SELF-PEROXIDATION OF METMYOGLOBIN RESULTS IN FORMATION OF AN OXYGEN-REACTIVE TRYPTOPHAN-CENTERED RADICAL

In the reaction between hydrogen peroxide and metmyoglobin, the heme i ron is oxidized to its ferryl-oxo form and the globin to protein radic als, at least one of which reacts with dioxygen to form a peroxyl radi cal. To identify the residue(s) that forms the oxygen-reactive radical , we utilized electron spin resonance (ESR) spectroscopy and the spin traps 2-methyl-2-nitrosopropane and 3,5-dibromo-4-nitrosobenzenesulfon ic acid (DBNBS), Metmyoglobin radical adducts had spectra typical of i mmobilized nitroxides that provided little structural information, but subsequent nonspecific protease treatment resulted in the detection o f isotropic three-line spectra, indicative of a radical adduct centere d on a tertiary carbon with no bonds to nitrogen or hydrogen. Similar isotropic three-line ESR spectra were obtained by spin trapping the ox idation product of tryptophan reacting with catalytic metmyoglobin and hydrogen peroxide. High resolution ESR spectra of DBNBS/(.)trp and of the protease-treated DBNBS/(.)metMb were simulated using superhyperfi ne coupling to a nitrogen and three non equivalent hydrogens, consiste nt with a radical adduct formed at C-3 of the indole ring. Oxidation o f tryptophan by catalytic metMb and hydrogen peroxide resulted in spin trap-inhibitable oxygen consumption, consistent with formation of a p eroxyl radical. The above results support self-peroxidation of a trypt ophan residue in the reaction between metMb and hydrogen peroxide.