title: Self-peroxidation of metmyoglobin results in formation of an oxygen-reactive tryptophan-centered radical.
abstract: 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.
authors.affiliation: Laboratory of Molecular Biophysics, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
authors.forename: M R
authors.initials: MR
authors.lastname: Gunther
authors.forename: D J
authors.initials: DJ
authors.lastname: Kelman
authors.forename: J T
authors.initials: JT
authors.lastname: Corbett
authors.forename: R P
authors.initials: RP
authors.lastname: Mason
mesh_terms: D000818:Animals; D001557:Benzenesulfonates; D003198:Computer Simulation; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008786:Metmyoglobin / Q000737:chemistry* / Q000187:drug effects; D008956:Models, Chemical; D009603:Nitroso Compounds; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D010545:Peroxides / Q000737:chemistry*; D013113:Spin Labels; D014364:Tryptophan / Q000737:chemistry* / Q000187:drug effects
chemical_list: D001557:Benzenesulfonates; D005609:Free Radicals; D009603:Nitroso Compounds; D010545:Peroxides; D013113:Spin Labels; D008786:Metmyoglobin; C049375:perhydroxyl radical; C048757:3,5-dibromo-4-nitrosobenzenesulfonate; D014364:Tryptophan; D006861:Hydrogen Peroxide; C034526:tert-nitrosobutane; D010100:Oxygen