Function and Organization of Photosystem I

光系统I，光依赖性质体青素-铁氧化还蛋白氧化还原酶，是叶绿体和蓝藻光合膜中的多亚基色素蛋白复合物。光系统I包含至少12种蛋白质、100种叶绿素、-胡萝卜素和一系列电子转移中心。为了确定单个多肽在该复合体的功能和组装中的作用，我们启动了一个程序，克隆和突变编码天然可转化蓝藻聚囊藻sp. PCC 6803光系统I蛋白亚基的基因。我们在nsf支持下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。本提案描述了我们继续研究的计划，重点关注(1)辅助亚基，特别是pal， PsaJ和PsaI在光系统I的调节和稳定组装中的作用；(2)光系统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。位点特异性突变和生化特性将被用来剖析PsaL作用的结构和调控基础。我们假设光系统I的小（4 kDa）疏水亚基（PsaJ, PsaM, PsaI）通过锚定和稳定其他蛋白质在复合物结构中起“螺母和螺栓”的作用。我们的PsaJ缺失突变体的特征表明，PsaJ对于PsaF（一个管腔暴露亚基）和PsaE（一个n侧外周亚基）的稳定组装是必要的。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，并进一步验证我们的假设。光系统I的末端还原反应包括电子从络合物上的还原位点转移到铁氧还蛋白。光系统em上的还原位点由PsaD、PsaE和PsaC亚基的表面暴露结构域组成。PsaD和PsaE是还原铁氧还蛋白所必需的，因此是铁氧还蛋白相互作用并接受PsaC电子的对接位点的基本拓扑组成部分。我们在蛋白酶可及性和nhs生物素标记的基础上确定了PsaD的溶剂暴露区域。这些结构域的位点特异性突变将用于鉴定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在光系统i的组装和功能中的作用。%%%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面对未来的食品和环境挑战至关重要。在蓝藻和植物中，光合作用需要膜内和可溶性酶的协调和有效的功能。光系统I是光合膜中两种色素-蛋白质复合物之一，可以利用光能产生高能生物化学物质。光系统I包含至少11种蛋白质、100种叶绿素、-胡萝卜素和一系列电子传递中心。我们的长期研究目标是确定光系统I中不同蛋白质在其功能和组织中的作用。我们启动了一项计划，通过基因操纵一种自然可转化的蓝藻的光系统I，这种光系统很容易适应基因工程。我们的nsf支持的研究提供了一些关于光系统I蛋白功能的见解。我们计划通过关注这些蛋白如何介导和稳定光系统I的组装来继续这些研究。我们还将研究光系统I蛋白在电子从光系统I转移到可溶性蛋白中的作用。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更好地理解植物如何利用光能产生化学能。＊＊＊

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. ***