Electron and Proton Transfer in Reaction Centers

描述（由申请人提供）：细菌反应中心（RC）是光合细菌中参与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电子转移链中的重要步骤。本项目是对光合细菌类球红细菌的细胞色素c2和RC之间的分子间电子转移的研究。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。阿克塞尔罗德等等人（2002）J. Mol. 319，501 - 515），其提出了关于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）参与两种蛋白质的结合的蛋白质-蛋白质缔合的结构和能量学，2）结合在RC表面上的细胞色素的蛋白质间电子转移和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，包括定点诱变的RC和细胞色素C2，结构测定的细胞色素：RC复合物，激光动力学测量的一阶和二阶电子转移速率和计算建模检查。 本课题的具体目标是：1）通过定点突变的方法，对RC与cyt相互作用表面的特定残基进行修饰。待靶向的残基包括参与两种蛋白质之间的氢键相互作用的残基和在cyt的前表面上的三个Thr残基的表面基序，所述表面基序对于结合似乎是重要的。将研究这些修饰对复合物的结合常数、从结合的细胞色素的电子转移速率和复合物的缔合速率的影响。2)将研究渗透压对结合和电子转移的影响，以确定结合水的作用。3)将研究电子转移速率的驱动力依赖性以表征电子转移机制。4)马心细胞色素c和RC之间的反应，这被认为是具有不同的结合构型，将进行研究。5)不同的细胞色素：RC复合物的X射线晶体结构将被确定。6)计算方法将被用来模拟的结合和动力学的细胞色素：RC复合物。

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.