ON THE MECHANISM OF ACTION OF CYTOCHROME-P450 - EVALUATION OF HYDROGEN ABSTRACTION IN OXYGEN-DEPENDENT ALCOHOL OXIDATION

The mechanism of oxidation of primary and secondary benzylic alcohols to the corresponding carbonyl compounds by purified rabbit liver cytochrome P450 forms 2B4 and 2E1 in a reconstituted enzyme system has been examined by linear free energy relationships, intramolecular and steady-state deuterium isotope effects, and the incorporation of an O-2-derived oxygen atom or solvent-derived deuterium. The k(cat) and K-m values were found to be relatively insensitive to the presence of electronic perturbations at the para position. The Hammett reaction constants for the oxidation of benzyl alcohols by P450s 2B4 and 2E1 are -0.46 and -0.37, respectively, and with 1-phenylethyl alcohols the corresponding reaction constants are -1.41 and -1.19, respectively. With [1-H-2(1)]benzyl alcohol, P450s 2B4 and 2E1 show similar intramolecular deuterium isotope effects of 2.6 and 2.8, respectively, whereas with [1-H-2(2)]benzyl alcohol under steady-state conditions, the deuterium isotope effects on the catalytic constants are 2.8 and 1.3, respectively. No significant isotope effect on the catalytic constant was noted for either form of P450 with 1-phenylethyl alcohol. In D2O, acetophenone formed by either form of P450 from 1-phenylethyl alcohol does not contain a deuterium atom at the methyl group, whereas under an atmosphere of O-18(2) approximately 30% of the labeled oxygen is incorporated into the carbonyl group with either form of the cytochrome. The results are consistent with a mechanism that involves stepwise oxidation of the alcohol to a carbon radical alpha to the alcohol function, followed by oxygen rebound to yield the gem-diol, dehydration of which gives the carbonyl product. However, the rate-determining step is dependent on the alcohol substrate and the form of cytochrome P450 that is examined. Carbon-hydrogen bond cleavage in benzyl alcohol is clearly rate-limiting with P450 2B4 and partially rate-limiting with P450 2E1, whereas in 1-phenylethyl alcohol this step-is not rate-limiting with either cytochrome.