Properties of crystalline reduced nicotinamide adenine dinucleotide phosphate-adrenodoxin reductase from bovine adrenocortical mitochondria. II. Essential histidyl and cysteinyl residues at the NADPH binding site of NADPH-adrenodoxin reductase.
The binding site of NADPH in NADPH-adrenodoxin reductase was examined using crystalline enzyme from bovine adrenocortical mitochondria by studies on the effects of photooxidation and chemical modifications of amino acid residues in the reductase. (1) Photoxication decreased the enzymatic activity of NADPH-adrenodoxin reductase. Photooxidation of the reductase was prevented by NADP+, adrenodoxin, or reduced glutathione, but not NAD+. Photoinactivation caused loss of a histidyl residue, but not of tyrosyl, tryptophanyl, cysteinyl, or methionyl residues of the reductase. It did not affect the circular dichroism spectrum of the reductase appreciably. (2) NADPH-adrenodoxin reductase activity was inhibited by diethyl pyrocarbonate and the inhibition was partially reversed by addition of hydroxylamine. The inhibition was prevented by NADP+, but not NAD+. (3) NADPH-adrenodoxin reductase activity was inhibited by 5,5'-dithiobis(2-nitrobenzoate) and the inhibition was reversed by reduced glutathione. It was also protected by NADP+, but not NAD+. The results indicate that a histidyl residue and a cysteinyl residue of NADPH-adrenodoxin reductase are essential for the binding of NADPH by the reductase.
D000302:Adrenal Cortex / Q000201:enzymology*; D000311:Adrenal Glands / Q000201:enzymology*; D000325:Adrenodoxin / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003545:Cysteine; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism* / Q000528:radiation effects; D005978:Glutathione / Q000494:pharmacology; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D006898:Hydroxylamines / Q000494:pharmacology; D008027:Light; D008751:Methylene Blue; D008928:Mitochondria / Q000201:enzymology; D009243:NAD / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP / Q000494:pharmacology*
D006898:Hydroxylamines; D009243:NAD; D000325:Adrenodoxin; D006639:Histidine; D009249:NADP; D005287:Ferredoxin-NADP Reductase; D009247:NADH, NADPH Oxidoreductases; D005978:Glutathione; D003545:Cysteine; D004047:Diethyl Pyrocarbonate; D008751:Methylene Blue