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Interaction of cytochrome b₅ and cytochrome c Eltis, Lindsay David 1989

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INTERACTION OF CYTOCHROME b  5  AND CYTOCHROME c by L I N D S A Y D A V I D ELTIS B . S c , The University of Toronto, 1984  A THESIS S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY in T H E F A C U L T Y O F G R A D U A T E STUDIES THE DEPARTMENT OF BIOCHEMISTRY  We accept this thesis as conforming to the required standard  T H E U N I V E R S I T Y O F BRITISH C O L U M B I A April 1989 © Lindsay David Eltis, 1989  In  presenting  degree freely  this  at the  thesis  in  partial  University  of  British Columbia, I agree  available for reference  copying  of  department publication  this or of  thesis for by  his  or  fulfilment  and study. I further  of  her  representatives.  this thesis for financial  iQ&t+eftl)  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  the  /  requirements that the  agree  scholarly purposes may  permission.  Department  of  be  It  gain shall not  is  for  an  Library shall make  that permission for granted  by the  understood  be allowed  advanced  that  it  extensive  head  of  copying  my or  without my written  ABSTRACT  The interaction and kinetics of electron transfer between cytochrome b  and cytochrome c, two  5  well  characterised  soluble  First, fluorescence c,  a fluorescent  electron  transfer  quenching experiments analogue  proteins,  have  been  investigated  were done with cytochrome b  of cytochrome c.  flavin  complex  yielded  second order  semiquinone  reduction  information  rate constant  about for  of  the  the  cytochromes  structure  ferrocytochrome  and b  within  the  electrostatics  of  evaluating  transfer  the  between  role  of  the  cytochrome  derivative of cytochrome b  s  b  cytochrome 5  and  c  techniques.  and porphyrin cytochrome  5  b  heme  s  through  Second, an analysis of the cytochrome die  propionates  performing  each  ^cytochrome  complex.  reduction of ferricytochrome  5  under a variety of solution conditions by stopped-flow spectroscopy. at  three  These quenching studies yielded association constants  and structural information for the cytochrome ^cytochrome c complex. rate of  by  Third,  c was  c the  determined  Particular effort  was directed  in  the  and  of  diese  interaction three  electron  studies  with  a  in which die heme propionates had been esterified (referred to as D M E -  cytochrome b ). 5  The  fluorescence  quenching  studies  on  the  interaction  of  cytochrome  b  5  and  porphyrin  cytochrome c and die kinetics of flavin semiquinone reduction of the proteins within the cytochrome ^•cytochrome propionates,  c  complex  influences  the  provided  evidence  docking geometry  that of  the  esterification two  proteins.  of  the These  cytochrome  b  findings  support  5  heme the  predictions of electrostatic calculations [Mauk, M . R . , Mauk, A . G . , Weber, P . C . & Matthew, J.B. (1986) Biochemistry,  25, 7085] in two respects.  First, esterification of the cytochrome b  s  heme  propionates  detectably  increases  the separation between die two heme groups in die cytochrome ^cytochrome c  complex.  Second, die cytochrome c heme group is not as sterically hindered in the DME-cytochiome  ^cytochrome c complex as in die cytochrome ^cytochrome c complex. The stopped-flow studies demonstrate that the bimolecular rate constant  for ferrocytochrome  b  5  reduction of ferricytochrome c is determined in part by the rate of association of the two proteins.  ii  This  rate  of  association  is  strongly  esterification of the cytochrome b between cytochromes b  s  5  influenced  by  electrostatic  forces.  The  principal effect  heme propionates on the second order rate of electron  transfer  and c is to influence complex formation between these two proteins.  stopped-flow studies further  of  The  suggest that the reduction potentials of native and DME-cytochromes b  5  are not significantly different when these proteins are bound to cytochrome c. The  nature  of  electron  transfer  protein-protein  transfer is discussed with respect to these findings.  iii  interactions  and  protein-protein  electron  T A B L E OF CONTENTS  page ABSTRACT  ii  T A B L E OF CONTENTS  iv  LIST O F T A B L E S  vi  LIST O F F I G U R E S  vii  ABBREVIATIONS  ix  ACKNOWLEDGEMENTS  x  INTRODUCTION  1  Overview  1  The Theory of Protein-Protein Electron Transfer Studies  2  Experimental Studies of Protein-Protein Electron Transfer Reactions  7  Protein-Protein Complexes  7  Electron Transfer Kinetics  16  Cytochromes Cytochrome b  24 26  5  Cytochrome c  30  Cytochrome ^ C y t o c h r o m e c Complex  34  Objectives  •  EXPERIMENTAL PROCEDURES  42  44  General Procedures  44  Interaction of Porphyrin Cytochrome c with Native and DME-Cytochrome b Preparation of protein  5  45 45  iv  Binding measurements  45  Energy Transfer measurements  46  Polarization measurements  47  Kinetics of Flavin Semiquinone Reduction of Cytochrome b and Cytochrome c  47  Ferrocytochrome b Reduction of Ferricytochrome c  49  5  5  RESULTS  55  Interaction of Porphyrin Cytochrome c with Native and DME-Cytochrome b  55  Kinetics of Flavin Semiquinone Reduction of Cytochrome b and Cytochrome c  65  Ferrocytochrome b Reduction of Ferricytochrome c  73  5  5  5  DISCUSSION  80  Interaction of Porphyrin Cytochrome c with Native and DME-Cytochrome b  80  Kinetics of Flavin Semiquinone Reduction of Cytochrome b and Cytochrome c  89  5  5  Reduction of cytochrome c by LfH-  89  Reduction of cytochrome c by F M N H "  92  Reduction of native and DME-cytochrome b by LfH-  93  5  Reduction of native and DME-cytochrome b by F M N H 5  Ferrocytochrome b Reduction of Ferricytochrome c 5  -  95 98  CONCLUSION  110  REFERENCES  112  APPENDIX A  122  APPENDIX B  124  v  LIST O F T A B L E S page I. Salt linkages of the model cytochrome ^cytochrome c complexes.  38  II. The p H dependence of the extinction coefficient of porphyrin cytochrome c at 505 nm.  55  III. Parameters used in fitting the ionic strength dependence of the association constant of the cytochrome b porphyrin cytochrome c complex to the Van Leeuwen equation. 5  62  IV. Second order rate constants for flavin semiquinone reduction of cytochrome c.  68  V . Second order rate constants for LfH- reduction of cytochrome b .  69  5  V I . Second order rate constants for F M N H - reduction of cytochrome b . 5  72  VII. Solvent exposure of cytochrome heme groups in the two predicted docking geometries of the cytochrome ^cytochrome c complex  73  VIII. Parameters used in fitting the ionic strength dependence of the second order rate constants of ferrocytochrome b reduction of cytochrome c to the Van Leeuwen equation. 5  76  I X . Calculated monopoles and dipoles of the cytochromes used in this study  79  X . The reported extinction coefficients of porphyrin cytochrome c at 505 nm.  81  X I . Properties of lumiflavin and F M N .  91  XII. Thermodynamic parameters of several second order protein-protein electron transfer reactions.  108  vi  LIST O F FIGURES page 1. ProtohemelX  25  2. Structure of cytochrome b  28  5  3. Electronic absorption spectrum of cytochrome b  29  4. Structure of cytochrome c  32  5. Electronic absorption spectrum of cytochrome c  33  6. Structure of Salemme (edge on) cytochrome ^cytochrome c complex  36  7. Structure of edge off cytochrome ^cytochrome c complex  37  8. Stopped-flow spectrophotometer mounted in a glove box  50  9. Simulated difference spectrum of cytochrome b5 and cytochrome c  52  10. Emission spectrum of porphyrin cytochrome c  56  11. The p H dependence of the fluorescence intensity of porphyrin cytochrome c  57  12. Fluorescence titration curve of porphyrin cytochrome c with cytochrome b  59  5  5  13. The ionic strength dependence of the K^, of cytochrome b and porphyrin cytochrome c  60  14. The p H dependence of  61  5  of cytochrome b and porphyrin cytochrome c 5  15. The ionic strength dependence of Q 16. The p H dependence of Q  63  m a x  64  m a x  17. Reduction of cytochrome c by lumiflavin  65  18. Reduction of cytochrome c by F M N H -  67  19. Reduction of cytochrome b by LfH-  69  20. Reduction of cytochrome b by F M N H -  71  21. Ferrocytochrome b reduction of ferricytochrome c  74  5  5  s  22. The ionic strength dependence of ferrocytochrome b reduction of ferricytochrome c  75  23. The p H dependence of ferrocytochrome b reduction of ferricytochrome c  11  24. Eyring plot of ferrocytochrome b reduction of ferricytochrome c  78  5  5  5  vii  25. Structure of flavins and their oxidation states  ABBREVIATIONS CD  circular dichroism  Co(dipic)  ammonium bis(dipiciolinato) cobaltate(III)  2  DME-cytochrome b  dimethyl esterified heme substituted cytochrome b  EDTA  ethylenediaminotetraacetic acid  EPR  electron paramagnetic resonance  EXAFS  extended x-ray absorption fine structure spectroscopy  FMN  flavin mononucleotide  FMNH-  flavin mononucleotide semiquinone  FTIR  fourier transform infrared spectroscopy  5  L  HNMR  5  proton nuclear magnetic resonance spectroscopy  Lf  lumiflavin (7,8,10 - trimethylisoalloxazine)  LfH-  lumiflavin semiquinone  MCD  Magnetic circular dichroism  Throughout  this  dissertation,  amino  acid  residues  are  referred  to  by  the  three  abbreviations recommended by the I U P A C - I U B Commission on Biochemical Nomenclature (1972).  letter A  number following a three letter abbreviation refers to the position of that residue in the amino acid sequence of the protein being discussed. For  example,  The amino terminal amino acid residue is numbered "1".  "horse heart cytochrome c His-18"  horse heart cytochrome c.  ix  refers  to the histidine residue at position 18 of  ACKNOWLEDGEMENTS  I will never be able to express my gratitude to all the people whose friendship and inspiration have contributed to bringing this project to fruition.  I am particularly thankful to my supervisor,  Grant Mauk, for his patience and guidance in developing my abilities as a scientist.  I am also very  grateful for the friendship, advice and assistance of my lab colleagues: Bhavini Sishta, Marcia Mauk, Ed Chang, Alf Gartner, Linda Pearce, Paul Barker, Steve Rafferty, and especially Tony Lim. Gordon Louie,  Albert Berghuis and Mike Murphy  provided valuable assistance with the  IRIS computer.  Finally, I should like to thank my family, David, Suzan, Christopher and Jonathan, for encouraging and supporting the various projects I have undertaken during my life.  x  INTRODUCTION  Overview  Electron  transfer  between  metallo- or  flavoproteins  is  central  to  many  metabolic  including respiration, photosynthesis, and the catalytic cycles of many enzymes. involve soluble or membrane bound proteins. to  modulate  the  rate of electron  transfer  processes,  These reactions can  In either case, the structural factors that are thought  between  the  proteins  are  the  same.  Electron  transfer  involving a soluble protein is facilitated by the formation of an intramolecular complex of the two reacting proteins directed  at  analyzing  Characterizing biological  prior to the electron transfer  the  electron  the  structural  interaction transfer  and  of electron  reactions,  but  event  (Scott et al,  functional  transfer also  properties  proteins  provides  is  not  1985). of  Much effort  these  only  precursor  essential  to  insight into protein-protein  has been complexes.  understanding interactions  in  general. The reaction between cytochrome b  and cytochrome c serves as an excellent model system for  study  transfer  5  the  of  dimensional  protein-protein  structures  of  these proteins  crystallography (Mathews et al, Williams et al., proposed  a model for a  complex (Ng et al,  1:1  have  mechanisms  been  1985).  for  determined  1979; Dickerson et al,  1985a; Moore et al.,  computer graphics.  et al,  electron  several  in both  1981a,b) and H l  reasons.  The  three  oxidation states by  x-ray  N M R (Veitch et al.,  1988;  Partly based on this information, Salemme (1976)  complex that might form between  these proteins  in solution using  This model stimulated several studies aimed at characterizing the nature of the 1977; Stoneheurner et al,  1988; Holloway  1979; Mauk et al,  1982; Eley & Moore, 1983; Rodgers  & Mansch, 1988), which have led in turn to further  computer  graphics  modelling (Mauk et al, 1986) and molecular dynamic calculations (Wendoloski et al., 1987). To characterize further to address some between  these  of the proteins  the complex that forms between cytochrome b  questions has  been  that have arisen from recent studied  in 1  the  current  5  and cytochrome c and  modelling studies,  work  by  three  the  new  interaction approaches.  Spectrofluorometry  has  been  porphyrin cytochrome c,  used  to  measure  die  binding  an analogue of cytochrome c.  information on die complex.  constant  between  Spectrofluoromety  cytochrome  b  5  and  also provided structural  Additional information about the cytochrome ^cytochrome c complex  was obtained through an analysis of the rates of reduction of the heme proteins within this complex by  flavin  semiquinones.  bimolecular  reduction  conditions.  of  Particular  cytochrome b  s  Finally,  stopped  ferricytochrome attention  has  c  been  flow by  spectrophotometry  ferrocytochrome  directed  at  the  b  role  5  was  used  to  under  a  range  of  the  heme  measure of  solution  propionates  in complex formation through studies widi a derivative of cytochrome b  5  the  of  in which the  heme propionates are esterified widi methyl groups (DME-cytochrome b ). 5  The Theory of Protein-Protein Electron Transfer Reactions  Although systems,  the  many  theory  for  electron  transfer  is  more  thoroughly  of the conceptual insights provided by this work are  electron transfer  (Devault,  1984; Marcus & Sutin, 1985).  developed  in nonbiological  applicable to  The transfer  protein-protein  of an electron from  one  protein to another can be represented by the following scheme (Scott et al., 1985). In diis  A" + B < - ^ - >  representation,  et  <-^->  A + B"  die two proteins, A and B , first come togedier to form a precursor complex, A " : B ,  with an association constant K j . rate k .  A':B — A : B "  The complex  Within this complex, an electron is transferred from A to B with a  dien dissociates  at  a  rate that  is assumed  to be  rapid.  The overall  measurable rate is given by equation 1. For die convenience of discussion, die reaction can be  k = K.k,,  envisaged to involve these three distinct processes, each of which is influenced by different factors. 2  [1]  A n expression for the rate of electron transfer can be derived by considering diat the rate is the  product  of  the  frequency  with  which  the  system  achieves  a  suitable  nuclear  configuration  (Franck-Condon factor) and the probability that an electron will move from one prosthetic group to the other (PAB) once this configuration has been achieved (Devault, 1984).  The potential energy of  the reactants and the surrounding medium is a function of all the nuclear coordinates of the system and can be represented represented  in a  by an n-dimensional surface.  similar manner.  The potential energy of the products can be  The Franck-Condon principle ensures that electron  only occur when the free energies of the reactants and products are equal. configuration,  the  rate  of  electron  transfer  is  determined  by  the  between the donor and acceptor orbitals as measured by the matrix element, H  A B  transfer  can  For a particular nuclear  extent  of  electronic coupling  . In equation 2, h  2ir P  AB= —  |H  | (4^Ak T)2  A B  [2]  1 / 2  B  h represents  Planck's  constant,  k  B  represents  Boltzmann's  constant  and  A  represents  the  reorganizational energy of the donor and acceptor sites (vide infra). In sites  biological  is often  referred  to  electron  very poor,  as  transfer hence  systems,  electronic  coupling between  die probability of electron transfer  being non-adiabatic.  is small,  1976) in which diis value depends exponentially on the distance separating In a subsequent  faster  electron  rather  than water.  transfer between  and  and  the  (Hopfield,  acceptor  reaction is Several  1974; Jortner,  the donor and acceptor  treatment, Larsson (1983) showed that the height of the tunnelling barrier  is significantly reduced by the presence of saturated or conjugated acceptor centres,  donor  In such cases a tunnelling mechanism is invoked.  expressions for the electronic coupling matrix element have been presented  orbitals.  die  over  greater  TT systems between the donor and  Such intervening material is predicted by Larsson to facilitate distances.  complex  formed  two electron transfer  between  the two proteins by minimizing  These proteins  theoretical  can optimize the  the distance between  significant separation is occupied by amino acid residues.  3  considerations  dictate  rate of electron  the prosdietic groups  and  that  a  transfer that any  Marcus  derived  an  expression  for  electron  transfer  rates  using  a  statistical  approximation of the nuclear motions as reviewed by Marcus and Sutin (1985). may be derived from a simplified one dimensional model (Devault, 1984). reactants and  products  as  harmonic oscillators, the  activation energy  mechanical  A similar equation  Treating the vibrations of  of the  reaction  ( E * ) can  be  expressed as a function of the reorganizational energy (A) and the driving force ( A E ) .  (A - A E )  2  [3]  E* =  4A Averaging over a Boltzmann distribution of energies, the rate of electron transfer is  -(A - A E ) 27T  2  [  kt = — h  |H  e  2  A B  ]  I (4T A k T ) " e  4Ak T  1/2  B  [4]  B  The Marcus relationship implies that proteins can regulate electron transfer reorganization energy and die reduction potential of the prosthetic group. arises from Marcus theory relates the rate of electron transfer  between  rates by modulating the Another equation (5) that  two species (k) to the rate  of self exchange of these two species ( k j j , k ) and the equilibrium constant of the reaction (Kgq). 2 2  k = (k k K /)'/2 ]1  22  [5]  eq  l o g / = (log K ^ / r ^ l o g t k , , k / Z ) ]  [5a]  2  22  In  this  relationship,  approaches This  unity, / ,  relationship  Z  is  the  collision  frequency.  the collection of constants  holds  providing  that  the  In  cases  where  the  equilibrium  given in equation 5a, is assigned  electron  transfer  reactions  are  constant  a value of one.  adiabatic  or  uniformly  nonadiabatic ( P ^ = P A A P B B ) 2  Alternative for  treatments of biological  temperature independent  Chromatium  rates,  as  electron  first  transfer  observed  incorporate  in the  photo-induced  vinosum at low temperatures (DeVault & Chance, 1966). 4  nuclear  tunnelling  cytochrome  Hopfield  to  account  oxidation in  (1974) developed a  semiclassical solution to this problem through an analogy with radiationless energy transfer 1948).  In the Hopfield  treatment, the vibrational energy  (Forster,  states of the reactants and products  are  quantized with a spacing equal to hw between these levels, but these states are populated according to a classical probability distribution.  A full quantum mechanical treatment of vibration coupled to  changes in electronic state as it applies to biological systems was presented by Jortner (1976). the  full  quantum  Arrhenius Devault  mechanical  thermally and  activated  Chance  that  treatment behaviour  prompted  and at  the high  semiclassical  equation  temperatures.  these theoretical  reproduce  Ironically,  developments  have  the  the  Both  classical  observations  since been  interpreted  by as  involving the oxidation of two cytochromes (Bixon & Jortner, 1986). With  an increasing number  development  of  molecular  of three dimensional protein structures  graphics  interaction of electron transfer  capabilities,  several  proteins have been reported.  detailed  being determined  hypodietical  models  and  the  for  the  Computer graphics has been used to  develop structural models of some of the relatively stable complexes that are known to form between electron transfer proteins including cytochrome c and cytochrome b  5  (Salemme,  1976), cytochrome c  peroxidase and cytochrome c (Poulos & Kraut, 1980), cytochrome c and flavodoxin (Simondsen et al., 1982), cytochrome b  5  and methemoglobin (Poulos & Mauk, 1983), and tetra-heme cytochrome c  3  with  both flavodoxin (Stewart et al., 1988) and ferredoxin (Cambrillau et al., 1988). More quantitative approaches  to the modelling of protein-protein interactions have made use of  electrostatics, Brownian dynamics and molecular mechanics calculations.  For example, calculations of  association constants based on modified Tanford-Kirkwood theory (Tanford & Kirkwood, et al.,  1974) reproduce the ionic strength dependence  cytochrome  c,  suggesting  that electrostatic  forces  of the association constants of flavodoxin and  constitute  the  energy of association of electron transfer proteins (Matthew et al., the complex formed between cytochromes b  5  all of the energy of association (Mauk et al.,  1957; Shire  principal contribution 1983).  and c, Tanford-Kirkwood  to  the  free  However, in the case of  dieory does not account  for  1986), implicating the importance of odier forces, such  as hydrophobicity, in stabilizing these complexes.  The considerable entropic effect of the release of  5  solvent molecules at the interface of the interacting proteins (Chothia & Janin, 1975) overcomes the loss  of  individual  molecular  degrees  of  freedom.  The  predominant  role  of  electrostatic  predicts a positive entropy and a negligible enthalpy of association (Ross & Subramanian,  forces  1981), as  observed in the case of cytochrome b and cytochrome c (Mauk et al., 1982). 5  Brownian  dynamics  calculations  have  been  used  to  simulate  cytochrome c and yeast cytochrome c peroxidase (Northrup et al., quadrupoles atom  of these proteins  in the  stochastically  structure  The  1988).  1986) and  usefulness  of  suggested  several  (Northrup et al., electrostatically  1988).  characteristics  of  these calculations  the  of  horse  is  cytochrome  c  to each  non-hydrogen  attested  to  by  their  stabilized docking geometries  be sampled during one encounter.  successful  Brownian Dynamics simulations peroxidase-cytochrome  c  that fulfill  the  requirements  for electron  The  complex  transfer,  In addition, although dipole-dipole interactions may be important  electrostatic  rates are close to the diffusion  and  Indeed, several docking geometries may  for steering the molecules towards each other from a distance, surface electrostatic effects close range.  were  Of particular note is the conclusion that the proteins can assume several  that no single complex appears to be energetically favoured.  at  heart  The monopoles, dipoles and  the Brownian motions of these models  prediction of the behaviour of chemically modified cytochrome c. have  association  were calculated by assigning a partial charge  (Northrup et al.,  simulated.  the  attraction  between  these proteins  ensures  that  their  dominate association  controlled limit, despite the fact that dieir active sites constitute a  low percentage of the surface area of the protein. water molecules, (2) consider the rearrangement  Notably, this study did not (1) explicitly include  of residue side chains during the docking process,  or (3) account for non-electrostatic forces that might contribute to complex formation. Molecular dynamics simulations of the cytochrome ^cytochrome time scale (Wendoloski et al.,  1987) support the notion of flexibility  once a particular complex is formed.  c complex on a picosecond in a given docking geometry  This work further suggests that rearrangement  of amino acid  side chains within a complex can lead to docking geometries that are more favourable for electron transfer  (vide  infra).  Each  of  these  three  approaches  6  (electrostatics,  Brownian  dynamics  and  molecular  dynamics)  interaction.  addresses  different,  yet  complementary,  Ultimately, it will be of interest  algorithm,  that also accounts  aspects  of  electron  to see these three approaches  for solvent effects,  transfer  protein  combined in a single  to simulate the entire encounter,  recognition and  realignment process. From  this discussion, it should be clear that the structures  of electron transfer  influence the reactions in which these proteins are involved in many ways. cases studied,  electrostatic  forces  modulate  the  proteins can  In most of the simple  rate and stability of precursor  complex  formation.  The rate of electron transfer widiin this complex depends on the diermodynamic driving force of the reaction,  the  reorganization  energies  of  the  prosthetic  groups  and  the  separation  and  relative  orientation of, and the nature of the intervening medium between the donor and acceptor sites.  Experimental Studies of Protein-Protein Electron Transfer Reactions  Electron various  transfer  mechanistic  determination  of  in biological features  the  transfer  between  proteins,  rates (vide supra),  discussed  association  elucidation of the structures  and  systems  has  above.  constants  been  studied  These  for  complex  by  studies  many  have  formation  of these complexes, (3) measurement  techniques  been  between  to  assess  the  directed  towards  (1)  soluble  proteins,  (2)  of the rate constants of electron  (4) evaluation of the magnitude of die parameters that may effect (5)  determination  of the  structural  features of the  proteins  that  these  influence  each of these characteristics.  (i) Protein-protein complexes The number  complexes of  complexes,  methods. evaluating  that  form  between  These -studies the  structural  stabilization, elucidating the structure  electron  have features  transfer  involved of  the  proteins  determining partner  have the  proteins  been  characterised  association that  by  constants of  contribute  to  a the  complex  of the complex, and determining how die complex influences  7  the  electron  techniques  transfer  between  including gel  the proteins.  filtration,  Association constants have  analytical ultracentrifugation,  been  electronic  measured  by  several  absorption  spectroscopy,  were  reported  spectrofluorometry, *H N M R , and, recently, microcalorimetric and pH-stat titrations. Physical  measurements of  cytochrome c peroxidase Mochan,  1971).  electron  transfer  protein-protein  complexes  and cytochrome c using ultracentrifugation  Although these initial  studies  did not  furnish  and gel an  first  filtration  association  for  (Nicholls &  constant  for  the  cytochrome c peroxidase-cytochrome c complex, these values have since been evaluated by these two techniques  (Kang et al.,  1977; Dowe et al.,  1984).  The dissociation constant  of the cytochrome  ^cytochrome c complex has also been estimated by both of diese techniques (Stoneheurner 1979).  