Crystalline reduced nicotinamide adenine dinucleotide phosphate-adrenodoxin reductase from pig adrenocortical mitochondria. Essential histidyl and cysteinyl residues of the NADPH-binding site and environment of the adrenodoxin-binding site.
Pig NADPH-adrenodoxin reductase was crystallized from pig adrenocortical mitochondria and its physicochemical properties were investigated. Pig NADPH-adrenodoxin reductase is a typical flavoprotein. Its optical absorption spectrum showed peaks at 272, 377, and 450 nm in the oxidized form. The adrenodoxin reductase contained one FAD per mol. The molecular weight was 49,000. The isoelectric points of the adrenodoxin reductase and its complex with adrenodoxin were 5.3 and 4.6, respectively. Pig NADPH-adrenodoxin reductase, unlike bovine NADPH-adrenodoxin reductase, was found to be free of carbohydrate. The fluorescences of tryptophanyl residues and FAD of the adrenodoxin reductase were quenched by holo- and apo-adrenodoxins. The NADPH-binding site of the adrenodoxin reductase was examined by photooxidation and selective chemical modifications with diethyl pyrocarbonate and sulfhydryl reagents. The results indicate that a histidyl and a cysteinyl residue of the adrenodoxin reductase are essential for the NADPH-binding site. The circular dichroism spectrum of the adrenodoxin reductase showed negative ellipticity in the visible region. Spur formation was observed between pig and bovine NADPH-adrenodoxin reductases against the antibody to bovine NADPH-adrenodoxin reductase in Ouchterlony double-diffusion agar plates. The antibody did not interact with spinach ferredoxin-NADP+ reductase.
D000302:Adrenal Cortex / Q000201:enzymology*; D000325:Adrenodoxin / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003545:Cysteine; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D005287:Ferredoxin-NADP Reductase / Q000276:immunology / Q000378:metabolism*; D005288:Ferredoxins / Q000494:pharmacology; D006639:Histidine; D008928:Mitochondria / Q000201:enzymology*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013552:Swine
D005288:Ferredoxins; D013439:Sulfhydryl Reagents; D000325:Adrenodoxin; D006639:Histidine; D009249:NADP; D005287:Ferredoxin-NADP Reductase; D009247:NADH, NADPH Oxidoreductases; D003545:Cysteine; D004047:Diethyl Pyrocarbonate