The overall goal of the proposed research is to identify and characterize molecular and physical determinants that regulate the catalytic efficiency of assimilatory NADH:nitrate reductase (E.C. 1.6.6.1). Comprising two identical subunits, each of which contains FAD, cytochrome b(557) and Mo-pterin prosthetic groups in a 1:1:1 stoichiometry in discrete, functional """"""""domains"""""""", the enzyme catalyzes the rate-limiting and regulated step in the process of inorganic nitrogen assimilation. Nitrate reductase represents an excellent model for investigating electron transfer reactions in complex, multi-center proteins containing diverse prosthetic groups and shares a number of common structural and mechanistic features with a variety of other flavin-, heme- and Mo-containing proteins including cytochrome b(5) reductase, cytochrome b(5) and sulfite oxidase. Heterologous expression systems have been developed for the flavin- and flavin/heme-containing domains to probe the regulation of FAD-heme energy transduction using site-directed mutagenesis. The proposed research will focus on defining the rates of the various inter-and intramolecular transfer steps using pre-steady-state and steady state kinetics, examining the effects of relative cofactor redox potential modulation on the efficiency of energy transduction and defining the roles of specific amino acid residues in NR functionality using a combination of chemical modification and site-directed mutagenesis. These studies will integrate molecular biological techniques with a wide array of biophysical methods to enhance our understanding of important structure-function relationships of nitrate reductase and other related metalloflavoproteins.
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