1. Introduction
One of the major mechanisms of toxicity from reactive oxygen species (ROS) is the direct oxidation of protein side chains [
1]. Although some oxidations are reversible, such as oxidation of the cysteine thiol to sulfenic acid, the majority are considered to be irreversible and to promote the destabilization of tertiary structure as well as the eventual loss of protein function [
2,
3]. One group of proteins that are particularly susceptible to oxidative damage is heme proteins [
4,
5,
6]. This is likely due to the redox activity of the porphyrin-centered iron. When present in the ferric state (III), heme proteins are prone to oxidation by endogenously produced H
2O
2, resulting in a highly unstable oxoferryl form, which can then oxidize protein side chains either internally or on another protein [
7].
One oxidative modification known to occur in myoglobin (Mb), especially under acidic conditions [
8], involves the covalent linkage between the heme group and protein side chains [
9,
10,
11]. It has been suggested that Mb cross-linked to heme be referred to as Mb-X to delineate it from the abbreviation for abnormal hemoglobin associated with Thalassemia (Mb-H) and the proposed sites of cross-linking, Mb-H when linked at a histidine and Mb-Y when linked via a tyrosine [
8]. This paper will use the Mb-X notation to refer to heme-to-protein covalent bonds. The Mb-X form of Mb has increased NADH oxidase activity [
12] and oxidizes low density lipoprotein (LDL), phospholipids, and cholesterol esters more rapidly than native Mb [
13]. Mb-X also promotes cell death when taken up by cultured fibroblasts [
14]. Mb-X and F2-isoprostanes, peroxidation products of arachidonic acid known to be produced by Mb [
15] were increased in the urine of rhabdomyolysis patients [
16], suggesting a role of Mb-X in the pathology of rhabdomyolysis.
Another oxidative modification shown to be present in H
2O
2-treated Mb is dityrosine. Dityrosine cross-links can be formed both intra- and intermolecularly [
17,
18] by the o,o’ coupling of two tyrosinyl radicals. Dityrosine cross-links have been viewed as markers of oxidative stress in vivo [
19,
20,
21,
22], although there is evidence that they might play a causal role in age-related pathologies such as Alzheimer’s [
23] and Parkinson’s disease [
24].
Here, we show that reaction of Mb with H2O2 increases peroxidase activity when ascorbate is the reducing co-substrate, a change that is associated with Mb-X formation. Furthermore, treatment of H2O2-reacted Mb with ascorbic acid reverses the Mb-X crosslink. We also show that Mb aggregates formed upon reaction of Mb with H2O2 are broken by subsequent treatment with ascorbic acid. In addition, it appears that Mb dimer reversal is protein catalyzed, as heat and detergent denatured Mb dimers were unable to reverse their cross-links. In summary, we find that oxidative modifications of Mb including formation of Mb-X and Mb aggregates are reversible by treatment with ascorbic acid, suggesting that Mb might serve a novel role of reversing oxidative modifications in proteins.
4. Discussion
The new information provided by this study includes the novel findings of reversible oxidative modifications of Mb upon treatment with ascorbic acid. For example, exposure to H2O2 increases Mb peroxidase activity and preference for ascorbate as the reducing co-substrate for Mb peroxidase activity. This increase of peroxidase activity was associated with Mb-X formed by reaction of Mb with H2O2, and both the increase in peroxidase activity and the Mb-X crosslink were reversed by treatment with ascorbic acid. While H2O2-reacted Mb forms intramolecular crosslinks to form dimers, trimers, and larger Mb aggregates, an important novel finding of the current study is that these interprotein bonds are broken by treatment with ascorbate. This action does not occur if the aggregates are first denatured by heat or incubation with SDS, suggesting that the native protein plays a role in reversal of interprotein crosslinks.
While the increase in peroxidase activity caused by exposure of Mb to H
2O
2 is a novel finding, there are reports of other Mb redox activities being enhanced by reaction of Mb with H
2O
2. For example, treatment of sperm whale or horse Mb with H
2O
2 reportedly increases NADH oxidase activity by up to 20-fold, and this activity when assessed for horse Mb was associated with Mb-X [
12]. The stoichiometry of this reaction was 1 mol of NADH oxidized per 1 mol of O
2 consumed, which is consistent with a two-electron transfer from NADH to O
2, forming H
2O
2 [
12]. Osawa and Korzekwa suggested that this NADH oxidase activity of Mb-X could contribute to toxicity by promoting further production of H
2O
2 [
12]. Other deleterious reactions mediated by H
2O
2-reacted Mb or Mb-X include peroxidation of lipids, phospholipids, LDL, and cholesterol esters [
13,
35]. Holt et al. reported increased presence of Mb-X and the free-radical-induced peroxidation of arachidonic acid, F2-isoprostanes, in urine of patients with rhabomyolysis [
16], suggesting a central role of Mb-X in rhabodomyolysis-related tissue damage. In contrast to the reactions described above, which are oxidative in nature (i.e., either promoting peroxidation or producing H
2O
2), an increase in peroxidase activity as shown in the current study would be a means to counteract an increase in reactive oxygen species.
