CRYSTALLOGRAPHIC STUDIES OF ELECTRON TRANSFER PROTEINS

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The fundamental biological processes of nitrogen fixation, nitrification and photosynthesis are catalyzed by complexes of electron transfer proteins. X-ray diffraction methods will be used to determine the three-dimensional structures of the component proteins in these systems. The structures will permit an evaluation of the protein - cofactor and cofactor - cofactor interactions that control the redox properties and electron transfer mechanisms of the proteins; the structural interactions that are responsible for the specificity in electron transfer between donor and acceptor proteins; and the mechanisms of energy transduction associated with the coupling of electron transfer to ATP hydrolysis (nitrogenase) and light absorption (photosynthesis). The specific structural objectives in these areas are: 1.Nitrogenase. Biological nitrogen fixation is catalyzed by the nitrogenase complex, which consists of iron (Fe-) protein and molybdenum iron (MoFe-) protein. The three-dimensional structures of Fe-protein from Azotobacter vinelandii and Clostridium pasteurianum will be completed. Structures of Fe-protein - nucleotide complexes and site directed mutants of Fe-protein will be determined to describe the mechanism of electron transfer, and the coupling of ATP hydrolysis to this process. Cocrystallizations and subsequent structure determinations of Fe-protein complexed with the physiological electron transfer partners MoFe-protein, ferredoxin and flavodoxin will be attempted. 2.Photosynthetic Reaction Center (RC). Refinement of the structures of the bacterial photosynthetic RCs from Rhodobacter sphaeroides strains R-26 and 2.4.1 will be completed. Structures of RCs in various oxidation states, from site directed mutants, and under low temperature conditions will be determined to provide a structural basis for understanding the efficiency of electron transfer in this system. The structure of the Rb. sphaeroides cytochrome c2 that serves as the physiological reductant for the oxidized RC will be determined, and co-crystallization of the cytochrome - RC complex will be attempted. As the only class of membrane proteins of known atomic structure, analysis of the RC will also focus on general implications for membrane protein structure. 3.Nitrification. The structure of the tetraheme cytochrome c-554, involved in nitrification reactions of Nitrosomonas europea, will be determined. Crystallization and structure determination of hydroxylamine oxidoreductase from this organism will be attempted.