Beef adrenal cortical cytochrome P-450 which catalyzes the conversion of cholesterol to pregnenolone. Oxidation-reduction potentials of the free, steroid-complexed, and adrenodoxin-complexed P-450.

A cytochrome P-450 (P-450,), active in the removal of the side chain from cholesterol to form pregnenolone, was isolated from bovine adrenocortical mitochondria in a cholesterol-containing (P-450a,,(CH)) and a steroidfree (P-45OeC,(SF)) form with a specific heme content of 9 2 1 nmol/mg of protein.Oxidized P-45OaCc(CH) forms a 1:l complex with oxidized adrenodoxin, as monitored by an increase in the amount of high spin heme as well as a decrease in the reduction potential of the minority low spin heme species upon adrenodoxin binding.Data are presented which make unlikely the presence of a tightly bound ternary complex between adrenodoxin reductase, adrenodoxin, and P-45OS,,(CH), at least when all three are oxidized; the stoichiometry determined is 1:2:1, implying either a quaternary complex or two binary complexes.Reduction potentials were measured for both P-45OS,(CH) and P-45OS,,(SF) as isolated and as complexed to steroid substrate, intermediates, and product of the cholesterol desmolase reaction.P-450,,(SF) is predominantly low spin with a reduction potential of -412 a 2 mV for this low spin form.The addition of cholesterol causes P-45OS,,(SF) to become predominantly high spin with a reduction potential of -305 1 mV for this high spin form; both properties are the same as for the protein isolated with cholesterol bound to it, i.e.P-450,,(CH).The reduction potentials of P-450a,,(CH) bound to any two sequential intermediates in the side chain cleavage reaction are the same within experimental error.However, there. is a slight but significant increase (10 to 30 mV) in reduction potential of the P-45OS,, complex with the final intermediate 20,22-dihydroxycholesterol over that of the P-450,,, complex with the substrate cholesterol.The reduction potential of the P-450,,, complex with the product pregnenolone is -30 mV more negative than that of the P-450.,,complex with substrate.