Mechanisms of reoxygenation injury in myocardial infarction: implications of a myoglobin redox cycle

The addition of ascorbate to ischemic rat hearts prevents the myocardial damage associated with reoxygenation. H2O2 oxidizes myoglobin (Mb+2) to higher oxidation states (Mb+4 and Mb+5) which are rapidly reduced by ascorbate. It is proposed that the operation of a myoglobin redox cycle, in which H2O2 causes the two-electron oxidation of myoglobin, is a critical determinant of reperfusion injury. Conversely, the reduction of myoglobin, in one-electron steps, may represent an essential protective mechanism against such injury in the heart.     

Oxidation of low-density lipoprotein by hemoglobin-hemichrome

Hemoglobin and myoglobin are inducers of low-density lipoprotein oxidation in the presence of H(2)O(2). The reaction of these hemoproteins with H(2)O(2) result in a mixture of protein products known as hemichromes. The oxygen-binding hemoproteins function as peroxidases but as compared to classic heme-peroxidases have a much lower activity on small sized and a higher one on large sized substrates. A heme-globin covalent adduct, a component identified in myoglobin-hemichrome, was reported to be the cause of myoglobin peroxidase activity on low-density lipoprotein. In this study, we analyzed the function of hemoglobin-hemichrome in low-density lipoprotein oxidation. Oxidation of lipids was analyzed by formation of conjugated diene and malondialdehyde; and oxidation of Apo-B protein was analyzed by development of bityrosine fluorescence and covalently cross-linked protein. Hemoglobin-hemichrome has indeed triggered oxidation of both lipids and protein, but unlike myoglobin, hemichrome has required the presence of H(2)O(2). In correlation to this, we found that unlike myoglobin, hemichrome formed by hemoglobin/H(2)O(2) does not contain a globin-heme covalent adduct. Nevertheless, hemoglobin-hemichrome remains oxidatively active towards LDL, indicating that other components of the oxidatively denatured hemoglobin should be considered responsible for its hazardous activity in vascular pathology.     

Intracellular O2 gradients in cardiac myocytes. Lack of a role for myoglobin in facilitation of intracellular O2 diffusion

The P₅₀ for oxygenation of myoglobin in intact cells was very high relative to that for isolated myoglobin, and was changed by addition of agents that altered respiratory rate. The P₅₀ for cytochrome $\text{a}$ oxidation in cells was very high relative to that for isolated mitochondria, but was unaffected by oxidation of myoglobin to metmyoglobin. These results demonstrate the existence of a substantial intracellular O₂ gradient in myocytes and indicate that myoglobin does not have a significant role in facilitation of O₂ diffusion to mitochondria.     

Enhanced lipid oxidation by oxidatively modified myoglobin: role of protein-bound heme

The formation of oxidized low density lipoprotein (LDL) is believed to play a significant role in the pathogenesis of atherosclerosis. Myoglobin in the presence of H(2)O(2) has been shown to catalyze LDL oxidation in vitro. It is established that an oxidatively altered form of myoglobin (Mb-H), which contains a prosthetic heme covalently crosslinked to the apoprotein, is a major product in the reaction of native myoglobin with peroxides. In the current study, we have shown for the first time that Mb-H, in the absence of exogenously added peroxides, oxidizes LDL and purified lipids, as determined by the formation of conjugated dienes, lipid peroxides, and thiobarbituric acid reactive substances. Moreover, the rate of oxidation of pure phosphatidylcholine by Mb-H was found to be at least sevenfold greater than that observed for native myoglobin. The current study strongly suggests a role for Mb-H in the lipid peroxidation observed with myoglobin.     

Acid and alkaline forms of the higher oxidation state of kangaroo, horse, and sperm whale myoglobin.

