Stopped-flow spectrophotometric analysis of intermediates in the peroxo-dependent inactivation of cytochrome P450 by aldehydes

The reaction of hydrogen peroxide and certain aromatic aldehydes with cytoc hrome P450(BM3)-F87G results in the covalent modification of the heme cofac tor of this monooxygenase. Analysis of the resulting heme by electronic abs orption spectrophotometry indicates that the reaction in the BM3 isoform is analogous to that in P450(2B4), which apparently occurs via a peroxyhemiac etal intermediate [Kuo et al., Biochemistry, 38 (1999) 10511]. It was obser ved that replacement of the Phe-87 in the P450(BM3) by the smaller glycyl r esidue was essential for the modification to proceed, as the wild-type enzy me showed no spectral changes under identical conditions. The kinetics of t his reaction were examined by stopped-flow spectrophotometry with 3-phenylp ropionaldehyde and 3-phenylbutyraldehyde as reactants. In each case, the pr ocess of heme modification was biphasic, with initial bleaching of the Sore t absorbance, followed by an increase in absorbance centered at 430 nm, con sistent with meso-heme adduct formation. The intermediate formed during pha se I also showed an increased absorbance between 700 and 900 nm, relative t o the native heme and the final product. Phase I showed a linear dependence on peroxide concentration, whereas saturation kinetics were observed for p hase II. All of these observations are consistent with a mechanism involvin g radical attack at the gamma-meso position of the heme cofactor, resulting in the intermediate formation of an isoporphyrin, the deprotonation of whi ch produces the gamma-meso-alkyl heme derivative. (C) 2000 Elsevier Science S.A. All rights reserved.