The recent achievement of atomic resolution crystal structures of cytochrome c oxidase provides the basis for more incisive analysis of its energy transduction mechanism by mutational and spectral methods. On the basis of the new structures and our previous studies on Rhodobacter sphaeroides cytochrome c oxidase (an excellent model of the mammalian enzyme), the CuA site and a closely associated Mg are proposed to have important roles in directing electron input and controlling proton output. Mutations have been made at the interface of subunits I and II, immediately above the heme a3 CuB site, where both these metals are ligated and proton output is predicted to occur. The mutants will be analyzed by transient kinetics, resonance Raman, EPR, FTIR, redox potential and proton pumping techniques to determine the roles of specific residues and the metal centers in mediating rapid, directed electron transfer and outward flow of pumped protons. This same region of the protein and additional residues in subunit II are predicted entry sites for electrons from cytochrome c. Cytochrome c interactions with oxidase mutants will be analyzed by steady state kinetics, binding, and rapid kinetic methods to determine the pathways of electron input. Our recent observation of chemical rescue by fatty acids of carboxylate mutants will be further investigated with the aim of understanding factors that affect the efficiency of energy coupling, and of distinguishing between entry sites for pumped versus substrate protons. Efforts to cyrstallize the Rhodobacter oxidase and selected mutants will be initiated. The proposed studies will critically test current models of electron transfer, proton transfer, and the energy coupling mechanism, and provide new insight into how efficiency of energy transduction may be controlled.
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