Although myoglobin in the presence of H
2O
2 can produce hydroxyl radical, scavengers of hydroxyl radical had no effect on peroxidation of either uric acid or arachidonic acid peroxidation in the presence of Mb and H
2O
2 [
35]. This suggests that the Mb itself—as opposed to Mb-produced hydroxyl radical—mediates uric acid or acachidonic peroxidation. In contrast, sulfhydryl reducing agents can prevent peroxidation of uric acid or arachidonic acid by myoglobin in the presence of H
2O
2 [
35]. A suggested mechanism of the protective effects of the reducing agents was that they prevented formation of a reactive derivative of Mb [
35], such as Mb-X. Mb-X, originally known as the green-pigmented species formed by reaction of Mb with H
2O
2, was found to be stable in solution at room temperature for months [
11]. The new information provided by the current study is that reducing agents can reverse—as opposed to simply prevent—formation of Mb-X. Because it seems apparent that Mb-X plays a toxic role in conditions such as rhabdomyolytis [
15,
16], this novel demonstration of reversal of the Mb-X crosslink by ascorbic acid has important therapeutic implications.
Catalano et al. reported that treatment of horse Mb with H
2O
2 causes a ~50% decrease in tyrosine (Y) content [
10]. Tryptic digests of H
2O
2-treated Mb contained a species not present in untreated Mb that had a molecular weight consistent with a heme group covalently bound to a peptide beginning at Y103 (YLEFISDAIIHVLHSK), though this peptide had virtually no Y content in H
2O
2-treated Mb compared to untreated Mb [
10]. Taking these data together, the authors suggested that reaction with H
2O
2 produces Mb with the heme covalently bound to Y103 [
10]. Reeder et al. [
8] re-examined the hypothesis that Y103 mediates the heme-to-Mb cross link by using site-directed mutagenesis of sperm whale Mb. While the Y103F mutation did not affect Mb-X formation, the H64V mutation of the E7 helix distal histidine almost fully prevented generation of Mb-X [
8]. Consistent with this, wild-type
Aplysia limacina that lacks the E helix distal histidine does not form Mb-X, while introduction of a histidine residue into
aplysia promotes Mb-X formation [
8]. We have detected a tryptic fragment of H
2O
2-reacted Mb that is consistent with the analysis of Reeder et al. [
8] that the cross-link of Mb-X can be mediated by histidine.
Reeder et al. reported that H
2O
2 reaction with Mb forms Mb-X at increasing rates when pH decreases [
36]. The authors suggested that formation of Mb-X requires both the protonated oxoferryl heme and a protein radical. It also appears that a protein radical is required for protein-to-protein Mb cross linking [
37]. Detweiler et al. used 3,4-dihydro-2,3-dimethyl-2H-pyrrole 1-oxide (DMPO) to trap radicals formed by reaction of sperm whale Mb with H
2O
2 [
37]. DMPO prevented formation of sperm whale Mb dimers and trimers [
37]. Use of DMPO followed by electrospray mass spectrometry showed that a peptide containing Y103 was the site of the radical [
37]. This is consistent with the suggestion of Svistunenko et al. that given the proximity of Y103 to the heme, the radical originates on Y103 before passing to other sites in Mb [
38]. Iodinization of horse Mb prevented subsequent formation of Mb dimers by reaction with H
2O
2, consistent of a role of a role of tyrosinyl side chains in generation of protein-to-protein cross links [
37]. Further, Y151 of sperm whale Mb, a tyrosine lacking in horse Mb, was required for Mb dimer formation [
37]. Our findings stand in contrast to those of Detweiler et al. [
37] in that tyrosine acetylation did not affect Mb dimer formation in the current study, suggesting that tyrosine-independent crosslinks can contribute to the Mb aggregation in the current study.
