The proposed research is a study of electron and proton transfer processes in bacterial reaction centers (RC). These processes are important in many other proteins and form the basis for energy conversion in biological membranes. The overall goal is to provide a description of the molecular basis for this protein in cellular machinery that couples electron transfer to proton pumping across the membrane. The work focuses on two key processes; 1) Proton-coupled electron transfer reactions that result in the reduction of a bound ubiquinone, QB, and 2) Inter-protein electron transfer reaction between cytochrome C2 and RC. The ability to initiate the electron transfer reactions using light pulses allows a detailed understanding of the molecular process involved in these reactions. Site directed mutagenesis will be used to investigate the mechanism of these reactions. The electron transfer rates for the proton-coupled reactions are complex and are believed to involve protein dynamics as well as proton and electron transfer. The molecular basis for the observed electron transfer rate will be examined by site directed mutagenesis to elucidate the factors that control the electron transfer rate. The proton transfer pathway into the QB site is believed to involve surface His residues and a chain of acidic residues and water molecules. The factors important for proton transfer will be investigated by modification of the environment of the residues in the proton transfer pathway and measurement of proton transfer rates using methods that we have developed. The inter-protein electron transfer reaction between cytochrome and RC will be examined to test ideas concerning dynamic docking mechanisms, which involves binding and electron transfer of cytochrome at the RC surface, and electron transfer through solvent water. The structures of the mutants with modified proton and electron transfer rates will be examined by X-ray crystallography, and spectroscopic techniques including electron paramagnetic resonance, electron nuclear double resonance and infrared spectroscopy. The proposed research would help elucidate the molecular basis for electron and proton transfer mechanisms found in many biological systems. The combination of functional assays, genetic information and structural biology provide an understanding of the molecular basis these important biological processes and should help lay the foundations for future advances in molecular medicine. ? ? ?
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