Gel filtration has been used to determine  cytochrome c oxidase (Osheroff et al.,  et  al.,  the dissociation constants of cytochrome c and  1980) and cytochrome c and sulphite oxidase (Speck et  al.,  1981). Spectrofluorometric proteins.  involve the use of a fluorescent  In native heme proteins,  coupled iron atom. and  studies  sometimes  derivative of one  form  a  partner  the porphyrin fluorescence is quenched by the paraniagnetically  Fluorescent derivatives of hemoproteins are generated by removing die iron atom  replacing it  with  another  metal  such as  tin or  zinc (Vanderkooi et  Quenching of the modified fluorescent protein is observed when another, to  of the  protein-protein  complex.  This approach  was  used  to  al.,  1976).  native heme protein binds  measure the  binding constants  between porphyrin cytochrome c peroxidase and cytochrome c (Leonard & Yonetani, 1974; Vitello & Erman, 1987), zinc substituted cytochrome c and cytochrome b  2  (Thomas et al.,  1983a), and porphyrin  cytochrome c and cytochrome c peroxidase (Kornblatt & English, 1986).  The dissociation constant of  cytochrome  be  c  to  porphyrin  cytochrome  c  peroxidase  was  found  to  strongly  ionic  strength  dependent (Vitello & Erman, 1987), varying from 0.4 ( ± 0 . 1 ) fiM at I = 0.01 M to 180 ( ± 1 4 0 ) nM at I = 0.1 M (pH 6.0, 25 °C).  The usefulness of fluorescent analogues in the measurement of protein-protein  association constants is attested to by how well these data compare  with determinations  on the native proteins by electronic absorption spectroscopy (Erman & Vitello,  8  1980) [for  performed instance,  K j = 0.2 ( ± 0 . 1 ) fj.M for native cytochrome c peroxidase and cytochrome c (I = 0.01 M , p H 6.0, 25°C)]. In  these studies,  derivative has  die use  of die modified cytochrome is based  similar hydrodynamic and electrostatic properties  on the  assumption  to the native protein.  that  In the case  of a porphyrin derivative, this assumption is imperfect because the removal of the iron may the tertiary structure and the thermal stability of the protein.  These effects  the  affect  might be expected to  be more pronounced in cytochrome c than in cytochrome c peroxidase given the smaller size and die greater  heme  exposure  of  the  former.  Nevertheless,  viscosity  measurements,  circular dichroism  spectra and tryptophan fluorescence studies indicate that the structure of porphyrin cytochrome c is very  similar  to  that  of  die native  protein  (Fisher et  al.,  1973).  Iron  reconstituted  porphyrin  cytochrome c has the same reduction potential and acts as a substrate for cytochrome b  with the  2  same affinity and velocity constants as native cytochrome c, indicating that removal of the iron does not irreversibly denature cytochrome c (Vanderkooi et al., steady  state kinetics widi cytochrome b , 2  1980).  This study also reports  porphyrin cytochrome c has an inhibition constant  is approximately  the same as the Michaelis-Menton binding constant  substantial  of  analogues  body  literature  of cytochromes  are  has  diat in  accumulated  interchangeable  with  the  implicit  which  for ferric cytochrome c.  assumption  with the native proteins  diat  (for example,  die  A  fluorescent  McLendon &  Miller, 1985; Cheung et al., 1986; Koloczek et al., 1987; Liang et al., 1988). The association constants for complex formation by four pairs of hemoprotein complexes have been arises  determined  by  monitoring die  when the proteins  small perturbation  form a complex.  5  electronic  absorption  These four pairs of hemoproteins  peroxidase and cytochrome c (Erman & Vitello, 1986); cytochrome b  in the  1980); cytochromes b  5  are:  spectrum  that  cytochrome c  and c (Mauk et al.,  1982,  and methemoglobin (Mauk & Mauk, 1982); and cytochrome c and cytochrome c  oxidase (Michel & Bosshard, 1984; Michel et al., 1989).  In each of these cases, the maximum change  in absorption caused by protein-protein association is no more than 2% of the total absorbance of the sample, limiting the determination of association constants by this technique to conditions under which complex formation is highly favoured.  9  Most  N M R studies  have measured  the off rates of protein-protein complexes and  which protons are affected by complex formation. complex between  cytochromes b  5  1983) as discussed below. strength other  of  the  However, *H N M R studies have detected a  and c with an association constant  of 10  3  M"  1  1:1  (Eley & Moore,  The high protein concentrations necessary for N M R work raise the ionic  solution to  techniques is difficult.  the  point  where  comparisons  with  association constants  measured  by  Two additional techniques have been used to measure the association  constant of a 1:1 complex formed between cytochrome c stat titrations (Guerlesquin et al., thermodynamic  determined  1987).  3  and ferredoxin, microcalorimetry and pH-  These techniques show considerable promise, because the  parameters and proton release  associated  with complex formation are  measured,  but  they have yet to be applied to other electron transfer protein-protein complexes. Each  of  these  techniques  monitors  a  different  characteristic  measurements are based on the broadening of proton resonances residues. prosthetic  Absorption and fluorescence groups.  Ultracentrifugation  spectroscopies and  properties of the free and bound proteins.  gel  of  the  complex.  NMR  that are associated with interacting  rely on an electronic interaction between  fdtration  detect  differences  in  the  the  hydrodynamic  These latter two techniques might detect complexes that  do not result in a perturbation of one of the characteristics upon which the first three tecluiiques are  dependent.  techniques detects.  might  More reflect  generally, differences  differences in the  in  the  populations  association  constants  of docking geometries  evaluated that each  N M R shows die most promise in discerning different docking geometries  involve proteins whose N M R resonances have been completely assigned.  by  these  technique  when complexes  It will be interesting to see,  in these cases, whether more than one docking geometry can be detected as a function of solution conditions. Information comes  about  die precise docking geometry  of electron  from an even greater range of techniques.  spectrofluorometry,  C D , M C D , chemical  reactivity with small electron transfer  Methods diat have  modification, chemical  reagents.  The U l  10  transfer  protein-protein been  used  crosslinking studies,  N M R studies reported  complexes  include N M R , and  variable  for the cytochrome  ^cytochrome c complex are discussed below.  N M R has also been used to characterize the complex  that forms between cytochrome c and flavodoxin 1985; Tollin et al, Hemoproteins  are  from two species of bacteria (Hazzard & Tollin,  1987) and cytochrome c peroxidase and cytochrome c (Satterlee et al, particularly  amenable  to  this  type  of  investigation  as  die  paramagnetic  1987). ferric  centre induces large chemical shifts in the heme resonances that make these protons easy to identify and  monitor.  While  complicating effects, the results of these  !  the  high concentrations  of  protein  required  for  N M R studies  can  create  such as high solution viscosity and protein-protein dimerization (Dixon,  1989),  H N M R studies are found to be in agreement with predictions of the computer  graphics models. Chemical modification studies have been developed to evaluate the role of individual amino acid residues in stabilising a protein-protein complex.  Two basic strategies have evolved based on this  approach, both of which have been used to map the electron transfer domain of cytochrome c widi its various electron transfer involves preparing  partners (Margoliash & Bosshard, 1983).  derivatives of the protein of interest,  testing  the  reactivities of  The first of these techniques  singly modified at  these modified proteins  with  a  a variety of  residues,  and  protein.  Trifluoroacetylation and trifluoromethylphenylcarbamoylation of cytochrome c lysine residues  were used to map die interaction domain of this protein with cytochromes b oxidase  (Ng  et  al,  1977;  Smith  et  al,  1980).  A  similar  dinitrophenylated lysine modified cytochromes c (Brautigan et al,  partner  electron  surface  and c  s  l  approach  and cytochrome  using  4-carboxyl-2,5-  1978a, b) has been used to map  the interaction domain of this protein with cytochrome c oxidase (Ferguson-Miller et al, ubiquinol-cytochrome c reductase (Speck et al, oxidase (Speck et al,  5  1968;  Dailey  &  1979), cytochrome c  {  (Konig et al,  1981), and yeast cytochrome c peroxidase (Kang et al,  has been undertaken with cytochrome b Strittmatter,  1979),  and NADHxytochrome b  though  the  individually modified proteins were not purified.  work  with  transfer  5  1978).  1978),  1980), sulphite Similar work  reductase (Loverde & Strittmatter,  cytochrome  b  5  is  less  satisfying  as  These chemical modification studies have been used  not only to evaluate the role of individual residues in stabilizing protein-protein complexes, but also  11  to implicate the role of higher order electrostatic interactions (Koppenol & Margoliash, 1982).  forces  (e.g.,  dipole moments)  in protein-protein  In each of these studies, die interaction of the proteins  is monitored by measuring a kinetic parameter, however, studies in which die dissociation constants were  measured  directly  yielded  similar  results  (Osheroff  et  al.,  1980;  Speck  et  al.,  1981).  Unfortunately, in some cases the modifying group is bulky, and it is not clear whether the altered behaviour of a modified protein can be attributed to electrostatic or steric effects. site directed mutagenesis  The advent of  has facilitated the introduction of more conservative changes,  as has  been  reported for the cytochrome ^cytochrome c complex (vide infra). A  second chemical modification strategy  has been to compare die susceptibility to modification  of residues on the protein free in solution with that of residues on the protein bound to an election transfer  partner.  interaction  The  should  premise  be  of  protected  this  from  strategy  is  that  modification.  residues  This  involved  strategy  of  in  protein-protein  differential  chemical  modification has made use of the methylation (Pettigrew, 1978) and acetylation (Reider & Bosshard, 1980)  of  lysine residues  of cytochrome  c,  and has  implicated the  same lysine residues  interaction domain of cytochrome c with cytochrome oxidase, cytochrome  reductase,  and cytochrome c peroxidase as the individually modified cytochromes c did. studies  of  tiiis  surface  when  type have been it  interacts  with  instrumental  transfer  partners.  the  cytochrome c,  Chemical modification  in establishing diat cytochrome c has  its physiological electron  in  one interaction  This surface  includes  most, i f not all of the exposed heme edge of the cytochrome as well as a number of hydrophobic residues (Margoliash & Bossshard, 1983). Chemical formed  by  crosslinking has  several  pairs  been  of electron  used  to study  transfer  the  proteins  structure  and  dynamics of the  including cytochrome  c  and  complexes  cytochrome  c  peroxidase (Bison & Capaldi, 1981; Waldmeyer & Bosshard, 1985; Moench et al., 1987), cytochromes b  5  and c (Mauk & Mauk, 1989), cytochrome c and plastocyanin (Geren et al., 1989) and cytochrome b  and NADPHxytochrome b  these studies,  proteins  5  the  partner  5  reductase (Hacked & Strittmatter,  were covalently coupled with zero  12  1983; Peerey & Kostic, 1984).  In  length crosslinking reagents,  such  as  l-emyl-3-[3-(dimediylamino)propyl]carrxxliimide hydrochloride, under  precursor  complex formation.  attachment  between  conventional  the  substrates  While it is theoretically possible to identify  proteins to  conditions  by  some  peptide  proteases  mapping, and  the  the  covalent  peptides  the  complexes  containing  die  that  sites  promote  of covalent  do • not crosslink  behave often  as  have  anomalous elution profiles on the chromatographic columns used to separate them (Mauk & Mauk, 1989).  This has made definitive identification of the sites of covalent attachment quite a challenging  task.  Apart  crosslinking geometries the  the  experiments  usefulness can  et  of  provide  that can be useful  complex.  (Erman  from  1987;  four  covalent  of covalent  Hazzard et al.,  cytochrome  ignore  the  characterizing  electron  transfer  the  precursor  proteins  in  multiplicity  1988) and a covalent  cytochrome  1984) have been reported. (Peerey  that are  formed  defined  transfer c  complexes  5  The lack of electron &  by  within  fo NADH:cytochrome  Kostic,  1989)  for protein-protein electron transfer.  of complexes  complexes,  fixed,  cytochrome c peroxidasecytochrome  cplastocyanin complexes  certain degrees of freedom are necessary generally  of  in  for evaluating the parameters that influence electron  reductase complex (Hackett & Strittmatter, within  studies  complexes  Some characterization  al.,  these  b  5  transfer  suggests that  While these studies  chemical crosslinking, as  the  technique develops it will be interesting to measure the reduction potentials of the proteins in these complexes, as has been done in cytochrome cplastocyanin complexes (Geren et al.,  1983), particularly  as the solution conditions diat promote noncovalent precursor complex formation (low ionic strength) are not conducive to electrochemical measurements. Spectrofluorometry  can  provide  structural  information  about  the  precursor  complex  if  the  mechanism of quenching of a modified hemoprotein by a native hemoprotein is interpreted in terms of Forster energy transfer theory (Forster, 1948).  According to the precepts of this theory, maximal  quenching is dependent on the separation and the relative orientation of the donor and the acceptor. This  treatment  has  permitted  the  estimation  of  the  distances  between  die  prosthetic  groups  of  cytochrome c and porphyrin cytochrome c peroxidase [15 A](Leonard & Yonetani, 1974), zinc and tin cytochrome c and cytochrome c oxidase [36 A](Vanderkooi et al.,  13  1977), and zinc cytochrome c and  cytochrome b  [18 A](McLendon et al.,  s  1985).  Three different fluorescent derivatives of cytochrome  c were used to estimate the distance between the heme of this protein and the heme of cytochrome b  2  [18 A](Vanderkooi et al.,  1980).  In another study, proteolytic fragments of cytochrome b  used to localise the interaction domain of this protein with cytochrome c (Thomas et al., Using two genetically engineered cytochromes b Stayton  et  al.  cytochrome b  (1988)  have  presented  in this study affects  was  evidence  attributed  to  that  interact  with  However, it is not clear how the fluorescent  probe  the properties of cytochrome b .  a  conformational  cytochromes  c  and  by cytochrome c peroxidase at least change  in  P-450  Interestingly, in one study of the quenching  5  of zinc cytochrome c phosphorescence  1983b).  singly modified at surface residues with acrylodan,  5  differently than myoglobin does.  5  were  2  cytochrome  c  that  part of the quenching  occurs  upon  binding  to  the  peroxidase (Koloczek era/., 1987). Another precursor  experimental  complexes  approach  involves  the  diat  has  kinetic  provided useful  analysis  of  the  information about reduction  of  die  the  individual  components of the complexes by exogenous, photolytically generated flavin semiquinones. of this approach is that alteration of the flavin different have  charges,  been  sizes and reduction potentials  reported  concerning the  reactions  electron transfer proteins (Tollin et al., electron transfer  substituents  1986).  of  protein  One appeal  can be used to generate reductants of  (Figure 25, Table X I ) .  of a  properties  Numerous kinetic studies  variety of flavins with  various small,  soluble  These studies have established that the mechanism of  from a semiquinone to a cytochrome involves the transfer  of an electron from the  N-5 dimethyl benzene ring portion and the C-4a position of the flavin to die exposed heme edge of the protein (Meyer et al., it possible to use flavin edge.  1984; Pryzsiecki et al.,  1985).  Using flavins widi different characteristics,  semiquinone reduction kinetics to probe the environment around the heme  A particularly striking example of this is the ionic strength dependence of the rate constant  of reduction of Rhodospirillum  rubrum cytochrome c  2  by F M N semiquinone (Meyer et al.,  indicating that the heme edge of this cytochrome has a positive electrostatic the net charge of the protein is zero.  surface,  even  1984), though  A n extension of the use of flavin semiquinones to probe the  14  active sites transfer  of electron  tranfer  proteins  protein bodi in the presence  observed  in the presence  involves performing  the  and absence of a partner  reduction kinetics of an electron protein.  Changes in the kinetics  of an interacting protein can be ascribed to the electrostatic  and  steric  influence of the partner protein on the heme edge. In studies of the flavodoxincytochrome c complex (Hazzard et al., constant from  of  7.1  (±0.5)  interpreted edge  in  reduction  as the  of  x  10  7  ferricytochrome M'V  arising from presence  of  to 2.9  1  c  by  (±0.3)  steric constraints flavodoxin.  the  A  x  and  electrostatically 10  7  M'V  reduced  similar  effect  1  1986), a decrease in the rate neutral  in the presence  7  M'V  negatively charged F M N and C 1 F M N 2  at  the  protein-protein  cytochrome dependence.  c  by  interface. these  1  on  the  rate  in the presence of flavodoxin].  of flavodoxin  constant 7  M'V  1  of  c  was heme  reduction  of  in the absence of  Reduction kinetics with the  semiquinones gave information about die electrostatic potential  Pseudo-first  negatively  semiquinone  accessiblity of die cytochrome  ferricytochrome c by riboflavin semiquinone was observed [5.4 ( ± 0 . 3 ) x 10 flavodoxin and 1.7 ( ± 0 . 2 ) x 10  lumiflavin  charged  This non-linearity was interpreted  order  rate  constants  semiquinones  showed  for  die a  reduction  of  bound  nonlinear  concentration  in terms of dynamic motions widiin  the complex.  Similar studies have provided insight into the complexes that form between cytochrome c and both cytochrome c oxidase (Ahmad et al., 1982) and cytochrome c peroxidase (Hazzard et al., and ferredoxin N A D P  +  1987; 1988),  reductase and a number of other electron transfer proteins (Bhattacharyya et  al., 1986; Bhattacharyya et al., 1987). The idea that the formation of an electron transfer protein-protein complex might modulate the parameters that influence the rate of electron transfer between the two proteins was suggested years ago (Nicholls,  1974), but there has been surprisingly little investigation into tliis area.  influence of charged groups on the reduction potential (Moore et al., energy (Warshel & Churg, 1983; Churg et al.,  some The  1986) and the reorganizational  1983) of a protein indicate that the proximity of an  oppositely charged electrostatic field of a partner protein could affect these parameters.  There have  been attempts to correlate the reduction potential with the overall charge of a protein (Rees, 1985),  15  but clearly, this is but one of many factors and is by no means the most important (Moore  &  Williams,  1977;  Moore et  al.,  1986).  Electrochemical studies  determinant  demonstrated  that  the  reduction potential of cytochrome c decreases from 290 ( ± 5 ) mV to 250 ( ± 5 ) mV upon binding to cytochrome c oxidase (0.02 M M E S , 0.2 M sucrose, p H 7.0) but is not altered by die presence of cytochrome b  or cytochrome c peroxidase (Vanderkooi & Erecinska, 1974).  5  performed  at an ionic strength  of 0.02  cytochrome ^-cytochrome c complex.  These studies  were  M , which may not be low enough to promote a  stable  Separate studies have shown that the reduction potential of  cytochrome c drops 25 m V on binding of the cytochrome to plastocyanin (Geren et al., mV on binding to flavodoxin (Hazzard et al.,  1986).  1983) and 30  Each of these studies was limited to a single  set of solution conditions (buffer composition, ionic strength, p H and temperature). CD, electron  M C D and phosphorescence transfer  protein-protein  measurements have provided some insight into the structure of  complexes.  Complexes  involving  cytochrome  (Tollin et al.,  1987), and cytochrome c and cytochrome c oxidase (Weber et al.,  MCD  that  spectra  complex  formation  changes  have  differ  from  induces  been  changes  interpreted  reorganizational free energy of  the  prosthetic  cytochrome c  groups  in the  the  sum of in as  the  the  electronic  facilitating  of electron transfer (Tollin  presence  et  al.,  spectra  of the  structure  electron  (Weber et al.,  1987).  partner  of  die  transfer  c  flavodoxin  1987) display C D or  proteins, prosthetic  eidier  and  by  suggesting groups.  that These  decreasing  1987) or increasing the accessibility  Recently, the  of cytochrome c oxidase has been  change  in the  interpreted  C D spectrum of  as a conformational  change involving the movement of phenylalanine-82 away from the cytchrome c heme (Michel et 1989a,b).  As discussed above, phosphorescence  the  al.,  quenching studies of zinc cytochrome c suggest that  this protein might undergo a conformational change upon binding cytochrome c peroxidase (Koloczek et al.,  1987).  The increasing number  of calculations based on structural  information of protein-  protein complexes may stimulate research in this area. The experimental studies reviewed above have supported the theoretical predictions discussed in the  previous  section  in at  least  two ways.  First, a  16  large number  of electron  transfer  protein  precursor  complexes  are  stabilized  by  electrostatic  forces.  Given  the  failure  of  electrostatic  calculations to account for all of energy of association of some of these complexes, it is surprising that few  experiments  complexes  have  been  complexes  bring  the  suggest  that  the  investigating the  role of uncharged  undertaken. prosthetic  computer  Second,  groups  graphics  docking geometry of these proteins.  of the  generated  amino acids in stabilizing protein-protein  experimental electron models  studies  tranfer are  provide  proteins  accurate  into  evidence close  representations  that  these  proximity and of  the  average  While it has been suggested that electron transfer proteins can  assume several docking geometries in which efficient electron transfer can occur (Maine et al., Northrup  et al.,  1988), there is no definitive experimental evidence for  although  two or  three studies  conformational  structure  of  suggest that complex formation  the  partner  proteins,  there  might  have  evaluate the effect of complex formation on the parameters,  been  this assertion.  influence few  the  1986; Finally,  electronic and  documented  attempts  to  such as the reduction potential and the  reorganizational energies of die partner proteins, that affect electron transfer.  Undoubtedly, part of  this deficiency arises from die practical difficulties involved in working at die low ionic strengths required for stable protein-protein complexes to form in most cases.  (ii) Electron transfer kinetics Various techniques such as  the  acceptor  centres.  rates between transfer  have  been  reduction potentials,  used to evaluate  reorganizational energies  Experimental studies  donor  and  acceptor  between soluble proteins.  the  factors  diat affect  fixed  rates,  and relative positions of the donor and  of protein-protein electron transfer  centres at  electron transfer  separation,  or  second  report  either  first  order  order  rates of electron  This discussion begins with the first case, which is conceptually  easier to envisage. To  measure the  rate of electron  transfer  between  sites  of fixed,  known separation,  several  laboratories have attached ruthenium compounds to specific histidine imidazoles (Mayo et al.,  1986).  These studies also provide information about the influence of the intervening medium, and in cases  17  where the ruthenium ligands and metal centres can be modified, electron  transfer.  transfer  depends  energy  is  These  studies  exponentially  increased  or  the  on  appear  to corroborate  distance  driving  and  force  that  the  the  is decreased  the driving force, on the rate of  predictions  rate decreases (Mayo  et  that  the  when  al.,  1986;  rate of  the  electron  reorganizational  McLendon,  1988).  Alternatively, some of these factors have been evaluated by measuring the rates of electron  transfer  within hemoglobin hybrids in which the iron atom of one of the subunits has been replaced widi zinc or tin (e.g., McGourty et al.,  1983).  Electron transfer between the metal centres is initiated by  a laser flash to photoproduce an excited triplet state.  This excited triplet state can either thermally  decay back to the ground state or, in the presence of a suitable acceptor, transfer an electron. thermodynamic transfer  driving  reactions,  forces  of  a consideration  some manner diat is difficult  these that  reactions  may  to assess.  also  are  much  influence  greater  the  than  mechanism  physiological of electron  The  electron  transfer  in  Nevertheless, these studies have provided insight into the  distances over which electron transfer can occur and the influence of the intervening medium on die rate of electron transfer. constants of electron  One question that all of these studies raise is why the first order rate  transfer  involving  proteins  that are  putatively  designed  for  electron  are so many orders of magnitude smaller than the rate constants of electron transfer comparable distances in non-protein systems (Miller, 1975; Heiler et al.,  transfer  measured over  1987) or in other biological  systems, such as the photosynthetic reaction centre (Parson et al., 1978). Measurements  of the  first order  rate of electron transfer  preformed complex have also been reported for cytochromes b  between  two soluble proteins  in a  and c (McLendon & Miller,  1985),  cytochrome c peroxidase and cytochrome c (reviewed in McLendon, 1988), and cytochromes b  and c  5  (McLendon et al.,  1987).  The attraction of these systems is tiiat diey involve donor and acceptor  sites located in physiological environments. complexes is unknown. transfer  reaction  may  One disadvantage  For each of these systems, be  2  evaluated  through  varying  is that die precise structures of these  the reorganizational energies the  thermodynamic  respective reactions and using the Marcus relationship (equation 3).  18  driving  of the forces  electron of  die  The best example involves the  electron transfer  reaction between cytochromes b  reorganization energy has been estimated  and c (McLendon & Miller,  5  to be 0.7 eV (16 kcal/mol).  