Reversible Oxidative Modifications in Myoglobin and Functional Implications

Abstract

Myoglobin (Mb), an oxygen-binding heme protein highly expressed in heart and skeletal muscle, has been shown to undergo oxidative modifications on both an inter- and intramolecular level when exposed to hydrogen peroxide (H2O2) in vitro. Here, we show that exposure to H2O2 increases the peroxidase activity of Mb. Reaction of Mb with H2O2 causes covalent binding of heme to the Mb protein (Mb-X), corresponding to an increase in peroxidase activity when ascorbic acid is the reducing co-substrate. Treatment of H2O2-reacted Mb with ascorbic acid reverses the Mb-X crosslink. Reaction with H2O2 causes Mb to form dimers, trimers, and larger molecular weight Mb aggregates, and treatment with ascorbic acid regenerates Mb monomers. Reaction of Mb with H2O2 causes formation of dityrosine crosslinks, though the labile nature of the crosslinks broken by treatment with ascorbic acid suggests that the reversible aggregation of Mb is mediated by crosslinks other than dityrosine. Disappearance of a peptide containing a tryptophan residue when Mb is treated with H2O2 and the peptide’s reappearance after subsequent treatment with ascorbic acid suggest that tryptophan side chains might participate in the labile crosslinking. Taken together, these data suggest that while exposure to H2O2 causes Mb-X formation, increases Mb peroxidase activity, and causes Mb aggregation, these oxidative modifications are reversible by treatment with ascorbic acid. A caveat is that future studies should demonstrate that these and other in vitro findings regarding properties of Mb have relevance in the intracellular milieu, especially in regard to actual concentrations of metMb, H2O2, and ascorbate that would be found in vivo.

Myoglobin expression by alternative transcript in different mesenchymal stem cells compartments

“…Molecular simulation of the apo-myoglobin folding suggests the occurrence of additional domain-swapping arising from partially denatured proteins [ 41 ]. Aggregation of Mb was reported to occur after exposure to H 2 O 2 , causing inter-monomers cross-links between the amino acid side chains of tyrosines and tryptophans [ 42 ]. The role of scavenger for reactive oxygen (ROS) and nitrogen species (RNS) is a well-established function of Mb [ 43 , 44 ], and a recent report has shown the protective role of Mb in breast cancer cells due to its ROS and NO scavenging properties [ 45 ].…”

A novel catalytic heme cofactor in SfmD with a single thioether bond and a bis-His ligand set revealed by a de novo crystal structural and spectroscopic study

“…Ascorbate reverses such oxidative modifications. 74 Ascorbate can facilitate the heme oxygenase reaction in place of NADPH/cytochrome P450 reductase. 75 The determined midpoint reduction potential of SfmD is an apparent redox potential, E app m because this is not the case of five-coordinate Fe( iii )–heme to five-coordinate Fe( ii )–heme, or six-coordinate to six-coordinate, instead it is six-coordinate Fe( iii ) to five-coordinate Fe( ii ) with associated heme and protein conformational changes.…”

Monitoring the Redox Status in Multiple Sclerosis

“…MPO also oxidizes tyrosine to tyrosyl radical [ 154 ]. MPO mediates protein nitrosylation, forming 3-chlorotyrosine (3-Cl-Tyr) and dityrosine crosslinks [ 155 , 156 ] ( Table 2 ).…”