9630215 Okamura The aim of this project is to study the molecular basis for the binding and electron transfer reactions between cytochrome c2 and the bacterial reaction center (RC) from the photosynthetic bacterium Rhodobacter sphaeroides. Site directed mutagenesis will be used to modify residues on both the RC and cytochrome in the interfacial region. The types of mutations to be constructed are: 1) Charge reversals: carboxyl groups on the RC will be changed to Lys and Lys on the cytochrome will be changed to carboxyls. The binding of Rcs and cytochromes with compensating mutations at interacting site should show improvement in the binding. 2) Formation of hydrophobic binding domains: polar residues on both proteins in the binding domain will be converted to hydrophobic residues to increase stability of the complex. 3) Site directed crosslinking: Cys residues will be introduced at putative interacting sites on both RC and cytochrome and used to crosslink the two proteins into a stable configuration. The binding constants and electron transfer rates of the complexes formed by mutated proteins will be measured and correlated with the structural changes in order to deduce the structure of the RC-cytochrome complex. Mutations will be made to improve the binding of the cytochrome to the RC to facilitate structural studies of the RC-cytochrome complex by x-ray diffraction. %%% Electron transfer reactions in biological membranes are essential to provide energy necessary for life processes. The reaction between cytochrome c2 and the RC in photosynthetic bacteria displays both the specificity in binding and high rate of interprotein electron transfer within the bimolecular complex necessary to maintain a fast electron transfer through the electron transfer chain. These studies of the binding and electron transfer reactions of between cytochrome c2 and the bacterial RC are aimed at determining fundamental principles of protein association and electron transfer in biology that should provide the basis for understanding energy conversion processes in other important biological systems. ***
