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Structure, function and biodynamics of photosystem II reaction center

光系统II反应中心的结构、功能和生物动力学

基本信息

批准号：
09440268
负责人：
SATOH Kimiyuki
金额：
$ 8.77万
依托单位：
Okayama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 2000
项目状态：
已结题

项目摘要

The structure, function and biodynamics of the photosystem II (PSII) reaction center were analyzed in this study from two different viewpoints.In the first approach, targeted in vitro random mutagenesis was applied to a wide region of the psbAII gene coding for the Ser148-Ala357 segment of the D1 subunit in Synechocystis sp.PCC 6803, in order to discover unexpected structure-function relationships in the PSII reaction center. A method based on the susceptibility of photosynthetic organisms to nitrofurantoin under illumination was established to screen mutants deficient in the function of photosynthetic electron transport. By this method we have succeeded in isolating more than 150 random mutants impaired in the function of PSII caused by 1-9 amino acid substitution (s) in the targeted amino acid region on D1 protein. By the analysis of these random mutants, several amino acid residues crucial for the function of water oxidation and charge separation in PSII were newly identified and the mechanism of functional modification of PSII by the amino acid substitution on D1 protein was investigated for each mutant.In the second approach, the phenomenon of carboxyl-terminal (C-terminal) processing of precursor D1 protein of PSII reaction center was analyzed from two aspects, i.e., enzyme and substrate. We have succeeded in over-expressing the enzyme, CtpA, responsible for the process in E.coli and purifying it in homogeneity. The enzymatic properties and the mode of action in vivo were analyzed by using the over-expressed enzyme and the substrate in different molecular environments. This analysis suggested the presence of specific interaction of CtpA with factor (s) in the PSII complex that modulates the proteolysis in response to physiological conditions.

本研究从两个不同的角度对光系统II(PSII)反应中心的结构、功能和生物动力学进行了分析。在第一种方法中，利用体外定向随机突变的方法，对聚球藻PcC6803中编码D1亚单位Ser148-Ala357片段的psbAII基因进行定向随机突变，以发现PSII反应中心中意想不到的结构-功能关系。根据光合作用生物在光照条件下对呋喃妥因的敏感性，建立了一种筛选光合作用电子传递功能缺失突变株的方法。通过这种方法，我们已经成功地分离到150多个随机突变体，这些突变体是由于D1蛋白上目标氨基酸区域的1-9个氨基酸替换(S)而导致PSII功能受损的。通过对这些随机突变体的分析，新鉴定了PSII中几个在水氧化和电荷分离功能中起关键作用的氨基酸残基，并对每个突变体的D1蛋白的氨基酸取代修饰机制进行了研究。在第二种方法中，从酶和底物两个方面分析了PSII反应中心前体D1蛋白的羧基末端(C端)加工现象。我们已经成功地在大肠杆菌中过度表达了负责这一过程的酶CTPA，并以同质性纯化了它。利用过表达的酶和底物在不同的分子环境中分析其在体内的作用方式和酶性质。这一分析表明，在PSII复合体中，CTPA与调节蛋白降解的因子(S)存在特异性相互作用，以响应生理条件。