Photoinduced electron-transfer reaction in a ternary system involving zinc cytochrome c and plastocyanin. Interplay of monopolar and dipolar electrostatic interactions between metalloproteins.

A carbodiimide promotes noninvasive cross-linking between amino groups surrounding the exposed heme edge in zinc cytochrome c and carboxylic groups in the acidic patch in plastocyanin. Eight derivatives of the covalent complex Zncyt/pc(I), which have similar structures but different overall charges because of different numbers and locations of N-acylurea groups, are separated by cation-exchange chromatography. Kinetics of electron transfer from the diprotein complex in the triplet excited state, 3Zncyt/pc(I), to free cupriplastocyanin at pH 7.0 and various ionic strengths is studied by laser flash spectroscopy. This reaction is purely bimolecular for all eight N-acylurea derivatives of the diprotein complex. The overall charges of the derivatives 1 and 2 at pH 7.0 are -2 and 0, respectively; both of them, however, have very large dipole moments of 410-480 D. The rate constants for their reactions with cupriplastocyanin, whose charge at pH 7.0 is -8 and whose dipole moment is 362 D, are determined over the range of ionic strengths from 2.5 mM to 3.00 M. The observed dependence of the rate constants on ionic strength cannot be explained in terms of net charges (monopole-monopole interactions) alone, but it can be fitted quantitatively with a theory that recognizes also monopole-dipole and dipole-dipole interactions [van Leeuwen, J. W. (1983) Biochim. Biophys. Acta 743, 408]. At ionic strengths up to ca. 10 mM monopole-monopole interactions predominate and Br?nsted-Debye-Hückel theory applies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron-transfer reactions of photoreduced flavin analogues with c-type cytochromes: quantitation of steric and electrostatic factors

We have found correlations between rate constants and the difference in redox potential of the reactants for electron-transfer reactions between oxidized cytochromes and either photoproduced riboflavin or flavin mononucleotide (FMN) semiquinones (the latter rate constants extrapolated to infinite ionic strength). The riboflavin-cytochrome rate constants are about 70% of those for reduction by lumiflavin, probably because of steric interference by the ribityl side chain. Reduction of cytochromes by FMN semiquinone was ionic strength dependent in all cases, due to electrostatic interactions. Extrapolation of rate constants to infinite ionic strength shows that the phosphate exerts a significant steric effect as well (rate constants average about 27% of those for lumiflavin, although part of this decrease is due to a difference in the semiquinone pK value). Differences in the magnitude of the FMN steric effect correlate well with surface topology differences for those cytochromes whose three-dimensional structures are known. Mitochondrial cytochromes c and the cytochromes c2 all showed attractive (plus-minus) interaction with FMN in spite of the fact that some of these proteins have large net negative charges. Four small c-type cytochromes (including Pseudomonas cytochrome c-551) show a weak repulsive interaction with FMN semiquinone. We conclude that flavosemiquinones interact at a site on the cytochromes that is near the exposed heme edge. There is a large positive electrostatic field at this site in mitochondrial cytochrome c and the cytochromes c2, but this region is primarily hydrophobic in Pseudomonas cytochrome c-551 and in the other small bacterial cytochromes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron self-exchange in Pseudomonas cytochromes

Electron self-exchange has been measured by an NMR technique for cytochromes c551 from Pseudomonas aeruginosa and Pseudomonas stutzeri. The rate for P. aeruginosa cyt c551 is 1.2 x 10(7) M-1 s-1 at 40 degrees C in 50 mM phosphate at pH 7. For P. stutzeri, under the same conditions, the rate is 4 x 10(7) M-1 s-1. For both cytochromes, the rate was independent of ionic strength up to 0.5 M in added NaC1, the enthalpy of activation was 20 +/- 4 kcal mol-1, and the entropy of activation was 38 +/- 10 cal mol-1 deg-1.     

