On the Mechanism of Action of Cytochrome

In previous studies in this laboratory, highly purified liver microsomal cytochrome P-450 was shown to catalyze the hydroperoxide-dependent hydroxylation of a variety of substrates in the absence of NADPH, NADPH-cytochrome P-450 reductase, and molecular oxygen, and evidence was obtained that the oxygen atom in the product was derived from the peroxide. To determine whether the cytochrome functions in such reactions by a peroxidase-type mechanism, the kinetics of its interactions with a variety of substituted hydroperoxides and peroxy acids have been determined by stopped flow spectrophotometry. The reaction of P-450m, with various peroxy compounds yields an intermediate with an absorption maximum at about 436 nm in the difference spectrum, with pseudo-first order or biphasic kinetics depending upon the individual rate constants and the concentration of the oxidant used. The results are in accord with a reversible two-step mechanism, as follows: P-450 + oxidant e C + D,