Properties of an adrenal cytochrome P-450 ( P-450 scc) for the side chain cleavage of cholesterol

Abstract

A highly purified (12 nmol of P-450-heme per milligram of protein) bovine adrenal cortex mitochondrial cytochrome P-450, termed P-450 sce, which cleaves the side chain of cholesterol to yield pregnenolone, is obtained in the substrate-bound ferric form with observed absorption maxima at 393 nm and 645 nm and a shoulder around 540 nm. The absorption spectra of the P-450 scc, whether in the substrate-bound ferric form or in the CO-complexed ferrous form, are subject to environmental perturbation. The addition of adrenal ferredoxin readily restores full ferric high spin type spectrum of the substrate-bound P-450 scc or, together with cholesterol and Tween 20, restores the CO-spectrum of the P-450 scc, exhibiting stable and typical spectra of cytochrome P-450. Tween 20, at concentration of 0.3%, remarkably increases the P-450 scc-catalyzed cholesterol side chain cleavage activity. Based on these findings, a highly reactive and reliable assay has been developed for the conversion of cholesterol to pregnenolone. The specific activity of the P-450 scc, thus determined in the presence of NADPH, NADPH:adrenal ferredoxin oxidoreductase (EC 1.6.7.1), adrenal ferredoxin, cholesterol, and molecular oxygen, is 16 mol of pregnenolone formed per minute per mole of P-450-heme and V of enzyme catalyzed reaction was 30 mol/min/mol of P-450-heme. Apparent K m values are 120 μ m for cholesterol and 1.5 μ m for adrenal ferredoxin. The P-450 scc has a pH optimum at pH 7.2 and is most active at ionic strength of 0.1.