Myoglobin(IV), the derivative of myoglobin at the formal oxidation state IV, prepared from kangaroo (Megaleia rufa), horse, or sperm whale myoglobin, when cooled to liquid nitrogen temperature, assumes acid and alkaline forms with different optical spectra. The essential features of the optical spectra of the acid forms are the same as those of leghemoglobin(IV) and are very similar to those of optical spectra of the red higher oxidation states of catalases and peroxidases. This shows that the configuration of the heme iron is the same throughout these compounds. That configuration is believed to be Fe(IV) in a porphyrin environment. The optical spectra of alkaline mammalian myoglobin(IV), like that of alkaline leghemoglobin(IV), resemble those of the alkaline low spin ferric proteins. Kangaroo myoglobin(IV) may be prepared by reaction of ferrous myoglobin with hydrogen peroxide. The acid forms of myoglobin(IV) are conveniently prepared by cooling solutions in borate buffers, initially pH 8.3, to liquid nitrogen temperature. At this temperature borate buffers become acidic.     

The Role of H2O2-Generated Myoglobin Radical in Crosslinking of Myosin

The mechanism of myoglobin/H₂O₂ derived peroxidation of myosin was studied by comparing the catalytic activity of myoglobin and horseradish peroxidase using O-dianisidine, N-acetyl tyrosine and myosin as substrates. It was found that both hemoproteins induced myosin crosslinking and concomitant tyrosines oxidation to bityrosines, suggesting inter-molecular coupling of tyrosines in the crosslinking. The enzymatic activity of both hemoproteins on myosin was weak compared to small substrates. While horseradish peroxidase was much more active than myoglobin on small substrates, the reverse was true for myosin peroxidation. Since the suicidal interaction of myoglobin with H₂O₂ forms unstable tyrosine radicals, we suggest that the increased activity of myoglobin on myosin results from an efficient electron transfer between surface tyrosines of myosin and myoglobin but not horseradish peroxidase. These conclusions were supported by evidence that sperm whale myoglobin, which contains two active tyrosines -the heme-adjacent (tyrosine-103) and the surface (tyrosine-151), is more active as a mediator of myosin peroxidation than horse heart myoglobin which is devoid of the surface tyrosine.     

Glutathione-dependent reduction of peroxides during ferryl- and met-myoglobin interconversion: a potential protective mechanism in muscle

Met-myoglobin is oxidized both by H2O2 and other hydroperoxides to a species with a higher iron valency state and the spectral characteristics of ferryl-myoglobin. Glutathione (GSH) reduces the latter species back to met-myoglobin with parallel oxidation to its disulfide (GSSG) but cannot reduce met-myoglobin to ferrous myoglobin. Under aerobic conditions, the GSH-mediated reduction of ferry-myoglobin is associated with O2 consumption and amounts of GSSG are formed far in excess over that of the peroxide added. Under anaerobic conditions, this ratio is close to unity. These results are interpreted in terms of a one-electron redox process involving the reduction of ferryl-myoglobin to met-myoglobin and the one-electron oxidation of GSH to its thiyl radical. Further reactions of thiyl radicals are influenced by the presence of oxygen which will be the determining factor in the ratio H2O2 added/GSSG formed. It is suggested that, when oxygen is limiting, myoglobin may serve as a protector of muscle cells against peroxides and other oxidants.     

Caffeine induces Ca2+ release by reducing the threshold for luminal Ca2+ activation of the ryanodine receptor