Redox protein electron-transfer mechanisms: electrostatic interactions as a determinant of reaction site in c-type cytochromes

The effect of ionic strength on the rate constant for electron transfer has been used to determine the magnitude and charge sign of the net electrostatic potential which exists in close proximity to the sites of electron transfer on various c-type cytochromes. The negatively charged ferricyanide ion preferentially reacts at the positively charged exposed heme edge region on the front side of horse cytochrome c and Paracoccus cytochrome c2. In contrast, at low ionic strength, the positively charged cobalt phenanthroline ion interacts with the negatively charged back side of cytochrome c2, and at high ionic strength at a positively charged site on the front side of the cytochrome. With horse cytochrome c, over the ionic strength range studied, cobalt phenanthroline reacts only at a positively charged site which is probably not at the heme edge. These inorganic oxidants do not react at the relatively uncharged exposed heme edge sites on Azotobacter cytochrome c5 and Pseudomonas cytochrome c-551, but rather at a negatively charged site which is away from the heme edge. The results demonstrate that at least two electron-transferring sites on a single cytochrome can be functional, depending on the redox reactant used and the ionic strength. Electrostatic interactions between charge distributions on the cytochrome surface and the other reactant, or interactions involving uncharged regions on the protein(s), are critical in determining the preferred sites of electron transfer and reaction rate constants. When unfavorable electrostatic effects occur at a site near the redox center, less optimal sites at a greater distance can become kinetically important.     

Partitioning of electrostatic and conformational contributions in the redox reactions of modified cytochromes c.

The reduction of acetylated, fully succinylated and dicarboxymethyl horse cytochromes c by the radicals CH3CH(OH), CO2.-, O2.-, and e-aq' and the oxidation of the reduced cytochrome c derivatives by Fe(CN)3-6 were studied using the pulse radiolysis technique. Many of the reactions were also examined as a function of ionic strength. By obtaining rate constants for the reactions of differently charged small molecules redox agents with the differently charged cytochrome c derivatives at both zero ionic strength and infinite ionic strength, electrostatic and conformational contributions to the electron transfer mechanism were effectively partioned from each other in some cases. In regard to cytochrome c electron transfer mechanism, the results, especially those for which conformational influences predominate, are supportive of the electron being transferred in the heme edge region.     

A study of the electron transfer properties of the heme undecapeptide from cytochrome c by 1H nmr spectroscopy

Nuclear magnetic resonance (nmr) spectroscopy has been used to investigate the heme undecapeptide from cytochrome c. Assignments of resonances to specific residues have been made based on spin decoupling, redox titration, and the pH and temperature dependence of resonance lines. An outline structure is presented based on the assignments, secondary shift data, and the x-ray crystal structure of cytochrome c. An equation is derived to relate the width of an nmr line during a redox titration to the percentage of each oxidation state. Using this equation the self-exchange rate constant for electron transfer for the heme peptide is 1.3 x 10(7) M-1 sec-1 at 330 degrees K. Discussion of the self-exchange rate constants of cytochrome c, cytochrome c3, and cytochrome c551 is related to this constant for the heme undecapeptide.     

Reduction of horse heart ferricytochrome c by bovine liver ferrocytochrome b5. Experimental and theoretical analysis.

The reduction of horse heart ferricytochrome c by the tryptic fragment of bovine liver cytochrome b5 and its dimethyl ester heme (DME)-substituted derivative has been studied as a function of ionic strength, pH, and temperature under solution conditions where the reaction is bimolecular. The rate constant for ferricytochrome c reduction by native ferrocytochrome b5 is 1.8 (+/- 0.2) x 10(7) M-1 s-1 (25 degrees C) with delta H++ = 7.5 (+/- 0.2) kcal/mol and delta S++ = -0.3 (+/- 0.6) eu (pH 7.0, I = 0.348 M). Under the same solution conditions, the reduction of ferricytochrome c by DME-ferrocytochrome b5 proceeds with a rate constant of 1.7 (+/- 0.1) x 10(7) M-1 s-1 with delta H++ = 7.9 (+/- 0.4) kcal/mol and delta S++ = 1 (+/- 1) eu. The rate constants for both reactions are strongly dependent on ionic strength. A detailed electrostatic analysis of the proteins has been performed. Two relatively simple Brownian dynamics simulation models predict rate constants for the reaction between the two native proteins that demonstrate a dependence on ionic strength similar to that observed experimentally. In one of these models, the proteins are treated as spheres with reactive surface patches that are defined by a 5 degrees cone generated about the dipole vector calculated for each protein and aligned with the presumed electron-transfer site near the partially exposed heme edge. The second model replaces the reactive patch assumption with an exponential distance dependence for the probability of reaction that permits estimation of a value for the distance-dependence factor alpha. Calculations with this latter model in combination with the aligned dipole assumption provide a reasonable approximation to the observed ionic strength dependence for the reaction and are consistent with a value of alpha = 1.2 A-1.     

