Discrete Species of Activated Oxygen Yield Different Cytochrome P450 Heme Adducts from Aldehydes

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Abstract
Aldehydes are known to inactivate cytochrome P450 in the reconstituted enzyme system containing NADPH and NADPH−cytochrome P450 reductase under aerobic conditions in a mechanism-based reaction involving heme adduct formation [Raner, G. M., Chiang, E. W., Vaz, A. D. N., and Coon, M. J. (1997) Biochemistry 36, 4895−4902]. In the study presented here, artificial oxidants were used to examine the mechanism of aldehyde activation by purified P450 2B4 in the absence of the usual O2-reducing system, and the adducts that were formed were isolated and characterized. With hydrogen peroxide as the oxidant, 3-phenylpropionaldehyde gives an adduct with a mass corresponding to that of native heme modified by a phenylethyl group, presumably arising from the reaction of a peroxy−iron species with the aldehyde to give a peroxyhemiacetal, which upon deformylation yields the alkyl radical. NMR analysis indicated that the substitution is specifically at the γ-meso position. In contrast, with m-chloroperbenzoic acid as the oxidant, an adduct is formed from 3-phenylpropionaldehyde with a mass that is consistent with the addition of a phenylpropionyl group, apparently arising by hydrogen abstraction from the aldehyde to give the carbonyl carbon radical. m-Chloroperbenzoic acid by itself forms a heme adduct with a mass corresponding to the addition of a chlorobenzoyloxy group apparently derived from homolytic oxygen−oxygen bond cleavage. These and other results with nonanal and 2-trans-nonenal support the concept that this versatile enzyme utilizes discrete oxidizing species in heme adduct formation from aldehydes.