1985), for which the  The driving force for the  reaction between these proteins was varied by removing the iron of cytochrome c and measuring the rate of electron transfer zinc cytochromes c.  within the complex formed by cytochrome b  with native, porphyrin and  5  The magnitude of the reorganization energy seems reasonable compared to the  calculated reorganization energy of cytochrome c [6.4 kcal/mol](Warshel & Churg, 1983; Churg et al., 1983) and considering that the reorganization energy of an electron transfer can be estimated to be the mean of the reorganization energies of the self exchange reactions. While  the  effects  of such  a modification on protein conformation  and  hence  the  precursor  complex geometry might be minimal (vide supra), the effect of such a substitution on the electronic configuration considering which  of the porphyrin is more difficult the  direction of  electron  the kinetics were performed.  assumed  that for the purposes  transfer  to evaluate.  within  In the studies  the  Additional  complex and  complications arise when die solution conditions in  of McLendon and Miller  of measuring reorganizational energies,  cited above,  electron transfer  a complex are equivalent no matter which direction the electron travels.  it is  rates within  Recent studies in which a  mutation of cytochrome c Phe-82 exerts a greater influence on the rate of electron transfer the  zinc cytochrome  electron radical  transfer (Liang  c peroxidase  from et  al.,  excited triplet  ferrocytochrome 1987)  indicate  c  to  the  state to ferricytochrome zinc  cytochrome  that this assumption  may  c  not  c  than  peroxidase be  valid.  on  the  rate of  porphyrin  different  docking geometries  cation  In light of  observation that the ionic strength of the solution can influence the rate of electron transfer the complex (Hazzard et al.,  from  the  within  1988) and the suggestion that different solution conditions can favour (Mauk et al.,  1986), care  must  be  taken  to reproduce  the solution  conditions in each case. Further  complexities of protein-protein  cytochromes b  2  and  c  and c (McLendon et al.,  discussed  above.  The  rate of  electron  transfer  are  apparent  from a  study  involving  1987) that is analagous to the study widi cytochromes electron  19  transfer  within  the  cytochrome  fc ' y c  2  tocnrome  b  5  c  complex was found to be independent of the driving force of die reaction.  The authors  suggested  that this observation can be taken as evidence of a conformational gating process controlling the rate of electron transfer. of cytochrome b  Intriguingly, U  is quite mobile in the holoenzyme (Labeyrie et al.,  2  timescale of this motion is unknown. the  structure  are  drawn.  N M R studies have suggested diat the cytochrome domain  l  of the cytochrome The  concept  Further characterisation of this conformational mobility and  fc2 y ^ c  that  1988), though die nature and  toc  rome  c  complex is required before  protein-protein  electron  transfer  might  definitive conclusions by  controlled  by  a  conformational change has been developed theoretically by Hoffman & Ratner (1987). A  first  order  rate  constant  for  the  nonphysiological reduction  ferrocytochrome c peroxidase has been reported [3.4 s"  1986; Cheung & English,  unimolecular at both high and low ionic strengths, vary  with  ionic  strength.  ferricytochrome  1987).  in  the  peroxidase  and to exhibit thermodynamic parameters  The authors have suggested  that  is rate limiting.  by  This reaction was observed to be  that at  low ionic strength,  transfer within the complex that is rate limiting and at higher ionic strengths, change  c  for the yeast proteins in 0.01 M phosphate,  1  p H 7.0, 24°C](Cheung et al,  of  While  these data  are  that  it is electron  it is a conformational  interesting,  kinetic data  involving cytochrome c peroxidase should be viewed with caution owing to die difficulty in isolating the  native,  pentacoordinated  (Yonetani & Anni, instances  1987).  form  of  the  enzyme  uncontaminated  with  Whatever die explanation of diese results,  a  six  coordinate  form  it is clear that even in  where a first order rate is measured between donor and acceptor  sites,  interpretation of  these rates solely in terms of electron transfer theory may be overly simplistic. Measurements difficulties oxidised  of  the  second  order  that make such measurements by  rates of electron  transfer  technically challenging.  molecular oxygen, necessitating  the  use  against a high background.  proteins  pose  several  Many reduced proteins are readily  of anaerobic  presented by intensely coloured heme proteins is the measurement  between  conditions.  Another  difficulty  of small changes in optical density  Despite these problems, the bimolecular rate constants of a number of  protein pairs have been measured including horse heart and Candida krusei cytochromes c (Yoshimura  20  et al.,  1981), horse heart cytochrome c and Pseudomonas cytochrome c  horse heart cytochrome c with plastocyanin and azurin (Augustin et al., were performed on stopped flow spectrophotometers,  (Morton et al.,  5 5 1  1983).  1970) and  These measurements  and all the bimolecular rate constants were on  the order of 10 -10 M ' V . 4  6  Electron  1  transfer  between  two  soluble  proteins  will  considerations discussed in the case of electron transfer the recognition and association process. binding  data  not  only  the  mechanistic  within a protein-protein complex, but also  There have been few attempts to correlate protein-protein  and. rates of electron transfer  order rate constants.  involve  within protein-protein complexes with measured  second  This analysis has been reported to a certain extent for the reactions between  cytochrome c and cytochrome c peroxidase (Summers & Erman, 1988) and between cytochrome c and plastocyanin (King et al., 1985). Evidence for the rate limiting protein conformational steps that have been invoked to explain the mechanism of protein-protein electron transfer within some complexes (vide supra) has also been seen  in  bimolecular  ferricytochrome  c  5 S 1  reactions.  The  kinetics  isolated from Pseudomonas  1973; Wilson et al.,  of  electron  aeruginosa  are  transfer  from  clearly  biphasic (Pecht  1975) and two forms of ferricytochrome c  The  of  cytochrome c al.,  1981).  transfer 5 5 1  reaction  azurin  isolated  from  Alcaligenes  5 5 1  Rosen,  with  suggests diat there is only one conformational form of Alcaligenes  Pseudomonas  azurin (Rosen et  These results were initially interpreted as involving the protonation of histidine residue  dependence  does not  &  to  (Rosen & Pecht, 1976).  faecalis  35 which is present in Pseudomonas azurin but not Alcaligenes (Rosen et al., pH  azurin  1975) and have been interpreted as involving an equilibrium between two forms  of reduced azurin (Wilson et al., electron  reduced  of the rate of electron transfer  suggest any  strong  dependence  between  Pseudomonas  of reactivity on the  1981).  However, the  azurin and cytochrome  state of protonation  c  5 5 1  of the protein  ( C o r i n g a/., 1983). In one of the most extensive studies of bimolecular protein-protein electron transfer, constant  the rate  for the reduction of several c-type cytochromes by flavodoxin semiquinone were determined  21  as a function of ionic strength fitting  the  scatter  in these data  (Tollin et al.,  electrostatically corrected illustrates  1984).  rate constants to  the  Although modest the  complexity of the  equations  success  was achieved in  of Hopfield  bimolecular electron  and  transfer  Marcus, the reaction.  In  fitting the data to the Marcus relationship, a value of 43 kcal/mol was used for the reorganization energy [A is 4 times A G J at zero driving force (equation 3)].  This is considerably larger than the  value of A (11 kcal/mol) used in a similar analysis of the second order rate constants of electron transfer  from free F M N to c-type cytochromes (Meyer et al.,  1984).  The authors equate structural  complexity with more extensive structural rearrangements and rationalise the difference in A by the greater structural complexity of flavodoxin as compared to F M N .  This interpretation is incompatible  with the suggestion that one of the roles of the polypeptide chain of electron transfer proteins is to reduce  the  discrepancy changes  reorganisational aside,  considering  in protein-protein  was observed  energy  between  the  of  the  the  electron  potential  electron transfer, second order  transfer  importance  centre  of  (Churg  complex  it is perhaps fortuitous  et  formation that any  rate constants of protein-protein  al.,  1983).  and  conformational  reasonable  This  correlation  electron transfer  and  the  driving force of the reaction. Wherland between  and  Pecht  (1978)  measured  several pairs of electron transfer  the  transfer  reactions  are  adiabatic  order  proteins at constant  constants to die Marcus relationship (equation electron  second  or  rate  constants  ionic strength  of  Wherland  and  Pecht,  there  is  uniformly nonadiabatic.  considerably  calculated from protein-protein electron transfer electron  transfer  reactions  between  proteins  less  agreement  rate constants measured and  Dixon et al.,  [1.2 x 10 M ' V 7  S5l  and  fitted  Although the  transfer  these rate  small  molecules  1  self  by other (Cummins 3  calculated  exchange  rates  workers, or from &  Gray,  1977).  M"'s"' (I = 0.13 M , pH  (0.05 M phosphate, p H 7.0, 2 5 ° Q ] (Gupta, 1973;  1989) agree reasonably well with the values calculated in this study [1.5 x 10  22  self  proteins studied in the  with  Interestingly, die measured self exchange rate of cytochromes c [6.7 x 10 7.0, 2 7 ° Q ] and cytochrome c  electron  5), even though it is not clear that protein-protein  exchange rates were remarkably consistent for the series of electron transfer work  of  2  M"'s"'  and 4.6 x 10 M ' s " respectively (0.05 M phosphate, p H 7.0, 25 °C)]. 7  1  Related  to  these  kinetic  metalloprotein electron transfer  measurements  has  self-exchange rates.  been  the  determination  of  a  1989a), cytochrome c  al., 1976; Dixon et al., 1989a), plastocyanin (Armstrong et al., 1985) and cytochrome b Studies of the ionic strength dependence  1989a,b)  1986)  have  electron  and the self-exchange been  transfer.  useful  Attempts  to  the  correlate  major these  role  of  electrostatic  self-exchange  rate  electron transfer  within the protein-protein complex (Dixon et al.,  (Dixon et al.,  1989b) are  interesting,  but  must  forces  constants  (Concar et in  al.,  protein-protein  with  distances  of  1989a) and reorganization energies  be viewed with caution until  protein-protein electron transfer has been established.  (Keller et  of these rate constants (Gupta, 1973; Dixon et  rate constants of chemically modified proteins  in establishing  5 5 1  (Dixon et al.,  5  al.,  of  Generally, diis has been accomplished using ' H  N M R , as in the case of cytochrome c (Gupta, 1973; Dixon et al.,  1989a,b).  number  the  mechanism of  Should this mechanism include a rate limiting  step involving complex formation or a change in protein conformation, dien the analysis of proteinprotein electron transfer by multiphonon electron transfer theory would not be appropriate. In these and related studies, dependence  several formalisms have been used to analyse the ionic  of the bimolecular rate.  The seminal work of Debye and Hiickel (1923) analyses  observed behaviour in terms of monopole-monopole interactions. Hiickel equations &  Gray,  to protein electron transfer  1976; Stoneheurner  situations, they are inadequate treat  proteins  strengths  of  et al.,  homogeneously  0.01  M , higher  Leeuwen, 1983).  1979).  Several adaptations  charged order  the  of the Debye-  reactions have been developed (for example, Wherland Although these treatments have been  to describe macromolecular interactions.  as  strength  spheres,  electrostatic  which  clearly  components  used  in many  Not only do these equation  they  contribute  are to  not, the  but  above  ionic  interactions  (Van  Two approaches that consider the contribution of protein dipole moments are those  developed by Koppenol et al. (1978), based on the Brdisted relationship, and Van Leeuwen (1983). Koppenol has revised his equation (Rush & Koppenol, 1988) and it is now virtually identical to the Van Leeuwen approach.  A n alternative strategy considers the interacting domains of the proteins as  23  two interacting charged plates.  A n empirical equation based on this approach works reasonably well  (Tollin et al., 1984), diough a detailed theoretical development of this model has not been published. These studies indicate that in the simplest cases, biological electron transfer interpreted  in terms of electron transfer  theory (DeVault, 1984; Marcus & Sutin, 1985).  whole, there remains a scarcity of data on the reorganization energies proteins within a precursor complex.  may be reasonably On the  and reduction potentials of  In many instances, electron transfer  between soluble proteins  seems to involve more than the collision of two proteins followed by an electron transfer event and may  be  determined  by  die rate of protein-protein  recognition and  association,  and conformational  rearrangements within the complex (Hoffman & Ratner, 1987).  Cytochromes  The cytochromes are a class of heme containing proteins whose principal biological function is electron transport 1973;  Pettigrew  nineteenth  century  by virtue of a reversible valency change of dieir heme iron (Leniberg & Barrett, & Moore, by  1987).  MacMunn  Although cytochromes  in the  late  (1884), dieir existence was not generally acknowledged until  they  were rediscovered by Keilin in the 1920's (Keilin, 1966). discovered  in all living  were  originally  discovered  Since that time, these proteins have been  organisms and have been broadly classified according to the  their prosthetic groups into a, b, c and d types.  structure of  Cytochromes have a distinctive red colour and a  characteristic four banded electronic absorption spectrum composed of an o band around 550-604 nm, a p band around 520-546 nm, a i  or Soret band around 400-450 nm, and a 5 band around 300-350  nm, in addition to the protein absorption bands in the ultraviolet region.  The extinction coefficients  of these bands were first determined in horse heart cytochrome c (Margoliash & Frohwirt, 1959). The majority of cytochromes studied consist of an iron porphyrin prosthetic group bound to a single polypeptide chain containing no disulphide linkages. ligated .to protoporphyrin IX (Figure 1) (Smith, 1975).  24  The heme  consists  of an  iron  atom  In some classes of cytochromes, the structure  ROOC Figure 1 : The structure of protoheme IX.  COOR In the native protoheme, R = H ; in dimethylesterified  protoheme IX, R = C H . 3  25  of the heme is modified. (Lemberg & Barrett,  For example, the heme of a type cytochromes contains a formyl side chain  1973).  Four of the octrahedrally coordinated iron ligands are provided by the  pyrrole nitrogens and the odier two axial ligands are derived from amino acid side chains. oxidised Fe(III) state, die iron has a single unpaired electron and is paramagnetic.  In the  As the iron  displaces two protons when it is incorporated into the porphyrin, ferriheme has a formal charge of + 1.  In the reduced Fe(II) state, the iron has no unpaired electrons, and the heme has no net  charge.  (i) Cytochrome b  5  As the name implies, cytochrome b bound heme.  5  is a fc-type cytochrome, and as such it has a noncovalently  The axial ligands of the iron atom are provided by the e-nitrogen atoms of histidine  residues 39 and 63.  Cytochrome b  5  occurs in several forms in vivo (Madiews, 1985).  bound form occurs in the smoodi endoplasmic reticulum of mammalian hepatocytes in steroid hydroxylation (e.g., Grinstead & Gaylor,  A membrane  where it functions  1982), fatty acid desaturation (Strittmatter et al.,  1974) and supports the reduction of a variety of cytochromes P-450 (e.g., Peterson & Prough, 1986; Tamburini & Schenkman, 1988).  This species is a monomer of about 16,000 daltons consisting of a  simgle polypeptide chain diat is folded into two domains.  The larger domain consists of the first 86  - 90 amino terminal residues and contains the heme prosthetic group.  This domain is hydrophilic  and extends into the aqueous environment of the cytoplasm (Tanford, 1980).  The second domain is  composed of the 37 predominantly hydrophobic, carboxyl terminal residues and is embedded lipid bilayer, anchoring die cytochrome to the membrane. residues links the two domains (Bendzko & Pfeil, 1983). liver  microsomal cytochrome b  5  was determined  in the  A flexible sequence of approximately 7 The complete amino acid sequence of bovine  (Ozols, 1975) but has recently  been  corrected at  three positions i n the hydrophilic heme binding region based on the sequence of the cytochrome b  s  gene (Cristiano & Steggles, 1989). A soluble form of cytochrome b  s  occurs in mature erythrocytes where it functions to reduce  26  methemeglobin (Passon & Hultiquist, 1971).  This species is apparently  derived from a microsomal  precursor by cathepsin digestion during erythroid development (Slaughter & Hultquist, 1979). erythrocytic cytochrome b  5  has 97 residues.  Although the first 96 residues  of die porcine and  human erythroid proteins are identical to their respective hepatocyte  species, a difference  97  derived  suggests  that  the  erythrocyte  closely related genes (Abe et al.,  and  hepatocyte  1985).  cytochromes  Cytochrome b  are  Mature  from  in residue  two  distinct  but  has also been isolated from two species of  5  sipunculids where it is believed to function in mememerythrin reduction (Utech & Kurtz, 1985; 1988). A second membrane bound species of cytochrome b membrane of mitochondria (Mathews, 1985).  5  is found on die inner surface of the outer  The amino acid sequence of die globular heme binding  domain of this protein shows 58% sequence homology to the corresponding microsomal heme binding domain (Lederer et al.,  1983).  in an electron shuttle  between  It has been suggested that this species of cytochrome b the inner and outer  mitochondrial membranes,  oxidation of exogenous N A D H (Bernardi & Azzone, 1981).  participates  s  effecting  the aerobic  In this proposed pathway, cytochrome b  5  reacts with intermembrane cytochrome c. Bovine microsomal cytochrome b  5  and trypsin solubilized form.  may be isolated in several forms including a lipase solubilized  The lipase solubilized form comprises residues 5 to 97 of the intact  microsomal sequence whilst the trypsin solubilized form is comprised of residues 7 to 88. current studies, the trypsin solubilized fragment crystal  structure  of the lipase solubilized  isolated from bovine liver was used.  In the  Currently, the  form of bovine microsomal ferricytochrome b  5  has  been  solved to a resolution of 2 A (Madiews et al., 1979), but two residues at die amino terminal and ten residues at the carboxyl terminal could not be detected in the electron density maps. the crystallographic structure is of a fragment solubilized species (Figure 2). tryptic fragment differences chain  diat approximately corresponds  Consequently,  to the smaller, trypsin  With resonance assignments for more than 40% of the protons of the  of oxidized and reduced cytochrome b , 5  two dimensional  *H N M R revealed that  between the crystal and solution structures are limited to small variations in a few side  orientations  (Veitch  et  al.,  1988).  Neither  27  technique  detected  significant  conformational  Figure 2 : The structure of the tryptic fragment of bovine hepatic ferrocytochrome b . 5  Tins image  was generated with the Raster3D package (D. Bacon, University of Alberta) on a Silicon Graphics IRIS 3130.  The heme atoms are shown in red and the aspartate and glutamate side chains, in  orange. The atomic coordinates where taken from the Brookhaven Data Bank (Mathews et al., 1979).  28  270  315  360  405  450  495  540  585  630  Wavelength (nm)  Figure  3  :  The  cytochrome b . 5  electronic  absorption  spectrum  the  tryptic  fragment  of  bovine  microsomal  The spectra are normalised to an extinction coefficient of 117 m M " at 412.5 nm of 1  the oxidised spectrum (Ozols & Strittmatter, Ferrocytochrome  of  b  s  was  produced  by  the  1964).  The oxidised and reduced  addition of a few  grains  forms are indicated.  of sodium  solution of ferricytochrome b . The conditions were 0.02 M sodium phosphate, p H 7.0, 2 5 ° C . 5  29  dithionite  to  the  differences  between  the  reduced  and  oxidized  forms  of  the  protein,  diough  x-ray  crystallography  detects the binding of a cation to heme propionate-7 in the reduced form (Argos & Matdiews, 1975). The crystallographic model of the cytochrome b  indicates that the molecule is cylindrical in shape,  measuring 37 A in height and 31 A in diameter.  The polypeptide backbone is folded into 6 short a  5  helices and a 5 stranded /? pleated sheet.  The heme is buried in a large hydrophobic core such that  the propionate groups (numbered 6 and 7) of the heme group are oriented towards the surface of die protein.  Propionate-6  extends into the  solvent  creating  a pronounced  surface of the molecule (Argos & Madiew, 1975; Mauk et al.,  1986).  not as prominent on the electrostatic  5  hydrogen bonds with serine 64.  J  surface of cytochrome b  the first by a 180° rotation about the 5  electrostatic  The second propionate group is  because it bends back to form two  H N M R studies suggest diat in 10% of the protein molecules, the  heme is bound in a second orientation (La Mar et al.,  Cytochrome b  pimple in the  1981).  0 - 7 - m e s o axis of the heme in the heme binding pocket.  is acidic, containing 17 aspartate and glutamate  9 basic (lysine and arginine) residues.  This second orientation differs from  residues,  two heme propionates,  and  The excess acidic residues are clustered on the surface of  the molecule around the mouth of die heme crevice, giving the molecule a substantial dipole moment. The trypsin solubilized fragment  of cytochrome b , 5  henceforth  referred  has a molecular weight of 10,063 daltons and a p i of 5.2 (Reid, 1984).  to as cytochrome  b, 5  The reduction potential has  been measured to be 5.1 (+0.6) mV (pH 7.0, I = 0.1 M , 25°C) versus the saturated hydrogen electrode (Reid et al.,  1982).  The electronic absorption spectra of ferro- and ferricytochrome b  5  are given in  Figure 3.  (ii) Cytochrome c Cytochrome c is one of the most extensively studied proteins. over 100 different species, including horse (Margoliash et al., structures of cytochrome c from several eukaryotes, et al., unpublished), bonito (Dickerson et al.,  The amino acid sequences from  1961), have been determined.  including horse (Dickerson et al.,  Crystal  1971; Bushnell  1971), tuna (Takano & Dickerson, 1981a,b), rice (Ochi  30  et al.,  1983), and yeast (Louie et al.,  studied,  the  bridges. of  heme  of cytochrome  c  1988) have been solved. is covalently linked  to  As with most c class cytochromes  die peptide  chain via two  thioether  Horse heart cytochrome c is made up of 104 amino acid residues and has a molecular mass  12,500 Daltons.  The axial ligands of the iron atom are provided by the  methionine (residue 80) and the e nitrogen atom of a histidine (residue 18).  6 sulphur atom of a  The iron is low spin in  both oxidation states of this protein. The  three  dimensional  resolution (Dickerson et al.,  structure 1971).  of  horse  heart  cytochrome  c  has  been  solved  to  2.8  A  The structure of this species of cytochrome c is very similar to  that of the tuna protein, which has been solved to 1.5 A and 1.8 A in the reduced and oxidized forms  respectively  (Takano & Dickerson, 1981a,  b)  (Figure 4).  The solution structure  cytochrome c in both oxidation states has also been studied by *H N M R (Williams et al., Moore et al.,  1985).  Apart from some small differences  carboxyl terminus, around heme propionate-7 well and the crystal structures  of tuna 1985a;  in three regions of die protein (at  and near thioether-2)  diese structures  accurately describe the solution structures  the  agree remarkably  of cytochrome c (Williams  et al.,  1985b).  Two dimensional ' H N M R studies of reduced and oxidised horse heart cytochrome c  (Wand  et  1989;  al.,  cytochrome and indicate  that  Feng  et  al.,  1989)  tuna cytochrome c are  there  are  few  indicate  minor.  conformational  that  Bodi differences  the  differences  between  this  species  ' H N M R and x-ray crystallography between  ferric  and  of  studies  ferrocytochrome  c  E X A F S studies indicate diat there is very little difference in die immediate coordination environment of the iron atom in the two oxidation states (Korszan et al.,  1982).  The molecule is a prolate  spheroid measuring 30 x 34 x 34 A with the peptide backbone folded into 6 helices that account for 48% of the structure.  Residues 1 to 47 are folded on one side of the heme while residues 48 to 91  are on the other.  The remaining residues (92 to 104) form a strap that passes across the molecule,  behind the heme.  The heme is deeply buried in a fairly hydrophobic pocket and is oriented such  that one of the heme edges is exposed to the solvent and the heme propionates point towards the bottom of the crevice.  31  Figure  4  generated 3130.  :  The three dimensional structure  of horse  heart  ferricytochrome  c.  The image  was  with the Raster3D package (D. Bacon, University of Alberta) on a Silicon Graphics IRIS The heme atoms are shown in red, the methionine and cysteine side chains are shown in  yellow, and the lysine and arginine side chains are shown in blue. generously provided by Prof. Gary D . Brayer.  32  The atomic coordinates  were  Wavelength (nm)  Figure  5  :  The  electronic  absorption  spectrum  of horse heart cytochrome  c.  The  spectra  are  normalised to an extinction coefficient of 106.1 m M " at 409 n M of the oxidised spectrum (Frohwirt 1  &  Margoliash, 1959).  produced  The oxidised and  reduced  species  are  indicated.  Ferrocytochrome  by adding a few grains of sodium dithionite to the solution of ferricytochrome  solution conditions were 0.02 M sodium phosphate, pH 7.2, 25°C.  33  c.  c  was The  Cytochrome c is a basic molecule with a pi of approximately 10.5. positively charged residues and 7 negatively charged residues.  The tuna protein has 18  The excess positively charged residues  are clustered around the mouth of die heme crevice, creating a significant charge asymmetry on the surface of the protein.  Horse heart cytochrome c has three additional positively charged  and three additional negatively charged residues.  residues  The reduction potential of cytochrome c is 261 mV  (pH 7.0, I = 0.01 M , 25°C) versus the saturated hydrogen electrode (Margalit & Schejter, 1973).  The  electronic absorption spectra of ferri- and ferrocytochrome c are presented in figure 5. Cytochrome c has several roles in vivo which have been reviewed recently (Pettigrew & Moore, 1987).  