Structural Basis for Electron Transfer Between the Bacterial Reaction Center and Cytochrome c2 in Rhodobacter Sphaeroides

9630215 Okamura本项目的目的是研究细胞色素c2和细胞色素c 2之间结合和电子转移反应的分子基础。 以及来自光合细菌球形红细菌的细菌反应中心（RC）。 定点诱变将用于修饰界面区域中RC和细胞色素上的残基。 待构建的突变类型为：1）电荷反转：RC上的羧基将变为Lys，细胞色素上的Lys将变为羧基。 在相互作用位点具有补偿突变的Rcs和细胞色素的结合应显示结合的改善。2)疏水结合结构域的形成：结合结构域中两种蛋白质上的极性残基将转化为疏水残基，以增加复合物的稳定性。 3)定点交联：Cys残基将被引入RC和细胞色素上的假定相互作用位点，并用于将两种蛋白质交联成稳定的构型。 将测量由突变蛋白形成的复合物的结合常数和电子转移速率，并将其与结构变化相关联，以推断RC-细胞色素复合物的结构。 将进行突变以改善细胞色素与RC的结合，以便于通过X射线衍射进行RC-细胞色素复合物的结构研究。 生物膜中的电子转移反应对于提供生命过程所需的能量是必不可少的。 光合细菌中细胞色素c2和RC之间的反应显示出结合的特异性和在双分子复合物内维持快速电子转移所必需的高速率的蛋白质间电子转移。 细胞色素c2和细菌RC之间的结合和电子转移反应的这些研究旨在确定生物学中蛋白质缔合和电子转移的基本原理，为理解其他重要生物系统中的能量转换过程提供基础。 ***

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. ***