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Peroxidation of a specific tryptophan of metmyoglobin by hydrogen peroxide
J A DeGray1, M R Gunther, R Tschirret-Guth
1Laboratory of Pharmacology and Chemistry, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Abstract:
Globin-centered radicals at tyrosine and tryptophan residues and a peroxyl radical at an unknown location have been reported previously as products of the reaction of metmyoglobin with hydrogen peroxide. The peroxyl radical is shown here to be localized on tryptophan through the use of recombinant sperm whale myoglobin labeled with 13C at the indole ring C-3. Peroxyl radical formation was not prevented by site-directed mutations that replaced all three tyrosines, the distal histidine, or tryptophan 7 with non-oxidizable residues. In contrast, mutation of tryptophan 14 prevents peroxyl radical formation, implicating tryptophan 14 as the specific site of the peroxidation.
Insights
Hydrogen peroxide reactions with metmyoglobin create radicals. This study identifies tryptophan 14 as the specific site of peroxyl radical formation in metmyoglobin.
Area of Science:
- Biochemistry
- Chemical Biology
- Protein Chemistry
Background:
- Metmyoglobin reacts with hydrogen peroxide, forming globin-centered radicals at tyrosine and tryptophan residues.
- A peroxyl radical has been previously detected but its location remained unknown.
Purpose of the Study:
- To identify the specific location of the peroxyl radical formed during the reaction of metmyoglobin with hydrogen peroxide.
- To elucidate the role of specific amino acid residues in peroxyl radical formation.
Main Methods:
- Utilized recombinant sperm whale myoglobin labeled with 13C at the indole ring C-3 for precise radical localization.
- Employed site-directed mutagenesis to replace key residues (tyrosines, distal histidine, tryptophans) with non-oxidizable alternatives.
- Analyzed the impact of these mutations on peroxyl radical formation.
Main Results:
- The peroxyl radical was localized to a tryptophan residue.
- Mutations in tyrosines, distal histidine, or tryptophan 7 did not prevent peroxyl radical formation.
- Mutation of tryptophan 14 specifically abolished peroxyl radical formation.
Conclusions:
- Tryptophan 14 is the precise site of peroxidation in metmyoglobin upon reaction with hydrogen peroxide.
- This finding clarifies the radical chemistry of metmyoglobin oxidation and provides insights into protein radical stability.