Function and Organization of Photosystem I

光系统 I 的功能和组织

• 批准号: 9405325
• 负责人: Parag Chitnis
• 金额: $ 18万
• 依托单位: Kansas State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9405325&HistoricalAwards=false
• 关键词: Function; Organization; Photosystem

Chitnis 9405325 Photosystem I, the light-dependent plastocyanin-ferredoxin oxidoreductase, is a multisubunit pigment-protein complex in the photosynthetic membranes of chloroplasts and cyanobacteria. Photosystem I contains at least twelve proteins, 100 chlorophylls, -carotenes and a series of electron transfer centers. To identify roles of individual polypeptides in the function and assembly of the complex, we initiated a program to clone and mutate genes that encode protein subunits of photosystem I of the naturally transformable cyanobacterium Synechocystis sp. PCC 6803. Our NSF-supported research has provided insights concerning the functions of several subunits of photosystem I. This proposal describes our plans to continue the investigations by focusing on (1) role of the accessory subunits, PsaL, PsaJ and PsaI in particular, in mediating and stabilizing assembly of photosystem I, and (2) mutational dissection and biochemical analysis of the reducing side of photosystem I. Recently we showed that PsaL is required for the formation of trimeric quaternary structure of photosystem I. PsaL binds calcium ions which stimulate trimerization. Site-specific mutations and biochemical characterization will be used to dissect the structural and regulatory basis for the role of PsaL. We postulate that the small (4 kDa) hydrophobic subunits of photosystem I (PsaJ, PsaM, PsaI) function as 'nuts and bolts' in the structure of the complex by anchoring and stabilizing other proteins. Characterization of our PsaJ-less mutant shows that PsaJ is necessary for the stable assembly of PsaF, a lumen-exposed subunit, and possibly PsaE, a peripheral subunit on the n-side. We will examine the interactions of PsaJ by biochemical and mutational analysis. We will also generate mutants lacking PsaI and further test our hypothesis. The terminal reduction reaction of photosystem I involves electron transfer from the reducing site on the complex to ferredoxin. The reducing site on photosyst em I consists of surface-exposed domains of PsaD, PsaE, and PsaC subunits. PsaD and PsaE are required for reduction of ferredoxin and therefore form essential topological components of the docking site where ferredoxin interacts and accepts electrons from PsaC. We have identified the solvent-exposed regions of PsaD on the basis of protease accessibility and labeling with NHS-biotin. Site-specific mutations in these domains will be used to identify residues that interact with ferredoxin. Physiological, biochemical and spectroscopic analyses will be conducted to investigate roles of mutations in specific subunits onthe assembly and function of photosystem I. %%%Photosynthesis is the major source of biological energy and oxygen on the earth. Betterunderstanding of this process is essential for facing the food and environmental challenges of the future. Photosynthesis requires coordinated and efficient functioning of membrane-embedded andsoluble enzymes in cyanobacteria and plants. Photosystem I is one of two pigment-proteincomplexes in the photosynthetic membranes, that can utilize light energy to produce high-energybiochemicals. Photosystem I contains at least eleven proteins, 100 chlorophylls, -carotenes and series of electron transfer centers. The long-term research objective of our research is to determine the roles of different proteins of photosystem I in its function and organization. We initiated a program to genetically manipulate photosystem I of a naturally transformable cyanobacterium that is easily amenable to genetic engineering. Our NSF-supported research has provided insights concerning the functions of several photosystem I proteins. We plan to continue these investigations by focusing on how these proteins mediate and stabilize the assembly of photosystem I. We will also study the role of photosystem I proteins in the transfer of electrons from photosystem I to soluble proteins. An interdisciplinary approach that uses molecular genetics, physiology , biochemistry and biophysics will lead to a better understanding of how light energy is utilized by plants to generate chemical energy. ***

Chitnis 9405325光系统I是叶绿体和蓝藻光合膜上的一种多亚单位色素-蛋白质复合体，是一种光依赖的叶绿体-铁氧还蛋白氧化还原酶。光系统I含有至少12个蛋白质、100个叶绿素、-胡萝卜素和一系列电子转移中心。为了确定单个多肽在复合体的功能和组装中的作用，我们启动了一个计划来克隆和突变自然转化的聚球藻的光系统I蛋白亚基的基因。PCC 6803。我们的NSF支持的研究已经提供了关于光系统I的几个亚基的功能的见解。这项建议描述了我们计划继续研究的重点：(1)辅助亚单位，特别是PSAL，PsaJ和PsaI在调节和稳定光系统I的组装中的作用，以及(2)光系统I的还原侧的突变解剖和生化分析。最近，我们发现PSAL是形成光系统I的三聚体四元结构所必需的。我们将使用定点突变和生化特征来剖析PSAL作用的结构和调控基础。我们推测，光系统I的小的(4 KDa)疏水亚基(PsaJ，PSAM，PsaI)通过锚定和稳定其他蛋白质，在复合体的结构中起到‘螺母和螺栓’的作用。对我们的PsaJ缺失突变体的鉴定表明，PsaJ对于稳定组装PSAF是必需的，PSAF是一个管腔暴露的亚基，可能还有PsaE，一个位于n侧的外围亚单位。我们将通过生化和突变分析来研究PSAJ的相互作用。我们还将产生缺乏PSAI的突变体，并进一步测试我们的假设。光系统I的末端还原反应涉及从络合物上的还原位置到铁氧还蛋白的电子转移。PSAD、PSAE和PSAC亚基的表面暴露区域构成了光合体em I上的还原部位。PSAD和PSAE是铁氧还蛋白还原所必需的，因此形成铁氧还蛋白与PSAC相互作用并接受来自PSAC的电子的对接位置的基本拓扑成分。我们已经根据蛋白酶的可及性和NHS-生物素标记确定了PSAD的溶剂暴露区域。这些区域的定点突变将被用来识别与铁氧还蛋白相互作用的残基。将进行生理、生化和光谱分析，以研究特定亚基突变对光系统I的组装和功能的作用。光合作用是地球上生物能量和氧气的主要来源。更好地理解这一过程对于应对未来的粮食和环境挑战至关重要。光合作用需要蓝藻和植物中的膜包埋酶和可溶性酶的协调和有效的功能。光系统I是光合膜中的两种色素-蛋白质复合体之一，可以利用光能产生高能生物化学物质。光系统I含有至少11种蛋白质、100种叶绿素、-胡萝卜素和一系列电子转移中心。我们研究的长期目标是确定光系统I的不同蛋白质在其功能和组织中的作用。我们启动了一个项目，对一种自然可转化的蓝藻的光系统I进行遗传操作，这种蓝藻很容易受到基因工程的影响。我们的NSF支持的研究提供了关于几个光系统I蛋白的功能的见解。我们计划通过重点研究这些蛋白质如何调节和稳定光系统I的组装来继续这些研究。我们还将研究光系统I蛋白在光系统I向可溶性蛋白质的电子转移中的作用。利用分子遗传学、生理学、生物化学和生物物理学的跨学科方法将导致对植物如何利用光能产生化学能的更好理解。***