The electrostatic character of cytochrome c-cytochrome c peroxidase complex formation has been studied by potentiometric titration between pH 5.5 and 7.75. Potentiometric data obtained at ionic strength > or = 100 mM were adequately analyzed in terms of 1:1 complex formation while the simplest model capable of fitting similar data obtained at lower ionic strength involves the assumption of two inequivalent binding sites for the cytochrome on the peroxidase. The stability of cytochrome c binding at the high-affinity site is ca. three orders of magnitude greater than that observed for the low-affinity site and is optimal between pH 6.75 and 7. The electrostatic properties of the two binding sites are distinctly different because, at most values of pH, binding of cytochrome c to the high-affinity site results in proton release while binding of the cytochrome to the low-affinity site results in proton uptake. Furthermore, binding of the cytochrome to the low-affinity site appears to be least stable in the pH range where binding to the high-affinity site is optimal. Interestingly, the binding parameters derived from these measurements were independent of temperature, consistent with a substantial entropic contribution to complex stability. Ferricytochrome c binds to the peroxidase with a slightly greater affinity than does ferrocytochrome c, and no evidence for specific anion effects on complex stability was observed. At low ionic strength (< or = 50 mM) and high pH (7.75), the interaction of the two proteins is more complex and cannot be adequately analyzed in terms of the two-site model.

A gene coding for lipase-solubilized bovine liver microsomal cytochrome b5 has been synthesized, expressed in Escherichia coli, and mutated at functionally critical residues. Characterization of the recombinant protein revealed that it has a reduction potential that is approximately 17 mV lower than that of authentic wild-type protein at pH 7 (25 degrees C). Structural studies determined that the recombinant protein differed in sequence from authentic wild-type cytochrome b5 owing to three errors in amidation status in the published sequence for the protein on which the gene synthesis was based. The structural origin of the lower reduction potential exhibited by the triple mutant has been investigated through X-ray crystallographic determination of the three-dimensional structure of this protein and is attributed to the presence of Asp-57 within 3.3 A of heme vinyl-4 in the mutant. In addition, the model developed by Argos and Mathews [Argos, P., & Mathews, F.S. (1975) J. Biol. Chem. 250, 747] for the change in cytochrome b5 oxidation state has been studied through mutation of Ser-64 to Ala. In this model, Ser-64 is postulated to stabilize the oxidized protein through H-bonding interactions with heme propionate-7 that orients this propionate group 6.2 A from the heme iron. Spectroelectrochemical studies of a mutant in which Ser-64 has been changed to an alanyl residue demonstrate that this protein has a reduction potential that is 7 mV lower than that of the wild-type protein; moreover, conversion of the heme propionate groups to the corresponding methyl esters increases the potential by 67 mV.(ABSTRACT TRUNCATED AT 250 WORDS)

The involvement of the heme propionate groups in the mechanism of the cytochrome b, oxidation-reduction transition has been evaluated by preparation and functional characterization of a derivative of the protein (DME-b5) which the native prosthetic group has been replaced by ferriprotoporphyrin IX dimethyl ester. Spectroelectrochemical studies of DME-b5 show that esterification of the heme propionate groups increases the potential of the protein from 5 mV observed for th-, native protein to 69 mV vs. N H E [pH 7.0 (phosphate), p = 0.1 M, 25 "C]. The dependence of E" on pH is analogous to that seen for the native protein and indicates that these propionate groups are not responsible for this effect. The ionic strength dependence of E" has been analyzed in terms of Debye-Hiickel theory to produce an apparent net electrostatic charge on oxidized DME-b5 of -8. The thermodynamic parameters for this equilibrium are AH" = -11 (1) kcal/mol and ASo = -31 (2) eu [pH 7.0 (phosphate), p = 0.1 MI. Kinetics analysis of DME-b5 reduction by Fe(EDTA)2-produces a second-order rate constant of 1.97 (2) x 103 M-1 s-I [pH 7.0 (phosphate), p = 0.1 M, 25 "C] and a pH dependence similar to that of the native protein.Activation parameters for this reaction are AH* = 4.05 (5) kcal/mol and AS* = -30 (2) eu. Analysis of the ionic strength dependence of DME-b5 reduction by Fe(EDTA)" in terms of the Marcus ionic strength equation yields an apparent net electrostatic charge on oxidized DME-bj of -6.9. These results have been used to calculate the apparent self-exchange rate constant demonstrated by DME-b5 in this reaction to produce a value (kllcorr) of 7.6 X lo2 M-I s-l , a value 70-fold greater than that observed for the native protein. We conclude that the present analysis of DME-b5 provides strong evidence for (1) the involvement of the partially exposed heme edge in heme protein electron transfer reactions in general, (2) short-range electrostatic effects in the Fe(EDTA)2-reduction of the native protein that are not adequately accommodated by available models for protein behavior, and (3) a major role of at least one heme propionate in determining the reduction potential of native cytochrome b5.

Experimental and theoretical investigation of the interaction of cytochrome c and cytochrome b5 performed over nearly twenty years has produced considerable insight into the manner in which these proteins recognize and bind to each other. The results of these studies and the experimental and theoretical strategies that have been developed to achieve these results have significant implications for understanding the behavior of similar complexes formed by more complex and less-well characterized electron transfer proteins. The current review provides a comprehensive summary and critical evaluation of the literature on which the current status of our understanding of the interaction of cytochrome c and cytochrome b5 is based. The general issues related to the study of electron transfer complexes of this type are discussed and some new directions for future investigation of such systems are considered.

The complex formed in solution by native and chemically modified cytochrome c with cytochrome b5 has been studied by 1H and 13C nuclear magnetic resonance spectroscopy (NMR). Contrary to predictions of recent theoretical analysis, 1H NMR spectroscopy indicates that there is no major movement of cytochrome c residue Phe82 on binding to cytochrome b5. The greater resolution provided by 13C NMR spectroscopy permits detection of small perturbations in the environments of cytochrome c residues Ile75 and Ile85 on binding with cytochrome b5, a result that is in agreement with earlier model-building experiments. As individual cytochrome c lysyl residues are resolved in the 1H NMR spectrum of N-acetimidylated cytochrome c, the interaction of this modified protein with cytochrome b5 has been studied to evaluate the number of cytochrome c lysyl residues involved in binding to cytochrome b5. The results of this experiment indicate that at least six lysyl residues are involved, two more than predicted by static model building, which indicates that cytochrome c and cytochrome b5 form two or more structurally similar 1:1 complexes in solution.