On the mechanism of action of cytochrome P-450. Role of peroxy spectral intermediates in substrate hydroxylation.

Abstract

Cytochrome P-450 functions as an oxygenase in 0 2 and NADPH-dependent substrate hydroxylations and also as a peroxygenase in a variety of peroxide-dependent substrate hydroxylation reactions. As shown previously, peroxy compounds react with the phenobarbital-induced isozyme of liver microsomal cytochrome P-450 to yield an intermediate (Complex D) with an absorption maximum at about 436 nm in the difference spectrum; a transient intermediate (Complex C) is also detected spectrally but only under certain conditions. The results are in accord with a reversible two-step mechanism, P-450 + oxidant e C e D, which differs from that of peroxidases in several important respects. In the present study, the effect of toluene as a hydroxylatable substrate on the kinetics was determined. The overall reaction is as follows: toluene + cumene hydroperoxide + benzyl alcohol + cumyl alcohol. Both the rate and amplitude of absorbance changes indicative of Complex D formation were shown to be functions of the toluene concentration, whereas only the amplitude is affected by perfluorocyclohexane, a pseudosubstrate. From these results it was concluded that the change in amplitude is due to binding of the hydroxylatable or pseudosubstrate and that the effect on the rate constant is due to the hydroxylation reaction. Reaction schemes in which either Complex C o r D yields free P-450 as well as benzyl alcohol and cumyl alcohol were evaluated by computer simulation and compared with the data obtained on the toluene-dependent spectral perturbation of the system containing P-450 and cumene hydroperoxide as well as on benzyl alcohol formation. Our findings indicate that Complex C is directly involved in the hydroxylation pathway and that Complex D is produced in a nonproductive side reaction.