On the mechanism of action of cytochrome P-450. Spectral intermediates in the reaction of P-450LM2 with peroxy compounds.

Abstract

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-450LM2 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 in equilibrium C in equilibrium D, where C represents a transient intermediate which is detected spectrally only under certain conditions and is probably an enzyme . oxidant complex, and D is the complex with an absorption maximum at about 436 nm in the difference spectrum. The absolute and difference spectra of C and D vary in magnitude and in the positions of maxima and minima with the organic moiety of the peroxy compound used. Whereas the kinetics of the reaction with cumene hydroperoxide and benzyl hydroperoxide is unchanged in the pH range 5.0 to 9.0, that of perbenzoic acids decreases markedly at higher pH, thus indicating that only the unionized compound reacts with the enzyme. Experiments with a variety of substituted cumene hydroperoxides, benzyl hydroperoxides, and perbenzoic acids indicated that the first equilibrium is driven to the right by hydrophobic bonding of the oxidant to P-450LM2 and that the rate of conversion of C to D is increased by electron-withdrawing substituents in the oxidant and decreased by electron donating substituents. Following the formation of Complex D, irreversible heme destruction occurs slowly. These results indicate that the reaction of liver microsomal P-450 with peroxides differs in two important respects from that of typical peroxidases; the intermediates arising from the reaction of P-450LM2 with peroxy compounds are formed reversibly, and the spectra of these intermediates vary with structural differences in the peroxy compounds. Furthermore, the absence of a common isosbestic point in the spectra observed with P-450LM2 rules out the possibility that they represent mixtures of Compounds I and II as reported for peroxidases.