Nitrogenase, the enzyme responsible for biological nitrogen fixation, consists of two metalloproteins, the Fe- protein and MoFe-protein, that mediate the coupling of ATP hydrolysis to the reduction of dinitrogen to ammonia. Nitrogenase is a prototypic example of an enzyme with multiple and varied iron-sulfur clusters that participate in electron transfer and substrate reduction, as well as providing an excellent model for energy transduction of ATP hydrolysis. We are pursuing coordinated biochemical and crystallographic approaches to determine the three-dimensional structures of defined states of the nitrogenase proteins individually and in complexes. Our research objectives emphasize biochemical, biophysical and structural characterization of nitrogenase proteins trapped under turnover conditions;an assessment of the oxidation states of metal sites in different forms of the nitrogenase proteins through crystallographic refinement at multiple wavelengths; and preparation and analysis of the nitrogenase proteins complexed with nucleotides and physiological electron donors. The structural and biochemical studies will be extended to other energy transduction systems, specifically the family of integral membrane protein ATP Binding Cassette (ABC) transporters, to assess the generality of our conclusions concerning the mechanisms of nucleotide dependent transduction processes. A high priority is to complete our current structural studies of ABC transporters mediating the import into cells of metal-chelate species and amino acids, followed by structural and biochemical characterization of defined complexes of these transporters with nucleotides and binding proteins. These studies will be extended to additional transporters implicated in metal metabolism. Electron transfer and ATP dependent transduction processes form the heart of cellular energy metabolism. Nitrogenase not only couples these elements to generate an essential nutrient, but also serves as an excellent structural model for proteins participating in signal and energy transduction pathways, including G- proteins and the ras oncogene. The study of ABC transporters will extend our structural and mechanistic focus on ATP dependent processes to a widespread family of integral membrane proteins, including biomedically relevant members responsible for multidrug resistance and cystic fibrosis.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
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Special Emphasis Panel (NSS)
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Smith, Ward
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California Institute of Technology
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