TAILIEUCHUNG - Báo cáo khoa học: Thermodynamic and kinetic analysis of the isolated FAD domain of rat neuronal nitric oxide synthase altered in the region of the FAD shielding residue Phe1395

In rat neuronal nitric oxide synthase, Phe1395 is positioned over the FAD isoalloxazine ring. This is replaced by Trp676 in human cytochrome P450 reductase, a tryptophan in related diflavin reductases (. methionine synthase reduc-tase andnovel reductase 1), and tyrosine inplant ferredoxin-NADP + reductase. Trp676 in human cytochrome P450 reductase is conformationallymobile, andplays akey role in enzyme reduction. Mutagenesis of Trp676 to alanine results in a functional NADH-dependent reductase. . | Eur. J. Biochem. 271 2548-2560 2004 FEBS 2004 doi Thermodynamic and kinetic analysis of the isolated FAD domain of rat neuronal nitric oxide synthase altered in the region of the FAD shielding residue Phe1395 Adrian J. Dunford Ker R. Marshall Andrew W. Munro and Nigel S. Scrutton Department of Biochemistry University of Leicester UK In rat neuronal nitric oxide synthase Phe1395 is positioned over the FAD isoalloxazine ring. This is replaced by Trp676 in human cytochrome P450 reductase a tryptophan in related diflavin reductases . methionine synthase reductase and novel reductase 1 and tyrosine in plant ferredoxin-NADP reductase. Trp676 in human cytochrome P450 reductase is conformationally mobile and plays a key role in enzyme reduction. Mutagenesis of Trp676 to alanine results in a functional NADH-dependent reductase. Herein we describe studies of rat neuronal nitric oxide synthase FAD domains in which the aromatic shielding residue Phe1395 is replaced by tryptophan alanine and serine. In steady-state assays the F1395A and F1395S domains have a greater preference for NADH compared with F1395W and wildtype. Stopped-flow studies indicate flavin reduction by NADH is significantly faster with F1395S and F1395A domains suggesting that this contributes to altered preference in coenzyme specificity. Unlike cytochrome P450 reductase the switch in coenzyme specificity is not attributed to differential binding of NADPH and NADH but probably results from improved geometry for hydride transfer in the F1395S- and F1395A-NADH complexes. Potentiometry indicates that the substitutions do not significantly perturbthermodynamic properties of the FAD although considerable changes in electronic absorption properties are observed in oxidized F1395A and F1395S consistent with changes in hydrophobicity of the flavin environment. In wild-type and F1395W FAD domains prolonged incubation with NADPH results in development of the neutral blue semiquinone .

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