The goal of the proposed research is to elucidate molecular and physical determinants that influence or control the thermodynamics and catalytic efficiency of assimilatory NADH:nitrate reductase (E.C. Comprising four identical subunits, each of which contains FAD, cytochrome b557 and Mo-pterin in a 1:1:1 stoichiometry, this enzyme catalyzes the rate-limiting and regulated step in the process of inorganic nitrogen assimilation and is an excellent model for investigating electron transfer reactions in complex, multi-center proteins containing diverse prosthetic groups. In addition, this enzyme shares a number of common structural and mechanistic features with a variety of other flavoheme- and Mo-containing proteins including cytochrome b5 reductase, cytochrome b5 and sulfite oxidase. The current grant period has focused on defining the thermodynamic and steady-state kinetic parameters of the native enzymes. The proposed research will focus on identifying determinants of redox potential of the various prosthetic groups within the enzyme and the effects of altering these potentials on catalytic function, in addition to identification of functional groups involved in binding and catalysis.
The specific aims of the proposed research are to establish the pathway of electron transfer within the enzyme and to determine the rates of the various intra- and intermolecular electron transfer reactions between the different prosthetic groups in active and substituted forms of Chlorella nitrate reductase using a combination of steady-state, rapid kinetic and electrochemical methods. Relative redox potential modulation of the FAD and Mo-pterin centers will be examined as a mechanism of increasing or decreasing catalytic efficiency. Redox properties will also be examined under various environmental conditions which relate to physiological function, such as in the presence of NADH/NAD+ and NO3-/NO2- or cyanide. In addition, amino acid residues crucial to the catalytic activity of the enzyme will be identified by specific chemical modification studies using both the native enzyme and various recombinant proteins corresponding to the individual functional domains. Specific amino acid modification reagents will be used together with protein sequencing studies to identify cysteine, lysine and arginine residues that are essential to the function of the FAD and Mo-pterin centers. These studies will enhance our understanding of important structure-function relationships of nitrate reductase and other related proteins.

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Physical Biochemistry Study Section (PB)
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University of South Florida
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Percy, M J; Crowley, L J; Boudreaux, J et al. (2006) Expression of a novel P275L variant of NADH:cytochrome b5 reductase gives functional insight into the conserved motif important for pyridine nucleotide binding. Arch Biochem Biophys 447:59-67
Percy, M J; Crowley, L J; Roper, D et al. (2006) Identification and characterization of the novel FAD-binding lobe G75S mutation in cytochrome b(5) reductase: an aid to determine recessive congenital methemoglobinemia status in an infant. Blood Cells Mol Dis 36:81-90
Roma, Glenn W; Crowley, Louis J; Barber, Michael J (2006) Expression and characterization of a functional canine variant of cytochrome b5 reductase. Arch Biochem Biophys 452:69-82
Roma, Glenn W; Crowley, Louis J; Davis, C Ainsley et al. (2005) Mutagenesis of Glycine 179 modulates both catalytic efficiency and reduced pyridine nucleotide specificity in cytochrome b5 reductase. Biochemistry 44:13467-76
Percy, M J; Crowley, L J; Davis, C A et al. (2005) Recessive congenital methaemoglobinaemia: functional characterization of the novel D239G mutation in the NADH-binding lobe of cytochrome b5 reductase. Br J Haematol 129:847-53
Marohnic, Christopher C; Crowley, Louis J; Davis, C Ainsley et al. (2005) Cytochrome b5 reductase: role of the si-face residues, proline 92 and tyrosine 93, in structure and catalysis. Biochemistry 44:2449-61
Davis, C Ainsley; Barber, Michael J (2004) Cytochrome b5 oxidoreductase: expression and characterization of the original familial ideopathic methemoglobinemia mutations E255- and G291D. Arch Biochem Biophys 425:123-32
Davis, C Ainsley; Crowley, Louis J; Barber, Michael J (2004) Cytochrome b5 reductase: the roles of the recessive congenital methemoglobinemia mutants P144L, L148P, and R159*. Arch Biochem Biophys 431:233-44
Pollock, Veronica V; Conover, Richard C; Johnson, Michael K et al. (2003) Biotin sulfoxide reductase: Tryptophan 90 is required for efficient substrate utilization. Arch Biochem Biophys 409:315-26
Bewley, Maria C; Davis, C Ainsley; Marohnic, Christopher C et al. (2003) The structure of the S127P mutant of cytochrome b5 reductase that causes methemoglobinemia shows the AMP moiety of the flavin occupying the substrate binding site. Biochemistry 42:13145-51

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