Electron and Proton Transfer in Reaction Centers

描述（由申请人提供）：细菌反应中心（RC）是参与光合细菌初始电子转移步骤的膜蛋白。随后从细胞色素c2 （cyt）到RC的电子转移反应是光合作用电子转移链中的重要步骤。本项目是研究光合细菌球形红杆菌细胞色素c2与RC之间的分子间电子转移。激发这项研究的最新进展是确定了cyt:RC复合物的高分辨率x射线晶体结构（H. Axelrod et. al. (2002) J. Mol. Biol. 319,501 -515），这提出了关于cyt:RC复合物在结合和电子转移反应中的作用的重要问题。将研究的反应的三个特征是；1)两种蛋白质结合过程中蛋白质-蛋白质结合的结构和能量学，2)细胞色素结合在RC表面的蛋白质间电子转移，3)蛋白质对接到电子转移位置的动力学。这些过程的分子基础将使用各种技术进行检查，包括RC和细胞色素c2的定点诱变，cyt:RC复合物的结构测定，一阶和二阶电子转移速率的激光动力学测量和计算模型。

DESCRIPTION (provided by applicant): The bacterial reaction center (RC) is the membrane protein involved in the initial electron transfer step in photosynthetic bacteria. The subsequent electron transfer reaction from cytochrome c2 (cyt) to the RC is an important step in the photosynthetic electron transfer chain. This project is a study of intermolecular electron transfer between cytochrome c2 and the RC from the photosynthetic bacterium Rhodobacter sphaeroides. A recent advance that motivates this study is the determination of the high-resolution x-ray crystal structure of the cyt:RC complex (H. Axelrod et. al. (2002) J. Mol. Biol. 319, 501-515) that raises important questions about role of the cyt:RC complex in binding and electron transfer reactions. Three features of the reaction that will be investigated are; 1) the structure and energetics of the protein-protein association involved in the binding of the two proteins, 2) the inter protein electron transfer of the cytochrome bound on the RC surface and 3) the dynamics of protein docking into position for electron transfer. The molecular basis for these processes will be examined using a variety of techniques including site directed mutagenesis of the RC and cytochrome c2, structural determination of cyt:RC complexes, laser kinetic measurements of first order and second order electron transfer rates and computational modeling. The specific aims of this project are to: 1) Modify specific residues on the interaction surface between RC and cyt by site directed mutagenesis. The residues to be targeted include residues involved in hydrogen bonding interactions between the two proteins and a surface motif of three Thr residues on the front surface of the cyt that appears to be important for binding. The effects of these modifications on the binding constant for the complex, rate of electron transfer from the bound cyt and the rate of association of the complex will be studied. 2) The effect of osmotic pressure on binding and electron transfer will be investigated to determine the role of bound water. 3) The driving force dependence of the rate of electron transfer will be studied to characterize the electron transfer mechanism. 4) The reaction between horse heart cyt c and the RC, which is believed to have a different binding configuration, will be studied. 5) X-ray crystal structures of different cyt:RC complexes will be determined. 6) Computational approaches will be used to model the binding and dynamics of the cyt:RC complex.