Protonation and Conformational Control of Photosynthetic Reaction Center Function

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MCB 9905672WraightThe primary and secondary acceptor quinones, QA and QB, in the photosynthetic reaction center (RC) of Rhodobacter sphaeroides are chemically identical but exhibit very distinct properties. Thus, they provide an ideal model for understanding how proteins determine the properties of bound cofactors. This project investigates the origin of these distinct properties, the mechanistic details of electron and proton transfer to QB, and the conformational events that accompany these transfers. Accommodation of the protein to buried charges will be examined through the decay of fluorescence emission from the flash-induced P+QA- state. Relaxation on the micro- to millisecond time scales will be determined, utilizing various quinones as QA as well as QA-site mutants. Long range interactions between the two quinone binding sites, that give rise to reciprocal perturbations of the free energy levels of QA and QB redox states, will be quantified via measurements of proton uptake, QA-to-QB electron transfer, and delayed fluorescence emission. Specific models of this interaction will be tested by site-directed mutagenesis and quinone analogues. Control of the redox potentials of QA and QB by electrostatic and steric interactions with the protein will be analyzed by site directed mutagenesis of QA and QB site residues and specifically synthesized quinones, to explore the role of the methoxy torsion angles in optimizing QA and QB function by ubiquinones. Structural implications will be investigated by Fourier-transform infra-red (FTIR) spectroscopy.Light energy conversion in the photosynthetic reaction center (RC) involves an initial electron transfer, followed by several more steps in which the separation of charge is stabilized and trapped. In bacterial RCs, the many reactions that the RC is capable of include at least 8 different electron transfer reactions, 2 distinct proton transfers, plus substrate (quinone) binding and protein (cytochrome) recognition, docking and binding. With many of the cofactors involved act as electrochromic reporter groups as well as reactants, and the RC provides unparalleled observability of charge movements and conformational relaxations and is exceptionally well suited for studying protein function and behavior. In some bacteria, two chemically identical quinones act as electron acceptors, in series, exhibiting very distinct properties. This project will use spectroscopic and biochemical methods to study how the protein modifies the properties of the two quinones, including the role of conformational relaxation in accommodating buried charges, and the effects of electrostatics and steric constraints in determining the redox properties of the quinones. The outcomes will provide general understanding of how proteins modify and determine the properties and reactivities of bound cofactors and substrates, essential to catalysis.