The goal of the proposed research is to elucidate molecular and physical determinants important for the catalytic efficiency and regulation of the enzyme, assimilatory NADH:nitrate reductase. This enzyme catalyzes the rate-limiting and regulated step in the process of nitrate assimilation which is the major pathway by which inorganic nitrogen is converted to a biologically usable form in eukaryotes and a variety of macroorganisms. Nitrate reductase is a multimeric enzyme which contains FAD, heme and molybdopterin prosthetic groups and transfers electron from NADH to nitrate. In addition to its biological importance, this enzyme represents and excellent model for investigating electron transfer reactions in complex proteins and shares several common mechanistic and structural features with other molybdo-, heme-, or flavo-enzymes. The specific objectives of the proposed research are to correlate the thermodynamic parameters and kinetic properties of the enzyme under various environmental conditions which relate to physiological functions. These conditions include pH, substrates, products, and known effectors such as phosphate and cyanide. Thermodynamic parameters and properties of individual prosthetic groups, identification and properties of important functional groups and kinetic parameters for individual steps will be determined by a combination of UV/vis, room temperature epr and CD spectroscopies; microcoulometry; stopped flow, freeze-quench epr and laser flash photolysis; and chemical methods. Conserved properties will be determined by selective comparative studies of nitrate reductases from various sources. Studies utilizing various substituted forms of nitrate reductase (FAD analogues, W and selenomethionine substituted forms) and limited proteolytic products will allow dissection and study of functional domains of the enzyme.
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