DIRECTED MUTAGENESIS OF PHOTOSYNTHETIC OXYGEN EVOLUTION

光合放氧的定向诱变

• 批准号: 2182035
• 负责人: RICHARD J DEBUS
• 金额: $ 15.68万
• 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 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2182035
• 关键词: Cyanophyta; DNA; calcium; carboxyl group; electron spin resonance spectroscopy; electron transport; genetic recombination; ligands; manganese; membrane proteins; oxidation; oxygen; photochemistry; photosystem; protein biosynthesis; protein structure; protein structure function; proteins; 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运行和 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。 充氧 光合作用提供分子氧和固定碳 维持动物的生命。 拟议的工作将确定配体 锰离子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 对锰集群运行模型的限制，并将 使此类模型能够置于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 膜蛋白和生物电子传递过程的研究 一般来说。 电子转移可以更容易、更精确地研究 光合作用比线粒体系统要好，因为电子转移可以 方便地用光引发并在单次翻转下进行研究 状况。 在氧气光合作用中，光系统 II (PSII) 利用光来提取氧气 来自水中的电子并将它们捐赠到电子传输链中 产生化学自由能和还原所需的当量 碳固定。 PSII 光化学发生在两个异二聚体中 称为D1和D2的多肽。 四个锰离子簇 响应这种光化学积累四个氧化当量， 然后用它们以协同机制氧化两个水分子 它需要钙并释放一个氧分子作为副产品。 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 识别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 psbA 和 psbD 基因的定点诱变来完成 单细胞蓝藻集胞藻属 sp。 PCC 6803，编码 分别为D1和D2多肽。 诱变筛选程序 将快速识别那些最有可能充当的羧基残基 配体。 这些残基的突变将通过生化特征来表征 和光谱方法，而剩余的突变体将被存档 未来分析。 我以前曾采用过定点诱变 集胞藻属的 psbA 和 psbD 基因。 PCC 6803 识别两者 PSII 核心中的氧化还原活性酪氨酸残基与 锰簇。