This project addresses the mechanisms of electron and proton transfer in the reaction center complex of the photosynthetic bacteria, Rhodobacter sphaeroides and Rhodopseudomonas viridis, for which the structure of the reaction center (RC) is known at atomic resolution. The main focus is the two quinone molecules that act as electron acceptors to the photochemical events. The quinones undergo one-electron reduction to the semiquinone, and double reduction to the quinol state, which also requires uptake of protons from the aqueous medium. The work aims to identify amino acid residues active in the binding of both quinones, in the stabilization of the semiquinone states, in the establishment of the functional redox potentials of the two quinones, and in the proton transfer pathways leading to formation of the quinol state. The effects of specific mutations on the functional redox and acid- base properties of the to-electron gate, in Rb. Sphaeroides, will be investigated by kinetic and equilibrium techniques based on optical spectroscopy and electrometric measurements. These will be supplemented by structural studies, including X-ray difference analysis where possible, and infra-red spectroscopy (FTIR), and by computational approaches, utilizing the comparative virtues of mutant RCs to maximum effect. %%% The primary goals of this work are (1) to demonstrate and understand how specific activities and properties are induced in biological cofactors by protein-ligand interactions, and (2) to investigate the mechanism of proton transfer through proteins. These are important general issues in enzymatic catalysis and biological specificity. The reaction center quinones are uniquely suited for these investigations, through the great experimental amenability of the system for biophysical measurements and for protein engineering.