Kinetic studies of the reduction of Pseudomonas aeruginosa ferricytochrome c551 by Fe(EDTA)2-

Kinetic studies of the reduction of Pseudomonas aeruginosa ferricytochrome c551 by Fe(EDTA)2- have been made. The reaction was found to follow a second-order rate law: k 4.2 x 10(3) M(-1) s(-1) [25 degrees, micro0.1 M, pH 7.0 (phosphate)]; deltaH+/+ 3.2 kcal/ mol; AS+/+ -30 cal/mol-deg. The electrostatics-corrected self-exchange rate constant (k11 corr) calculated for cytochrome c551 based on the Fe(EDTA)2- cross reaction is 2 M(-1) s(-1), as compared to a value of 6 M(-1) s(-1) for horse heart cytochrome c. The close correspondence of the two k11 corr values is taken as an indication that the two proteins employ very similar electron transfer mechanisms in their reactions with Fe(EDTA)(2-). It is proposed that this mechanism involves reagent contact, but little protein conformational change, at the partially exposed heme edge.     

Evolutionary change of the heme c electronic structure: ferricytochrome c-551 from Pseudomonas aeruginosa and horse heart ferricytochrome c.

Individual assignments of the ¹H n.m.r. lines of heme c in reduced and oxidized cytochrome c-551 from $\text{Pseudomonas aeruginosa}$ were obtained by nuclear Overhauser enhancement and saturation transfer experiments. Comparison with the corresponding data on horse heart cytochrome c showed that the locations of high spin density on the heme c periphery as well as the in-plane principal axes x and y of the electronic g-tensor are rotated by approximately 90° in ferricytochrome c-551 relative to horse ferricytochrome c. High spin density in ferricytochrome c-551 is thus localized on the pyrrole ring III. While this pyrrole ring is well shielded in the interior of mammalian-type cytochromes c, it is more easily accessible in cytochrome c-551. It is suggested that this evolutionary change of the heme c electronic structure would be compatible with the hypothesis that the electron transfer in both species is via solvent exposed peripheral ring carbon atoms.     

The modulation of cytochrome c electron self-exchange by site-specific chemical modification and anion binding

The site-specific chemical modification of horse heart cytochrome c at Lys-13 and -72 using 4-chloro-3,5-dinitrobenzoic acid (CDNB) increases the electron self-exchange rate of the protein. In the presence of 0.24 M cacodylate (pH* 7.0) the electron self-exchange rate constants, kex, measured by a 1H NMR saturation transfer method at 300 K, are 600, 6 X 10(3) and 6 X 10(4) M-1 X s-1 for native, CDNP-K13 and CDNP-K72 cytochromes c respectively. Repulsive electrostatic interactions, which inhibit cytochrome c electron self-exchange, are differentially affected by modification. Measurements of 1H NMR line broadening observed with partially oxidised samples of native cytochrome c show that ATP and the redox inert multivalent anion Co(CN)3-6 catalyse electron self-exchange. At saturation a limiting value of approximately 1.4 X 10(5) M-1 X s-1 is observed for both anions.     

Electrostatic interactions of 4-carboxy-2,6-dinitrophenyllysine-modified cytochromes c with physiological and non-physiological redox partners

An analysis of the effect of electrostatic properties of 4-carboxy-2,6-dinitrophenyllysine (CDNP-lysine) cytochromes c on their reactions with strongly and weakly binding redox partners is given. For strongly binding systems (cytochrome-c oxidase, cytochrome-c reductase, sulphite oxidase and yeast cytochrome-c peroxidase) the magnitude of the dipole moments of the CDNP cytochromes c determines their relative reactivities. For weakly binding redox agents, such as hexacyanoferrate(III), cobalt(III)tris(1,10-phenanthroline), azurin and plastocyanin, the electrostatic potential at the haem edge accounts for the greater part of the relative activities. Relative rate data were obtained from the literature. It is concluded that the dipole moment of native cytochromes c may account for an approx. 50-fold increase in the efficiency of its physiological activity towards membrane-bound enzymes. A correction on a formula to describe the contribution of a molecular dipole moment to the ionic strength dependence of a bimolecular rate constant (Koppenol, W. H. (1980) Biophys. J. 29, 493-508) leads to an equation nearly identical to that obtained by Van Leeuwen et al. (Van Leeuwen, J.W., Mofers, F.J.M. and Verrman, E.C.I. (1981) Biochim. Biophys. Acta 635, 434-439).     

The dipole moment of cytochrome c.