Mb-X formation from metMb is modestly faster than Mb-X formation from oxygen-bound Mb (oxyMb) [
36]. However, Mb-X formation from metMb is inhibited by presence of oxyMb [
36]. This brings into question whether Mb-X formation could occur intracellularly under normoxic conditions in which oxyMb would be the predominant Mb form. On the other hand, it has been shown by magnetic resonance spectroscopy of human skeletal muscle that moderate aerobic exercise (about 50% or 60% of maximum oxygen consumption rate (
O
2max) causes about 50% of Mb to be in its deoxygenated form (deoxyMb) in the contracting skeletal muscle [
39,
40]. This deoxygenation of Mb sets in at moderate exercise intensity but does not further increase as exercise intensity increases up to
O
2max [
39,
40]. At the same time that exercise increases deoxyMb [
39,
40], muscle contractions also increase intracellular NADPH oxidase-generated superoxide [
41], which rapidly dismutates to H
2O
2. Thus, it appears that aerobic exercise could create conditions under which Mb-X could potentially form (i.e., increases in both H
2O
2 and deoxyMb).
The dityrosine western blot showing reversal of dityrosine cross-links should be treated with caution, as dityrosine has such a high bond dissociation energy [
42] that it would be unlikely to be broken. Consistent with a side chain other than tyrosine participating in protein crosslinking, acetylation of tyrosine residues did not prevent formation of Mb dimers. As a potential alternative mechanism of Mb–Mb cross-linking, it is possible that some other amino acid side chain, such as tryptophan, participates in protein-to-protein linkages. For example, radical-induced generation of ditryptophan (W–W) and tryptophan-tyrosine (W–Y) crosslinks has been shown to mediate protein and peptide dimerization [
43,
44,
45]. W–W crosslinks have a fluorescence excitation and emission profile [
46] that is similar to that of dityrosine [
17] and so could potentially contribute to the fluorescence changes (e.g.,
Figure 8A and
Figure 9B) in Mb exposed to H
2O
2. Mb contains a tryptophan residue that becomes a tryptophanyl radical after reaction with H
2O
2 [
33]. Our MALDI-TOF data show that the peptide containing this W residue (and also the other W in horse Mb) disappears after treatment of Mb with H
2O
2 and reappears after subsequent treatment with ascorbic acid. Given the position of this W residue in Mb, it seems unlikely to be able to participate in binding to heme. However, the data are consistent with W being a candidate for forming reversible protein-to-protein crosslinks between Mb proteins, perhaps involving W–W bonds, W–Y bonds, or both. Our findings suggest the vital importance of denaturing H
2O
2-reacted Mb with heat and/or SDS before running blots under reducing conditions to ensure both detection of Mb dimers and larger aggregates and subsequent reversal, given the labile nature of the cross links when exposed to reducing agents in the presence of native (i.e., non-denatured) Mb.
The nature of ditryptophan bonds is not yet fully elucidated [
47]. Available data suggest that C and N participate in ditryptophan crosslinking [
47], giving the possibility of either C–C or C–N crosslinks. Notably, the ditryptophan dimer in superoxide dismutase or lysozyme can cleave under MS/MS conditions [
47], suggesting that it is not as stable as a dityrosine link and thus is similar to the labile crosslinks we have detected in the current study. Paviani et al. have suggested that the susceptibility to cleavage of ditryptophan is more consistent with a C–N bond than a C–C bond [
47]. Accordingly, the crosslink that is reversed by ascorbate in the current study is likely a C–N bond.
Our finding of a peptide corresponding to the mass of a peptide containing both Y103 and Y146 suggests that both tyrosines can participate in dityrosine crosslinks. We did not collect data on which tyrosine residues were acetylated by treatment of Mb with N-acetylimidazole. When horse heart Mb is incubated with a 100-fold excess of N-acetylimidazole, both tyrosines are acetylated, though the amount of tyrosine acetylated can be varied by titration with N-acetylimidazole and assessed by changes in tyrosine absorbance at 280 nm [
48]. Our N-acetylimidazole-to-Mb molar ratio was 10-fold lower than that used by Giulivi et al. [
49], so it is possible that Y103 was unaffected under these conditions. The finding that dimerization was not prevented by acetylation suggest that Y103 was not acetylated by N-acetylimidazole in our study. For example, Y103 is likely the site of radical initiation before the radical is passed to other residues [
38], which would be necessary for radical-induced dimerization involving residues such as tyrosine or tryptophan. Interestingly, iodination of horse heart Mb prevents Mb dimerization [
37]. This suggests that the dimerization reported by Detweiler et al. [
37] is truly mediated by dityrosine, as opposed to the labile crosslink found in the current study.
