Phospholipid vesicle-reconstituted cytochrome P-450SCC. Mutually facilitated binding of cholesterol and adrenodoxin.

reduction of adrenodoxin promotes its dissociation from the reductase.Adrenodoxin is now shown to associate preferentially with cytochrome P-450,,, to which cholesterol is bound, to form an adrenodoxincytochrome P-450 *cholesterol ternary complex.T h e Kd for adrenodoxin binding to cholesterol-free cytochrome is 600 1 1 ~ compared with 30 nM for cholesterol-bound hemoprotein.To vary the binding of cholesterol to cytochrome, the cytochrome was reconstituted into liposomes of varying cholesterol/phospholipid ratios.In the absence of cholesterol, the preparation had a low spin optical spectrum; the fraction of hemoprotein in the high spin form was proportional to the cholesterol/ phospholipid ratio rather than to the total cholesterol concentration (calculated on the basis of the total aqueous volume).Adrenodoxin caused a further conversion to high spin when cholesterol was present in the membrane and a smaller conversion when cholesterol was absent; adrenodoxin-induced spin state changes occurred rapidly (<3 ms) when both hemoprotein and cholesterol were membrane-associated, suggesting that neither interaction of cholesterol nor of adrenodoxin with cytochrome P-450.,, is rate-limiting in the overall hydroxylation.The data indicate that cholesterol and adrenodoxin both bind preferentially to the same form of cytochrome P-450.,, and calculations show that the binding of either to cytochrome increases the binding of the other by a factor of -20.We suggest that this mechanism causes preferential interaction of adrenodoxin with cytochrome P-450 to which substrate is bound, and may function to prevent the hemoprotein from operating as a futile NADPH or adrenodoxin oxidase.Results are also consistent with previous findings (Seybert,