The cytochrome c-cytochrome c peroxidase (CcP) system provides an exceedingly rich opportunity to investigate the intimate details of protein-protein interactions and long-range electron transfer between physiologically related heme proteins. The objectives of this research proposal lie in two areas. This first area is to characterize the interaction between both horse and yeast iso-1 cytochrome c with yeast CcP. Experiments designed to clarify the stoichiometry of binding between cytochrome c and CcP will be performed to determine if, and under what conditions, more than one cytochrome c may bind to CcP. The high-affinity cytochrome c binding site on CcP will be mapped through site-directed mutagenesis. The role of individual amino acids at the binding site will be assessed through thermodynamic measurements of the binding affinity. The location of any secondary interaction sites will also be determined through site-directed mutagenesis. The second area of interest is to characterize electron transfer between the heme groups of cytochrome c and CcP. Transient-state kinetic techniques will be used to investigate the rate of electron transfer from ferrocytochrome c to the oxidized intermediate of CcP and the rate of redox equilibration between the heme iron and Trp- 191 in CcP compound II. The specificity of the reaction dynamics and the electron transfer pathway will be explored through site- directed mutagenesis of CcP. %%% This work should lead to a better understanding of protein-protein recognition in general, and of heme proteins, which include hemoglobin and the proteins in energy metabolism, in particular
