SPECTRAL INTERMEDIATES OBSERVED IN THE CUMENE HYDROPEROXIDE-DEPENDENT HYDROXYLATION OF TOLUENE CATALYZED BY P-450LM2

Abstract

The mechanism of the P-450 LM 2 -catalyzed, cumene hydroperoxide-dependent hydroxylation of toluene has been studied by stopped flow spectrophotometry. Addition of cumene hydroperoxide to purified P-450 LM 2 in the absence of toluene caused a pseudo first-order change in the absorbance of the enzyme. Observation of changes at various wavelengths provided the difference and absolute spectra of the intermediate formed. Addition of both cumene hydroperoxide and toluene to P-450 LM 2 produced a type I spectral change in the dead time of the instrument, followed by a pseudo first-order change in the absorbance of the enzyme to give the same spectral intermediate. In these and other studies we have seen no evidence of a classical Compound I of the type seen with peroxidases. In contrast, with P-450 LM 2 the intermediate is formed reversibly and the spectrum varies with the organic moiety of the peroxy compound.