The catalytic cycle of cytochrome P-450scc and intermediates in the conversion of cholesterol to pregnenolone

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Abstract
Cytochrome P-450scc as isolated is a cholesterol-depleted low-spin haemoprotein; addition of cholesterol results in formation of a high-spin complex. Cytochrome P-450scc-cholesterol is a one-electron acceptor on titration with NADPH. Cytochrome P-450scc-cholesterol can be anaerobically reduced to the ferrous state which, on oxygenation, forms an oxygenated cytochrome P-450scc-cholesterol complex. This oxygenated complex in the absence of adrenodoxin autoxidises to ferric cytochrome P-450scc-cholesterol without oxidation of cholesterol. The decay of the oxygenated complex is first-order, k= 9.3 × 10−3s−1 at 4°C. The rate of autoxidation is influenced by pH, ionic strength and the chemical nature of bound sterol. The activation energy of autoxidation is 75 kJ mol−1. Addition of equimolar amounts of adrenodoxin to cytochrome P-450scc-cholesterol followed by stoichiometric reduction under anaerobic conditions and subsequent oxygenation, allows single catalytic turnover cycles of cytochrome P-450scc to be observed. This has led to detection of intermediates in the conversion of cholesterol to pregnenolone and a precusor/product sequence of cholesterol → 22-hydroxycholesterol → 20,22-dihydroxycholesterol → pregnenolone has been established. Addition of oxidised adrenodoxin to oxygenated cytochrome P-450scc-cholesterol results in formation of 22-hydroxycholesterol.