The reaction between metmyoglobin and hydrogen peroxide produces both a ferryl-oxo heme and a globin-centred radical(s) from the two oxidizing equivalents of the hydrogen peroxide. Evidence has been presented for localization of the globin-centred radical on one tryptophan residue and tyrosines 103 and 151. When the spin-trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO) is included in the reaction mixture, a radical adduct has been detected, but the residue at which that adduct is formed has not been determined. Replacement of either tryptophans 7 and 14 or tyrosines 146 and 151 with phenylalanine has no effect on the formation of DMPO adduct in the reaction with hydrogen peroxide. When tyrosine 103 is replaced with phenylalanine, however, only DMPOX, a product of the oxidation of the spin-trap, is detected. Tyrosine-103 is, therefore, the site of radical adduct formation with DMPO. The spin trap 2-methyl-2-nitrosopropane (MNP), however, forms radical adducts with any recombinant sperm whale metmyoglobin that contains either tyrosine 103 or 151. Detailed spectral analysis of the DMPO and MNP radical adducts of isotopically substituted tyrosine radical yield complete structural determinations. The multiple sites of trapping support a model in which the unpaired electron density is spread over a number of residues in the population of metmyoglobin molecules, at least some of which are in equilibrium with each other.

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.