ANALYSIS OF THE BIMOLECULAR REDUCTION OF FERRICYTOCHROME-C BY FERROCYTOCHROME B(5) THROUGH MUTAGENESIS AND MOLECULAR MODELING

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, others 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- cytochrcome b(5) precursor complex on the basis of Brownian dynamics simulations of the bimolecular encounter event.