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

ESR spin trapping was used to investigate the reaction of rabbit cytoc hrome 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 radic al adduct to the composite ESR spectrum were influenced by the concent ration of the spin trap. Computer simulation of the experimental data obtained at various 5,5-dimethyl-1-pyrroline N-oxide (DMPO) concentrat ions was used to quantitate the contributions of each radical adduct t o the composite ESR spectrum. The alkoxyl radical was the initial radi cal produced during the reaction. Experiments with 2-methyl-2-nitrosop ropane identified the carbon-centered adducts as those of the methyl r adical, hydroxymethyl radical, and a secondary carbon-centered radical . The reaction did not require NADPH-cytochrome P450 reductase or NADP H. It is concluded that the reaction involves the initial homolytic sc ission 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 oxyge n. We conclude that the DMPO peroxyl radical adduct detected in the pr esence of oxygen was due to the methylperoxyl radical formed by the re action of the methyl radical with oxygen. At a higher P450 concentrati on, a protein-derived radical adduct was also detected.