Role of cysteine residues in human NADH-cytochrome b5 reductase studied by site-directed mutagenesis. Cys-273 and Cys-283 are located close to the NADH-binding site but are not catalytically essential.

摘要: Human NADH-cytochrome b5 reductase (EC 1.6.2.2) contains 4 cyteine residues (Cys-203, -273, -283, and -297). Cys-283 was previously proposed to be involved in NADH binding by chemical modification (Hackett, C. S., Novoa, W. B., Ozols, J., Strittmatter, P. (1986) J. Biol. Chem. 261, 9854-9857). In the present study role of cysteines enzyme probed replacing these Ser, Ala, or Gly employing site-directed mutagenesis modification. Four mutants, which 1 Cys replaced retained comparable kcat Km values those wild type. All mutants were as sensitive type treatment with SH modifiers, while a double mutant, C273S/C283S resistant. Since inhibition modifiers protected NADH, Cys-273 implicated close NADH-binding site. C273A C273A/C283A showed approximately one-fifth enzyme-FAD reduction rate revealed steady-state kinetics stopped-flow analysis. Anaerobic titration has shown that re-oxidation processes including formation red semiquinone not significantly altered from From results it concluded none are essential catalytic reaction, but conserved among enzymes homologous plays role(s) facilitating reaction. A difference spectrum peak at 317 nm, formerly considered derived interaction between NAD+ reduced enzyme, appeared upon only also mutant both site replaced.