title: Glu318 and Thr319 mutations of cytochrome P450 1A2 remarkably enhance homolytic O-O cleavage of alkyl hydroperoxides. An optical absorption spectral study.
abstract: 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.
authors.affiliation: Institute for Chemical Reaction Science, Tohoku University, Sendai, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Shimizu
authors.forename: Y
authors.initials: Y
authors.lastname: Murakami
authors.forename: M
authors.initials: M
authors.lastname: Hatano
mesh_terms: D000438:Alcohols / Q000378:metabolism; D001555:Benzene Derivatives / Q000378:metabolism; D019388:Cytochrome P-450 CYP1A2; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D005971:Glutamates / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid; D006861:Hydrogen Peroxide / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010545:Peroxides / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013057:Spectrum Analysis; D013912:Threonine / Q000235:genetics / Q000378:metabolism*; D020122:tert-Butylhydroperoxide
chemical_list: D000438:Alcohols; D001555:Benzene Derivatives; D005971:Glutamates; D010545:Peroxides; D013912:Threonine; D018698:Glutamic Acid; D003577:Cytochrome P-450 Enzyme System; D020122:tert-Butylhydroperoxide; D006861:Hydrogen Peroxide; D010088:Oxidoreductases; D019388:Cytochrome P-450 CYP1A2; C007164:cumene hydroperoxide