Mauk Ag

Analysis of the bimolecular reduction of ferricytochrome c by ferrocytochrome b5 through mutagenesis and molecular modelling

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 b5. In total, 18 variants have been studied by kinetics 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- cytochrome b5 precursor complex on the basis of Brownian dynamics simulations of the bimolecular encounter event.

Binding and oxidation of mutant cytochromes c by cytochrome-c oxidase

Mutation of conserved Phe‐82 of yeast iso‐1 cytochrome c to Tyr, Gly, Ser, Leu, or Ile affects binding to and reaction with cytochrome‐c oxidase from beef heart. The observed changes of binding and kinetic constants reflect mutation‐induced rearrangements in the heme vicinity brought about by the replacement of Phe‐82. Such conformational rearrangements are also revealed by altered circular dichroism spectra of the oxidase‐bound mutant cytochromes c. Variations in K m for cytochrome c oxidation do not parallel variations in K d, the dissociation constant for binding of cytochrome c to the oxidase. This observation does not support an enzymatic mechanism in which the rate of cytochrome c oxidation is governed by product dissociation.