In a broad sense, the objectives of this research are to understand biological processes on a molecular level, and to further expand the range of molecular genetics techniques available for exploring such questions. The specific problem addressed in this proposal concerns the mechanism of photochemical energy conversion in bacteria. The site of the primary photochemistry is an integral membrane pigment-protein complex called the reaction center. Our goal is to extend our understanding of the role of specific amino acid residues and cofactors in the function of the reaction center. The methodologies used in this proposal combine the ability to alter proteins in specific ways by molecular genetics and the elucidation of structural and functional changes by an interdisciplinary range of biochemical and spectroscopic techniques. An investigation of the effect of systematic alterations of amino acid residues by site-directed mutagenesis of the reaction center genes is planned. Molecular genetic techniques will be used to develop a system in the purple bacterium, Rhodobacter sphaeroides, in which any amino acid residue of the reaction center can be changed and the resulting mutation analyzed with a minimum of difficulty. The role of the non-heme iron atom in the reaction center will be addressed by characterizing a set of mutant reaction centers containing changes in the ligands to the iron, with regard to the specificity of the metal binding site, the electronic environment of the iron, electron transfer rates involving nearby cofactors, and the three dimensional structures. Construction and analysis of mutations in other residues that are highly conserved in related proteins from other photosynthetic organisms are planned. Some of the findings and methodologies are expected to be relevant to the binding of metals in other proteins, the structure of membrane proteins, and the process of electron transfer, most notably in the respiratory chain in mitochondria.
