Structural and Functional Analysis of Photosystem I

蓝藻光系统I反应中心是一个光驱动氧化还原酶，催化电子从细胞色素c533传递到铁氧还蛋白。这项研究计划的长期目标是了解光系统I反应中心的结构和功能。在这个项目中，生化分解和重组与大肠杆菌的定点诱变和蛋白质过量生产相结合，产生具有改变光谱和电子传递特性的反应中心。基于我们最近的成果和在当前资助期内开发的工具和构建的突变体，将解决以下长期目标：1。FA和/或FB在FNR室温光还原铁氧还蛋白（或黄氧还蛋白）和NADP+中的作用。2. 聚珠球菌pcc7002中两种FNR的生化特性研究。3. 聚珠球菌pcc7002中PS I多肽编码基因的分析。4. PsaD和PsaE在PS I结构和功能中的作用。5. PS I复合物与铁氧还蛋白和黄氧还蛋白特异性相互作用的机制。6. PsaC、PsaD、PsaE和FNR蛋白的三维结构。7. P700+再还原过程中依赖于PsaE-和ndhf的电子传递路径的性质地球上的所有生命最终都依赖于光合作用中对太阳能的捕获，这一过程导致二氧化碳的减少和固定，以及作为副产物的氧气的进化。光能捕获和转导发生在称为反应中心的多蛋白复合物中，该反应中心利用光能启动氧化还原反应和电子转移事件，从而产生化学能（以ATP的形式）和化学还原剂（以NADPH的形式）。在氧光合作用中——发生在绿色植物、藻类和被称为蓝藻的细菌中的类型——两个反应中心的合作是必需的，称为光系统I和II。本文提出的长期研究目标是了解光系统I反应中心的结构组织，并揭示导致该反应中心中电荷分离、电荷稳定和电子向可溶载流子转移的电子转移事件的具体细节。拟议的项目是两个不同研究学科的结合，是我们与约翰·h·戈德贝克博士（内布拉斯加州大学生物化学系）实验室的研究活动密切合作和协调的结果。结合现代分子遗传学、重组DNA技术、位点定向诱变、蛋白质过量生产和蛋白质生物化学（我的实验室），结合生化重建、现代超快激光光谱学、电子自旋共振（ESR）光谱学和其他生物物理测量（Golbeck实验室）。我们还将共同表征涉及光系统I反应中心的替代电子传递途径（例如，循环电子传递）以及替代电子受体铁氧还蛋白和黄氧还蛋白的功能。通过我们的工作，我们希望对光合作用太阳能捕获和光化学转换提供更好的理解。

The cyanobacterial Photosystem I reaction center is a light-driven oxidoreductase catalyzing electron transport from cytochrome c533 to ferredoxin. The long-term goal of this research program is to understand the structure and function of the Photosystem I reaction center. In this project biochemical resolution and reconstitution are combined with site-directed mutagenesis and protein overproduction in E.coli to produce reaction centers with altered spectroscopic and electron transport properties. Building upon our most recent results and the tools developed and mutants constructed in the current funding period, the following long-term goals will be addressed: 1. The roles of FA AND/OR FB in the room-temperature photoreduction of ferredoxin (or flavodoxin) and NADP+ via FNR. 2. Biochemical characterization of the two forms of FNR identified in Synechococcus sp. PCC 7002. 3. Characterization of the remaining genes encoding PS I polypeptides in Synechococcus sp. PCC 7002. 4. The roles of PsaD and PsaE in PS I structure and function. 5. The mechanism of the specific interactions between the PS I complex and ferredoxins and flavodoxin. 6. The 3-dimensional structures of the PsaC, PsaD, PsaE, and FNR proteins. 7. The nature of the PsaE- and NdhF-dependent paths of electron transport in P700+ re-reduction. %%% All life on Earth ultimately depends on solar light energy capture during photosynthesis, a process which leads to the reduction and fixation of carbon dioxide and the evolution of oxygen gas as a by- product. Light energy capture and transduction takes place in multiprotein complexes known as reaction centers, which utilize the light energy to initiate oxidation-reduction reactions and electron transfer events which result in the production of chemical energy (in the form of ATP) and chemical reductant (in the form of NADPH). In oxygenic photosynthesis--the type which occurs in green plants, algae, and bacteria known as cyanobacteria--the cooperation of two reaction centers, denoted Photosystems I and II, are required. The long-term goal of the research here proposed is to understand the structural organization of the Photosystem I reaction center and to unravel specific details of the electron transfer events that lead to charge separation, charge stabilization, and electron transfer to soluable carriers in this reaction center. The proposed project is a marriage of two different research disciplines and is the result of a close collaboration and coordination of our research activities with those of the laboratory of Dr. John H. Goldbeck (Dept. of Biochemistry, University of Nebraska). A combination of modern molecular genetics, recombinant DNA technology, site directed mutagenesis, protein overproduction and protein biochemistry (my lab) is combined with biochemical reconstitution, modern ultra-fast laser spectroscopy, electron spin resonance (ESR) spectroscopy, and other biophysical measurements (the Golbeck laboratory). We will also jointly characterize alternative electron transport pathways involving the Photosystem I reaction center (e.g, cyclic electron transport) and function of the alternative electron acceptors ferredoxin and flavodoxin. Through our work we hope to provide a better understanding of photosynthetic solar energy capture and photochemical conversion.