DIRECTED MUTAGENESIS OF PHOTOSYNTHETIC OXYGEN EVOLUTION

光合放氧的定向诱变

基本信息

批准号：
2734651
负责人：
RICHARD J DEBUS
金额：
$ 17.25万
依托单位：
UNIVERSITY OF CALIFORNIA RIVERSIDE
依托单位国家：
美国
项目类别：
财政年份：
1989
资助国家：
美国
起止时间：
1989-12-01 至 2001-06-30
项目状态：
已结题

项目摘要

The long-term objective is to understand the operation of metal-ion clusters and electron/proton transfer processes in biology. The specific goal is to understand the mechanism of photosynthetic water oxidation. Oxygenic photosynthesis provides the molecular oxygen and fixed carbon required to sustain all animal life. The proposed work will identify amino acid residues that coordinate the manganese and calcium ions at the catalytic site of water oxidation or whose protonation properties influence the reactivity of the manganese cluster. Identification of these residues 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 and proton transfer processes in general. Electron and proton transfer reactions can be studied more easily and precisely in photosynthetic than in mitochondrial systems because both processes can be conveniently initiated with light. 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, then oxidizes two molecule of water by a process that requires calcium and releases molecular oxygen as a by-product. The proposed work will (l) determine if specific amino acid residues of the D1 polypeptide (e.g, Asp- 170, His-332, Glu-333, His-337, and Asp-342) ligate the Mn cluster, (2) characterize the role of specific amino acid residues of the D1 polypeptide in the binding of calcium (e.g., Asp-59, Asp-61, His-332, Glu- 333, His-337, and Asp-342), (3) further characterize the influence of particular residues of the D1 polypeptide on the operation of the intact Mn cluster (e.g., Asp61, His-92, and Glu-189), and (4) identify amino acid residues whose protonation properties influence the energetics and mechanism of water oxidation. Mutants with substitutions of the latter residues would contain Mn clusters that are significantly perturbed or are non-functional and that exhibit altered patterns of proton release. The required mutations have already been constructed by us in the cyanobacterium Synechocystis sp. PCC 6803.

长期目标是了解金属离子的运作生物学中的簇和电子/质子转移过程。具体的目标是了解光合水氧化的机制。产氧光合作用提供分子氧和固定碳维持所有动物生命所必需的。拟议的工作将鉴定氨基协调锰离子和钙离子的酸残基水氧化的催化位点或其质子化性质影响锰簇的反应活性。识别这些残留物将对模型的运行施加新的限制锰簇，并将使此类模型能够放置在上下文中蛋白质结构。所获得的信息将适用于其他金属离子簇，以及膜蛋白和生物的研究一般电子和质子转移过程。电子和质子转移反应可以更容易、更精确地研究光合作用比线粒体系统更重要，因为这两个过程都可以方便地用光引发。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，然后通过需要钙和的过程氧化两个水分子释放分子氧作为副产品。拟议的工作将 (l) 确定 D1 多肽的特定氨基酸残基（例如，Asp- 170、His-332、Glu-333、His-337 和 Asp-342) 连接 Mn 簇，(2) 表征 D1 的特定氨基酸残基的作用钙结合中的多肽（例如，Asp-59、Asp-61、His-332、Glu- 333、His-337 和 Asp-342），（3）进一步表征了 D1多肽的特定残基对完整序列的操作 Mn 簇（例如 Asp61、His-92 和 Glu-189），以及 (4) 识别氨基酸其质子化性质影响能量的残基和水氧化机理。后者取代的突变体残留物将含有受到显着扰动或非功能性的并且表现出改变的质子释放模式。这我们已经构建了所需的突变蓝藻集胞藻属 sp. PCC 6803。