Electron Transfer in the Cytochrome c - Cytochrome c Peroxidase Complex

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The cytochrome c - cytochrome c peroxidase system provides an outstanding opportunity to uncover the intimate details of the structural requirements for peroxidase activity, as well as those for long range electron transfer between physiologically related heme proteins. The objective of this research proposal is to elucidate the detailed mechanism of cytochrome c peroxidase catalysis with special emphasis on characterizing the interaction between cytochrome c and cytochrome c peroxidase. Experiments designed to clarify the stoichiometry of binding between cytochrome c and cytochrome c peroxidase will be performed to determine if, and under what conditions, more than one cytochrome c may bind to cytochrome c peroxidase. The specificity of binding at the high affinity binding site will be explored by utilizing a variety of cytochrome cs isolated from different sources and through site-directed mutagenesis of cytochrome c peroxidase. Transient state kinetic techniques will be used to investigate the rate of electron transfer between ferrocytochrome c and the oxidized intermediates of cytochrome c preoxidase. The specificity of the reaction dynamics will be evaluated by using cytochrome c from various sources and the role of specific amino acid residues in the enzyme will be explored by site-directed mutagenesis.