DIRECTED MUTAGENESIS OF PHOTOSYNTHETIC OXYGEN EVOLUTION

长期目标是了解金属离子 团簇和生物学中的电子/质子转移过程。具体 目的是了解光合水氧化的机制。 光合作用提供了分子氧和固定碳 维持所有动物的生命。拟议的工作将确定氨基 酸残基，协调锰和钙离子在 水氧化的催化位点或其质子化性质 影响锰团簇的反应性。识别这些 残留物将对模型的操作施加新的限制， 锰集群，并将使这些模型被放置在背景下， 蛋白质结构。所获得的信息将适用于其他 金属离子簇，并研究膜蛋白和生物 电子和质子转移过程。电子和质子 转移反应可以更容易和精确地研究， 光合作用比线粒体系统，因为这两个过程都可以 方便地用光启动。 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， 然后通过一个需要钙的过程氧化两个水分子， 释放出分子氧作为副产品。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特定氨基酸残基的作用 多肽结合钙（例如，Asp-59、Asp-61、His-332、Glu- 333，His-337和Asp-342），（3）进一步表征 D1多肽的特定残基对完整的 Mn簇（例如，Asp 61、His-92和Glu-189），和（4）鉴定氨基酸 其质子化性质影响能量学的残基， 水的氧化机制。 具有后者取代的突变体 残基将含有Mn簇，其被显著扰动或被 非功能性的并且表现出改变的质子释放模式。 的 所需的突变已经由我们在 蓝细菌集胞藻属PCC 6803。

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.