The Mechanism of Action of Cytochrome P-450. Occurrence of the 'NIH Shift' during Hydroperoxide-Dependent Aromatic Hydroxylations

Abstract

The mechanism of liver microsomal aromatic hydroxylation was investigated by using cumene hydroperoxide as the hydroxylating agent and comparing this reaction with the NADPH-dependent reaction. The conversion of [4-3H]acetanilide to 4-hydroxyacetanilide by rat liver microsomes (or purified cytochrome P-450) in the presence of either cumene hydroperoxide or NADPH is attended by comparable NIH shifts [intramolecular migrations with concomitant retentions of aryl substituents]. This indicates that hydroxylation in the 2 systems proceeds via a common intermediate, presumably an arene oxide. The intermediacy of an arene oxide, phenanthrene-9,10-oxide, is established by incubating [3-3H]-phenanthrene with rat-liver microsomes and cumene hydroperoxide in the presence of either non-radioactive phenanthrene-9,10-oxide as a trap or in the presence of cyclohexene oxide, an inhibitor of the enzyme epoxide hydrase. Incubation of phenanthrene with cumene hydroperoxide in an 18O-enriched medium has confirmed that the O2 atom in phenanthrene-9,10-oxide is derived from the hydroperoxide and not from the medium.