Peroxidation of a Specific Tryptophan of Metmyoglobin by Hydrogen Peroxide

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.

Human Indoleamine 2,3-Dioxygenase Is a Catalyst of Physiological Heme Peroxidase Reactions

“…It is also plausible that the initial reaction involves the reaction of L-Trp with an IDO protein radical (77). Upon formation, the neutral tryptophanyl radical is known to react with O 2 to form a peroxyl radical (75,77,78), which upon forming a hydroperoxide can rearrange via yet to be characterized chemistry into N-formylkynurenine that hydrolyzes into kynurenine (79,80). Further evidence for compound I-mediated oxidation of L-Trp is the observation that tempol and ascorbate both inhibited H 2 O 2 -induced oxidation of the amino acid by IDO (Fig.…”

H2O2-mediated Cross-linking between Lactoperoxidase and Myoglobin

“…Tyr-146, the third tyrosine in the protein, forms a radical, but it may not participate in chemical reactions (13). Of the two tryptophan residues, Trp-7 and Trp-14, only Trp-14 appears to be involved in the formation of a protein-peroxy radical that can co-oxidize styrene (13,19). Evidence has also been obtained for the formation of a radical located on Cys-110 (20), which is unique to human myoglobin, and on an aliphatic MetMb amino acid residue (21).…”

“…Preparation and Expression of Site-directed Protein Mutants-The mutant SwMb proteins and wild-type recombinant rLPO were expressed and purified as described previously (12,13,19,28). Recombinant SwMb proteins were oxidized to the met form using a slight excess of K 3 Fe(CN) 6 and then passed over a PD-10 column eluted with 50 mM potassium phosphate buffer, pH 6.8.…”

“…The early observation by EPR of a protein radical in the reaction of MetMb with H 2 O 2 has been more precisely defined by recent work (12,19). Site-specific mutagenesis, in conjunction with protein digestion, peptide sequencing, mass spectrometry, and EPR spectroscopy, has established that MetMb cross-linking involves the formation of Tyr-103 and Tyr-151 dityrosine cross-links (10,12,13).…”

Cardiovascular Redox and Ox Stress Proteomics

“…The former group includes the direct attack by a hydroxyl radical on glutamate residues, which converts the amino acid into pyruvic acid and ammonia or a reaction of the hydroxyl radical with a proline residue, forming aminobutyric acid and carbon dioxide (CO 2 ). Also included in this category are the initial amino-acid side-chain modifications such as aldehyde production, which transfers the radical to the protein backbone via alkoxyl radicals (62,77,118,119,199,202,221,312). Meanwhile, the latter includes direct oxidation of the amino-acid side chains as well as lipid and sugar oxidation by-products that form adducts with the side chains.…”