The oxyferro complex of adrenal cytochrome P-450scc. Effect of cholesterol and intermediates on its stability and optical characteristics.

The binding of O2 to reduced cytochrome P-450 is the initial step in the activation of oxygen; subsequent addition of a second electron leads to substrate hydroxylation. Without the second electron, the complex between reduced cytochrome and O2 (oxyferro) undergoes internal electron transfer to regenerate the oxidized enzyme and, presumably, superoxide. We have used 38% ethylene glycol and subzero temperatures to stabilize the oxyferro complex of cytochrome P-450scc and have examined the effect of cholesterol, and hydroxycholesterol intermediates in the conversion of cholesterol to pregnenolone, on the complex. The binding of cholesterol or the intermediates 20 alpha-hydroxycholesterol, 22R-hydroxycholesterol, and 20 alpha, 22R-dihydroxycholesterol to the cytochrome perturbed the optical spectra of the oxyferro complex with Soret maximums varying from 416 to 423 nm. Activation energies for the autooxidation of each of these sterol-oxyferro complexes were similar (approximately 22 kcal/mol). The half-time for autooxidation of the oxyferro complex was increased 15-fold by cholesterol over substrate-free cytochrome, and the hydroxycholesterols caused a further 3-17-fold increase in the stability of the oxyferro complex over that observed for cholesterol, the stability increasing with the number of hydroxyl groups on the cholesterol side chain. This was observed in both 38% ethylene glyco at -17 degrees C and dioleoyl phosphatidylcholine vesicles at 2 degrees C. The data indicate that the 1-electron-reduced-oxygenated complex of cytochrome P-450scc is kinetically stabilized by the binding of the reaction intermediates, preserving the complex for the arrival of the second electron.