DIRECTED MUTAGENESIS OF PHOTOSYNTHETIC OXYGEN EVOLUTION

光合放氧的定向诱变

• 批准号: 3302536
• 负责人: RICHARD J DEBUS
• 金额: $ 14.72万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-12-01 至 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3302536
• 关键词: Cyanophyta; DNA; calcium; carboxyl group; electron spin resonance spectroscopy; electron transport; genetic recombination; ligands; manganese; membrane proteins; oxidation; oxygen; photochemistry; photosynthesis; protein biosynthesis; protein structure; protein structure function; site directed mutagenesis; water

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.

总体目标是了解金属离子簇的操作和 生物系统中的电子传输过程。 具体目标是 了解光合水氧化的机制。 氧气 光合作用提供了分子氧和固定碳 维持动物的生活。 拟议的工作将确定配体 位于光合作用的催化位点的锰和钙离子 水氧化。 这些配体的识别将施加新的 对锰簇操作的模型的限制，并将 使此类模型能够放置在蛋白质结构的背景下。 这 获得的信息将适用于其他金属离子簇，并适用于 膜蛋白和生物电子转移过程的研究 一般来说。 电子传输可以更轻松，精确地研究 光合作用比线粒体系统中的光合作用，因为电子转移可以 便利地用光线启动，并在单个转换下进行研究 状况。 在充氧光合作用中，光系统II（PSII）使用光来提取 来自水的电子并将其捐赠到电子传输链中，该链 产生化学自由能并减少相同 碳固定。 PSII光化学发生在两个的异二聚体中 多肽称为D1和D2。 四个锰离子的簇 响应于这种光化学的四个氧化等效物， 然后用它们在协同机构中氧化两个水分子 这需要钙并释放一个分子作为副产品。 据信锰和钙的配体主要是 D1和D2多肽上的羧基残基。 拟议的工作将 确定特定的羧基配体给锰和钙，以及 表征它们对氧气进化的影响。 这将是 通过从位置定向的PSBA和PSBD基因的诱变完成 单细胞蓝细菌合成细胞杆菌sp。 PCC 6803，该编码 D1和D2多肽。 诱变筛选程序 将迅速识别那些最有可能用作的羧基残基 配体。 这些残基的突变将以生化为特征 和光谱方法，而其余的突变体将被存档 未来分析。 我以前曾采用过地点指导的诱变 Synechocystis sp。的PSBA和PSBD基因。 PCC 6803确定这两个 PSII核心中的氧化还原活性酪氨酸残基与 锰簇。