Glu318 and Thr319 mutations of cytochrome P450 1A2 remarkably enhance homolytic O-O cleavage of alkyl hydroperoxides. An optical absorption spectral study.

Interactions of putative distal mutants of cytochrome P450 1A2 (P450 1A2) with hydroperoxides were studied with optical absorption spectroscopy. The Soret absorption at 393 nm of the wild-type P450 1A2 decreased by adding H2O2, tert-butyl hydroperoxide (TBHP), and cumyl hydroperoxide (CHP), whereas the absorption decrease was accompanied by the appearance of a new band around 423 nm for Glu318 and Thr319 mutants with similar treatments. Spectral simulation based on Gaussian analyses and visible bands indicate that H2O2- and TBHP-induced intermediates' spectra of the wild type are close to that of Compound I of horseradish peroxidase, whereas TBHP- and CHP-induced intermediates' spectra of the mutants are close to that of Compound II of horseradish peroxidase. Based on kinetic parameters of the spectral changes, it is suggested that: 1) TBHP and CHP mainly form porphyrin+.FeIV = O due to heterolytic O-O scission in the wild type, while forming porphyrinFeIV = O for the mutants owing to homolytic O-O scission; and 2) heterolytic O-O scission of H2O2 is not changed by the mutations. Heterolytic/homolytic cleavage ratios for the P450 1A2 reactions with CHP obtained from product analysis were consistent with those obtained from spectral changes.