The role of the essential sulfhydryl group in assimilatory NADH: nitrate reductase of Chlorella.
Incubation of the complex metalloflavoprotein, assimilatory nitrate reductase with N-ethylmaleimide, or a spin-labeled analog, 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl, resulted in a time-dependent inactivation of NADH:nitrate reductase and NADH: cytochrome-c reductase activity with no effect on reduced methyl viologen:nitrate reductase activity. Inactivation of the enzyme, which could be prevented by incubation in the presence of NADH, was achieved following modification of a single sulfhydryl group determined from [3H]N-ethylmaleimide incorporation and quantitation of the EPR spectrum of the spin-labeled enzyme. Sulfhydryl group modification precluded reduction of the enzyme by NADH and NAD+ binding. The EPR spectrum of the spin-labeled enzyme revealed the presence of a single species with the nitroxide retaining substantial motional freedom. Cleavage of the spin-labeled enzyme using corn-inactivating protease and separation into its flavin and molybdenum/heme domains followed by EPR spectroscopy revealed the modified sulfhydryl group to be associated with the latter fragment suggesting a close interaction of these domains in the region of the nucleotide-binding site.
D002708:Chlorella / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D050895:Nitrate Reductase (NADH); D009565:Nitrate Reductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D013816:Thermodynamics
D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D009565:Nitrate Reductases; D050895:Nitrate Reductase (NADH)