This protein is found in the periplasmic space between die outer and inner mitochrondrial  membranes.  Its primary role is to shuttle electrons between ubiquinol-cytochrome c  oxidoreductase  (fccj complex, complex III) and cytochrome c oxidase (complex IV) in eukaryotic respiration. periplasmic space,  cytochrome c can react  with other  intermembrane  electron transfer  as sulphite oxidase in animals and cytochrome c peroxidase and cytochrome b related cytochromes occur in other electron transfer  pathways,  shuttle  oxidoreductase  electrons  between  ubiquinol-cytochrome  c  2  2  proteins  in yeast.  such as in photosynthesis and  the  In the such  Closely  where they  photochemical  reaction  centre protein.  The Cytochrome / ^ C y t o c h r o m e c Complex  The wealth of structural and functional data for cytochromes b  s  choices addition,  for  use  in  the  study  of  protein-protein  interaction  genes encoding for both of these proteins  Bodman et al.,  1986; Pielak et al.,  1985).  and  have been  and c makes them excellent  electron expressed  transfer  reactions.'  In  in high efficiency (von  This development allows for the production of specifically  mutated forms of diese proteins that should be of considerable value in elucidating the contributions of individual amino acid residues to the interaction of and electron transfer  between  these proteins.  Although the physiological significance of the cytochrome ^cytochrome c complex remains a moot  34  point  (vide  supra),  physiologically relevant  (Johnson & Rajagapolan, contain  a  cytochrome  cytochromes  b  oxidoreductases. cytochrome b Second, been  5  c  have  with  been  each  documented.  other  bimolecular  rate  cytochrome  of  with  sulphite  the  oxidase  1980), two proteins that  Two lines of evidence  in a manner constant  c  (Briquet et al.,  2  interact the  of  1980) and cytochrome b  domain,  s  First,  analogous  to  reduction  suggest  diat  that of physiological of  cytochrome  c  is very similar to that of physiological electron transfer reactions (Strittmatter,  by  1964).  the cluster of positively charged residues around the heme crevice of cytochrome c have  implicated  in  Bosshard, 1983). thought  b  and  5  1977,  interactions  its  interactions  with  physiological electron  transfer  partners  Similarly, die acidic groups clustered around the heme edge of cytochrome b  5  to mediate  the  interaction between  1979),  stearyl  CoA  are  this protein and its various physiological partners such  as hemoglobin (Mauk & Mauk, 1982; Poulos & Mauk, 1983), NADHrcytochrome b Strittmatter,  (Margoliash &  reductase  and  NADPHxytochrome  P-450  reductase (Dailey &  5  reductase  (Dailey  &  Strittmatter, 1980). The charge complementarity  between  cytochrome c and cytochrome b  is thought  s  to stabilise  the complex that forms between these proteins in a manner analogous to the physiological examples. Salemme (1976)  proposed  a model of the  complex formed  by  these two proteins  structural coordinates of tuna cytochrome c and bovine microsomal cytochrome b  5  intermolecular  charge  and  steric  least squares fitting process. be coplanar, in the  final  interactions  between  these two  proteins  were  based  on  (Figure 6).  The  optimized using a  Aldiough the hemes of the cytochromes were initially constrained  model they lie at an angle of 15° to each other.  charge pairs in the model are listed in Table I.  the  to  The complementary  The closest approach of die TT bonded atoms of the  respective prosthetic groups is 8.4 A (iron to iron distance of 18 A ) . Evidence  supporting  this  model  has  come  from  several  laboratories.  reduction of ferricytochrome c was shown to be strongly ionic strength al.,  1979), suggesting  that the  reaction is mediated by complementary  Ferrocytochrome  dependent (Stoneheumer charge  interactions.  studies were performed with microsomal preparations containing cytochrome b and NADHxytochrome 5  35  b  5  et  These  Figure 6 : The structure of the cytochrome ^cytochrome c complex as proposed by Salemme (1976). The heme atoms are shown in red, the side chains of the negatively charged cytochrome b  5  positively  charged  residues  of cytochrome c that are proposed to form  shown in orange and blue respectively.  specific salt  and the  linkages are  This image was generated with the Raster3D package (D.  Bacon, University of Alberta) on a Silicon Graphics IRIS 3130. were provided by Dr. James B. Matthew.  36  The coordinates for this structure  Figure 7 : The structure of the edge off cytochrome ^cytochrome  c complex (Mauk et al.,  1986).  The image was generated with the Raster3D package (D. Bacon, University of Alberta) on a Silicon Graphics IRIS 3130.  The colour scheme is the same as that used in Figure 6.  this structure were provided by Dr. James B . Matthew.  37  The coordinates for  Table  I  :  Charged  groups  diat  form  specific salt  linkages  in computer  graphic  models  of  the  cytochrome ^cytochrome c complexes (Salemme, 1976; Mauk et al., 1986).  Edge on model  Off edge model  cytc  cyt b$  cyt c  cyt fr  Lys-13  Glu-48  Lys-72  Glu-48  Lys-27  Glu-44  Lys-79  Glu-11  Lys-72  Asp-60  Lys-86  Glu-56  Lys-79  heme propionate-6  Lys-87  Asp-60  Lys-25  GIu-43*  5  * Although not proposed in die original Salemme model, diis salt linkage has been suggested by the work of N g et al. (1977), Rodgers et al. (1988) and Holloway & Mansch (1988).  b  s  reductase and  ferricytochrome formation  of  low enough  c a  by 1:1  ultracentrifugation.  concentrations  ferrocytochrome complex  b  between  5  of modified cytochromes  was  rate  cytochromes  A dissociation constant of 20  limiting. b  5  and  These c  in  to  evaluate  /^cytochrome complementary  die  role  c complex. charge  pairs  of  specific  audiors  vitro  by  also gel  reduction of detected  die  permeation  and  (0.01 M Tris-HCl, p H 7.5, 25°C) was measured.  Derivatives of cytochrome c, modified at individual lysine residues, system  c so that the  charge  pair  interactions  The same lysines of cytochrome c with carboxyl groups  (cytochrome c Lys-25 to Glu-43 of cytochrome b ), s  were used in the same kinetic in  diat  on cytochrome b , 5  stabilizing  the  cytochrome  Salemme postulated in addition to a  were implicated (Ng et al.,  to form fifth  one  1977; Smith et al.,  1980). Using difference absorption spectroscopy, Mauk et al. (1982) were able to detect a cytochrome ^cytochrome c complex with a 1:1  stoichiometry.  The association constant  of this complex was  shown to be strongly dependent on ionic strength, decreasing from 4 ( ± 3 ) x 10 M " at I = 0.001 M 6  38  1  to 8 ( ± 4 ) x 10  M  at I = 0.01 M , providing more evidence of the role of complementary charge  pairs in the formation of a complex between these proteins. which is midway between the p i of the two proteins.  The optimum p H of interaction was 7.5,  Further studies using absorption spectroscopy  have expanded and refined the view of the precursor complex. b  To evaluate the role of cytochrome  heme propionate-6 that in the interaction with cytochrome c, the stability of the complex formed  s  between cytochrome c and DME-cytochrome b (Mauk et al., complex  1986).  formed  with  5  was determined under a variety of solution conditions  As with the complex formed between the native proteins, the stability of the DME-cytochrome  b  5  was  found  to  be  strongly  ionic  strength  dependent.  However, the association constant for the DME-cytochrome ^cytochrome c complex [3 ( + 1) x 10 M"  1  (I =  0.005 M , p H 8.0, 25 °C)] exhibited a higher optimal p H (8.0) and was actually slightly  greater than  that of the native proteins  [7 ( ± 3 ) x  Electrostatic  calculations  graphic  describes neutral  6  the pH.  and  computer  10  5  M  modelling  - 1  (I  =  0.005  confirmed  protein-protein orientation that is electrostatically favoured However, a  second  docking geometry,  referred  to  as  that by the  M , p H 7.5, the the  25°C)].  Salemme  native proteins  off-edge  complex,  predicted to be favoured at higher p H and by elimination of the charges of the cytochrome b propionates  (Figure 7).  The surface  residues  proposed salt bridges of diis second geometry  of cytochromes are  b  5  and  c  listed in Table I.  model at was heme  5  that participate  in  the  In the off edge docking  geometry, the distance between the iron of cytochrome b and the iron of cytochrome c is 26 A . s  Recently,  it has become apparent that the residues of cytochrome b  5  that were proposed  participate in specific salt bridges were based on incorrect sequence information (vide supra).  to The  original model proposed a salt bridge between Lys-79 of cytochrome c and residue 13 of cytochrome b. 5  Residue 13 was originally thought to be glutamate but the sequence of the cytochrome b  s  has since indicated that residue 13 is glutamine (Cristiano & Steggles, 1989). can form a hydrogen bond with cytochrome c Lys-79. close to residue 13 in the primary sequence. graphics terminal indicates that residue  Cytochrome b  5  gene  Gln-13  Alternatively, Glu-11 of cytochrome b  5  is  Visual examination of this substitution on a molecular  11 is considerably further  39  away than residue  13 from  the  proposed interface of die two proteins in die off edge docking geometry,  and that an interaction  with Lys-79 of cytochrome c would only be possible after considerable side chain rearrangement.  A  more thorough reassessment of alternate docking orientations is in progress. *H N M R measurements also detect the formation of a 1:1 complex between cytochromes b c (Eley & Moore, 1983).  The lower association constant that these workers measured (10  be reconciled with the higher ionic strength used in the N M R experiment. that the p K of a cytochrome b c.  3  5  and  M " ) can 1  These data also suggest  carboxyl group decreases upon complex formation with cytochrome  5  With the assignment of the heme resonances of cytochrome b  5  (McLachlan et al.,  1986), this  group was identified as heme propionate-6, which is the group that in die crystal structure is showm to extend into the solvent.  This finding is consistent with the participation of cytochrome b  propionate-6 in a salt linkage with Lys-79 of cytochrome c.  5  heme  It is also possible that die pKg of the  heme propionate is lowered in the complex by a reduction of the negative electrostatic potential of its  environment  that  would  be  experiments (Hartshorn et al., chromium (ethylenediamine)^  3  expected  to  1987) suggest  accompany  binding  that cytochrome b  when cytochrome b  s  to  cytochrome  c.  Subsequent  heme propionate-6 is free to bind  5  is bound to cytochrome c.  This result suggests  that heme propionate-6 is not involved in a specific salt linkage with a corresponding group on the surface of cytochrome c or that the additon of the chromium compound induces a different proteinprotein docking geometry, in which heme propionate-6 is free to bind exogenous cations. edge complex is compatible with the availability of cytochrome b Using  site directed mutagenesis,  residues of cytochrome b with  cytochrome c  cytochrome  c  spectroscopy formation.  was to  to  s  heme propionate-6 in this maimer.  Rodgers and coworkers (1988) converted each of the acidic  that the Salemme models predicts to be involved in a specific salt linkage  the  corresponding amide.  monitored  determine  5  The off  under  free  hyperbaric  energies  and  Complex formation between conditions  specific  using  volume  electronic  changes  these mutants  absorption  associated  with  and  difference complex  The linear free energy relation between A G and P A V observed in this study suggested  that a major  contribution to the  volume change  40  accompanying complex formation is provided by  these salt linkages.  The results of these experiments confirm the modelling studies of Mauk et al.  (1986).  It would be interesting do a similar study to determine  mutations  on the  stability of and volume changes  the effect  of a wider range of  accompanying die formation of the  cytochrome  fe cytochrome c complex. 5  Fourier  transform  infrared  spectroscopy  (FTIR)  measurements  indicate  that  3  glutamic  acid  residues  of cytochrome b  1988).  Although the original cytochrome ^cytochrome c model complex (Salemme, 1976) implicated  two  are involved in salt bridges with cytochrome c (Holloway & Mansch,  s  glutamate  residues  of cytochrome  b  in this  s  role, the  FTIR  finding  is" consistent  with  the  involvement of Glu-43 of cytochrome b  in the formation of a salt linkage with a surface residue of  cytochrome  al.  5  glutamic transfer  c,  acid  as proposed residues  partners  in  (Dailey  by the  &  Ng et  formation  Strittmatter,  (1977). of  salt  1979;  Chemical bridges  1980).  modifications implicate these three  widi The  three  other  participation  physiological electron of  Asp-60 and  heme  propionate-6 of cytochrome b in complex stabilization was not investigated in the FTIR study. s  Molecular dynamic simulations have been reported for die complex formed by cytochromes and c (Wendoloski et al.,  1987).  These calculations took die edge on docking geometry  b  5  of the  complex (Salemme, 1976) as the starting orientations of the cytochromes and suggest that within this one docking conformation, the side chains of the protein residues can rearrange, more  favourable  to  electron  transfer.  Rearrangement  of amino  acid  creating a structure  side chains  at  the  protein-  protein interface predicted by these calculations resulted in inter-iron distances that are 1.1 to 2.1 A smaller than in the Salemme model.  In addition, they predict that phenylalanine 82 of cytochrome c  is reoriented so diat it bridges the two hemes in the complex and actually moves back and forth between the heme groups of the two proteins widiin the complex. Despite  the  increased  attention  directed  toward  characterization  ^cytochrome c complex, several critical issues remain to be elucidated. the  exact  identity  of which  residues  are  involved  in salt  linkages,  (2)  of  die  cytochrome  These issues include (1) the  contribution of non-  electrostatic forces to the stability and docking geometry of the complex, (3) whether an alternative  41  docking geometry  or microheterogeneity  of the docking geometries  in the cytochrome  ^cytochrome  c complex can be detected, and (4) the relevance of alternate docking geometries of the complex to electron transfer.  Objectives  To  characterise  ferrocytochrome b  the  protein, 5  mechanism  of cytochrome b  particular  cytochrome b  the  of  interaction  and  electron  transfer  and ferricytochrome c, several biophysical studies were undertaken.  s  heme propionates  better  effort  was  directed  in these processes.  derivative of cytochrome b  create such a prominent  5  at  evaluating  Because the  feature in the electrostatic  the  role  of  the  heme  surface of  propionates  of  The role of these groups has been assessed dirough the use of a  in which the native heme  5  between  was substituted  widi a dimethyl esterified  heme (DME-cytochrome b ). s  The interaction between cytochromes b  and c was studied by fluorescence quenching titrations  5  of  porphyrin  c,  a  fluorescent  measurements yielded energy of  transfer  the  stoichiometry,  theory  cytochrome  measurements were  analogue  (Forster, b  performed  cytochrome  c,  association constants and  1948) was used to estimate  heme  5  of  and under  the  cytochrome  c  with  ferricytochrome  b.  These  values.  Forster  the relative orientation and  separation  maximal quenching  porphyrin  within  the  5  complex.  a variety of solution conditions to evaluate  the  These  influence of  ionic strength and p H on the stability and die conformation of the complex. The complex that forms between cytochromes b  5  kinetics  of  approach other  reduction  has  provided  complexes  environments  tiiat  near  performed  components  of the  information regarding has  the  (Bhattacharya et al., were  of the  been  interpreted  prosthetic  groups  and c was studied further  complex by  the  rate of electron  in terms of prosthetic and  dynamic  1986, 1987; Hazzard et al., 1986, 1987).  with  die  neutral  various  lumiflavin  semiquinone 42  motions  flavin  transfer group within  by measuring  semiquinones. to  and  within  accessibility, the  the This  several  electrostatic  protein  complex  In the present study, reduction kinetics and  the  negatively  charged  flavin  mononucleotide semiquinone. cytochrome b  5  Experiments were repeated at pH 7 and pH 8 with native and D M E -  in an attempt to detect any difference between the reactivities off edge and the edge  on conformations that may be present under different conditions (Mauk et al., 1986). The final native and strength,  set  of experiments  involved  DME-ferrocytochrome b . 5  p H , and  protein-protein  temperature  to  association constant  measuring  These permit  the  experiments  correlation  of  rate of ferricytochrome c reduction by were performed the  kinetic  as  results  a function of with  measurements under similar solution conditions.  the  ionic  restdts of  The results of  these studies provide a more detailed understanding of the mechanism of cytochrome ^cytochrome c recognition and the mechanism of electron transfer from ferrocytochrome b to ferricytochrome c. 5  43  EXPERIMENTAL PROCEDURES  General Procedures  Glass distilled  water  purified with a Barnstead  Mohms was used in all experimental work.  NANOpure  system  to a resistivity of  A l l p H measurements were performed widi a Radiometer  Model P H M 84 meter equipped with a Radiometer Type G K 2321C combination electrode. where  noted,  reagent  16-17  grade chemicals were used.  Phosphate  buffers  Except  of specified p H and  ionic  strength were prepared from monobasic and dibasic sodium salts. Most of the spectrophotometric spectrophotometer holder,  and  equipped  a  with  thermostatted  measurements were performed on a Shimadzu UV-250 recording  an  OPI-2  ( ± 0 . 1 °C)  Optional  Program  interface,  a  circulating Brinkmann R M 3 water  absorption spectra were collected on a Cary-219 spectrophotometer  thermostatted bath.  The  cuvette electronic  interfaced to a Zenith ZW-248-82  microcomputer by On Line Instruments Systems, Jefferson, Georgia, U . S . A . (OLIS).  Data acquisition  and manipulation were performed with OLIS model 4000 Data System Spectrophotometry version 7.05 software. The  tryptic  fragment  of  bovine  hepatic  cytochrome  b  5  and  its  dimethylesterheme-substituted  derivative were prepared as described previously (Reid & Mauk, 1982; Reid et al.,  1984).  Horse heart  cytochrome c (Type VI) was purchased from Sigma Chemical Co. and purified by anion exchange chromatography concentrated  to remove deamidated cytochrome (Brautigan et al.,  by  ultrafiltration  using  Amicon-52  Centripreps and Centricons (Amicon). 106.1 m M  - 1  and  « 412.5 =  stirred  cells  1978c). fitted  Protein solutions were  with  YM-5  membranes,  Protein concentrations were determined using values of  £410=  117.0 m M " for ferricytochromes c (Margoliash & Frohwirt, 1959) and 1  (Ozols & Strittmatter, 1964) respectively.  44  b  5  Interaction of Porphyrin Cytochrome c with Native and DME-Cytochrome b  5  Preparation of Protein. Porphyrin cytochrome c was prepared as described previously (Kornblatt & English,  1986) from horse heart cytochrome c, with the assistance of Prof. Jack A . Kornblatt at  the University of Concordia, Montreal. possible.  Porphyrin cytochrome c was protected from light as much as  The extinction coefficient of porphyrin cytochrome c was determined by both the Bradford  assay (Bradford, 1976) and by amino acid analysis.  The Bradford assay was performed with a kit  purchased from Bio-Rad Laboratories, using horse heart cytochrome c as the standard. method  entailed  absorbance. that  of  determining  the  amino acid composition of a sample  The second  of porphyrin c of known  The amino acid composition of porphyrin cytochrome c was assumed to be identical to  horse  heart  cytochrome  c  The  extinction coefficient  calculated by averaging the values predicted for each residue.  of  the  porphyrin derivative  was  Amino acid analyses were performed  on a Durrum Amino Acid Analyzer Model D-500 with the assistance of Dr. Robert L . Cutler.  The  extinction coefficient at 505 nm and the relative fluorescence emission of porphyrin cytochrome c were measured from p H 6.0 to 8.0 (I = 0.01 M , 25°C). Binding  measurements.  Fluorometry  measurements  fluorometer interfaced to an IBM-PC A T microcomputer.  were  wavelength.  The absorbance  widi  an  S L M SPF-500C  A l l measurements were performed at 25 °C.  Fluorescence samples were excited at 505 nm (1 nm bandpass) 624.5 nm (20 nm bandpass).  made  and die emission was recorded at  of the sample never exceeded 0.1 at the excitation  Binding measurements were performed over a p H range of 6 to 8 and an ionic strength  range of 2 to 50 m M in sodium phosphate buffer.  To an appropriately buffered solution of porphyrin  cytochrome c (~2 i*M) was added 15 to 25 10 / i L aliquots of a similarly buffered stock cytochrome b solution (~50 b. s  /xM).  5  The fluorescence of the sample was recorded after each addition of cytochrome  To minimise die exposure of the sample to light, the excitation and emission shutters were  closed between measurements,  and the room lights were shut off while the sample compartment  open.  45  was  Inner filter correction and quenching values were calculated according to the formulae derived by Hwang and Greer (1979). program (Duggleby, 1984). and  Binding data were fitted to equation (6) using a nonlinear regression In this equation, B and C represent the concentrations of cytochrome b  porphyrin cytochrome  c  respectively, Q  is the  degree of quenching  observed,  Q  m a x  is  5  the  quenching at infinite cytochrome fc concentration, and K , is the binding constant. 5  Qmax  1  Q =  1 ((C + B + — ) K, . K,  (C + B + 2B  Energy transfer amounts of  measurements.  cytochrome  b  5  to  2  - 4BC)*)  [6]  Maximal quenching values, obtained by addition of saturating  solutions  conditions of p H and ionic strength  of porphyrin cytochrome  were  determined  under  the  The theory of Forster (1948)  was applied to  calculate the distance of separation between the porphyrin groups in the complexes.  Based on this  theory,  the  characteristic  distance  listed above.  c,  of  separation  between  the  donor  fluorophore  and  the  acceptor  chromophore at which 50% quenching of the fluorescence signal is observed, R , may be calculated 0  9000 In 10 re $ J 2  Ro =  PI 128ir « N 6  according to equation 7, where «  2  4  is the orientation factor, $ is the quantum yield of the donor, J  is the overlap integral, n is the refractive index of the intervening medium, and N is Avogadro's number. 1980).  The quantum yield and K  2  were taken to be 0.01 and 2/3 respectively (Vanderkooi et al.,  As the intervening medium can be approximated by a light o i l , n is given a value of 1.4.  The overlap integral was evaluated between 560 nm and 760 nm from the electronic spectra of the f (v)E (v) d  a  J = /  dv v  two proteins using equation 8, in which f wavenumber v and E  a  d  [8]  4  is the fraction of total donor fluorescence occurring at  is the corresponding extinction coefficient of the acceptor.  With these values,  the separation, R, from the efficiency of energy transfer, Q ^ , may be calculated using equation 9.  46  R = R (  1-Qn  Q  )  [9]  1 / 6  Qn  Polarization measurements. cytochrome c in the presence  Fluorescence anisotropy measurements were performed on porphyrin and absence of cytochrome b .  The measurements were  5  performed  with an S L M Aminco SPF-500 polarization accessory at an ionic strength of 2 m M , p H 7.0, 2 5 ° C . The equations used in analysing these data are given in appendix A .  Kinetics of Flavin Semiquinone Reduction of Cytochrome bS and Cytochrome c  The kinetics of flavin semiquinone reduction of the components of the 1:1 complex formed by cytochrome c with either cytochrome b  5  or DME-cytochrome b  were performed at the University of  s  Arizona with the collaboration of Profs. Michael A . Cusanovich, Gordon Tollin and Dr. James T. Hazzard (Eltis et al.,  1988).  Tuna cytochrome c (Type XI) was purchased from Sigma Chenucal Co.  and purified by anion exchange chromatography (Brautigan et al.,  1978d).  Lumiflavin was prepared  as described by Guzzo and Tollin (1963) and F M N purchased from Sigma Chemical C o . was purified by size exclusion chromatography on a BioRad P2 column (Nagy et al., 1982). Electron transfer was initiated by a 300 ps pulse of light from a nitrogen laser (PRA LN100) pumped dye solution (10 m M C-450 from P R A , Inc. in ethanol) in a 1 cm path length cuvette.  The  dye solution has an emission maximum at 450 nm which is close to the absorption maximum at 442 nm of lumiflavin and F M N . monochromator  A quartz-iodide projection lamp focused through a Bausch & Lomb  onto the sample and then onto an R C A 4463 photomultiplier tube (S-20  comprised the monitoring beam.  response)  A minimum of four decay curves were averaged on a Nicolet 1170  signal averager (Simondsen & Tollin, 1983). The reaction mixtures, containing 50 / x M flavin and 0.5 m M E D T A in phosphate buffer (total ionic strength Scientific),  of 4 m M ) , were made up in 3 m L cuvettes,  and bubbled for 30 minutes  sealed widi rubber stoppers  with argon that had passed 47  (Thomas  through an oxygen scrubbing  solution (Stone & Hume, 1939) to remove dissolved oxygen. introduced  with  additional  five  gas  tight  syringes,  minutes.  and  argon  was passed  The photo-generated  flavin  The appropriate cytochromes were then over  triplet  top  of the  solution for  state in the  reaction  mixture  with the E D T A to produce the flavin semiquinone (Ahmad et al.,  the  1982).  an  reacted  Under these conditions, less  than 0.1 i t M of semiquinone was produced. Experiments  were  conducted  at  24°C  at  p H 7.0  and  8.0.  The  reduction of horse  heart  ferricytochrome c by LfH- and F M N H - was studied in the absence and presence of ferricytochrome b  5  as a decrease in absorption at 565 nm, an isosbestic point in die cytochrome b  5  difference evidence observed. the  spectrum. of  Cytochrome  cytochrome  b  5  c  reduction  was prior  preferentially to  or  reduced  coincident  The reduction of D M E - and native ferricytochrome b  5  presence  and  ferricytochrome b  s  absence  of  ferrocytochrome  c.  In  under with  these  oxidation-reduction  conditions,  cytochrome  c  and  reduction  no was  by LfH- and F M N H - was studied in  experiments  involving  die  bound to ferrocytochrome c, the cytochrome c was photoreduced  reduction  of  by steady-state  illumination of the flavin in the reaction mixture prior to the anaerobic addition of ferricytochrome b, s  and  the  reaction  was  monitored as a decrease in the  absorption  at  580 nm.  