Hydroperoxysterols as a probe for the mechanism of cytochrome P-450scc-mediated hydroxylation. Homolytic versus heterolytic oxygen-oxygen bond scission.

The interaction of 20-, 23-, and 25-hydroperoxy derivatives of cholesterol with various heme proteins, including the cholesterol side-chain-cleaving enzyme, cytochrome P-450scc, was studied by means of product and spectral analyses. Quasi-Fenton homolytic decomposition via intermediate alkoxy radicals appears to prevail during nonspecific interaction. Highly stereospecific hydroperoxide-driven hydroxylations suggest the absence of free radical species and are interpreted as resulting from a heterolytic type of peroxide decomposition or, alternatively, homolytic decomposition assuming proximal base effect to stabilize a putative intermediate alkoxy radical. Spectral aberrations during the early stages of the latter interaction indicate formation of a ternary iron-peroxo-substrate complex. Decomposition of this complex results in multiple product formation suggesting that peroxide cleavage reverts to regular homolytic decomposition upon denaturation of the enzyme. The implications of these observations for the mechanism of hydroxylation and oxidative carbon-carbon bond scission during enzymic side-chain cleavage of cholesterol are discussed.