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团簇的运行和 生物系统中的电子转移过程。具体目标是 了解光合作用水氧化的机理。含氧的 光合作用提供所需的分子氧和固定碳 维持动物的生命。这项拟议的工作将确定 位于光合作用催化部位的锰和钙离子 水氧化。对这些配体的识别将带来新的 对锰群组运作模式的限制，并将 使这样的模型能够放在蛋白质结构的背景下。这个 所获得的信息将适用于其他金属离子团簇，以及 膜蛋白与生物电子传递过程的研究 总体而言。电子转移的研究可以更容易和更精确地在 光合作用比线粒体系统更大，因为电子转移可以 可以方便地用光启动并在单次翻转下研究 条件。 在氧合光合作用中，光系统II(PSII)利用光来提取 把水中的电子送入电子传输链， 产生所需的化学自由能和还原当量 碳固定。PSII光化学发生在两个杂二聚体中 多肽被称为d1和d2。由四个锰离子组成的团簇 积累了四个氧化当量以响应这种光化学， 然后用它们协同机制氧化两个水分子 这需要钙，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 D_1和D_2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 描述它们对放氧的影响。这将是 通过定点诱变水稻的PSBA和PSBD基因实现 单细胞蓝藻聚球藻。PCC 6803，它编码 分别为D_1和D_2多肽。诱变筛选程序 将快速识别那些最有可能作为 配基。这些残基的突变将以生物化学为特征 和光谱方法，而剩下的突变体将被存档 未来的分析。我之前曾使用过定点突变技术 聚球藻的psba和psbD基因。PCC 6803来识别这两个 PSII核心中的氧化还原活性酪氨酸残基与 锰团簇。