The protein  concentrations were sufficiently high (>  5 /*M) to ensure pseudo-first order conditions relative to the  concentration of semiquinone generated  (vide supra) and to ensure that flavin disproportionation did  not compete with protein reduction.  Kinetic experiments were performed at 6 protein concentrations  by adding successive samples (5 - 10 /xL) of stock cytochrome solutions to the reaction mixture with a gas tight syringe. data.  First order rate constants, k  o b s  These were linear for at least 4 half lives.  linear plots of k  o b s  versus protein concentration.  , were derived from semilog plots of the kinetic Second order rate constants were calculated from  On the basis of multiple determinations, the error  in the second order rate constants was estimated to be ± 10%. The solvent accessible surface areas of the hemes of cytochromes b  5  and c, unbound and in  each of the proposed cytochrome ^cytochrome c complex docking geometries, an algoridim that uses the coordinates of the structure,  48  were calculated with  van der Waal's radii of the atoms and a  spherical solvent probe with a radius of 1.4 A (Connolly, 1983).  Ferrocytochrome b Reduction of Ferricytochrome c 5  Rapid mixing experiments were performed with a stopped flow spectrophotometer design of Gibson and Milnes (1964) as marketed by Dionex (Model D-103). reactant delivery system (Scott, 1984).  based on the  Modifications to the  1980) and syringe plungers were described previously (Reid,  To achieve greater anaerobicity of the reacting solutions, the entire reactant flow  1982; system  and the photomultiplier tube were mounted in a Vacuum Atmospheres C o . Model HE-243-2 glove box equipped with a HE-63-P pedatrol, a HE-493 Dri-Train and a Sargent Welch Scientific Co. model 1402 vacuum pump.  A similar apparatus has recently been described (Leung et al.,  1989).  Light from an  Osram Xenophot H L X 64250 lamp passed through a J - Y Model DH-10 dual grating monochromator residing outside the glove box was delivered to the stopped flow cuvette by a fibre optic bundle. The drive syringes and stopped  flow  cuvette  were bathed  with water  thermostatted  Model T-41 water bath and circulated by a Gorman-Rupp Industries M877 pump. filled with prepurified argon and maintained at less than 5 ppm oxygen.  with a Haake  The glove box was  Although water was added  to the cooling bath immediately prior to the experiment and removed immediately afterward,  the use  of  frequent  an  open  water  bath  for  the  thermostatting  of  the  drive  syringes  necessitated  regeneration of the catalyst. The remainder of the stopped flow apparatus was designed by OLIS. 3 amps by an OLIS XLI50 power supply.  The lamp was operated at  The output from an EMI-Gencome model RFI/QL-30F  photomultiplier tube was amplified by an OLIS amplifier equipped with a variable (0.05 - 100 ms) time constant.  The data acquisition and instrument  control signals were collected and manipulated  with OLIS model 3920Z Data System Stopped Flow version 6.12 software run on a Zenith Z-100 microcomputer.  The apparatus is shown in Figure 8.  determined to be approximately 7 ms.  49  The dead time of the stopped flow  was  Lamp  Microcomputer  Monochromator Water bath  Signal amplifier  Fibre optic cable  B  Water pump  Argon  Copper tubing  Glove box  4  Reactant flow system Antechamber  CH  Photomultiplier tube  HE-493 Dri-traln  Butyl rubber glove  Figure 8 : Stopped-flow spectrophotometer  mounted in a glove box.  (A) The fibre optic cable was  introduced into the glovebox through a port in the side of the box.  Outside of the glove box, the  cable was sealed in a stainless steel bellows.  (B) The cables for the photomultiplier tube signal,  power and the stopped-flow trigger (labelled 1, 2 and 3 respectively) and the argon gas line for the pneumatic  flow  actuator plunger  fourth glove port.  were  introduced through  a modified steel  plate  mounted  on  the  (C) The copper pipes for the cooling water were introduced through ports in the  top of the glove box.  50  The reduction of ferricytochrome c by ferrocytochrome b  5  conditions with the reactants at equimolar concentrations (~1.7 by  the  addition  prepared  as  of  an  described  excess in  of  was performed under  A*M).  Ferricytochrome c was prepared  ammonium bis(dipiciolinato)cobaltate(III)  Schenkein  et  al.  (1977).  Excess  second order  (Co(dipic) ), previously 2  Co(dipic)  was  2  removed  from  ferricytochrome c by passing the sample down a 10 x 0.5 cm column of Sephadex G-25, superfine equilibrated with the buffer in which stopped flow experiment was to be performed.  The protein  solutions were prepared in glass serum bottles that were sealed with cleaned rubber serum and secured  with copper  wire.  The solutions were degassed  for 30 minutes  under  stoppers  a stream of  prepurified argon that had been passed through a 20 cm column of Chemalog Catalyst R3-11 and a water bubbler.  After 30 minutes, 0.2 equivalents of lumiflavin from a 200 i * M stock (~40  introduced  the  into  reduced by steady  cytochrome  b  s  solution with  a gas  tight  syringe.  state illumination of tiiis solution for 30 minutes.  itL) was  Ferricytochrome b  5  The needle punctures  serum caps were then covered with electrical tape, and the serum bottles were transferred glove box.  The antechamber  was  in the into the  was evacuated for a minimum of 1 hour after the introduction of an  aerobic atmosphere. The oxidation of ferrocytochrome b  5  the  change  monitored  in absorbance at  416  ferrocytochrome b  s  nm,  was monitored at 428 nm, where the total absorbance and  of cytochrome c are an  (Figure 9).  isosbestic  point  small.  in  the  The reduction of ferrocytochrome electronic  absorption  spectra  of  c was  ferri-  and  A minimum of 5 decay curves were averaged at each wavelength.  The data were transferred to an I B M - P C A T microcomputer where they were fit to a second order rate equation (10) using M I N S Q , a least squares fitting program (MicroMath Scientific Software). In AQ -  Aoo  A =  [10]  t  1 + kc t 0  this equation, A is the absorbance at time t, A Q and Aoo are the initial and final absorbances of the t  protein solution respectively, CQ is the initial concentration of each cytochrome species and k is the second  order  rate constant.  At  the  lower  ionic 51  strengths,  the  initial  portion  of  the  reaction  Figure 9 : Simulated difference spectrum of cytochrome b  5  the  sum of the  molar extinction coefficients  ferricytochrome  c  minus  ferricytochrome  b  and  s  the  sum  of  ferrocytochrome  the c.  and cytochrome c.  The curve represents  of an equimolar mixture of ferrocytochrome extinction  coefficients  Electronic absorption  used in this simulation are presented in Figures 3 and 5.  of spectra  an  equimolar  of the  52  5  mixture  respective  and of  species  The arrows indicate the wavelengths (416  nm and 428 nm) at which ferrocytochrome b reduction of ferricytochrome c was monitored. 5  b  occurred within the dead time of the instrument.  In these cases,  die initial concentration of die  cytochromes (c ) was determined by normalising the overall change of absorbance  (AA )  0  tot  to  AA  t o (  observed for the reaction performed at high ionic strength. The second order rate constants obtained as described above were compared with rate constants obtained by two alternative methods. under  pseudo-first  ferrocytochrome  order  b  (1  5  measurements owing  conditions /*M).  In the first of these methods, with  Greater  ferricytochrome excess  of  c  in 5  cytochrome  to the high background absorbance  inherent  and to the limited time resolution of the stopped flow technique.  to c  die reactions were performed 30  fold  molar  could  not  be  excess  used  at such cytochrome  generating  lumiflavin.  ferrocytochrome  b  by anaerobic  5  reduction with  these  concentrations  In die second alternative method,  the second order rate constants were measured under conditions of equimolar reactant but  in  over  concentrations  sodium dithionite rather than  Several grains of dithionite were added to a sample (~10  with  itL) of cytochrome b .  The  5  dithionite was removed by passing the sample over a small Sephadex G-25 column in the glove box. Ferricytochrome b  s  was collected in a serum bottle containing  concentration of ferricytochrome b  5  was determined  this solution to an anaerobic cuvette.  12 m L of anaerobic  spectrophotometrically  buffer.  by transferring  The  1 m L of  Both control kinetic experiments were performed at I =  0.5  M , p H 7.0, 2 5 ° C . Ferricytochrome c reduction of ferrocytochrome b  5  was studied over a p H range of 6.0 to 8.0,  an ionic strength range of 0.1 to 1.0 M , and a temperature range of 4 ° C to 32°C. activation  parameters  dependence (1983).  data.  of  The  the  reaction  were  ionic  strength  dependence  In this analysis, Z j and  the components cytochrome b  5  calculated  from  an  was analysed  Eyring by the  plot  Thermodynamic  of  method  the  temperature  of Van Leeuwen  are the monopole charges of die proteins, and P[ and P  of the dipoles of the proteins  through  their respective exposed  heme  2  edges.  the heme edge was taken to be the 7 carbon of die methylene bridge between  are In the  heme pyrroles with the propionate substituents and in cytochrome c the heme edge was taken to be the methyl carbon of the most solvent exposed heme pyrrole. Rj and R are the radii of die two 2  53  q  ln(k) = ln(koo) - ( Z , ^ + ZP(1 + §R) + PP(1 + §R) )  2  f(§)  2  [11]  411« e k T R 0  Z P X  2  B  + Z2P1  ZP =  [11a] qR P,P  2  PP=  [lib] (qR)  2  R = R,+R  [11c]  2  l-2e $ 2 2  R  f(§)=  [Hd] 2 § R ( 1 + §R,) 2  § = 0.329(1)* A ' (at 294 K)  [1 le]  1  proteins, q is the elementary  charge,  e  0  is the dielectric permittivity, e is the dielectric constant  of water, and k is Boltzmann's constant. B  The monopoles and dipoles of the proteins were calculated on a Silicon Graphics IRIS 3130 computer using Brookhaven data bank coordinates of ferric cytochromes b  5  program Northrup.  (Northrup et al.,  and c with a F O R T R A N  1986) generously provided to Prof. Gary D . Brayer by Prof. Scott H .  This program used a C H A R M M library to assign a charge and mass to each non hydrogen  atom in the protein structure. ferrous heme only.  The C H A R M M library contains the charge and mass distribution for a  Dipoles for the oxidised and reduced cytochromes were calculated from the same  structures,  but the hemes of the oxidised proteins were assigned an additional positive charge, 75%  of  was assigned  which  nitrogens. removing  to the  iron and the  remaining 25% was distributed amongst  The dipole of porphyrin cytochrome c was calculated from a structure the  ferrous  atom  from  the  ferrocytochrome  c  structure  and  the pyrrole generated  redistributing  the  density of the iron (0.24), and the mass of two hydrogen atoms, over the pyrrole nitrogens. the dipole of DME-cytochrome b  s  was calculated from a structure  generated  charge Finally,  by placing a methyl  group on the least sterically hindered oxygen of each of the heme propionates of the cytochrome structure. A partial charge was assigned to the various atoms based on the dipoles of the ester bonds. 54  by  b  s  RESULTS  Interaction of Porphyrin Cytochrome c with Native and DME-Cytochrome b  5  The extinction coefficient of porphyrin c at 505 nm was determined to be 13.1 ( ± 0 . 2 ) m M " (I = 1  0.01 M , p H 7.0, 25°C) by the Bradford assay, and 12.9 ( ± 0 . 6 ) m M " (I = 0.01 M , p H 7.0, 25°C) by 1  amino acid analysis.  The value of 13.1 m M " was used in subsequent analysis. 1  The extinction  coefficient at 505 nm was found to vary from 12.6 m M " at p H 6 to 14.0 m M " at p H 8.0 (Table II). 1  1  At p H 7, the absorption spectrum of porphyrin cytochrome c was found to be essentially invariant with ionic strength.  The appropriate  value was used to determine  the concentration of porphyrin  cytochrome c in the analysis of the binding curves determined at various values of p H . The emission spectrum of die porphyrin cytochrome c synthesized and used in this study shown in Figure 10. 11.  is  The p H dependence of the fluorescence emission spectrum is depicted in Figure  The fluorescence intensity of porphyrin cytochrome c is considerably greater at p H 6.0 than at  p H 7.0. The fluorescence anistropy of porphyrin cytochrome c was determined to be 0.048, while in  Table II : The extinction coefficients of porphyrin cytochrome c at 505 nm at the pH's used in this work.  pH  « (mM" )  6.0  12.6 ± 0 . 6  6.5  12.7 ± 0 . 5  7.0  13.1 ± 0 . 5  7.5  13.5 ± 0 . 5  8.0  14.0 ± 0 . 7  1  55  450  525  600  675  750  Wavelength (nm)  Figure  10  :  The  visible  absorption  and  uncorrected  fluorescence  cytochrome c (1 = 0.01 M sodium phosphate, p H 6.0, 25 °C).  emission  spectra  porphyrin  The sample was excited at 505 nm.  fluorometer settings of the excitation and emission bandpasses were 1 and 20 nm respectively.  56  of  The  540  600  660  720  780  Wavelength ( n m )  Figure  11 : The p H dependence  of the fluorescence  intensity of porphyrin cytochrome c in the  absence (panel A ) and presence (panel B) of cytochrome b  5  concentration of porphyrin cytochrome c was 5.0 i t M . cytochrome c and cytochrome b  5  (I = 0.002 M , 25°C).  In panel B , the concentrations of porphyrin  were 0.74 nM and 3.6 (iM respectively.  not corrected for instrument response or the inner filter effect.  57  In panel A , the  The emission spectra are  the presence of saturating amounts of cytochrome b it was 0.084. 5  Figure 12 depicts a fluorescence quenching titration curve of porphyrin cytochrome c by D M E cytochrome b .  The curve was generated  s  porphyrin cytochrome c (2 nM). analyzed by equation 5.  by normalizing the data to a constant  concentration of  In general, the data were not normalized in tiiis way, but were  In all cases, quenching was completely reversed at the end of a titration by  the addition of 100 /zL of 3.3 M sodium chloride. The association constant  for the formation of the cytochrome 6 porphyrin cytochrome c was 5  strongly dependent on ionic strength, decreasing from 3.3 ( ± 0 . 4 ) x 10 25°C) to 1.4 ( ± 0 . 5 ) x 10 M " (I = 0.05 M , p H 7.0, 25°C)(Figure 13). 5  association constant  for  1  the  6  M " (I = 0.002 M , p H 7.0, 1  Under the same conditions, the  formation of the DME-cytochrome ^ p o r p h y r i n cytochrome c complex  [1.2 ( ± 0 . 3 ) x 10 M " (I = 0.002 M , p H 7.0, 25°C)] was slightly lower than that of native cytochrome 6  b  5  and  1  porphyrin cytochrome  c.  The association constant  of the  DME-cytochrome & porphyrin 5  cytochrome c complex was too small to measure by this technique at ionic strengths of 0.05 M and greater. fitting  The curves in Figure 13 represent fits of the data to equation 11. routine  (MINSQ)  failed  to converge after  As the least squares  100 iterations when all seven parameters in the  equation were allowed to vary simultaneously, two different fits were performed. monopoles and dipoles, as calculated from the crystal structures in the second, die radii and the dipoles were held constant. Table III.  In die first,  (Table IX) were held constant  the and  The results of both fits are shown in  The calculated association constants at infinite ionic strength  cytochrome ^ p o r p h y r i n cytochrome c complex and 50 M "  1  were  6000  M"  1  for  the  for the DME-cytochrome ^ p o r p h y r i n  cytochrome c complex. The p H dependence complex  varied with  the  of the association constant availability  of the  for the cytochrome ^ p o r p h y r i n cytochrome c  cytochrome b  5  heme  propionates  (Figure  14).  The  association constant of the complex formed between DME-cytochrome b$ and porphyrin cytochrome c was essentially independent of p H (I = formed by native cytochrome b  5  0.01 M , 25 °C).  The association constant for the complex  and porphyrin cytochrome c at the extreme  58  values of p H studied  Figure  12  :  Fluorescence titration curve of porphyrin cytochrome c  experiment was performed with cytochrome b fit of the data to equation 6. The value of Q„  ax  5  with cytochrome b .  (I = 0.002 M , p H 7.0, 25 °C).  represents an extrapolated value.  59  s  This  The curve represents a  0.00  0.08  0.16  Figure 13 : The ionic strength dependence of the association constant of D M E (•) cytochrome b  5  and porphyrin cytochrome c (pH 7.0, 25 °C).  Leeuwen equation (11), using the parameters given in Table III.  60  0.24  and native  (•)  The lines represent fits to die Van  1  I  1  I  I  6.0  1  7.0  1  1  1  1  1  8.0  PH Figure 14 : The p H dependence  of the association constant  native ( • ) or DME-cytochrome b ( • ) (I = 0.01 M , 25°C). 5  61  of porphyrin cytochrome c and either  was about half the magnitude that it was over the p H 6.5 to 7.5 range. The  maximal quenching,  Q  m a x  greatest at p H 6.0 (0.38 ( ± 0 . 0 1 ) ) ,  ,  of porphyrin cytochrome and decreases to a constant  porphyrin cytochrome c in the presence of DME-cytochrome b  5  strength of 0.01 M (Figure 15). native cytochrome b  s  cytochrome b ,  the Q  s  M  (Figure 16).  c  by  native  cytochrome  value at p H 7.0.  b,  was  s  The Q  m a x  of  was independent of p H at an ionic  In contrast, the Q , ^ of porphyrin cytochrome c in die presence of  was insensitive to ionic strength at p H 7, while in the presence of D M E m a x  of porphyrin cytochrome c increased with ionic strengdi, plateauing at 0.01  The value for the overlap integral (equation 8, the units of wavenumber were cm" ) 1  used in calculating the separation of the donor and the acceptor sites from Q  was 2.65 x 10" . 14  m a x  The value of the characteristic distance of separation (equation 7) was determined to be 15.3 A .  Table III :  Parameters used in fitting the ionic strength dependence of the association constants of  native and DME-cytochrome b widi porphyrin cytochrome c to the Van Leeuwen equation (11). 5  parameter  cyt ^ p o r p h y r i n cyt c  DME-cyt ^ p o r p h y r i n cyt c  L n Koo  7.7 (1.5)  8.7(1.6)  2(100)  4(20)  Ri  8(20)  17(f)  4(100)  17(f)  R  9 (20)  17(f)  8(25)  17(f)  - 9(f)  - 150(150)  - 7(f)  -300 (100)  2  Zl Z2  6.2(0  Pi  - 468 (f)  - 468 (f)  - 391 (f)  - 391 (0  255 (f)  255 (f)  255(f)  255 (f)  .06  .05  2  p  2  s.s.  .6(15)  S.S. refers to the sum of squares of the fit.  6.2 (f)  .3 (10)  1.4  The numbers in parandieses are errors; an "f" denotes  that the parameter was held constant.  62  Figure 15 : The ionic strength dependence of maximal quenching ( Q by DME-cytochrome b  5  (pH 7.0, T = 25°C).  m a x  ) of porphyrin cytochrome c  The quenching of porphyrin cytochrome c by native  cytochrome b showed no ionic strength dependence. 5  63  Figure 16 : The p H dependence of maximal quenching ( Q ^ ) ( • ) and DME-cytochrome b  s  ( • ) (I = 0.01 M , 25°C).  theoretical significance.  64  of porphyrin cytochrome c by native  The lines are drawn for visual aid and have no  Kinetics of Flavin Semiquinone Reduction of Cytochrome b and Cytochrome c. s  Pseudo-first order rate constants for ferricytochrome c reduction by LfH- at p H 7 and 8 as a function  of  protein  ferricytochrome b  concentration  in  the  presence  are shown in Figure 17.  5  plots are  given in Table I V .  unaffected  by  the presence  First,  the  Second,  binding  At p H 7, the rate constant  of native ferricytochrome b ,  native  of  and  of  stoichiometric  amounts  of  for ferricytochrome c reduction was  while it was decreased  5  rate constant to  absence  The second order rate constants obtained from these  complex formed with DME-ferricytochrome by effect.  and  by 35% in the  Increasing the p H to 8 produced two types of kinetic  ferricytochrome  c  reduction  DME-ferricytochrome  b  by  L f H ' increased  decreased  5  the  rate  substantially. constant  for  ferricytochrome c reduction by LfH- by 60% and 24% respectively. The  dependencies  reduction by F M N H amounts  of  the  rate constants  derived  0  for  horse  heart  on cytochrome c concentration in the presence  of native or DME-ferricytochrome b  constants  (k bs)  from  these  plots  are  5  listed  and  tuna  ferricytochrome c to native or DME-ferricytochrome b  5  IV.  c  and absence of stoichiometric  are shown in Figure 18. in Table  ferricytochrome  The second order  Binding  of  tuna  significantly decreases the  or  rate  horse  heart  rate constant of  cytochrome c reduction by F M N H - at p H 7 (by 73% and 60% respectively). The variations in observed rate constants for native and DME-ferricytochrome b LfH  -  conditions, the  DME-cytochrome  The  rate constant With  5  5  5  The corresponding second order rate constants are listed in Table V .  b.  ferricytochrome b b  reduction by  in the presence and absence of stoichiometric amounts of ferrocytochrome c (pH 7 and 8) are  shown in Figure 19. all  s  of reduction of native ferricytochrome b  an  increase  in  pH  from  7  to  8,  the  is lower than  5  rate  reduction by LfH- is unchanged while the rate constant  constant  for  Under that of native  for DME-ferricytochrome  is increased to an extent (~1.7 fold) similar to that observed for LfH- reduction of cytochrome c rate  constants  stoichiometric amounts  for  reduction  of  of ferrocytochrome  both c.  ferricytochromes  b  5  increase  For native ferricytochrome  in  b, 5  the  the  increase  reduction rate constant is more pronounced at p H 8, while for DME-ferricytochrome b , the increase s  65  presence  of  in the  [Cytochrome c] Figure 17 : Plots of k  o b s  versus cytochrome c concentration for the reduction of horse cytochrome c  by LfH- at p H 7 (A) and 8 (B) in the absence ( • ) and the presence of native ( • ) or DME-cytochrome b  5  (•).  66  [Cytochrome c] (pM) Figure 18 : Plots of k  o b s  versus cytochrome c concentration for the reduction of horse (A) and tuna  (B) cytochromes c by F M N H - in the absence ( • ) and the presence of native ( • ) or DME-cytochrome  67  Table I V : Second order  rate constants  for flavin  semiquinone reduction of cytochrome c in the  presence and absence of cytochrome b . 5  Reduction by LfH-  protein  k (xlO' M ' V ) 7  1  2  pH 7  pH 8  horse cytochrome c  6.6  11.0  + cytochrome b  7.0  6.7  4.3  8.4  5  + DME-cytochrome b  5  Reduction by F M N H - at p H 7  k (xlO" M ' ! " ) 7  8  1  2  horse cytochrome c 14.7  free + cytochrome b  4.4  5  + DME-cytochrome b  5.6  5  tuna cytochrome c 12.5  free  3.2  + cytochrome b5 + DME-cytochrome b  5.3  5  As noted i n the Methods, die standard error in these rate constants is ± 10 %  68  2000  [Cytochrome b ] (pM) 5  Figure  19  :  concentration  Dependence of  cytochrome b  s  cytochrome  of b  5  the  rate  constant  at  pH  7  of  (A) and  in the absence (O) and presence  (•)  cytochrome 8  (B).  b  5  Rates  69  were  by  LfH- on  determined  for  the  native  of horse heart cytochrome c and for D M E -  cytochrome b in the absence ( • ) and presence ( • ) of horse heart cytochrome c. 5  reduction  Table V : Second order rate constants for reduction of cytochrome b  by LfH- in the presence and  5  absence of ferrocytochrome c.  protein  k (xlO  7  2  M-V ) 1  pH7  pH8  free  1.3  1.3  + horse cytochrome c  1.5  2.1  free  1.8  3.0  + horse cytochrome c  2.5  4.2  cytochrome b  5  DME-cytochrome b  5  As noted in the Methods, the standard error of these rate constants is ± 10%.  on binding to ferrocytochrome c was significant at both values of p H . The kinetic results for the reduction of native and DME-ferricytochrome b  5  presence and absence of tuna and horse ferrocytochromes c are presented forms of free cytochrome b$, reduction by F M N H -  by F M N H  in Figure 20.  in die  For both  was slower than semiquinone disproportionation,  so cytochrome reduction proceeded primarily via the fully  reduced species, F M N H " .  rate constants for tiiis reduction are given in Table V I .  The rate constant of F M N H " reduction of  free  native  cytochrome  ferricytochrome b, 5  as  b  5  was  only slightly smaller than  was also the case for  LfH- reduction (cf.  reduction kinetics of native and DME-ferricytochrome b with either horse or tuna cytochrome c.  5  Table V ) .  for  die  free D M E -  Large effects  on  the  by reduced F M N occur upon complexation 5  -  5  obtained  In all cases, reduction of the cytochrome b  the complex occurs via the semiquinone species, F M N H , b.  that  The calculated  heme within  in marked contrast to die free cytochromes  Contrary to the data obtained for cytochrome c reduction in the complex (cf. Table IV), plots  of k ^ versus complex concentration for cytochrome b are not linear. These kinetics were fit to s  70  800  [Cytochrome b ] (uM) 5  Figure  20  :  Variation  of  the  concentration of cytochrome b  5  rate constants of cytochrome  b  5  reduction  by  FMNH-  with  in the presence of horse heart (A) and tuna (B) cytochrome c  panel A , the rates of unbound native cytochrome b rates for unbound DME-cytochrome b  5  respective cytochromes c to native ( • )  5  In  reduction (O) are shown, and in panel B , the  (0) are shown.  In both panels, the effects of binding the  and DME-cytochrome b  represent the theoretical fits to the data described in the Discussion.  71  the  5  (•)  are shown.  The solid lines  Table V I : Second order rate constants for the reduction of cytochrome b by reduced F M N at p H 7. 5  kjtxlO^M-V )  protein  k, (s- )  1  1  cytochrome b  5  free  0.14 (fully reduced)  + horse cytochrome c  2.7 (semiquinone)  460  + tuna cytochrome c  5.9 (semiquinone)  1040  DME-cytochrome b  The  5  free  0.22 (fully reduced)  + horse cytochrome c  6.4 (semiquinone)  840  + tuna cytochrome c  8.1 (semiquinone)  1390  oxidation state of the reductant  is indicated in paranthesis.  As  noted in the Methods, the  standard error in these rate constants is ± 10%.  first order rate limiting processes, the mechanisms of which are presented in the discussion.  72  Table VII : Solvent exposure of cytochrome heme groups in the two predicted docking geometries for the cytochrome ^cytochrome c complex.  docking geometry  cytochrome c ( A )  cytochrome b ( A )  unbound heme  514  485  2  2  5  unbound proteins  59.0(11.5)  161.0(33.2)  edge on (Fig. 6)  63.2(12.3)  166.6(34.4)  off edge (Fig. 7)  38.2(7.4)  161.0(33.2)  The  numbers  in  paratheses  represent  die  percentage  of  the  heme  surface  area  that  is  solvent  accessible.  Ferrocytochrome b Reduction of Ferricytochrome c 5  Examples of decay curves (and fits to equation 10) observed at 416 nm and 428 nm upon the mixing of ferrocytochrome b  5  and ferricytochrome c are presented  in Figure 21.  rate constants determined at the two wavelengths agreed within 10%. the  second  order  rate  constant  for  ferrocytochrome  b  5  At I = 0.5 M (pH 7.0, 25°C)  reduction  of  conditions of equimolar concentration was determined to be 18 ( ± 2 ) x 10 was used to generate ferrocytochrome b .  the second order rate constant for ferrocytochrome b  5  of  6  M'V . 