Cross-linking studies of adrenocortical cytochrome P-450scc. Evidence for a covalent complex with adrenodoxin and localization of the adrenodoxin-binding domain.
A cleavable cross-linking reagent, dimethyl-3,3'-dithiobispropionimidate, was used to study the molecular organization of adrenocortical cytochrome P-450scc. Extensive cross-linking was found to occur, resulting in the formation of heterologous oligomers up to octamer. The covalently cross-linked complex of adrenocortical cytochrome P-450scc with adrenodoxin has been obtained by using dimethyl-3,3'-dithiobispropionimidate. In the presence of NADPH and adrenodoxin reductase, electron transfer to cytochrome P-450scc occurs in the complex, and, in the presence of cholesterol, the latter effectively oxidizes to pregnenolone. By using covalently immobilized adrenodoxin and heterobifunctional reagent, N-succinimidyl-3-(2-pyridyldithio)propionate, the adrenodoxin-binding site was shown to be located in the heme-containing, catalytic domain of cytochrome P-450scc. The data obtained indicate the existence of two different sites on the adrenodoxin molecule that are responsible for the interaction with adrenodoxin reductase and cytochrome P-450scc. This is consistent with the model mechanism of electron transfer in the organized complex.
D000302:Adrenal Cortex / Q000201:enzymology*; D000325:Adrenodoxin / Q000032:analysis*; D000818:Animals; D001665:Binding Sites; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000032:analysis*; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis*; D004579:Electron Transport; D007096:Imidoesters / Q000494:pharmacology; D008970:Molecular Weight; D010088:Oxidoreductases / Q000032:analysis*; D011817:Rabbits
D003432:Cross-Linking Reagents; D007096:Imidoesters; D000325:Adrenodoxin; C007471:dimethyl dithiobispropionimidate; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D002786:Cholesterol Side-Chain Cleavage Enzyme