The protein-centered radicals formed in the reaction between hydrogen peroxide and metmyoglobin have been studied since the 1950s without a specific localization of the site(s) of protein modification. In work completed in 1995 we provided evidence for the formation of a peroxyl radical centered on one of the two conserved tryptophan residues of mammalian myoglobins. In a collaboration with the group of Paul R. Ortiz de Montellano at UCSF, we obtained recombinant sperm whale metmyoglobin labelled at the tryptophan residues with carbon 13. As predicted, the electron spin resonance (ESR) spectrum obtained from the labelled protein contained additional lines compared to that from the native protein, consistent with the peroxyl radical being centered on C-3 of the indole ring of a tryptophan residue (the site of the label). Site-directed mutant sperm whale myoglobins were prepared in which tryptophan 7, tryptophan 14, or both were replaced with phenylalanine residues. The ESR spectra of the tryptophan 7 mutant but not those of mutant proteins in which tryptophan 14 was replaced by phenylalanine were characteristic of a peroxyl radical, indicating that the site of peroxyl radical formation is tryptophan 14. We are continuing the research into metmyoglobin radicals using the site-directed mutant proteins to investigate whether the site of peroxyl radical formation is the same as that of reaction with spin-traps.
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