OxyFile #215

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

Author:   Gunther MR; Kelman DJ; Corbett JT; Mason RP

Source:   J Biol Chem 1995 Jul 7; 270(27):16075-81


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.