Y103 is local to the heme, and it appears that formation of a radical occurs at Y103 before transferring to other residues [
37,
38]. We have detected a peptide containing both Y103 and Y146 in H
2O
2-treated Mb. This suggests that both the heme-localized tyrosine and the tyrosine in a helix closer to the protein surface can participate in dityrosine bonding. The distal histidine is in close proximity to the heme and has previously been demonstrated to form a cross link with heme [
8]. W14, on the other hand, is distant from both the heme and Y103 but still reportedly forms a radical when Mb reacts with H
2O
2 [
33]. For reference regarding positions of the heme and amino acids in Mb, a 3D structure of horse heart metMb (MMDB ID 57734) is available in the Molecular Modeling Database (MMDB) [
50] housed by the National Center for Biotechnology Information.
Dityrosine occurs in various functional, structural elements such as silk proteins [
51,
52], elastin [
53], and sea urchin eggs [
54]. On the other hand, dityrosine can be a marker for both aging-associated oxidative damage [
55] and acute bouts of oxidative stress, such as in myocardial infarction [
56]. While the aforementioned studies consider dityrosine to be a biomarker for oxidative stress, others have proposed that it might play more of a harmful role in certain mammalian tissues. For example, dityrosine-mediated cross-linking of β-amyloid peptide [
23,
57] and α-synuclein protein [
24] promotes the stabilization of their respective aggregates. Interestingly, overexpression of neuroglobin (Ngb), an oxygen-binding globin expressed mainly in neurons, has been shown to reduce Aβ fibril formation in vivo [
58], and low Ngb levels correlate with Alzheimer’s disease [
59]. The data from the current study suggest that ascorbic acid can break protein-to-protein crosslinks caused by reaction of Mb with H
2O
2. Thus, it seems possible that actions of globins might be a means through which deleterious protein aggregates could be broken in vivo.
Reaction schemes for reduction of ferrylMb (Mb with Fe
4+ in an oxo complex) by ascorbate such as would occur in peroxidase activity [
60], pH dependence of Mb redox reactions [
61], generation of Mb-X [
36], formation of dityrosine [
17], and formation of ditryptophan [
43,
47] are presented in the literature. The increased peroxidase activity once Mb is treated with H
2O
2 is most likely due to formation of the heme-to-protein crosslink. The mechanism for the increased redox activity of Mb-X has not been elucidated, but it has been suggested to be attributable to a change in protein structure surrounding the heme [
12]. Although we do not know the mechanism by which ascorbate becomes a preferred reducing substrate of Mb-X, we speculate that Mb-X retains the ability to be reduced by ascorbate at both sites for electron donation on Mb as described by Reeder et al. [
60].
Translation of the in vitro data presented in this study to physiological conditions relies on the assumption that metMb would be present in vivo. While Mb protein is expressed in mammalian heart and skeletal muscle at about 400 µmol/kg [
62,
63], metMb concentration is relatively low in tissues in vivo if it is present at all. For example, Kreutzer et al. reported that metmyoglobin is undetectable by NMR in perfused rat heart [
64]. On the other hand, it has been suggested that 1% of Mb in cardiac tissue is in the metMb form [
65]. As measured spectroscopically in anoxic pig heart, metMb content was 32 µmol/dm
3, which was about 6% of total Mb [
66]. Ascorbate concentrations in human skeletal muscle are about 170 µmol/kg [
67]. Unfortunately, H
2O
2 levels in skeletal muscle have been difficult to measure [
68]. Palomero et al. [
69] used extracellular H
2O
2 to calibrate intracellular H
2O
2 levels in isolated rat skeletal muscle fibers using the reactive oxygen species probe chloromethyl-2′,7′-dichlorofluorescin. They used this extracellular H
2O
2 standard to estimate that intracellular H
2O
2 reached about 1 µM during contractile activity [
69]. Jackson later suggested that this H
2O
2 concentration would be closer to 0.1 µM due to a trans plasma membrane H
2O
2 gradient that would result from H
2O
2 being applied to the extracellular medium [
68]. In summary, the in vitro concentrations of metMb and H
2O
2 used in the current study are much greater than would be found in the intracellular environment. Future study should be done to determine whether reversible modifications of Mb, such as Mb-X or labile Mb aggregates, can be found in skeletal muscle or heart under normal and pathological conditions. Of course, Mb can be also be found extracellularly, in conditions such as rhabdomyolysis. In the extracellular milieu, where serum H
2O
2 concentration and plasma ascorbate concentration are both about ~50 µM [
67,
70], the reversible modifications described in the in vitro studies of the current paper seem possible. Future work should investigate whether reversible modifications of Mb occur in vivo in both intracellular and extracellular spaces.