Analysis of the bimolecular reduction of ferricytochrome c by ferrocytochrome b 5 through mutagenesis and molecular modelling

Abstract

Site-directed mutagenesis has been used to produce variants of cytochrome c in which selected structural or functional properties of this protein are altered that have been implicated previously in contributing to the rate at which ferricytochrome c is reduced by ferrocytochrome b 5. In total, 18 variants have been studied by kinetic and electrochemical methods to assess the contributions of thermodynamic driving force, surface charge and hydrophobic interactions, and redox-linked structural reorganization of the protein to the rate of electron transfer between these two proteins under conditions where the reaction is bimolecular. While some variants (those at position-38) appear to affect primarily the driving force of the reaction, other appear to influence the rearrangement barrier to electron transfer (those at positions-67 and -52) while the interface between electron donor and acceptor centers is the principal effect of substitutions for a conserved aromatic heme contact residue at the surface of the protein (position-82). Interpretation of these results has been facilitated through the use of energy minimization calculations to refine the hypothetical models previously suggested for the cytochrome c- cytochrome b 5 precursor complex on the basis of Brownian dynamics simulations of the bimolecular encounter event.