Electron Transfer Kinetics of Mutant Cytochrome C

Stewart, John

Abstract

The objective is to determine whether and in what ways some 50 alternations at any of fifteen residue positions of yeast iso-1-cytochrome c, at which characterized single amino acid replacements have been obtained in revertants of cyc1 yeast mutants, affect the kinetics of electron transfer reactions between cytochrome c and cytochrome c reductase (c1) and cytochrome c oxidase (a a3) from beef and yeast mitochondria and cytochrome c peroxidase from yeast mitochondria. The essential nature of residues at altered sites found to affect the reactions may become evident directly, because various substitutions at most of the sites are available, as is knowledge of several of the substitutions in the defective mutants lacking active iso-1-cytochrome c, from which these revertants were prepared. Characteristics of reaction of steady state and pre-steady state kinetics of cytochrome c with oxidases and reductases from both beef and yeast mitochondria and with yeast peroxidase will be followed spectrophotometrically, and will be analyzed in terms of mechanism of reaction. The cytochromes c found defective will constitute reagents for future, faster kinetic studies of these reactions using equilibrium perturbation methods that may expose individual reaction steps. Systematic examinations of other properties of these altered cytochromes c are intended in future, to evaluate heme-linked equilibria and reaction kinetics with protons and iron-binding ligands, oxidation reduction midpoint potentials, and kinetics of reactions with small oxidants and reductants. The objective is to probe the properties of cytochrome c and of the electron transfer reactions that it enters, central to mitochondrial function and bioenergetics.