1  From a linear plot of k  ferrocytochrome b  s  ferricytochrome 6  M"V  c  under  when lumiflavin  When dithionite was used to produce ferrocytochrome  5  x 10  The bimolecular  o b s  b, 5  reduction of ferricytochrome c was 18.5 ( ± 0 . 9 )  versus the concentration of ferricytochrome c, the mixing  and ferricytochrome c under pseudo-first  order  conditions yielded  a second  order rate constant of 20 ( ± 2 ) x 10 M ' V . 6  1  The reduction of ferricytochrome c by ferrocytochrome b ionic strength (Figure 22).  5  showed a marked dependence  The second order rate constant of DME-ferrocytochrome b 73  5  on  reduction of  -0.20 I 0.0  , 0.8  _,  , 1.6  I  time (s)  Figure 21 : The change in absorbance at 428 run (A) and 416 nm (B) associated with ferrocytochrome b  5  reduction of ferricytochrome c (I =  represents an average of 5 decay curves.  0.5 M sodium phosphate,  p H 7.0, 25°C).  Each data set  The smooth lines represent second order fits of the data  (equation 10).  74  21  19 CM T  17  15 0.0  0.4  0.8  1.2  ionic s t r e n g t h (M)  Figure 22  : The  ionic strength dependence of the bimolecular rate constant of die  ferricytochrome c by native ( • )  and DME-ferrocytochrome b  represent non-linear least squares fits to equation  11.  order rate constants is less than the size of the data symbols.  75  5  (•)  (pH 7.0, 25.0°C).  reduction of  The solid lines  The error in the natural log of the second  ferricytochrome c was lower than that of native ferrocytochrome b except at an ionic strength of 1.0 M (pH 7.0, 25°C).  reduction of ferricytochrome c  5  The curves in Figure 22 represent fits of the  data to the Van Leeuwen equation (11) using the parameters summarised in Table I X . ionic strength  dependence  of the association constants  of cytochrome b  5  As with the  and porphyrin cytochrome  c, the fits were done in two ways. The results of these fits are presented in Table VIII. At an ionic strength of 0.5 M , 25°C, reduction  by  either  Attempts to measure  D M E - or  native  the second order rate constants  ferrocytochrome  this p H dependence  at 0.1  b  s  were  for ferricytochrome c  independent  M ionic strength  under  of  pH  (Figure 23).  conditions of equimolar  cytochrome concentrations were unsuccessful because the reaction was too fast to monitor.  Table VIII constant  of  :  Parameters  used in fitting  ferrocytochrome  b  reduction  5  the ionic strength of  dependence  ferricytochrome  c  to  of die second order rate  the  Van  Leeuwen  (equation 11).  parameter  cyt & cyt c  DME-cyt & c y t c  5  5  In koo  13.7 (.1)  13.5 (.4)  14.3 (3)  15.1 (.2)  Ri  8(5)  17(f)  3(13)  17(f)  R  9(5)  17(0  15 (10)  17(f)  - 10(f)  - 280 (50)  - 8(f)  - 100(20)  2  Zi  (0  Z2  7.2(f)  Pi  - 343 (f)  - 343 (f)  - 187 (f)  - 187 (f)  246 (f)  246 (f)  246 (f)  246 (f)  .01  .01  .06  .01  p  2  s.s.  •2 (.3)  7.2  1.5 (.5)  The numbers i n parantheses are errors; an "f" denotes that the parameter was held constant. S.S. refers to the sum of squares of the fit.  76  equation  24 CD  I O  18  -  12  -  x  6 CM  0  6.0  7.0  8.0  PH Figure 23 : The p H dependence of the second order rate constant of ferricytochrome c reduction by native ( • ) and DME-ferrocytochrome b  s  ( • ) (I = 0.5 M , 2 5 ° C ) .  aid and have no theoretical significance.  77  The solid lines are drawn for visual  3.3  3.4  3.5  3.6  1/T ( K ~ x 1 0 ) 1  3  Figure 24 : Eyring plots of the bimolecular rate constant of ferricytochrome c reduction by native (•)  and D M E ( • )  ferrocytochrome b  5  (I = 0.5 M , p H 7.0).  linear least squares fits to the data.  78  The straight lines represent weighted  The Eyring plots of the temperature dependence DME-ferricytochrome b parameters for AHt=  5  of ferricytochrome c reduction by native and  (I = 0.5 M , p H 7.0) are shown in Figure 24.  ferricytochrome  c  reduction  by  native  ferrocytochrome  7.5 ( ± 0 . 2 ) kcal/mol and A S t = -0.3 ( ± 0 . 6 ) e.u..  The thermodynamic activation b  s  were  determined  to  be  For ferricytochrome c reduction by D M E -  ferrocytochrome b , values of A H J = 7.9 ( ± 0 . 4 ) kcal/mol and A S f = 1 ( ± 1 ) e.u. were calculated under 5  the same solution conditions. The results  of the dipole calculations of the cytochromes and their derivatives used in this  work are listed in Table I X .  Table I X . The calculated dipoles and monopoles of the proteins used in this work.  oxidation state  protein  cytochrome b  DME-cytochrome b  s  cytochrome c  porphyrin cyt c  dipole  8  - 10  - 501  47  - 343  ferric  - 9  - 468  51  - 295  ferrous  - 8  - 412  63  - 187  ferric  - 7  - 391  69  - 139  ferrous  + 6.2  + 255  24  + 232  ferric  + 7.2  + 275  26  + 246  n/a  + 6.2  + 255  26  + 229  ferrous  5  monopole  P  is the angle between the dipole and the vector from the centre of mass of the protein the heme edge.  79  DISCUSSION  The  three  different  techniques  that  have  been  used  to  characterise  the  interaction  cytochromes b  and c are discussed independendy of one another in the following sections.  facets  discussion  s  of  this  overlap,  such  as  those  sections  dealing  with  the  calculations  electrostatic dipoles of the proteins and the solvent accessibility of the heme groups.  between Some of  the  These aspects  are discussed when they are first referred to.  Interaction of Porphyrin Cytochrome c with Native and DME-Cytochrome b  5  The absorption spectrum of porphyrin cytochrome c exhibits five maxima in the visible region, including the Soret (Figure 10) which is characteristic of porphyrins in which two of the pyrrole nitrogens  are  protonated  (Falk,  1964).  Some discrepancies  extinction coefficient of porphyrin cytochrome c.  exist  in the  literature  regarding  The values that have been measured  and the method of determination are summarised in Table X . the values determined by Flatmark and Robinson (1968).  the  at 505 nm  The current results agree best with  Based on this result, a value of 13.1 m M "  1  (I = 0.01 M , p H 7.0, 25 °C) was used for the extinction coefficient of porphyrin cytochrome c at 505 nm in the current work. The fluorescence emission spectrum of porphyrin cytochrome c (Figure 10) corresponds closely with that observed by previous workers (Vanderkooi & Erecinska, 1975; Strottman Kornblatt & Laberge,  1988).  et al.,  1984;  The increase in the fluorescence intensity of porphyrin cytochrome c  with acidic p H (Figure 11) agrees with the reported p H dependence this protein (Kornblatt & Laberge,  1988).  of the fluorescence intensity of  These workers have measured  an apparent pK of 6.2  associated with the change in intensity and have attributed this increase in fluorescence the protonation of His-18 of porphyrin cytochrome c.  In native cytochrome c, His-18 provides one  of the axial ligands of the iron atom. The conclusion that the increase in fluorescence intensity  80  intensity to  Table X : The extinction coefficient of porphyrin cytochrome c at 505 nm.  e (mM* ) 1  13.0* 7.4  1  b  method of determination  reference  amino acid analysis  Flatmark & Robinson, 1968  dye binding (Lowry assay)  Vanderkooi et al., 1976  13.1 ± 0 . 2  C  dye binding (Bradford assay)  current work  12.9 ± 0 . 6  C  amino acid analysis  current work  0.04 M phosphate, p H 6.9; 0.01 M phosphate, p H 7.2; I = 0.01 M phosphate, p H 7.0 b  c  involves a residue that is in a different environment in porphyrin cytochrome c as compared to its state in the native protein is consistent with the observation that no residues in native cytochrome c titrate between p H 6.0 and 8.0 (Matthew, 1978). Several plausible mechanisms could account for the observed quenching of porphyrin cytochrome c by  cytochrome  b, s  including dipole-dipole energy  transfer  and  electron  transfer.  heavy atoms can quench fluorescence by spin orbital coupling and paramagnetic fluorescence studies  by  promoting intersystem crossing (McGlynn  involving  the  interaction  of  porphyrin  or  et al.,  metal  1964).  substituted  cytochromes conclude diat this quenching is due to dipole-dipole resonance  In addition,  centres can quench  The authors of several cytochromes energy  with  native  transfer (Leonard  & Yonetani, 1974; Vanderkooi et al., 1977, 1980; McLendon et al., 1985; Vitello & Erman, 1987).  In  the most closely related system to the current work, Vanderkooi et al. (1980) found that consistent with a resonance  energy  transfer  mechanism of fluorescence quenching, the fluorescence  porphyrin cytochrome c is decreased in the presence of cytochrome b . 2  cytochrome b 1983).  2  is structurally homologous to the tryptic fragment  lifetime of  The heme binding domain of  of cytochrome b  s  (Le & Lederer,  In the present work, the spectral overlap between the emission of porphyrin cytochrome c  and the absorption of cytochrome b  5  are favourable for energy  porphyrin cytochrome c fluorescence by cytochrome b  s  to dissociate the complex (high ionic strength).  transfer  to occur.  Quenching of  is inhibited by conditions that are expected  In addition there is no evidence that cytochrome b 81  5  was  reduced  mechanism. quenching.  during  die  It  concluded  is  course  Nevertheless,  it  of that  die  experiment,  ruling  out  dipole-dipole (Forster)  would  be  useful  to  electron  energy  perform  transfer  transfer  fluorescence  is  as  the  a  quenching  mechanism  of  lifetime measurements of  porphyrin cytochrome in the presence and absence of cytochrome b . s  Previous  reports  have  established  that  the  complex  cytochrome c has a 1:1 stoichiometry (Stoneheurner et al., 1983).  formed  between  1979; Mauk et al.,  cytochrome  b  5  and  1982; Eley & Moore,  Two lines of evidence in the current work indicate that this is also true for die cytochrome  ^porphyrin  cytochrome c complex.  First, at the lower ionic stengths, diere is a distinct  point in the titration curves at a 1:1  stoichiometry.  Second, the results of die polarization study  indicate that the complex has 2.3 times the volume of free porphyrin cytochrome c. volume of cytochrome b  from its crystal structure (Mathews et al.,  5  break  Calculating the  1979), which indicates that the  molecule is a cylinder measuring 37 A in height and 31 A in diameter, estimates the volume of this protein at 2.8 x 10 A . 4  The calculated volume of the complex is 4.6 x 10 A , which is 2.5 times  3  4  that of porphryin cytochrome c. cytochrome proteins  The agreement of these values argues that the stoichiometry of the  ^ p o r p h y r i n cytochrome  nor  the  complex  are  3  c complex is  perfect  spheres,  1:1.  an  While  assumption  it  is true that neither  made  in  calculating  the  of these rotation  correlation time from the volume of the protein, the error introduced by this assumption does not affect the conclusion. The Q M,  m a x  p H 7.0).  of porphyrin cytochrome c in the presence of native cytochrome b  5  is 0.29 (I =  0.01  Using the values for the constants given in the Results section, calculations according  to Forster theory indicate that a Q  m a x  of 0.29 corresponds to an average separation of 18 A between  the donor and acceptor dipoles of the cytochrome ^ p o r p h y r i n cytochrome c complex.  Although the  locations of these dipoles have not been unambiguously assigned, they are generally taken to be the centres of the porphyrin groups (Vanderkooi et al., between  the  centres of  the  porphyrin and  the  1977).  heme  This result indicates that the separation  prosthetic  ^ p o r p h y r i n cytochrome c complex is 18 A (I = 0.01 M , p H 7.0).  82  groups  in the  native  cytochrome  This result agrees remarkably well  with  the  intersite  separation  of the  donor  and acceptor  in the  cytochrome  & zinc  cytochrome  5  c  complex which was calculated to be 18 A also (McLendon et al., 1985). The complex formed by DME-cytochrome b (I = 0.01 M , p H 7.0, 25 °C).  s  and porphyrin cytochrome c yields a Q  m a x  of 0.18  Using Forster energy transfer theory as described above, this value  corresponds to a centre to centre intersite separation of 20 A .  This result suggests that the average  intersite separation is different in the native cytochrome ^ p o r p h y r i n cytochrome c complex than in the  DME-cytochrome ^ p o r p h y r i n cytochrome  geometry of the two complexes is different.  c complex, and  therefore  that  the  average  The largest source of error in Forster energy  docking transfer  calculations is introduced by estimating the relative orientation of the donor and acceptor dipoles. The value of K  (2/3) used in the current study is often used when the relative orientation between  2  the donor and acceptor diffusion prior to energy  dipoles is unknown, or when this orientation is randomised by rotational transfer  (Lakowicz,  when the range of donor-acceptor  calculation of R . these  molecules  (Northrup et al., cytochrome b  5  2  Alternatively, a value of 0.476 may be used  does not change during die lifetime of the excited of 0.667 and 0.476 introduces an error of 5% in the  Modelling studies of the cytochrome c peroxidase-cytochrome c complex suggest  Q  that  orientations  The difference in K  state (Steinberg, 1971).  1983).  rotate  1988).  with  respect  to  one  However, the current  another  while they  are  close  to  one  fluorescence polarization measurements suggest that  and porphyrin cytochrome c do not rotate significantly with respect  within the cytochrome ^ p o r p h y r i n cytochrome c complex.  to one  2  c  (Vanderkooi et al., 1980).  values  ^porphyrin of  of  the  separation  between  the  cytochrome c might not be accurate,  cytochrome  b  5  heme  propionates  induces  donor  cytochrome ^ p o r p h y r i n cytochrome c complex.  83  and  acceptor  the variation of Q  a change  would not  In general, the indeterminancy of K  not resulted in serious errors in the calculation of distances (Lakowicz, absolute  another  A discussion of the relative orientations  of the donor and acceptor dipoles of two hemes suggests that the indeterminancy of K cause more than 20% error in R  another  in the  m a x  average  1983). sites  2  has  Finally, while the in  the  cytochrome  indicates that esterification docking geometry  of  the  The  apparent  increase  in  the  Q  of  m a x  the  native  cytochrome  i •porphyrin  cytochrome  5  c  complex below p H 7.0 (Figure 16) can be attributed to the increase in the fluorescence yield of free porphyrin cytochrome c at acidic p H .  The lack of a p H dependence of the fluorescence of porphyrin  cytochrome c in the presence of cytochrome b effect.  Cytochrome b  5  could be due to either a kinetic or thermodynamic  could shield His-18 of porphyrin cytochrome c from die solvent, preventing  5  the protonation of His-18.  This mechanism is analogous to the inhibition of cyanide binding of  cytochrome c by cytochrome c peroxidase (Hoth & Erman, 1984). shift the p K of His-18. exogeneous  reductants (vide infra),  effect  on  the  it seems likely that the principal effect of cytochrome b  A p H titration of His-18 measured by pH  dependence  seen for DME-cytochrome b  of  the  fluorescence  of  !  H N M R would be interesting. porphyrin cytochrome  evidence  that  modes  has  been  This effect is not 5  the p K of porphyrin cytochrome c His-18. the  c  A  and porphyrin cytochrome c, presumably because DME-cytochrome b  s  shift  is to  s  observed i n the presence of cytochrome c oxidase (Kornblatt & Laberge, 1988).  does not  might  5  Considering that the heme of cytochrome c is sterically accessible to small,  shift the p K of His-18. similar  Alternatively, cytochrome b  of  interaction  between  native  This result can be taken as or  DME-cytochrome b  5  and  further  porphyrin  cytochrome c are not equivalent. The  ionic  strength  dependence  of  Q  m a x  for  native  cytochrome  ^porphyrin  cytochrome  c  complex (pH 7.0, 25°C) (Figure 15) indicates that the average docking geometry of this complex does not vary appreciably under these conditions. ^porphyrin  m a x  of the DME-cytochrome  cytochrome c complex varies between 0.11 and 0.18 over an ionic strength  0.002 M to 0.02 M (pH 7.0, 25 °C). to 22 A .  On the other hand, the Q  Changes in Q  m a x  These values of Q , ^ correspond to intersite separations of 20  could arise from factors other than changes in the average separation of  the porphyrin donor and die heme acceptor dipoles. a particular docking geometry  range of  (vide infra),  If electrostatic forces predominate in stabilising  it is easy to envisage how increasing the ionic  strength  lowers the energy barrier of the proteins to rotate relative to one another.  Rotation in this manner  would  m a x  alter Q  m a x  through changing  /c . 2  Nevertheless,  84  the variation of Q  with  ionic  strength  indicates that the average  docking conformation of the DME-cytochrome i -porphyrin cytochrome c 5  complex changes in some manner with ionic strength. a recent  This is an interesting observation in light of  report in which the first order rate constant of electron transfer  within the cytochrome c  peroxidase-cytochrome c complex has been shown to be dependent on ionic strengdi (Hazzard et 1988).  Why the Q  max  al.,  of the native cytochrome ^ p o r p h y r i n cytochrome c complex is independent  of ionic strength is not clear. The  ionic  strength  dependence  of  the  association  constants  of  the  cytochrome  ^porphyrin  cytochrome c complex (Figure 13) suggests that electrostatic forces play a large role in mediating the interaction of these proteins.  The fit of the data to the Van Leeuwen equation (Table III) is  much better for the interaction of porphyrin cytochrome c and native cytochrome b  than porphyrin  5  cytochrome c and DME-cytochrome b . 5  This could be due in part to the small number of data  points collected for the interaction between DME-cytochrome b  s  and porphyrin cytochrome c.  dipole calculations suggest that esterification of the cytochrome b substantial  change  heme  in the electrostatic dipole of the protein (Table I X ) .  vector of ferrocytochrome b  5  heme group.  5  propionates  results  For instance,  passes through the surface of the molecule between  The in a  the dipole  Asn-57 and die  Esterification of the propionates not only diminishes the magnitude of the vector by  20%, but also shifts the vector away from exposed heme edge, towards Glu-49 and Asp-53. calculations should be viewed as approximations for several reasons.  Specifically,  These  the calculations  treat the protein as a uniform dielectric, and dipoles are assigned to bonds in residues irrespective of the residue's relative location in the tertiary structure. strength  dependence  The uncertainty in the fit of the ionic  data is too great to determine whether or not the difference in die behaviour  of native and DME-cytochrome b  5  to porphyrin cytochrome c can be adequately  explained by  the  Van Leeuwen treatment. The p H dependences of the association constants of cytochrome b  s  and porphyrin cytochrome c  (Figure 14) indicate that either none of the groups involved in the interaction titrate over this p H range,  or that any  changes  that do occur compensate  85  for each other.  The p H optimum of the  association constant  of cytochrome b  and porphyrin cytochrome c (7.0) is similar to that reported  s  for the native proteins (Mauk et al., between  DME-cytochrome b  1982).  The lack of p H dependence of the association constant  and porphyrin cytochrome c is different  5  dian what  is observed  for  DME-cytochrome b and cytochrome c. This discrepancy is discussed below. 5  The  current  work provides  two  lines of evidence  in addition to  die points  made  in  the  Introduction, that indicate that porphyrin cytochrome c is a good analogue for native cytochrome c in binding assays.  First, neither the magnitude nor the orientation of the dipole of cytochrome c is  appreciably perturbed by the removal of the iron atom (Table I X ) , assuming that at p H 7.0, His-18 is not protonated.  Perhaps this is not surprising as the iron atom is close to the centre of mass of  cytochrome c and positive charges of ferrous iron are replaced by two protons at neutral p H . calculations  were  preparation).  based  It  on  the  is reasonable  crystal  structure  to expect  of  native  cytochrome  that some structural  occur upon the removal of the iron atom.  c  These  (Bushnell et  rearrangement  of the  al.,  protein  in  would  This has not been accounted for in diese calculations.  Second, the association constants of the cytochrome  fe porphyrin  cytochrome c complex measured by  5  fluorescence quenching agree well with the measurements performed by absorption spectroscopy using the native cytochromes (Mauk et al., obtained  for  measured  by the two methods  between  the  spectroscopy  the  association  constants  the  cytochrome  (Erman & Vitello,  constants  determined  c  peroxidase-cytochrome  1980; Vitello & Erman, 1987). by  the  fluorescence  quenching  c  complex  The agreement and  absorption  That porphyrin cytochrome c is a good analogue for native cytochrome c is useful  because fluorescence measurements  quenching is a more sensitive technique than absorption spectroscopy, of  association  constants  association constants of the cytochrome magnitude  of  This agreement is not quite as good as that  indicates that the two techniques detect the same population of docking geometries of  the complexes.  the  association  1982; 1986).  to  those determined  by  over  fc porphryin 5  absorption  a  wider  of  conditions.  Although  the  cytochrome c complex agree within an order of  spectroscopy  solution conditions, the association constants measured  86  range  allowing  for  the  native  proteins  by the two techniques  imder  similar  display different  pH  optima. were  Whether this difference  done  at  different  ionic  represents differences  strengths),  differences  in experimental  between  techniques  (the  porphyrin cytochrome  c  pH  studies  and  native  protein, or experimental error is difficult to say. As discussed in the Introduction, electrostatic calculations of the complexes that form between cytochrome b  and cytochrome c (Mauk et al.,  5  1986) predict that the overall binding energy of the  complex that forms between these proteins is insensitive to p H (over a range of 6.75 to 8.25), but that two docking geometries exist (Figures 6 & 7).  For the native proteins, the edge on docking  geometry is the more stable of the two orientations below p H 8.0.  Above p H 8, the two docking  geometries  the  are  orientations.  isoenergetic  and  The electrostatic  cytochrome b  there  are  equimolar  calculations further  amounts  predict  of  that in the  edge  on  and  complex formed  off  edge  by D M E -  and cytochrome c, the two docking geometries are isoenergetic at neutral p H , but as  5  the p H rises, histidine 26 of cytochrome b  5  deprotonates, . stabilizing the off edge geometry  and  increasing the overall association constant of the complex. The the  current  cytochrome  cytochrome  results b  5  heme  ^cytochrome  are  consistent  propionates c  with  induces  complex.  The  the  electrostatic  a change pH  calculations in that esterification of  in the  dependence  average of  the  docking geometry docking geometries  of  the  that  is  predicted by the electrostatic calculations is not observed in the fluorescence quenching experiments. In making these comparisons between the calculations and the experimental observations, it is useful to  recall  association ones.  that  the  constants  binding  energies  that  two orders  are  derived of  from  the  magnitude  electrostatic smaller  than  calculations the  correspond  experimentally  to  observed  This suggests that there are forces in addition to electrostatic interactions diat contribute to  the stability of the complex formed by cytochrome b  s  contribute to stabilising different  docking geometries  and c.  These additional forces  may also  within the complex, and may explain in part  why the fluorescence quenching experiments do not reproduce the p H dependence  predicted by the  calculations.  of the predicted  In addition, the calculated difference  in electrostatic  docking geometries at p H 7.0 and 8.5 is approximately 1 kcal/mol.  87  energy  (AG ) e)  This suggests that even under  conditions that the calculations predict to favour one docking geometry, shallow energy  minimum in the energy  surface  this geometry  of potential conformations  represents a  and may only exist in  solution slightly longer than a myriad of other docking geometries as the cytochromes tumble around each other.  This corresponds closely to the view of the interaction of cytochrome c peroxidase and  cytochrome c that emerges from Brownian dynamics simulations (Northrup et al., or  three docking geometries  are  slightly favoured  over  the  many  1988) in which two  docking geometries  that these  proteins sample. The  fluorescence  quenching  data  indicate that several  within the complex formed by cytochrome b  5  different  docking geometries  and porphyrin cytochrome c.  can  exist  It is likely that under  any given conditions, a population of docking geometries exist, all differing slightly with respect intersite separation and orientation. determine between  Solution conditions and the electrostatic surface of die proteins  where this equilibrium lies. the native cytochromes,  to  If this is taken as a model for the complex that  the question arises as to which docking geometries  forms  correspond  to  productive electron transfer complexes. In conclusion, cytochrome b  and porphyrin cytochrome c form a 1:1 complex in solution that  5  has approximately the same stability as previously observed for the complex formed by the native proteins.  The  stability  electrostatic forces. with a average  of  the  cytochrome  ^porphyrin  cytochrome  c  complex  is  Maximal quenching of porphyrin cytochrome c by cytochrome b  5  docking geometry  of the proteins in which the centre  to centre  mediated  by  is consistent  separation  of the  prosthetic groups is 18 A , in good agreement with the distance between die iron atoms in the edge on  model  of  cytochrome b  5  the  cytochrome  heme propionates  ^cytochrome  c complex  (Salemme,  1976).  Esterification of  does not appreciably decrease the binding strength  but does induce an altered average docking geometry. on the interaction of cytochrome b  s  the  of the complex  This effect of heme propionate esterification  and porphyrin cytochrome c can be taken as a reasonable model  the effect that the esterification would have on the interaction of the native proteins.  88  Kinetics of Flavin Semiquinone Reduction of Cytochrome b and Cytochrome c 5  The  characterisation  of  a  large  number  of  electron  transfer  proteins  and  protein-protein  complexes by flavin semiquinone reduction kinetics was reviewed in the Introduction. used in the current study are illustrated in Figure 25. are provided in Table X I . the  two  flavins  are  The flavins  Some of the properties of these two flavins  As can be seen from this information, the electrochemical properties of  similar  while  the  electrostatic  properties  are  not.  