Vertebrate cytochromes c and the cytochromes c of insects and plants have, on average, dipole moments of 320 and 340 debye, respectively. The direction of the dipole vector with respect to the haem plane, at the solvent-accessible edge of which electron transfer presumably takes place, is conserved in these two groups--at 32 degrees +/- 7 degrees and 22 degrees +/- 10 degrees, respectively. The variation of dipole orientations and magnitudes observed in these species is compared with the results of a model in which charge distributions occur randomly. Since this model does not generate the observed charge asymmetries of the various cytochromes c, it is concluded that the dipole moment of cytochrome c is a feature that is evolutionarily conserved, apparently because it has an important influence on the interaction of this mobile electron carrier with its physiological electron donors and acceptors in the intermembrane space of mitochondria.     

The electric potential field around cytochrome c and the effect of ionic strength on reaction rates of horse cytochrome c.

1. The electric potential fields around tuna ferri- and ferrocytochrome c were calculated assuming that (i) all of the lysines and arginines are protonated, (ii) all of the glutamic and aspartic acids and the terminal carboxylic acid are dissociated, and (iii) the haem has a net charge of +1e in the oxidized form. 2. Near the haem crevice high values for the potential (greater than +2.5 kT/e) are found. Consequently, electron transfer via the haem edge is favored if the oxidant or reductant is negatively charged. 3. The inhomogeneous distribution of charges leads to a dipole moment of 244 and 238 debye for oxidized and reduced tuna cytochrome c, respectively. Horse cytochrome c has dipole moments of 303 (oxidized) and 286 (reduced) debye. 4. A line through the positive and negative charge centres, the dipole axis, crosses the tuna cytochrome c surface at Ala 83 (positive part) and Lys 99 (negative part). The direction of the dipole axis of horse cytochrome c is very similar. Since the centre of the domain on the cytochrome c surface, which is involved in the binding to cytochrome c oxidase, is found at the beta-carbon of the Phe 82 in horse cytochrome c (Ferguson-Miller, S., Brautigan, D.L. and Margoliash, E. (1978) J. Biol. Chem. 253, 149--159) it is suggested that the direction of the dipole is of physiological importance. 5. The activity coefficients of horse ferri- and ferrocytochrome c were calculated as a function of ionic strength using a formula derived by Kirkwood (Kirkwood, J.G. (1934) J. Chem. Phys. 2, 351--361). 6. Due to the high net charge at pH 7.5 the influence of the dipole moments of horse ferri- and ferrocytochrome c on the respective activity coefficients can be neglected at I less than or equal to 50 mM. 7. Using the Br?nsted relation the effect of ionic strength on reaction rates of horse cytochrome c was calculated. Good agreement is found between theory and experimental results reported in the literature.     

Kinetics of reduction by free flavin semiquinones of the components of the cytochrome c-cytochrome c peroxidase complex and intracomplex electron transfer

The kinetics of reduction by free flavin semiquinones of the individual components of 1:1 complexes of yeast ferric and ferryl cytochrome c peroxidase and the cytochromes c of horse, tuna, and yeast (iso-2) have been studied. Complex formation decreases the rate constant for reduction of ferric peroxidase by 44%. On the basis of a computer model of the complex structure [Poulos, T.L., & Finzel, B.C. (1984) Pept. Protein Rev. 4, 115-171], this decrease cannot be accounted for by steric effects and suggests a decrease in the dynamic motions of the peroxidase at the peroxide access channel caused by complexation. The orientations of the three cytochromes within the complex are not equivalent. This is shown by differential decreases in the rate constants for reduction by neutral flavin semiquinones upon complexation, which are in the order tuna much greater than horse greater than yeast iso-2. Further support for differences in orientation is provided by the observation that, with the negatively charged reductant FMNH., the electrostatic environments near the horse and tuna cytochrome c electron-transfer sites within their respective complexes with peroxidase are of opposite sign. For the horse and tuna cytochrome c complexes, we have also observed nonlinear concentration dependencies of the reduction rate constants with FMNH.. This is interpreted in terms of dynamic motion at the protein-protein interface. We have directly measured the physiologically significant intra-complex one electron transfer rate constants from the three ferrous cytochromes c to the peroxide-oxidized species of the peroxidase. At low ionic strength these rate constants are 920, 730, and 150 s-1 for tuna, horse, and yeast cytochromes c, respectively. These results are also consistent with the contention that the orientations of the three cytochromes within the complex with CcP are not the same. The effect on the intracomplex electron-transfer rate constant of the peroxidase amino acid side chain(s) that is (are) oxidized by the reduction of peroxide was determined to be relatively small. Thus, the rate constant for reduction by horse cytochrome c of the peroxidase species in which only the heme iron atom is oxidized was decreased by only 38%, indicating that this oxidized side-chain group is not tightly coupled to the ferryl peroxidase heme iron. Finally, it was found that, in the absence of cytochrome c, neither of the ferryl peroxidase species could be rapidly reduced by flavin semiquinones.(ABSTRACT TRUNCATED AT 400 WORDS)     

A complex of cardiac cytochrome c1 and cytochrome c.

