Detection of peroxyl and alkoyl radicals produced by reaction of hydroperoxides with heme-proteins by electron spin resonance spectroscopy

Abstract

ESR spin trapping using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) has been used to directly detect alkoxyl radicals (with hyperfine coupling constants a N 1.488, a H 1.600 mT and a N 1.488, a H 1.504 mT for the t BuO . and PhC(CH 3) 2O . adducts, respectively) and peroxyl radicals ( a N 1.448, a H 1.088, a H 0.130 mT and a H 1.456, a H 1.064, a H 0.128 mT for the t BuO . and PhC(CH 3) 2O . adducts, respectively) produced from t-butyl or cumene hydroperoxidases by a variety of heme-containing substances (purified cytochrome P-450, metmyoglobin, oxyhemoglobin, methemoglobin, cytochrome c, catalase, horseradish peroxidase) and the model compound hematin. The observed species exhibit a complicated dependence on reagent concentrations and time, with maximum concentrations of the peroxyl radical adducts being observed immediately after mixing of the hydroperoxide with low concentrations of the heme-compound. Experiments with inhibitors (CN −, N 3 −, CO, metyrapone and imidazole) suggest that the major mechanism of peroxyl radical production involves high-valence-state iron complexes in a reaction analogous to the classical peroxide pathway. The production of alkoxyl radicals is shown to arise mainly from the breakdown of peroxyl radical spin adducts, with direct production from the hydroperoxide being a relatively minor process.