Under  the  conditions  employed in this study, the association constant of complexes involving die two cytochromes b cytochrome c is approximately 10 M " (Mauk et al., 6  in the rate constants the  1  1982; 1986; this work).  5  and  In general, differences  for semiquinone reduction of one cytochrome in the absence and presence of  second cytochrome can be interpreted  in terms of steric and electrostatic changes  induced by  complex formation. Reduction of Cytochrome c by L f H : -  The observation that the rate constant for cytochrome c  reduction by L f H - is unaffected by the presence of cytochrome b  5  at p H 7, suggests that there is no  change in the accessibility of the cytochrome c heme when this protein binds to native cytochrome b.  When DME-cytochrome b  s  5  kinetically  detectable  manner,  binds to cytochrome c this heme accessibility is decreased  indicating that the  stereochemical environments  heme edge are not equivalent in the two complexes at p H 7.  of the  in a  cytochrome c  This difference could arise from the  increased steric bulk introduced by esterification of the heme propionates or from changes that this modification  induces in the geometry  essentially uncharged at p H 7,  of the complex formed by the two proteins.  the observed kinetic difference cannot  be attributed  As L f H - is to electrostatic  effects. The substantial increase in the rate constant of cytochrome c reduction by L f H - at p H 8 has been observed previously by Przysiecki et al. (1985). observed  for  several  small  electron  transfer  proteins  Similar rate enhancement of varying net  electrostatic  reactions with this flavin. This rate enhancement has been attributed to an increase in the  89  at p H 8 has been charge  in  their  Figure 25 :  The structure of the three oxidation states of flavin.  ribityl phosphate for lumiflavin and F M N respectively.  R is a methyl group ( C H ) and  The reduction potential of die quinone and  the semiquinone (E°) and the pKj's of the semiquinones of these flavins are given in Table X I .  90  3  Table X I : Some relevant properties of lumiflavin and F M N .  Property  lumiflavin  FMN  R (in F i g . 25)  methyl  ribityl phosphate  charge  0  E°  pH 7  - 231  - 238  pH 8  - 259  - 269  pR,  8.27  disp. rate  3  8.55  pH 5  6.2 x 10  pH9  1.1 x l O  rate of semiquinone disproportionation.  a  - 2  9  9  2.6 x 10  9  1.9 x l O  9  Equilibria data were taken from Draper & Ingram (1968)  and disproportionation rates were taken from Vaish & Tollin (1970).  intrinsic reactivity of the lumiflavin 25, Table X I ) .  radical upon deprotonation to form an anionic species (Figure  The increased reactivity is a manifestation of the lower reduction potential and a  change in the electron spin density distribution, both of which are characteristic of die deprotonated semiquinone.  Electrostatic effects  that result from  this deprotonation could  also contribute  increase in the rate constant of reduction, but in previous studies, this effect was less  important  (Przysiecki  et  al.,  1985).  Interestingly,  reduction in its complex with native cytochrome b  5  the  increase  in L f H - reactivity is perhaps offset  cytochrome c heme. DME-cytochrome b  5  In contrast,  the  rate  constant  of  observed to be ferricytochrome  was unaffected by the increase in p H . by an increase  in the  steric  to an  c  At p H 8,  hindrance  of  the  the rate constant of cytochrome c reduction in its complex with  approximately doubles on raising the p H from 7.0 to 8.0, which is similar to the  p H induced increase observed for free cytochrome c. Overall, b  5  these results  heme propionate groups  are consistent  with the suggestion that esterification of the cytochrome  induces a change in the docking geometry  91  for the interaction of this  protein  with  reduction  cytochrome  of  the  c  at  p H 7.  In  kinetic accessibility of  addition,  the  partially  cytochrome ^-cytochrome c complex at higher p H . exposed native cytochrome b heme  propionate  groups  5  (cf.  the  current  exposed  findings  are  cytochrome  c  consistent heme  with  edge  in  a the  The electrostatic environment of the partially  edge is highly negatively charged owing to die presence of the two Figure 2).  This characteristic  may  inhibit the  interaction  of  the  partially anionic lumiflavin semiquinone with the heme of cytochrome c in its complex with native cytochrome b  at p H 8.  5  Such an electrostatic effect is not expected to contribute to the kinetics of  cytochrome c reduction in its complex with DME-cytochrome b  5  as the electrostatic environment of  the heme edge in this derivative is effectively neutral (Mauk et al., strength  dependence  of these reactions is required to clarify  1986).  Analysis of the ionic  this latter point, though the sensitivity  of the association constant of the complex to ionic strength precludes an extensive investigation. Reduction of cytochrome c by F M N H - : permits  evaluation of the  sign and magnitude  near the site of electron transfer (Meyer et al., species of cytochrome c (horse  Use of F M N semiquinone ( F M N H ) as a reductant of the effective  electrostatic  1984; Hazzard et al.,  charge  1987).  of the protein  In addition, use of two  and tuna) can provide information regarding  die effects  of amino  acid substitution in the region surrounding the cytochrome c heme edge [cf. Table V I in Hazzard et al.  (1987)]  that  are  thought  to  be  important  in  the  formation  of  electron-transfer  complexes  (Salemme, 1976; Mauk etal., 1986). The minimal consistent observed  with with  differences  previous FMNH-,  results  1984).  reduction behaviour of the  (Hazzard et  relative to  negatively charged F M N H partially exposed heme  in the  L f H - , can  al., be  1987).  The  attributed  to  two species greater the  of cytochrome c is  reduction  electrostatic  rate  attraction  constants of  the  to the highly positively charged surface of cytochrome c surroimding its  edge at the low ionic strengths used in these experiments  (Meyer et  al.,  Other properties of the semiquinones which might affect the kinetics of reduction, such as  the reduction potential, are very similar (Table IV). The sensitivity of the rate constants of cytochrome c reduction by die anionic flavin  92  (FMNH)  to the presence of cytochrome b  contrasts with the identity of the rate constants of cytochrome c  5  reduction by the neutral flavin ( L f H ) in the presence or absence of native cytochrome b  at pH 7.  5  The dependence  of kinetic behaviour on the electrostatic properties of die flavin employed probably  arises  differential  from  positive  a  electrostatic  cytochrome b .  diminished  potential  of  surface  the of  two  flavins  cytochrome  c  to  the  that  appreciable  occurs  neutralisation  when  it  binds  of  to  the  native  This conclusion correlates with the smaller effect of cytochrome c binding to D M E -  5  cytochrome b  response  on the  5  negative  exposed heme  reduction rate constants for F M N H . -  electrostatic  potential  edge (Mauk et al.,  surface  1986).  of the  That  is, it is consistent  D M E derivative near  Furthermore,  this interpretation  the  with  the  region of die  implies that the  heme  propionate groups are in sufficiently close proximity to the site of F M N H - approach to cytochrome c that  they  exert  a  kinetically  significant  electrostatic  influence.  Finally,  dependence  of cytochrome c reactivity within the complexes is interesting.  to extend  these experiments  to include yeast  iso-1-cytochrome c as  species of cytochrome may bind to cytochrome b a  much  larger  dipole moment  bimolecular rate constant  than  the  horse  heart  species  lack  (results  of  species  It might be worthwhile  two factors  somewhat differently.  5  the  suggest diat  this  First, this cytochrome has not  shown).  of ferricytochrome c reduction by ferrocytochrome b  Second,  the  is much greater for  5  yeast iso-1-cytochrome c than the horse heart protein (Eltis, Barker & Mauk, unpublished). Reduction of Native and DME-Cytochrome b  5  and DME-cytochrome b  5  reduction by LfH- vary directly with the thermodynamic driving forces of  the reactions (Meyer et al., b  5  by L f H - : At p H 7, die rate constants for native  1983).  For example, the rate constants for reduction of free cytochrome  are 25-45% lower than those obtained for free DME-cytochrome b , 5  as expected from the finding  that heme propionate group esterification increases the reduction potential of this protein by 60 mV (Reid et al.,  1982, 1984).  Similarly, the rate constants for cytochrome c ( E  reduction are significantly faster b. 5  Other,  differences  less  between  readily  than the rate constant  correlated  cytochrome  c  and  factors,  are  cytochrome  93  5  = 260 mV vs SHE)  of reduction of either form of cytochrome  expected b  m 7  to  derivatives  contribute observed  to  die  here.  rate These  constant factors  include the extent of heme exposure to solvent [12% in cytochrome c, 33% in cytochrome b ;  Table  s  VII][cf. Table I and Figure 1 of Tollin et al. (1986)] and the Franck-Condon activation energies of the two proteins. calculated  by  The solvent exposure of cytochromes  Stellwagen  (1978)  simply  because  in  b  5  the  and c hemes are  current  study,  the  larger  surface  dian diose areas  were  calculated from the edge of the probe sphere rather than from its centre. The differential response cytochrome b  to an increase in p H of the rate constants of reduction of the two  derivatives may be attributed to the sum of two opposing effects.  5  electrostatic effects  On one hand, the  of partial deprotonation of LfH- to generate the anionic form of the flavin at  alkaline p H should decrease the rate constant for the native cytochrome reduction to a much greater extent than in the case of the modified protein as a consequence solvent exposed heme propionate  groups  (Reid et al.,  1984).  reactivity of the anionic form of the flavin (Przysiecki et al., reduction of both cytochrome b  s  derivatives.  of the electrostatic effect of the  On the other  hand,  the  increased  1985) will tend to increase the rate of  For the native protein, this second effect appears to  be exactly offset  by the inhibitory electrostatic influence of the heme propionate groups, while for  DME-cytochrome  b  5  elimination of the  electrostatic  contribution of the  the increased reactivity of LfH- at alkaline p H to prevail. two  forms  of  cytochrome  b  is not  5  related  to  changes  reduction potentials of native and DME-cytochrome b (Reid etal,  5  propionate  groups  permits  Clearly, the differential response of die in thermodynamic  are largely unaffected  driving  force  as  by this change in pH  1982, 1984).  While the mechanistic basis for the increase in the reduction rate constant of cytochrome b the presence barrier  to  the  5  in  of cytochrome c is unknown, it may be related to a decrease in the reorganizational electron  cytochrome b  5  transfer  or  to  an  increase  in  the  reduction  potentials  that is induced by binding of either protein to cytochrome c.  of  both  forms  of  This latter possibility  could be related to the increase in reduction potential observed for native and DME-cytochrome b  s  with increased ionic strength (Reid et al.,  1982, 1984), the origin of which is not fully  The increase in reactivity of cytochrome b  5  understood.  associated with protein-protein complex formation is not  94  without precedent.  For the reduction of bovine cytochrome c oxidase (Ahmad et al.,  yeast cytochrome c peroxidase (Hazzard et al.,  1987) by free flavin semiquinones, electron transfer is  not fast enough to compete with semiquinone disproportionation. however, though  rapid electron the  mechanism  electron transfer. effective  transfer  from flavin  involves direct  1982) and of  to either  reduction  of  In the presence of cytochrome c,  the oxidase or the  cytochrome  the peroxidase c  followed  does occur,  by  intracomplex  In these cases, it has been argued that binding to cytochrome c establishes  electron-transfer  acceptor protein.  pathway  through  structural  changes  induced  in  the  ultimate  an  electron  Whether a similar mechanism is responsible for the present observation  requires  further study. Reduction of Native and constant  for F M N H -  reduction affected  of  strong  cytochrome b 1986).  s  c  (Table  electrostatic  by F M N H - :  5  reduction of cytochrome b  cytochrome  by  DME-Cytochrome b  V)  5  suggests  The observation that the rate  (Table VI) is so much slower than that for the that  repulsion between  FMNH"  the  reduction  negatively  charged  kinetics  are  FMNH-  significantly  and  bodi  free  species, as would be expected from previous electrostatic calculations (Mauk et  al.,  The small magnitude of the difference between the rate of reduction of native and D M E -  cytochromes b  5  by F M N H - suggests that although the electrostatic repulsion between F M N H  and die  protein may be affected by the neutralisation of the heme propionate charge by esterification, other factors  such  as  heme  accessibility and  the  change  in the  reduction  potential  also  reduction kinetics, resulting in a complex mixture of partially cancelling effects. rate constants for the reduction of native and DME-cytochrome b  5  reductant with a net charge of -2, are 112 and 1970 M ' V et al.,  1984).  kinetics  of  The difference in the effect  these two  reductants  [2-fold  1  influence  The  by Fe(EDTA) ",  the  second-order another small  2  (I = 0.1 M and p H 7) respectively, (Reid  of the heme propionate esterification on the reaction  for  FMNH"  versus  10-fold  fundamental difference in the mechanism of reduction of cytochrome b  for 5  Fe(EDTA) "] 2  suggests a  by these reductants, possibly  involving requirement for * -orbital overlap between the flavin and the heme prosthetic group. The non-linearity of the plots of k  o b s  versus complex concentration for cytochrome b  95  5  in the  presence of cytochrome c (Figure 20) suggests that there is a first order rate limiting process in the reaction mechanism.  One possible explanation for this is represented  by [a] and  fe (III)-cyt c(II)* represents a form of the complex with which F M N H -  [b] where cyt  preferentially reacts, perhaps  5  as the result of increased accessibility of the cytochrome b heme group. Thus, the rate limiting 5  cyt fe (III)-cyt c(II)  ^ - > cyt & (TiI)-cyt c(U)*  5  [a]  5  cyt & (HI)-cyt c(IJ.)* + F M N H - - ^ - > cyt fc (II)-cyt c(II) + F M N 5  process at high concentrations combination  with  our  [b]  5  is the first-order isomerisation of the complex.  current  results  implies that  the  active  form  of  This mechanism in  the  complex  must  have  electrostatic properties that are distinct from those of the inactive form and to which reduction by F M N H - (but not L f H ) is sensitive. A  second  plausible  mechanism  involves the  formation  of  a  kinetically  detectable  precursor  complex between the protein-protein complex and F M N H - as represented by equations [c] and [d].  cyt fc (ffl)-cyt cQl) + F M N H -  k  5  2  > cyt & (ffl)-cyt c(II)-FMNH-  [c]  5  cyt & (III)-cyt c(II)-FMNH- — ^ - > cyt i> (II)-cyt c(II) + F M N 5  Inasmuch  as  these  two  mechanisms  are  mathematically  equivalent  (Stricklan et  numerical values for the first- and second-order rate constants are the same. the  second-order  bound  rate constant  cytochrome  b$  and  [d]  5  reflects  is  thus  to  the  effective  1975),  the  For either mechanism,  the interaction of the negatively charged sensitive  al.,  electrostatic  FMNH-  with  the  environment  near  the  interaction domain. The  large  ferricytochrome considerable  values b  5  for  compared  neutralisation  of  the to the  second-order  rate  those observed cytochrome  constants  with  b  5  96  the  negative  of  free  semiquinone proteins  charge,  and  reduction  indicate hence  a  that  of there  bound is a  greater reactivity  with  the  reduced  cytochrome b  5  consistent  F M N , in the  complex.  The increased  rate constant  for  reduction  of D M E -  in complexes with horse or tuna cytochrome c, relative to native cytochrome b ,  with the results obtained with the free proteins.  Whereas no difference  between  and tuna cytochrome c reduction was observed in the complexes with cytochrome b , marked species dependent differences in the cytochrome b  5  DME-cytochrome b ,  the  s  values for  the second-order  reduction kinetics.  rate constants in the  For bodi native and complexes  cytochrome c were significantly smaller than those obtained with tuna cytochrome c.  cytochrome b  there is 5  either  a  less  negative  or a  more  horse  there were  5  suggests that  is  5  positive electrostatic  with  horse  This finding  potential  near  the  reduction site in the complex with tuna cytochrome c than is the case in the complex  with horse cytochrome c. The interpretation the mechanism. Clostridium  of the effects  on the rate-limiting first-order  rate constant  is dependent on  In one previous case, F M N H - reduction of ferricytochrome c in a 1:1 complex with  pasteurianum  flavodoxin,  it  was  shown  unambiguously  that  the  mechanism  given by  equations [a] and [b] was correct, based on the independence of the first-order rate-limiting process of the identity of the reductant (Hazzard et al., made.  However,  mechanism  some  given by  1986).  discussion concerning the  equation  [a]  and  [b],  a  In the present case, no such comparison was  mechanism can be  larger  value for  kj  given.  According  implies that  ^cytochrome c complex can explore different docking geometries more readily. values obtained with complexes involving DME-cytochrome b  cytochrome b  is that esterification of the heme propionates makes the cytochrome  5  the  cytochrome  One explanation for  the larger kj  5  the  to  as opposed to native fe cytochrome 5  c  complex more dynamic (i.e., the barriers to rotation between different docking geometries are lower in  the  DME-cytochrome ^cytochrome  c complex).  Similarly,  the greater value of k] for  tuna  cytochrome c complexes would imply that a more dynamic complex is formed with this cytochrome than with horse cytochrome c. small ( A A G t results  Admittedly, the differences  ~0.3 kcal/mol based on the ratio of kj).  in terms of the  mechanism of equations  97  in these barriers  to rotation are quite  Although it is more difficult to rationalise the  [c] and  [d],  such arguments  are  of course  not  definitive and further study is required. Calculation of the solvent exposure of the heme groups in the two static docking geometries that have been proposed for the cytochrome ^cytochrome c complex (Salemme, 1976; Mauk et  al.,  1986)  unaffected  by  On the other hand,  the  indicates  cytochrome b exposure  5  that the  exposure  of cytochrome b  heme  5  to solvent is essentially  binding of cytochrome c in either orientation (Table VII).  of the cytochrome c heme  is unaffected  on binding to cytochrome b  in the edge on  s  docking geometry (Figure 6) while it is reduced ~ 3 5 % on binding to this protein in the off edge docking geometry (Figure 7). constant  for  the  cytochrome b  s  reduction  The current results are consistent with these models in that the rate of  cytochrome  c  by  LfH  -  is  unaffected  by  the  binding  at p H 7.0 but is reduced by 25% upon binding to DME-cytochrome b  5  to  native  at p H 8.0.  Nevertheless, the value of estimating the effect of complex formation on solvent exposure is clearly limited  in light  (Wendoloski  of  et  al.,  peroxidase-cytochrome  molecular dynamics calculations of 1987)  and  Brownian  the  Dynamics  c complex (Northrup et al.,  cytochrome calculations  1988) which  ^cytochrome of  the  c complex  cytochrome  suggest that there may be  c  some  flexibility in the solution conformations of electron transfer protein-protein complexes.  Ferrocytochrome b Reduction of Ferricytochrome c 5  Pseudo-first order conditions for analysis of a second order reaction are satisfied when one of the reactants is i n at least a 40 fold excess over the other (Moore & Pearson, 1981). extinction reduction  coefficients of  exhibited  ferricytochrome  c  by  cytochromes  severely  restricted  b  and  5  the  order kinetics could be performed in the present study. order reaction in which one of the reactants is in a first order process underestimates  c  and  the  concentration  rate  of  range over  ferrocytochrome which  b  5  pseudo-first  Calculations showed that treating a second five-fold  excess over the other as a pseudo-  the second order rate constant by 10%.  A similar treatment of a  reaction in which one of the reactants is in a ten-fold excess over the other  98  The large  underestimates  the  rate constant  by 5% (data not shown).  These errors are within the reproducibility of the second  order rate constants measured in this work. of and  ferrocytochrome the  second  concentrations  of  rate  argues that  determined  b  and  used  to  5  that  constants  the  obtained  rate constant  under  b  s  reduction  obtained  prepare  with  of  pseudo-first  conditions b  involving reduction  5  ferricytochrome  c  b  does  5  not  obtained  Although it is preferrable  to  conditions  equimolar  reactant c  the second order rate  using  cytochrome  contribute  order  of ferricytochrome  The agreement between  photolytically reduced  ferrocytochrome  observed in the stopped flow cuvette.  under  of ferrocytochrome  by either technique.  ferrocytochrome  cytochrome lumiflavin  reduction of ferricytochrome c obtained  5  order  can be accurately constant  b  The agreement between the second order rate constants  dithionite  b  indicates  5  die  reduced diat  absorbance  the  changes  to keep die reaction mixture as  simple as possible, it was difficult to perform the reaction in the absence of lumiflavin because once the reductant was removed from the ferrocytochrome b ,  up to 10% of the protein auto-oxidised.  s  the presence  of lumiflavin,  any ferrocytochrome  b  s  that oxidised was re-reduced  until  In  all of the  oxidant, presumably oxygen (Berman et al., 1976) was purged from the protein solution. Strittmatter to be 4.7 x 10  (1964) first reported the rate of ferrocytochrome b 7  M'V  novel stopped flow  1  reduction of ferricytochrome c  (0.1 M phosphate, p H 7.0, 25°C) in a brief communication that described a  technique.  This value is difficult  to compare with the current  ionic strength used in the earlier study is ambiguous. was probably used in this early report this is not stated clearly either. was 0.25 M .  s  results as the  Furthermore, lipase solubilised cytochrome  rather than the trypsin solubilised form used here,  b  5  tiiough  If the buffer was 0.1 M phosphate (pH 7), then the ionic strength  In this event, the current results [4.7 ( ± 0 . 4 ) and 9.5 ( ± 0 . 6 ) x 10  7  M'V  at ionic  strengths of 0.3 and 0.2 M respectively] are in good agreement with Strittmatter's measurement. The extremely  bimolecular large.  rate  constant  None of the  for  reported  ferrocytochrome second order  b  5  reduction  of  rate constants of electron  soluble electron transfer proteins are as great at 0.1 M ionic strength, p H 7.0. unexpected  considering  that  the  exclusive biological  99  ferricytochrome  function  of  these  two  transfer  c  is  between  Perhaps diis is not proteins  is  electron  transfer.  Hemoproteins such as cytochrome c peroxidase and hemoglobin, whose rates of electron  transfer  with other proteins are considerably slower (Cheung et a l . , 1986; Summer & Erman, 1988;  Mauk et al., expect  1984), do not function primarily as electron transfer  that factors  proteins.  It is reasonable  to  such as the reorganisational energy of cytochromes would be optimised for the  purpose of electron transfer in a way that could not be achieved in a protein that has evolved to perform other functions.  The reorganisation energies of cytochrome c peroxidase and myoglobin are  greater than those of cytochromes b constants of electron transfer Augustin et al.,  ferrocytochrome b  5  transfer  7  soluble electron transfer  Comparison of bimolecular rate  proteins  (Wherland & Pecht,  1978;  1986) reveals that one of the few measured second order rate  between  1  pairs  of electron  transfer  proteins  that  (pH 7.0, I = 0.1 M , 25°C)] (Takana et al., 1981).  of ferrocytochrome b  between  1986).  approaches  diat of  reduction of ferricytochrome c is that of the oxidation of ferrocytochrome / by  plastocyanin [6.0 x 10 M ' V  measured  between  1983; Tollin et al.,  constants of electron  rate constant  and c (Mayo et al.,  s  either  The second order  reduction of ferricytochrome c is also much greater than that  s  of these proteins and a wide variety of small electron transfer  reagents  (Hodges et al., 1974; Wherland & Gray, 1976; Cummins & Gray, 1977; Mauk et al., 1979; Reid & Mauk, 1982; Reid et al., 1984; Chapman et al., 1984). This observation is discussed below. The unimolecular rate constant of electron transfer from cytochrome b  5  the complex has been determined by pulse radiolysis to be 1600 ( ± 7 0 0 ) s"  1  phosphate,  p H 7](McLendon & Miller,  study (1.7 pM),  (t^  = 0.43 ms)[l m M  At the concentrations of protein used in the current  the half life of the bimolecular reaction (I = 0.5 M , p H 7.0) is about 30 ms.  second order rate constant process  1985).  to cytochrome c within  would have to be 1.4 x 10  to be equal to the half-life  9  M'V  1  of the first order process.  The  for the half-life of the bimolecular If electron transfer  between  the  heme groups within the cytochrome ^cytochrome c complex is comparable at 1 m M phosphate and I =  0.5 M , and ferrocytochrome b  5  reduction of cytochrome c involves a protein-protein binding step  prior to electron transfer (Scott et al., ferrocytochrome  b  5  1985), then at I = 0.5 M , p H 7 the association constant of  and ferricytochrome c is M O  100  4  M" . 1  This value is larger than experimentally  determined values [e.g. 10  3  M " measured at I = 0.04 M (Eley & Moore, 1983)] suggesting diat the 1  rate of electron transfer within the protein-protein complex is ionic strength dependent. Neutralising the charge of the heme propionates of cytochrome b bimolecular rate constant of ferrocytochrome b  5  is expected to influence the  5  reduction of ferricytochrome c in at least two ways:  (1) through perturbation of the electrostatic potential surface of the cytochrome b  and (2) through  5  alteration of the thermodynamic driving force for electron transfer. charges protein.  from  the  surface  of the  protein would  alter  both  the  First, removing two negative  monopole and  the  Theoretical calculations indicate that the dipole moment of cytochrome b  is reduced by  5  this modification (Table IX).  According to the model developed for the reaction between cytochrome  c peroxidase and cytochrome c (Northrup et al.,  1988), the principal effect of diminishing die dipole  moment is to decrease the rate at which the proteins come together in solution. monopole might affect the geometry of the complex.  