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

蓝藻、藻类和植物的光合作用中的细胞色素bf复合物在两个光化学反应中心之间传递电子，并为ATP的合成建立质子梯度。此外，BF复合物在氧化还原传感和光合作用的调节中起着至关重要的作用。催化机理涉及膜一侧的质体醌氧化和另一侧的质体醌还原。醌还原酶位点的功能知之甚少，但似乎是必要的BF复合物，循环电子流的营业额，并对氧自由基的生产有影响。一个独特的，最近发现的血红素（ci或x）驻留在醌还原酶口袋。本计画将探讨BF复合物与醌还原酶位功能对氧化还原调控的机制与结果。大多数研究将使用蓝细菌集胞藻PCC 6803，这是服从遗传学，时间分辨光谱学和微阵列分析。具体目标是：1）研究现有的Rieske铁硫蛋白和bf复合物的低和高电位链突变体的状态转换（光捕获天线的再分布）和电子传递动力学。2)通过微阵列杂交、2D凝胶分离和质谱研究特定突变体，以表征bf复合物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氧化还原调控突变体。4)在细胞色素bf醌还原酶结构域构建基于结构的定点突变，以研究其催化机制和在循环电子传递中的作用。突变蓝藻的特点是电子转移动力学，调节状态转换，氧自由基的生产，和性质的ci（x）血红素。这项研究是及时的，因为细胞色素bf的结构，并将提供重要的新见解蛋白质的结构功能和光合作用中的氧化还原信号。更广泛的影响：研究是在UW奥什科什许多学生的教育经验的重要组成部分。氧化还原调节，催化机制和蓝藻光合作用的细胞色素bf复合物中的氧自由基产生的研究是多方面的，充分利用了NSF资助的蛋白质组学和功能基因组学核心设施，并将在项目的各个方面为学生提供众多，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.