The interactions of cytochrome c1 and cytochrome c from bovine cardiac mitochondria were investigated. Cytochrome c1 and cytochrome c formed a 1:1 molecular complex in aqueous solutions of low ionic strength. The complex was stable to Sephadex G-75 chromatography. The formation and stability of the complex were independent of the oxidation state of the cytochrome components as far as those reactions studied were concerned. The complex was dissociated in solutions of ionic strength higher than 0.07 or pH exceeding 10 and only partially dissociated in 8 M urea. No complexation occurred when cytochrome c was acetylated on 64% of its lysine residues or photooxidized on its 2 methionine residues. Complexes with molecular ratios of less than 1:1 (i.e. more cytochrome c) were obtained when polymerized cytochrome c, or cytochrome c with all lysine residues guanidinated, or a "1-65 heme peptide" from cyanogen bromide cleavage of cytochrome c was used. These results were interpreted to imply that the complex was predominantly maintained by ionic interactions probably involving some of the lysine residues of cytochrome c but with major stabilization dependent on the native conformations of both cytochromes. The reduced complex was autooxidizable with biphasic kinetics with first order rate constants of 6 X 10(-5) and 5 X U0(-5) s-1 but did not react with carbon monoxide. The complex reacted with cyanide and was reduced by ascorbate at about 32% and 40% respectively, of the rates of reaction with cytochrome c alone. The complex was less photoreducible than cytochrome c1 alone. The complex exhibited remarkably different circular dichroic behavior from that of the summation of cytochrome c1 plus cytochrome c. We concluded that when cytochromes c1 and c interacted they underwent dramatic conformational changes resulting in weakening of their heme crevices. All results available would indicate that in the complex cytochrome c1 was bound at the entrance to the heme crevice of cytochrome c on the methionine-80 side of the heme crevice.     

Ionic strength dependence of the kinetics of electron transfer from bovine mitochondrial cytochrome c to bovine cytochrome c oxidase.

The effect of ionic strength on the one-electron reduction of oxidized bovine cytochrome c oxidase by reduced bovine cytochrome c has been studied by using flavin semiquinone reductants generated in situ by laser flash photolysis. In the absence of cytochrome c, direct reduction of the heme a prosthetic group of the oxidase by the one-electron reductant 5-deazariboflavin semiquinone occurred slowly, despite a driving force of approximately +1 V. This is consistent with a sterically inaccessible heme a center. This reduction process was independent of ionic strength from 10 to 100 mM. Addition of cytochrome c resulted in a marked increase in the amount of reduced oxidase generated per laser flash. Reduction of the oxidase at the heme a site was monophasic, whereas oxidation of cytochrome c was multiphasic, the fastest phase corresponding in rate constant to the reduction of the heme a. During the fast kinetic phase, 2 equiv of cytochrome c was oxidized per heme a reduced. We presume that the second equivalent was used to reduce the Cua center, although this was not directly measured. The first-order rate-limiting process which controls electron transfer to the heme a showed a marked ionic strength effect, with a maximum rate constant occurring at mu = 110 mM (1470 s-1), whereas the rate constant obtained at mu = 10 mM was 630 s-1 and at mu = 510 mM was 45 s-1. There was no effect of "pulsing" the enzyme on this rate-limiting one-electron transfer process. These results suggest that there are structural differences in the complex(es) formed between mitochondrial cytochrome c and cytochrome c oxidase at very low and more physiologically relevant ionic strengths, which lead to differences in electron-transfer rate constants.     

