Reaction of Cytochrome P450 with Cumene Hydroperoxide:  ESR Spin-Trapping Evidence for the Homolytic Scission of the Peroxide O−O Bond by Ferric Cytochrome P450 1A2

Abstract

ESR spin trapping was used to investigate the reaction of rabbit cytochrome P450 (P450) 1A2 with cumene hydroperoxide. Cumene hydroperoxide-derived peroxyl, alkoxyl, and carbon-centered radicals were formed and trapped during the reaction. The relative contributions of each radical adduct to the composite ESR spectrum were influenced by the concentration of the spin trap. Computer simulation of the experimental data obtained at various 5,5-dimethyl-1-pyrroline N-oxide (DMPO) concentrations was used to quantitate the contributions of each radical adduct to the composite ESR spectrum. The alkoxyl radical was the initial radical produced during the reaction. Experiments with 2-methyl-2-nitrosopropane identified the carbon-centered adducts as those of the methyl radical, hydroxymethyl radical, and a secondary carbon-centered radical. The reaction did not require NADPH-cytochrome P450 reductase or NADPH. It is concluded that the reaction involves the initial homolytic scission of the peroxide O-O bond to produce the cumoxyl radical. Methyl radicals were produced from the beta-scission of the cumoxyl radical. The peroxyl adduct was not observed in the absence of molecular oxygen. We conclude that the DMPO peroxyl radical adduct detected in the presence of oxygen was due to the methylperoxyl radical formed by the reaction of the methyl radical with oxygen. At a higher P450 concentration, a protein-derived radical adduct was also detected.

Functional Characterization of Human Cytochrome P450 2S1 Using a Synthetic Gene-Expressed Protein inEscherichia coli

“…Many P450s can use organic hydroperoxides to carry out oxidation of their substrates in the absence of cytochrome P450 reductase. This peroxide-dependent pathway is known as the "peroxide shunt" (Barr et al, 1996). Cumene hydroperoxide can support several P450-catalyzed reactions, including aromatic and aliphatic hydroxylation, N-or O-dealkylation, and alkene epoxidation (Nordblom et al, 1976;Griffin et al, 1980;Barr and Mason, 1995).…”

Oxyfunctionalization of unactivated C–H bonds in triterpenoids with tert-butylhydroperoxide catalyzed by meso-5,10,15,20-tetramesitylporphyrinate osmium(II) carbonyl complex

“…An ion intermediate is not involved (Barr et al, 1996;Trotta et al, 1983). In this study, particularly noteworthy was the predominant formation of oxo-derivatives by ketonization of an unactivated methylene C-H bond in the substrates 1, 3, and 5.…”

High warfarin sensitivity in carriers of CYP2C9*35 is determined by the impaired interaction with P450 oxidoreductase

“…21 To validate this hypothesis NADPH was replaced by CuOOH that can supply electrons to CYPs, circumventing POR ('peroxide shunt' 3 ). The metabolism of both substrates in the presence of CuOOH was monitored for 15 min as longer incubations led to a sharp decline of enzymatic activity (Figure 3), apparently due to a rapid heme inactivation by CuOOH.…”

“…2 This peroxidedependent pathway is known as a 'peroxide shunt'. 3 The CYP2C subfamily accounts for about 18% of total CYP proteins in human liver microsomes. 4 CYP2C9 is one of the most abundant CYP enzymes in the liver responsible for approximately 12.8% of the metabolism of clinically important drugs including warfarin, losartan and diclofenac.…”