charge distribution on the surface of cytochrome b ^cytochrome c docking geometries. transfer  between  the  1986) indicate that a change in die  stabilises a different  s  two  proteins  through  alterations  of DME-cytochrome b  is 60 mV higher (Reid et al.,  the  of cytochrome c is 261  reduction  potential  population of cytochrome  Different docking geometries can readily influence the rate of in  protein structure intervening between the donor and acceptor sites. 5  A change in the  The present studies combined with the binding  studies previously reported from this laboratory (Mauk et al.,  electron  dipole of die  s  separation,  orientation  and  Second, die reduction potential  1984) than that of native cytochrome b .  As  s  mV (Margolit  increasing the reduction potential of cytochrome b  the  &  Schejter,  1973),  the  effect  of  is to decrease the driving force of the reaction  and thereby reduce the rate of electron transfer within the complex. In the current experiments, the second order rate constant for ferrocytochrome b ferricytochrome  was  decreased  by  esterification  of  the  heme  propionates  when  5  the  reduction of reaction  was  performed at ionic strengths less than 0.5 M .  However, there was no difference in die bimolecular  rate constants for  by  ferricytochrome c  strengths of 0.5 M and greater.  reduction  native  and  D M E - ferrocytochrome  b  5  Several mechanisms could account for these observations.  101  at  ionic First,  the  reduction  cytochrome  potentials  c.  of  native  Alternatively, the  and  DME-cytochromes b  might  s  bimolecular rate constant  for  be  similar  ferrocytochrome  when b  5  bound  to  reduction of  ferricytochrome c might involve a rate determining step such as protein-protein coniplexation or a protein conformational heme  groups  within  change.  In such a mechanism,  the complex would not  the  rate of electron  transfer  between  the  influence the bimolecular rate of ferrocytochrome  reduction of ferricytochrome c and hence factors influencing the rate of electron transfer  b  5  widiin die  complex would not affect the bimolecular rate constant. Measurements of the bimolecular rate constant of ferrocytochrome b ferricytochromes c (Eltis et al., nature of  the  protein-protein  between cytochromes b  5  heme propionates of  ferricytochrome  interface  and c.  through  reduction of mutant  yeast  unpublished) indicate that factors such as the driving force and the influence the  bimolecular rate constant  of electron  transfer  This observation indicates that esterification of the cytochrome  probably decreases the bimolecular rate constant c  5  perturbation  of the  interaction of  for ferrocytochrome these two  proteins  b  5  and  b  5  reduction diat  die  reduction potential of native and DME-cytochromes are similar when bound to cytochrome c Several points regarding the reduction potentials and docking geometry c are worth considering.  of cytochromes b  The reduction potentials of the cytochromes used in these studies  measured for the proteins free in solution.  5  and were  It is not at all clear that die reduction potential of a  cytochrome bound to another protein is the same as the reduction potential of that same cytochrome free in solution.  Charged groups and the exclusion of solvent from the heme, factors that change  upon complex formation, have a documented effect on the reduction potential of cytochromes (Moore & Williams, 1977; Stellwagen, 1978).  The reduction potential of cytochrome c has been determined to  be unaffected by the presence of cytochrome b  5  performed  at  an  complex formation.  ionic  strength  that  is too  (Vanderkooi & Erecinska, 1974), but this study was high to  promote  Electrostatic calculations (Mauk et al.,  reduction kinetics of cytochrome b  5  complex, the cytochrome b  s  stable  cytochrome  ^cytochrome  c  1986) and the current flavin semiquinone  and cytochrome c argue that in the cytochrome ^cytochrome c  heme propionates  are neutralised by the positive electrostatic  102  field of  lysine  residues  on  cytochrome b  s  solution.  the  surface  potential  of  within the complex might approach the reduction potential of DME-cytochrome b  in  The principal effect  on  cytochrome  of the  heme  c.  For  propionate  this  reason,  the  reduction  esterification on cytochrome  b  s  behaviour  5  might be to leave a residue on cytochrome c unpaired in the complex, and thus to perturb reduction potential of cytochrome c in the cytochrome ^cytochrome c complex. the difference in reduction potentials of native and DME-cytochromes b another  factor  interesting  that  to  influences  measure  electron  the  transfer,  reduction  such  potentials  as of  It is possible that  is offset by a difference in  s  reorganisational the  the  energies.  It  within  the  cytochromes  would  be  complex.  Alternatively, it would be interesting to investigate the effect of the reduction potential and docking geometry  on  transfer  the  within  rate of electron a  covalently  transfer  crosslinked  within  a complex by  measuring  the  rate of electron  DME-ferrocytochrome Zj ferricytochrome 5  c  complex  of  defined structure. With respect to the docking geometries of the cytochrome ^cytochrome c complex, it should be remembered  that the models (Figs. 6 & 7) represent shallow minima in the energy  possible docking orientations  1986) and, as is suggested  by  Brownian dynamic simulations of the cytochrome c peroxidase-cytochrome c complex (Northrup et  al.,  1988),  movement  strength.  between  Cytochrome  formation,  and hence  at low ionic strengths (Mauk et al.,  surface of  these geometries  b  5  heme  decrease  the  rate  of  propionates  might  transfer  complexes  as  distinguish  between  propionate  the second order  by one of two mechanisms.  occur  very  esterification  readily,  could  particularly  influence  rate of ferrocytochrome b  5  at  high  protein-protein  the  formed  association.  formation by  the  of  native  these two possibilities.  Second,  complexes  reduction of ferricytochrome  cytochromes. In either  esterification  that  are  not  At this  case,  ionic  complex  First, reduction of the magnitude of the dipole of cytochrome b  protein-protein  promote  could  of as  time,  increasing the  cytochrome  b  5  s  might heme  favourable  to  it  possible  is not  ionic  strength  c,  electron to  of die  protein solution would dampen these effects, consistent with the current observations. Protein conformational rearrangements are not likely  103  to contribute significantly to the rate of  ferrocytochrome  b  reduction of ferricytochrome c.  5  Several studies  the reactants are very similar to those of the products. cytochromes  b  nor  s  oxidation state. change  a change  As discussed in the Introduction,  in spin state nor  Some conformational changes  axial  ligation with  of ferric and ferrocytochrome b  the  three dimensional  The M C D and C D spectra of cytochrome c are  upon binding to cytochrome c oxidase (Weber et al., interpreted  as a conformational change  1989b).  Measurements  1987).  This spectral perturbation  in the heme environment  of phosphorescence  lifetime have  there  are  no  ferrocytochrome b  5  significant  protein  conformational  perturbed has been  of cytochrome c (Michel et  suggested  that the  conformation  cytochrome c changes upon binding to cytochrome c peroxidase (Koloczek et al., processes  diat  affect  1987).  electron  al.,  of zinc The view  transfer  in  reduction of ferricytochrome c is supported by a preliminary report on ' H N M R  studies that indicates that the structures of cytochromes b solution (Veitch et al., changes  die  There are several reports of complex formation affecting the environment of die heme  groups of the interacting hemoproteins.  that  change of  are reported to be identical to each odier (Mathews et  s  1979).  the  of  neither  in the side chains of cytochrome c accompany  in oxidation state of the protein (Takano & Dickerson, 1980), but  structures al.,  c undergoes  indicate that the structures  1988).  in ferrocytochrome  b  5  s  and c in the complex are the same as in  It is not possible to rule out completely the role of conformational reduction  of ferricytochrome  c,  particularly as  the  mechanisms of  reactions involving azurin (Corin et al., 1983), cytochrome c peroxidase (Cheung & English, 1987) and cytochrome b  2  (McLendon et al.,  1987) remain so poorly defined.  However, there is no kinetic  evidence for a conformational change in the present case. Several  authors  have  analyzed  second  order  protein-protein  electron  transfer  electron transfer theory as developed by Marcus and others (Wherland & Pecht, 1986; Dixon  et al.,  1989).  bimolecular rate constant Dixon et al. b  5  The Marcus relationship (equation  for ferrocytochrome b  s  1  by  1978; Tollin et  al.,  5) can be used  to calculate  the  reduction of ferricytochrome c in a similar maimer.  (1989) have measured the electron transfer  and c to be 2600 M ' V  reactions  self-exchange rate constants of cytochromes  (I = 0.12 M , 25 °C) and 5400 M ' V  104  1  (I = 0.12 M , 27 °C) respectively,  and /  can be estimated to be 0.43 if the collision frequency  1966).  is 1 0  The second order rate constant for ferrocytochrome b  5  M s  10  reduction of ferricytochrome c, using  a thermodynamic driving force of 255 mV, is calculated to be 3.5 x 10 value is several orders  of magnitude  (Reynolds & Luniry,  _ I  smaller than the observed  5  M s~ A  (equation 5).  l  rate constant.  This  Variation of die  collision frequency by two orders of magnitude does not affect / by more than 30%.  It is not clear  whether the Marcus relationship fails in the present case because the electron transfer  reactions  are  not uniformly nonadiabatic or because electron transfer is not rate limiting. The observed ionic strength dependence  of the bimolecular rate constant  reflects die large role  of electrostatic forces in the association process.  Unfortunately, the electron transfer  association constants cannot  as they cannot  strengths with  the  and the current the  be directly compared  techniques  currently available.  Nevertheless,  rates and die  be obtained at comparable the results  ionic  of die binding studies  second order rate studies are consistent in that both the association constants and  second order  rate constant  dependent on ionic strength  of ferrocytochrome  b  reduction of ferricytochrome c are  s  strongly  and that bimolecular kinetics are observed under conditions where no  complex is detected by electronic absorption spectroscopy (Mauk et al.,  1982; 1986) or fluorescence  quenching titrations. The fits of the data to the Van Leeuwen model (equation 11) appear to be quite good, but it is worth noting two points about the best fit parameters. the cytochromes were  fixed,  First, in the fits in which the monopoles of  the radii of cytochromes b  5  the size that the crystal data suggest (Mathews et al., in  the  fits  in which  the  radii  were  fixed,  Z{Z^  1979; Takano & Dickerson, 1980a,b). for  approximately twice that for the native cytochromes needs  some  refinement.  It  is  constants for DME-cytochrome b  5  interesting  to  and c (Table VIII) are approximately half  note  DME-cytochrome b  (Table Vffl). that  s  Second,  and cytochrome c is  This suggests that the model  Wherland-Gray  fits  of  the  association  and cytochrome c yielded higher Z j / ^ than for native cytochrome  b and cytochrome c (Mauk et al., 1986). 5  The equivalence of the rate constant  for reduction of ferricytochrome c by native and D M E -  105  ferrocytochrome b  s  b  5  at high ionic strength has been discussed above.  The rate of ferrocytochrome  reduction of (trifluoromethyl)phenylcarbamyl-Lys-13 cytochrome c has been measured to be ~37%  that of native ferricytochrome c at 0.1 M ionic strength (Ng et al., 1977), but at I = 1.5 M , the two forms of cytochrome c are reduced at the same rate by ferrocytochrome b 1979).  (Stoneheurner  et  al.,  Although the rate constant that these workers report is estimated from steady state kinetics  and was not observed  5  measured  directly, the ionic strength  in the present study.  dependence  that they  report  is similar to that  It is also intriguing that cytochrome c lysine modifications diat  decreased this rate the most at I = 0.1 M (Lys-13, Lys-72) exhibited rate constants that where 35% to  40%  that  of  native  cytochrome  c  (Stoneheurner  ferricytochrome c reduction by DME-ferrocytochrome b  5  et  al.,  1979).  In  the  present  (I = 0.1 M ) yielded a rate constant that is  70% smaller than that of ferricytochrome c reduction by native ferrocytochrome b .  While D M E -  5  cytochrome b$ is modified at two positions, these results are consistent cytochrome b  with  the  involvement of  heme propionate-6 in stabilizing the cytochrome ^cytochrome c complex to die same  5  extent as cytochrome c lysines 13 and 72. cytochrome  fc cytochrome 5  This result is clearly consistent with the model of the  c complex proposed by Salemme (1976)(Figure 6, Table I).  kinetic studies that detect differences  Steady state  in reactivity between native cytochrome c and cytochromes c  singly modified at various surface lysines with a number of enzymes (Ferguson-Miller et al., Kang et al.,  study,  1978;  1978; Speck et al., 1979; Smith et al., 1980; Speck et al., 1981) were performed at ionic  strengths below 0.2  M.  The second order  rate constants of plastocyanin reduction by modified  cytochromes c were measured at 0.1 M ionic strength (Augustin et al.,  1983).  The greatest decrease  in the second order rate constant of plastocyanin reduction was 50%, which was observed for the 4carboxy-2,6-dinitrophenyl-Lys 13 cytochrome c derivative. the current  observations  These previous studies are consistent with  that chemical modifications that alter die surface  the protein only decrease the bimolecular rate constant of electron transfer  electrostatic below I  properties of =  0.5 M and  that the magnitude of this decrease is at most 70%. The p H independence of the bimolecular rate constant of ferricytochrome c reduction by either  106  D M E - or native ferrocytochrome b  5  is not altogether surprising.  Any differences  in reactivity that  might arise would be due to the titration of a group on one of the proteins that would alter the electrostatics  of  this  protein  and  hence  its  capability  to  interact  Electrostatic calculations indicate that His-26 of cytochrome b , 5  8.0 (Mauk et al., little  difference  1986). between  cytochromes b ,  other  protein.  or c that titrates over the p H range 6.0 to  However, at the ionic strength of the current study (I = 0.5 M ) diere was the  rate constant  of ferricytochrome  which differ in charge by -2.  5  the  a residue found on die surface of  s  the protein, is the only residue on either cytochromes b  with  c  This finding  reduction  by  D M E - and  native  suggests that it is unlikely  diat a  difference of a single charge would affect the rate constant of reduction of ferricytochrome c at I = 0.5 M .  It was not possible to repeat the p H dependence at 0.1 M ionic strength as die reaction was  too fast to monitor under these conditions.  While it is not clear what the reduction potentials of  the proteins are in the cytochrome ^cytochrome c complex, it is worth noting diat the reduction potentials  of native and DME-cytochromes b  and cytochrome c,  s  free  in solution, do not  change  more than 10 m V between p H 6.0 and 8.0 (Margalit & Schejter, 1973; Reid et al., 1982; 1984). The thermodynamic activation parameters for ferricytochrome c reduction by native and D M E ferrocytochrome b ferrocytochrome the  are very similar (I = 0.5 M , p H 7.0).  5  b  5  cytochrome b  5  This result suggests that the mechanism of  reduction of ferricytochrome c is not fundamentally heme  propionates.  With the notable  activation enthalpy and entropy of ferrocytochrome b  5  by esterification of  exception of self-exchange  reactions,  the  reduction of ferricytochrome c fall within the  values measured for other bimolecular protein-protein electron transfer the activation enthalpy of ferrocytochrome b  altered  reactions (Table XII).  While  reduction of ferricytochrome c is comparable to that  5  of the bimolecular oxidation or reduction of either cytochromes b  5  (Reid & Mauk, 1982; Reid et al.,  1984; Chapman et al., 1984) or c (Hodges et al., 1974; Cummins & Gray, 1977; Mauk et al., 1979) with a is  variety of small electron transfer considerably  electron transfer  smaller  than  reagents, the activation entropy  those of  the  protein-reagent  between either cytochromes b  5  reactions.  of the protein-protein The  reaction  activation entropy  for  or c and a small reagent varies from -11 e.u. for  107  ferricytochrome c oxidation by r^ntaanmiinepyridineruthenium(III)  (I = 0.5 M , p H 5.3, 25°C) (Cummins  & Gray, 1977) to -30 e.u. for DME-ferricytochrome b reduction by Fe(EDTA)" (I = 0.1 M , p H 7.0, 2  5  Table  XII :  Thermodynamic  parameters  of several  second  order  protein-protein  electron  transfer  reactions.  reactants  A H 1 (kcal/mol)  ferrocytochrome/ + cupriplastocyanin  A S t (e.u.)  reference  ld  10.5  11.0  ferrocytochrome/ + cupriplastocyanin  2b  8.4  4.9  N i w a e r a / . , 1980  ferricytochrome / + cuproplastocyanin  2b  8.6  3.9  ibid  3  13.4  21.5  ferrocytochrome c + cupriplastocyanin  7.6  - 2.4  King etal., 1985 Rosen & Pecht, 1976  ferrocytochrome/ + cupriplastocyanin  ferrocytochrome c ferricytochrome c  5 5 1  ferrocytochrome c ferricytochrome c  5 5 1  5 5 1  5 5 1  8  Wood, 1974  Tanakaer al., 1981  + cupriazurin  40  7.8  - 1.1  + cuproazurin  40  13.7  18.8  ibid  + cupriazurin  5d  18.1  31.0  Rosen etal., 1981  + cuproazurin  5d  15.2  22.5  ibid  ferrocytochrome c + ferricytochrome c  8.4  - 11  Barbush & Dixon"  ferrocytochrome b + ferricytochrome b  5.5  - 23  Dixon et al., 1989  ferrocytochrome b + ferricytochrome c  7.5  - 0.3  this work  7.9  1.0  diis work  5  5  e  s  DME-ferrocytochrome b + ferricytochrome c  e  5  Solution conditions were: I = 0.1 M , p H 7.5; I = 0.2 M , p H 7.0; I not stated, p H 7.0; I = 0.1 M , a  b  p H 7.0; I = 0.5 M , p H 7.0. e  ^Petroselinum ^Alcaligenes  sativum; faecalis.  C  d  The sources of proteins isolated from non-mammalian tissues were:  Brassica  komatsuna;  In  instances  2  those  ^Raphanous  when  the  errors  parameters are reported, they are not greater than 1 unit, "unpublished.  108  sativus; in  ^Pseudomonas the  thermodynamic  aeruginosa; activation  25°C) (Reid et al., transfer each  between  of  1984).  This result suggests that the principal reason diat the rate of electron  cytochromes  these proteins  and  b  and c so greatly  s  small  reagents  is  electron transfer  reaction is so much greater.  transfer  small  between  exceeds  that  the  the  rate of electron  activation  entropy  of  transfer  die  between  protein-protein  The large, negative activation entropies of electron  reagents and proteins  has been  partly  explained in terms of the  loss of  translational and rotational degrees of freedom accompanying formation of the collisional complex of the reactants (Bennett, 1973).  The magnitude of this effect has been estimated to be -13 e.u..  possible that in the case of ferrocytochrome b  It is  reduction of ferricytochrome c, these entropic losses  5  could be compensated by the release of solvent molecules at the protein-protein interface in complex formation (Chothia & Janin, 1975).  This interpretation is consistent  the rate limiting step in ferrocytochrome b  5  with complex formation being  reduction of ferricytochrome c.  of experimental data for protein-protein electron transfer  reactions,  the  variation in  generality  of  this  phenomenon.  Nevertheless,  the  Given the limited range  it is premature the  to comment on  activation parameters of  protein-protein electron transfer  reactions is remarkable, even amongst  different  It may be that different electron transfer protein pairs have evolved  sources (Table XII).  similar proteins isolated from  slightly different mechanisms to enhance their reactivities. The  present  results  indicate  that  the  second  reduction of ferricytochrome c is determined largely  controlled by  between  cytochromes  cytochrome  b, s  electrostatic  forces.  b  was  5  suggesting  and  c  that  the  similar when bound to cytochrome c. heme propionates of  protein-protein  electron  transfer.  order  rate  in part by the At high ionic  unaffected  reduction  by  potentials  constant  ferrocytochrome  b  rate of complex formation which  strength,  esterification of native  The principal effect  for  die  rate of electron heme  5  is  transfer  of  the  propionates  and  DME-cytochromes b  of are  5  of esterification of die cytochrome  b  5  is to influence complex formation with cytochrome c, either by reducing the rate association or promoting the formation of complexes that are The  results  are  consistent  with  the  participation  of  not  conducive to  cytochrome  propionate-6 in an intermolecular salt bridge that stabilises the cytochrome ^cytochrome c complex.  109  b  5  heme  CONCLUSION  The  work outlined in this dissertation  has  evaluated  kinetics of electron transfer between cytochrome b to  consideration  of  the  influence of the  aspects of the  and cytochrome c.  5  given  several  cytochrome  b  5  interaction  and  Particular emphasis has been  heme  propionate  groups  in these  phenomena and on the use of porphyrin cytochrome c as a fluorescent analogue for cytochrome c in such investigations.  In addition, the dependence of the mechanism of protein-protein interaction and  electron  solution conditions (pH, ionic  transfer  on  strength,  temperature)  has  been  evaluated  for  these proteins. The  fluorescence  cytochrome b  5  quenching  studies  on  the  interaction  of  porphyrin  cytochrome  c  and  combined with the kinetics of flavin semiquinone reduction of the cytochromes within  the cytochrome i cytochrome c complex provide evidence that esterification of the cytochrome 5  heme  propionate  groups  influences the docking geometry  of these two proteins.  These  b  5  findings  extend the conclusions obtained through electrostatic calculations (Mauk et al.,  1986) by establishing  two  heme  principal  cytochrome  points.  First,  ^cytochrome  the  c  distance  complex  propionate groups are esterified.  is  of  separation  detectably  between  increased  the  when  two the  groups  cytochrome  in  b  the heme  s  Second, die cytochrome c heme group is not as sterically hindered  in the complex formed by this protein with DME-cytochrome b  5  as in the complex formed between  the two native proteins. The stopped-flow studies demonstrate that the bimolecular rate constant  for ferrocytochrome  b  s  reduction of ferricytochrome c is determined in part by the rate of association of the two proteins. This  rate of  cytochrome b  association 5  heme  is  strongly  propionate  between cytochromes b  s  influenced by  electrostatic  forces.  The  esterification on the bimolecular rate constant  principal  effect  of electron  transfer  and c is to influence complex formation between diese two proteins.  stopped-flow studies suggest that the reduction potentials of native and DME-cytochromes b  s  significantly different when these proteins are bound to cytochrome c.  110  of  The  are not  Much  of the  literature  describing complexes formed  between  such complexes as discrete and structurally definable entities.  electron transfer  proteins  treats  The overall picture that emerges from  this work is one in which these protein-protein complexes may exist in a variety of structurally similar  orientations.  Furthermore,  it  appears  that  the  relative  population  of  a  given complex  geometry is influenced by surface modifications on either of the interacting proteins and by solution conditions.  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Fluorescence anisotropy is defined by equation 12 where Ip is the intensity of the emission when the observing s  Ip-Io  r =  Ip + 2I  [12] C  polarizer is parallel to the direction of the polarized excitation and I is the intensity of the emission when the observing polarizer is perpendicular to the direction of the polarized excitation. I is corrected for the inability of the spectrofluorometer to detect I and I with equal efficiency. 0  0  p  Anisotropy is related to the rotational correlation time, the fluorophore by the Pen-in equation (13). R  c  and the fluorescence lifetime, r , of  0  r =  [13] 1 +  T/$  The rotational correlation time of the fluorophore is related to the volume, V , of the molecule in which the fluorophore is located by equation 14 where n is the viscosity of the solution (.00894 flV $ = — k T  • [14]  B  poise), k is Boltzmann's constant and T is the temperature. The relative volumes of free and complexed porphyrin cytochrome c will be the ratio of the rotational correlation lifetimes. B  The rotational correlation of free porphyrin cytochrome c, 3>, may be calculated by first estimating the volume of the molecule. Approximating the molecule as a sphere of radius 16 A , $ evaluates to 3.7 ns. The fluorescence lifetime of porphyrin cytochrome c, r , is 6.5 ns (Vanderkooi, 19). Knowing the maximal steady state fluorescence quenching ( Q = 0.25) of porphyrin cytochrome c by bound cytochrome b , the fluorescence lifetime of bound porphyrin cytochrome c, T , is calculated to be 4.9 ns by the equation 15. f  f  f  m a x  5  c  r  Q =  f  —  [15]  Assuming that the orientation of the dipole in the free and complexed porphyrin cytochrome c is the same (i.e., r is unaffected by cytochrome b binding) then the rotational correlation time of bound porphyrin cytochrome c may be calculated by taking the ratio of die Perrin equation of bound and free protein (equation 16). This yields a value of 8.5 ns. 0  5  122  r  c  1 + 7y$  r  f  1 + r /<J> c  f  c  The ratio of the rotational correlation times, and hence the volumes is 2.3.  123  APPENDIX B Second order rate constants for ferrocytochrome b reduction of ferricytochrome c 5  Cytochrome b  s  M'V )  pH  T(°C)  7.0 7.0 7.0 7.0 7.0  25.0 25.0 25.0 25.0 25.0  0.1 0.2 0.3 0.5 1.0  540 95 47 18.1 8.2  90 4 4 1.5 .8  6.0 6.5 7.5 8.0  25.0 25.0 25.0 25.0  0.5 0.5 0.5 0.5  18.3 17.6 18.7 20.0  .9 .9 .8 1.6  7.0 7,0 7.0 7.0  3.9 9.6 15.1 32.3  0.5 0.5 0.5 0.5  I(M)  k (x 10-  1  6  6.2 8.4 11.3 24.0  s.e.m.*  1.8 .7 • 1.1 .4  DME-Cytochrome b  5  I(M)  k (x 1Q-  M'V ) 1  pH  T(°C)  7.0 7.0 7.0 7.0 7.0  25.0 25.0 25.0 25.0 25.0  0.1 0.2 0.3 0.5 1.0  157 51 27 16.5 10.7  26 2 1 .6 .6  6.0 6.5 7.5 8.0  25.0 25.0 25.0 25.0  0.5 0.5 0.5 0.5  15.2 15.7 16.5 17.8  .1 .2 .5 .4  7.0 7.0 7.0 7.0 7.0  4.1 10.1 14.8 19.9 31.5  0.5 0.5 0.5 0.5 0.5  5.5 7.1 9.9 13.1 21.2  .3 1.5 .6 1.4 .8  'Standard error of the mean  124  6  


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