6.45 ± 0.040.20 10.33 ± 0.060.25 10.84 ± 0.070.30 13.1 ± 0.10.40 16.34 ± 0.070.50 19.7 ± 0.1161Protein [Azide] (M) k (s1)Phe82Gly 0.05 26.3 ± 0.327.1 ± 0.30.10 44.3 ± 0.444.1 ± 0.40.20 83±180.4 ± 0.60.30 106±1102 ± 10.40 131 ± 2139 ± 20.5 142±2140 ± 2Phe82Leu 0.05 16.1 ± 0.215.5 ± 0.20.10 27.2 ± 0.525.7 ± 0.20.20 40.2 ± 0.340.4 ± 0.30.30 53.9 ± 0.555.8 ± 0.50.40 68.6 ± 0.771.0 ± 0.80.50 82±188 ± 1Phe82Ile 0.05 9.6 ± 0.19.6 ± 0.1162Protein [Azide] (M) k (s1)Phe82Ile 0.10 14.5 ± 0.115.4 ± 0.114.8 ± 0.10.15 18.6 ± 0.118.9 ± 0.10.25 27.6 ± 0.226.6 ± 0.20.30 34.6 ± 0.332.3 ± 0.20.40 45.3 ± 0.544.0 ± 0.50.50 44.7 ± 0.452.0 ± 0.5Phe82Ser 0.05 22.6 ± 0.224.0 ± 0.10.10 37.7 ± 0.237.3 ± 0.30.20 60.5 ± 0.658.2 ± 0.60.30 77.9 ± 0.578.3 ± 0.60.40 96±1102 ± 10.50 117 ± 2115 ± 1Phe82Tyr 0.05 6.46 ± 0.076.70 ± 0.060.10 9.30 ± 0.069.44 ± 0.08163Protein [Azide] (M) kthS (s’)Phe82Tyr 0.20 13.90 ± 0.0712.99 ± 0.080.30 17.39 ± 0.0816.76 ± 0.090.40 20.4 ± 0.121.0 ± 0.20.50 23.7 ± 0.123.7 ± 0.2164

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