DIRECTED MUTAGENESIS OF PHOTOSYNTHETIC OXYGEN EVOLUTION

总体目标是了解金属离子簇的运作， 生物系统中的电子转移过程。 具体目标是 了解光合水氧化的机制。 含氧 光合作用提供所需的分子氧和固定碳， 维持动物生命。 拟议的工作将确定配体的 锰和钙离子位于光合作用的催化位点， 水氧化 这些配体的鉴定将带来新的 限制锰集群的运作模式，并将 使这些模型能够放在蛋白质结构的背景下。 的 所获得的信息将适用于其他金属离子簇， 研究膜蛋白和生物电子传递过程的学科 梗概. 电子转移可以更容易和精确地研究， 光合作用比线粒体系统，因为电子转移可以 可方便地用光引发，并在单次翻转下进行研究 条件 在产氧光合作用中，光系统II（PSII）利用光提取 电子从水中分离出来，并将它们送入电子传输链， 产生化学自由能和还原当量， 碳固定 PSII光化学发生在两个 称为D1和D2的多肽。 四个锰离子簇 响应于这种光化学作用积累四个氧化当量， 然后用它们在协同机制中氧化两个水分子 它需要钙并释放一个氧分子作为副产品。 锰和钙的配体被认为主要是 D1和D2多肽上的羧基残基。 拟议的工作将 确定锰和钙的特定羧基配体，以及 表征它们对氧气释放的影响。 这将是 通过来自以下的psbA和psbD基因的定点诱变完成： 单细胞蓝细菌集胞藻属PCC 6803，其编码 D1和D2多肽。 诱变筛选程序 将快速识别那些羧基残基最有可能作为 配体。 这些残基的突变将通过生物化学方法表征。 和光谱方法，而其余的突变体将被存档， 未来分析 我以前曾采用定点诱变的方法， psbA和psbD基因的集胞藻PCC 6803，以确定这两个 PSII核心中的氧化还原活性酪氨酸残基， 锰团簇

The overall goal is to understand the operation of metal-ion clusters and electron transfer processes in biological systems. The specific goal is to understand the mechanism of photosynthetic water oxidation. Oxygenic photosynthesis provides the molecular oxygen and fixed carbon required to sustain animal life. The proposed work will identify the ligands to the manganese and calcium ions located at the catalytic site of photosynthetic water oxidation. Identification of these ligands will impose new constraints on models for the operation of the manganese cluster, and will enable such models to be placed in the context of protein structure. The information obtained will be applicable to other metal-ion clusters, and to the study of membrane proteins and biological electron transfer processes in general. Electron transfer can be studied more easily and precisely in photosynthetic than in mitochondrial systems, because electron transfer can be conveniently initiated with light and studied under single turn-over conditions. In oxygenic photosynthesis, Photosystem II (PSII) uses light to extract electrons from water and donate them into an electron transport chain that generates the chemical free energy and reducing equivalents required for carbon fixation. PSII photochemistry takes place in a heterodimer of two polypeptides known as D1 and D2. A cluster of four manganese ions accumulates four oxidizing equivalents in response to this photochemistry, and then uses them to oxidize two molecules of water in concerted mechanism that requires calcium and releases one molecule of oxygen as a by-product. The ligands for both manganese and calcium are believed to be predominantly carboxyl residues on the D1 and D2 polypeptides. The proposed work will identify the specific carboxyl ligands to manganese and calcium, and characterize their influence on oxygen evolution. This will be accomplished by site-directed mutagenesis of the psbA and psbD genes from the unicellular cyanobacterium Synechocystis sp. PCC 6803, which encode the D1 and D2 polypeptides, respectively. A mutagenic screening procedure will rapidly identify those carboxyl residues most likely to serve as ligands. Mutations of these residues will be characterized by biochemical and spectroscopic methods, while the remaining mutants will be archived for future analysis. I have previously employed site-directed mutagenesis of the psbA and psbD genes of Synechocystis sp. PCC 6803 to identify the two redox-active tyrosine residues in the PSII core that interact with the manganese cluster.