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.
