Oxidase and oxygenase function of the microsomal cytochrome P450 monooxygenase system.
The rates of the NADPH-dependent formation of superoxide radicals and hydrogen peroxide have been measured in liver microsomes from phenobarbital-pretreated rats. Correcting a quenching of O2(-) radicals by microsomes, a stoichiometry of O2(-) to H2O2 close to 2:1 was obtained. This, and the fact that pseudo-substrates of microsomal cytochrome P450 like perfluoro-n-hexane and perfluorinated cyclohexane did not increase H2O2 formation in a catalase-inhibited assay, rules out a two-electron reduced oxygen species as the source of H2O2. The rates of O2(-) as well as H2O2 generation in the presence of 7-ethoxycoumarin were equally inhibited by carbon monoxide (75%) and resulted in photochemical action spectra with a maximum reactivation at 450 nm. Using the same conditions the monooxygenation was inhibited to a high degree (83%) but without exogenous substrate the inhibition of H2O2 formation dropped to 55%. It was concluded that most of the O2(-) originated from the oxycomplex of cytochrome P450 and that substrates can modify the rates of its decomposition and sensitivity to carbon monoxide. No correlation of H2O2 formation or of substrate monooxygenation with the optical substrate binding spectra could be observed. From the pH dependence a proton-assisted decomposition of oxy-cytochrome P450 appears likely. H2O2 formation was only slightly decreased at 20 microM dioxygen suggesting that H2O2 formation via cytochrome P450 should also occur in vivo.
D000818:Animals; D002248:Carbon Monoxide / Q000494:pharmacology; D055598:Chemical Phenomena; D002621:Chemistry; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification*; D006861:Hydrogen Peroxide / Q000096:biosynthesis; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D009243:NAD / Q000494:pharmacology; D010088:Oxidoreductases / Q000502:physiology*; D010101:Oxygen Consumption; D010105:Oxygenases / Q000502:physiology*; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D009243:NAD; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D006861:Hydrogen Peroxide; D010088:Oxidoreductases; D010105:Oxygenases