Reactions of the Protein Radical in Peroxide-treated Myoglobin

TLDR

Reaction of horse myoglobin with H₂O₂ oxidizes the iron to a ferryl (Fe(IV)=O) state and generates a protein radical that is rapidly dissipated by poorly understood mechanisms. Amino‑acid analysis, terminal sequencing, and liquid secondary ion mass spectrometry reveal that the radical induces covalent attachment of the heme to the peptide YLE‑FISDAIIHVLHSK (residues 103‑118), primarily via the tyrosine residue. The study shows that up to 18 % of the heme becomes covalently bound to the protein, the heme‑peptide complex retains a chromophore similar to free heme, and cross‑linking occurs at tyrosine 103 to a meso‑carbon of the heme, independent of the vinyl groups.

Abstract

Reaction of horse myoglobin with H2O2 oxidizes the iron to the ferryl (Fe(IV) = O) state and produces a protein radical that is rapidly dissipated by poorly understood mechanisms. As reported here, the reaction with H2O2 results in covalent binding of up to 18% of the prosthetic heme group to the protein. The chromophore of the protein-bound prosthetic group is very similar to that of heme itself. High performance liquid chromatography of tryptic digests indicates that the formation of heme-bound peptides is associated with disappearance of the peptide with the sequence YLE-FISDAIIHVLHSK corresponding to residues 103-118 of horse myoglobin. Amino acid analysis, terminal amino acid sequencing, and liquid secondary ion mass spectrometry establish that the heme is primarily attached to this peptide. The heme appears to be bound to the tyrosine residue because the tyrosine is the only amino acid that disappears from the amino acid analysis. The mass spectrometric data indicates that the heme-peptide is formed without addition or loss of an oxygen or other major structural fragment. The site of attachment to the heme group has not been unambiguously determined, but the heme vinyl groups are not essential for the reaction because equal cross-linking is observed in H2O2-treated mesoheme-reconstituted myoglobin. The results are most consistent with binding of tyrosine 103 to a meso-carbon of the prosthetic heme group.