Self-peroxidation of metmyoglobin results in formation of an oxygen-reactive tryptophan-centered radical.

In the reaction between hydrogen peroxide and metmyoglobin, the heme iron is oxidized to its ferryl-oxo form and the globin to protein radicals, at least one of which reacts with dioxygen to form a peroxyl radical. 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-nitrosobenzenesulfonic acid (DB-NBS). Metmyoglobin radical adducts had spectra typical of immobilized nitroxides that provided little structural information, but subsequent nonspecific protease treatment resulted in the detection of isotropic three-line spectra, indicative of a radical adduct centered on a tertiary carbon with no bonds to nitrogen or hydrogen. Similar isotropic three-line ESR spectra were obtained by spin trapping the oxidation 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 superhyperfine coupling to a nitrogen and three non-equivalent hydrogens, consistent with a radical adduct formed at C-3 of the indole ring. Oxidation of tryptophan by catalytic metMb and hydrogen peroxide resulted in spin trap-inhibitable oxygen consumption, consistent with formation of a peroxyl radical. The above results support self-peroxidation of a tryptophan residue in the reaction between metMb and hydrogen peroxide.