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J. Biol. Chem., Vol. 261, Issue 10, 4562-4567, Apr, 1986

The role of the essential sulfhydryl group in assimilatory NADH: nitrate reductase of Chlorella

MJ Barber and LP Solomonson

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.
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