Characterization of the interaction of Rhodobacter capsulatus cytochrome c peroxidase with charge reversal mutants of cytochrome c(2)

Steady-state kinetics for the reaction of Rhodobacter capsulatus bacterial cytochrome c peroxidase (BCCP) with its substrate cytochrome c(2) were investigated. The Rb. capsulatus BCCP is dependent on calcium for activation as previously shown for the Pseudomonas aeruginosa BCCP and Paracoccus denitrificans enzymes. Furthermore, the activity shows a bell-shaped pH dependence with optimum at pH 7.0. Enzyme activity is greatest at low ionic strength and drops off steeply as ionic strength increases, resulting in an apparent interaction domain charge product of -13. All cytochromes c(2) show an asymmetric distribution of surface charge, with a concentration of 14 positive charges near the exposed heme edge of Rb. capsulatus c(2) which potentially may interact with approximately 6 negative charges, localized near the edge of the high-potential heme of the Rb. capsulatus BCCP. To test this proposal, we constructed charge reversal mutants of the 14 positively charged residues located on the front face of Rb. capsulatus cytochrome c(2) and examined their effect on steady-state kinetics with BCCP. Mutated residues in Rb. capsulatus cytochrome c(2) that showed the greatest effects on binding and enzyme activity are K12E, K14E, K54E, K84E, K93E, and K99E, which is consistent with the site of electron transfer being located at the heme edge. We conclude that a combination of long-range, nonspecific electrostatic interactions as well as localized salt bridges between, e.g., cytochrome c(2) K12, K14, K54, and K99 with BCCP D194, D241, and D6, account for the observed kinetics.     

The reaction between cytochrome c1 and cytochrome c

The kinetics of electron transfer between the isolated enzymes of cytochrome c1 and cytochrome c have been investigated using the stopped-flow technique. The reaction between ferrocytochrome c1 and ferricytochrome c is fast; the second-order rate constant (k1) is 3.0 . 10(7) M-1 . s-1 at low ionic strength (I = 223 mM, 10 degrees C). The value of this rate constant decreases to 1.8 . 10(5) M-1 . s-1 upon increasing the ionic strength to 1.13 M. The ionic strength dependence of the electron transfer between cytochrome c1 and cytochrome c implies the involvement of electrostatic interactions in the reaction between both cytochromes. In addition to a general influence of ionic strength, specific anion effects are found for phosphate, chloride and morpholinosulphonate. These anions appear to inhibit the reaction between cytochrome c1 and cytochrome c by binding of these anions to the cytochrome c molecule. Such a phenomenon is not observed for cacodylate. At an ionic strength of 1.02 M, the second-order rate constants for the reaction between ferrocytochrome c1 and ferricytochrome c and the reverse reaction are k1 = 2.4 . 10(5) M-1 . s-1 and k-1 = 3.3 . 10(5) M-1 . s-1, respectively (450 mM potassium phosphate, pH 7.0, 1% Tween 20, 10 degrees C). The 'equilibrium' constant calculated from the rate constants (0.73) is equal to the constant determined from equilibrium studies. Moreover, it is shown that at this ionic strength, the concentrations of intermediary complexes are very low and that the value of the equilibrium constant is independent of ionic strength. These data can be fitted into the following simple reaction scheme: cytochrome c2+1 + cytochrome c3+ in equilibrium or formed from cytochrome c3+1 + cytochrome c2+.     

Effects of amino acid replacements in yeast iso-1 cytochrome c on heme accessibility and intracomplex electron transfer in complexes with cytochrome c peroxidase

The kinetics of reduction of wild type and several site-specific mutants of yeast iso-1 cytochrome c (Arg-13----Ile, Gln-16----Ser, Gln-16----Lys, Lys-27----Gln, Lys-72----Asp), both free and in 1:1 complexes with yeast cytochrome c peroxidase, by free flavin semiquinones have been studied. Intramolecular one-electron transfer from the ferrous cytochromes c to the H2O2-oxidized peroxidase at both low (8 mM) and high (275 mM) ionic strengths was also studied. The accessibility of the cytochrome c heme within the electrostatically stabilized complex and the rate constants for intramolecular electron transfer at both low and high ionic strength are highly dependent on the specific amino acids present at the protein-protein interface. Importantly, replacement by uncharged amino acids of Arg or Lys residues thought to be important in orientation and/or stabilization of the electron-transfer complex resulted in increased rates of electron transfer. In all cases, an increase in ionic strengths from 8 to 275 mM also produced increased intramolecular electron-transfer rate constants. The results suggest that the electrostatically stabilized 1:1 complex is not optimized for electron transfer and that by neutralization of key positively charged residues, or by an increase in the ionic strength thereby masking the ionic interactions, the two proteins can orient themselves to allow the formation of a more efficient electron-transfer complex.