Protonation and Conformational Control of Photosynthetic Reaction Center Function

球形红杆菌（Rhodobacter sphaeroides）光合反应中心（RC）的主受体醌QA和仲受体醌QB化学性质相同，但性质截然不同。因此，它们为理解蛋白质如何决定结合辅因子的特性提供了一个理想的模型。该项目研究了这些独特性质的起源，电子和质子向QB转移的机械细节，以及伴随这些转移的构象事件。蛋白质对埋藏电荷的调节将通过闪光诱导的P+QA-态的荧光发射衰减来检测。将利用各种醌类作为QA和QA位点突变体，确定微至毫秒时间尺度上的松弛。两个醌结合位点之间的远程相互作用，会引起QA和QB氧化还原态自由能级的互反扰动，将通过测量质子摄取、QA到QB的电子转移和延迟荧光发射来量化。这种相互作用的具体模型将通过定点诱变和醌类似物进行测试。通过对QA和QB位点残基的定点诱变和特异性合成醌，分析QA和QB与蛋白质的静电和空间相互作用对氧化还原电位的控制，探讨甲氧基扭转角在泛醌优化QA和QB功能中的作用。结构意义将由傅里叶变换红外光谱（FTIR）研究。光合反应中心（RC）的光能转换包括最初的电子转移，随后是电荷分离稳定和捕获的几个步骤。在细菌RCs中，RCs能够进行的多种反应包括至少8种不同的电子转移反应，2种不同的质子转移反应，以及底物（醌）结合和蛋白质（细胞色素）识别、对接和结合。由于许多辅助因子作为电致变色报告基团以及反应物，RC提供了无与伦比的电荷运动和构象弛豫的可观察性，非常适合研究蛋白质的功能和行为。在一些细菌中，两种化学性质相同的醌作为电子受体，串联在一起，表现出非常不同的性质。该项目将使用光谱和生化方法来研究蛋白质如何改变两种醌的性质，包括构象松弛在容纳埋藏电荷中的作用，以及静电和空间约束在确定醌的氧化还原性质中的作用。这些结果将提供对蛋白质如何修饰和决定结合辅因子和底物的性质和反应性的一般理解，对催化至关重要。

MCB 9905672WraightThe primary and secondary acceptor quinones, QA and QB, in the photosynthetic reaction center (RC) of Rhodobacter sphaeroides are chemically identical but exhibit very distinct properties. Thus, they provide an ideal model for understanding how proteins determine the properties of bound cofactors. This project investigates the origin of these distinct properties, the mechanistic details of electron and proton transfer to QB, and the conformational events that accompany these transfers. Accommodation of the protein to buried charges will be examined through the decay of fluorescence emission from the flash-induced P+QA- state. Relaxation on the micro- to millisecond time scales will be determined, utilizing various quinones as QA as well as QA-site mutants. Long range interactions between the two quinone binding sites, that give rise to reciprocal perturbations of the free energy levels of QA and QB redox states, will be quantified via measurements of proton uptake, QA-to-QB electron transfer, and delayed fluorescence emission. Specific models of this interaction will be tested by site-directed mutagenesis and quinone analogues. Control of the redox potentials of QA and QB by electrostatic and steric interactions with the protein will be analyzed by site directed mutagenesis of QA and QB site residues and specifically synthesized quinones, to explore the role of the methoxy torsion angles in optimizing QA and QB function by ubiquinones. Structural implications will be investigated by Fourier-transform infra-red (FTIR) spectroscopy.Light energy conversion in the photosynthetic reaction center (RC) involves an initial electron transfer, followed by several more steps in which the separation of charge is stabilized and trapped. In bacterial RCs, the many reactions that the RC is capable of include at least 8 different electron transfer reactions, 2 distinct proton transfers, plus substrate (quinone) binding and protein (cytochrome) recognition, docking and binding. With many of the cofactors involved act as electrochromic reporter groups as well as reactants, and the RC provides unparalleled observability of charge movements and conformational relaxations and is exceptionally well suited for studying protein function and behavior. In some bacteria, two chemically identical quinones act as electron acceptors, in series, exhibiting very distinct properties. This project will use spectroscopic and biochemical methods to study how the protein modifies the properties of the two quinones, including the role of conformational relaxation in accommodating buried charges, and the effects of electrostatics and steric constraints in determining the redox properties of the quinones. The outcomes will provide general understanding of how proteins modify and determine the properties and reactivities of bound cofactors and substrates, essential to catalysis.