RUI: The Cytochrome bf complex: Role in Redox Regulation and Function of the Quinone-reductase Site

蓝藻、藻类和植物氧化光合作用的细胞色素bf复合体在两个光化学反应中心之间传递电子，并为ATP合成建立质子梯度。此外，bf复合体在氧化还原感觉和光合作用的调节中起着至关重要的作用。催化机理包括膜一侧的质酚氧化和膜的另一侧的质酚还原。对苯醌还原酶部位的功能知之甚少，但似乎对bf络合物的周转，循环电子流是必要的，并与氧自由基的产生有关。一种最近发现的独特的血红素(Ci或x)存在于苯醌还原酶口袋中。本项目将研究bf复合体和苯醌还原酶位点功能对氧化还原调节的机制和结果。大多数研究将使用符合遗传学、时间分辨光谱分析和微阵列分析的蓝藻聚球藻PCC6803进行。具体目标是：1)研究可利用的Rieske铁硫蛋白和bf复合体的低电位链和高电位链突变体的状态转变(捕光天线的重新分布)和电子传输动力学。2)通过基因芯片杂交、2D凝胶分离和质谱学研究特定突变体，以确定bf复合体在基因和蛋白质调控中的作用。3)通过化学诱变和荧光筛选获得额外的氧化还原调节突变体。4)构建细胞色素bf醌还原酶结构域的定点突变，研究其催化机制及其在循环电子传递中的作用。突变蓝藻的特征包括电子转移动力学、状态转变的调节、氧自由基的产生以及Ci(X)亚铁血红素的性质。由于细胞色素bf的结构，这项研究是及时的，并将为光合作用中的蛋白质结构功能和氧化还原信号提供重要的新见解。广泛影响：研究是UW Oshkosh许多学生教育经验的重要组成部分。关于蓝藻光合作用细胞色素bf复合体中氧化还原调节、催化机制和氧自由基产生的研究是多方面的，充分利用了美国国家科学基金会资助的蛋白质组学和UW Oshkosh大学的功能基因组学核心设施，并将在该项目的各个方面为学生提供大量、优秀和令人兴奋的研究机会。

The cytochrome bf complex of cyanobacterial, algal, and plant oxygenic photosynthesis transfers electrons between the two photochemical reaction centers and establishes a proton gradient for ATP synthesis. In addition, the bf complex plays a crucial role in redox sensing and regulation of photosynthesis. The catalytic mechanism involves plastoquinol oxidation on one side of the membrane and plastoquinone reduction on the other side. Quinone-reductase-site function is poorly understood but seems necessary for turnover of the bf complex, cyclic electron flow, and has implications for oxygen radical production. A unique, recently discovered heme (ci or x) resides in the quinone-reductase pocket. This project will investigate the mechanism and consequences of redox regulation by the bf complex and quinone-reductase-site function. Most studies will be performed using the cyanobacterium Synechocystis PCC 6803, which is amenable to genetics, time-resolved spectroscopy, and microarray analysis. Specific objectives are: 1) To investigate available Rieske iron-sulfur protein and low and high-potential chain mutants of the bf complex for state-transitions (the redistribution of light harvesting antenna) and electron transport kinetics. 2) To investigate specific mutants by microarray hybridizations, 2D gel separations, and mass spectrometry to characterize the role of the bf complex in gene and protein regulation. 3) To obtain additional redox-regulation mutants by chemical mutagenesis and fluorescence screening. 4) To construct structure-based, site-directed mutations in the cytochrome bf quinonereductase domain to investigate its catalytic mechanism and role in cyclic electron transport. Mutant cyanobacteria will be characterized for electron transfer kinetics, regulation of state-transitions, oxygen radical production, and properties of the ci (x) heme. The research is timely because of the cytochrome bf structures and will provide important new insights into protein structure-function and redox signaling in photosynthesis.Broader impacts: Research is an important component of the educational experience for many students at UW Oshkosh. The research on redox regulation, catalytic mechanisms, and oxygen radical production in the cytochrome bf complex of cyanobacterial photosynthesis is multifaceted, takes full advantage of the NSF-funded Proteomics and Functional-Genomics Core Facility at UW Oshkosh, and will provide numerous, excellent and exciting research opportunities for students in all aspects of the project.