Identification of the essential cysteine residue of NADH-cytochrome b5 reductase.
The soluble catalytic domain of NADH-cytochrome b5 reductase was radiolabeled with [14C]N-ethylmaleimide. Reaction for a limited time resulted in incorporation of 0.41 eq of N-ethylmaleimide and loss of 36% of the enzyme activity. Chromatography on a 5'-ADP affinity column separated the reductase which was modified with N-ethylmaleimide from the unreacted enzyme; the isolated derivative constituted 37% of the total material, was completely inactivated, and contained 1.00 eq of N-ethylmaleimide. Cyanogen bromide cleavage of the derivative demonstrated that radioactivity was limited to a single peptide which contained both Cys-283 and Cys-297. Tryptic hydrolysis of this cyanogen bromide peptide showed that the radioactivity was associated with Cys-283. Automated sequenator analysis confirmed that Cys-283 was the radiolabeled residue. These data demonstrate unambiguously that Cys-283 provides the essential thiol group of cytochrome b5 reductase.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide / Q000494:pharmacology; D003545:Cysteine / Q000032:analysis*; D003579:Cytochrome Reductases / Q000032:analysis*; D042966:Cytochrome-B(5) Reductase; D005033:Ethylmaleimide / Q000494:pharmacology; D008099:Liver / Q000201:enzymology; D008297:Male; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism
D000596:Amino Acids; D009247:NADH, NADPH Oxidoreductases; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase; C014227:ferricyanide reductase; D003545:Cysteine; D005033:Ethylmaleimide; D003488:Cyanogen Bromide