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 double-ended dagger = 7.5 (+/- 0.2) kcal/mol and DELTA-S double-ended dagger = -0.3 (+/- 0.6) eu (pH 7.0, I = 0.348 M). Under the same solution conditions, the reduction 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 double-ended dagger = 7.9 (+/- 0.4) kcal/mol and DELTA-S double-ended dagger = 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 angstrom-1.