Myeloperoxidase, released by activated phagocytes, forms reactive oxidants by catalysing the reaction of halide and pseudo-halide ions with H(2)O(2). These oxidants have been linked to tissue damage in a range of inflammatory diseases. With physiological levels of halide and pseudo-halide ions, similar amounts of HOCl (hypochlorous acid) and HOSCN (hypothiocyanous acid) are produced by myeloperoxidase. Although the importance of HOSCN in initiating cellular damage via thiol oxidation is becoming increasingly recognized, there are limited data on the reactions of HOSCN with other targets. In the present study, the products of the reaction of HOSCN with proteins has been studied. With albumin, thiols are oxidized preferentially forming unstable sulfenyl thiocyanate derivatives, as evidenced by the reversible incorporation of (14)C from HOS(14)CN. On consumption of the HSA (human serum albumin) free thiol group, the formation of stable (14)C-containing products and oxidation of tryptophan residues are observed. Oxidation of tryptophan residues is observed on reaction of HOSCN with other proteins (including myoglobin, lysozyme and trypsin inhibitor), but not free tryptophan, or tryptophan-containing peptides. Peptide mass mapping studies with HOSCN-treated myoglobin, showed the addition of two oxygen atoms on either Trp(7) or Trp(14) with equimolar or less oxidant, and the addition of a further two oxygen atoms to the other tryptophan with higher oxidant concentrations (> or = 2-fold). Tryptophan oxidation was observed on treating myoglobin with HOSCN in the presence of glutathione and ascorbate. Thus tryptophan residues are likely to be favourable targets for the reaction in biological systems, and the oxidation products formed may be useful biomarkers of HOSCN-mediated protein oxidation.     

Redox cycling of myoglobin and ascorbate: a potential protective mechanism against oxidative reperfusion injury in muscle

Metmyoglobin catalyzes the decomposition of H2O2 as well as other hydroperoxides by using ascorbic acid as a substrate. The ratio of H2O2 reduced to ascorbate oxidized is close to one, whereas the rate of oxidation is directly proportional to both H2O2 and metmyoglobin concentrations. Ascorbate also prevents the protein modifications and the O2 evolution that accompany the reaction of metmyoglobin with hydroperoxides. In the absence of ascorbate, myoglobin and H2O2 promote the peroxidation of unsaturated fatty acids and, thus, may cause damage to cellular constituents. However, lipid peroxidation is inhibited in the presence of ascorbate and, for this reason, it is suggested that this heme protein functions in the opposite manner. The redox cycling of myoglobin by ascorbate may act as an important electron "sink" and defense mechanism against peroxides during oxidative challenge to muscle.     

Participation of tyrosine residues of myoglobin in the disproportionateness reaction of heme iron (II) and (IV)]

By means of the comparison of the constant oxidation reactions of both the myoglobin modified by N-acetylimidazole and the intact myoglobin in the presence of H2O2 or ferrylmyoglobin we characterized the role of the tyrosine residues (Tyr) of myoglobin in the synproportionation reaction between heme iron (II) of one molecule and heme iron (IV)--of another. It was demonstrated that Tyr derivatization resulted in the decrease of the velocity of redox interaction between deoxymyoglobin (II) and ferrylmyoglobin (IV) and led to the decrease of the efficiency of oxymyoglobin deoxygenation. The effects were shown to be independent on Tyr quantity in myoglobin molecule and to have the same character for both the sperm-whale myoglobin and the horse myoglobin.     

Pyruvate but not lactate prevents NADH-induced myoglobin oxidation

In this work, we investigated the influence of NADH on the redox state of myoglobin and the roles of pyruvate and lactate in this process. NADH increased the autoxidation rate of myoglobin. Both a drop in pH and partial deoxygenation markedly stimulated the autoxidation process and the influence of NADH. A correlation between met-Mb formation rate and NADH oxidation rate was always observed. The increased rate of Mb autoxidation caused by NADH was inhibited by catalase and pyruvate but not by l-lactate. The antioxidant activity versus H2O2 of both pyruvate and lactate was evidenced by chemiluminescence experiments. The antioxidant activity of lactate disappeared completely in the presence of myoglobin or apo-myoglobin, whereas it was only reduced for pyruvate. These results could be of interest in preventing autoxidation of myoglobin that can contribute to ischemia-reperfusion injury during infarction or high-intensity exercise.     

The interaction of Trolox C, a water-soluble vitamin E analog, with ferrylmyoglobin: reduction of the oxoferryl moiety.

The oxidation of the heme iron of metmyoglobin by H2O2 yields an oxo ferryl complex (FeIV = O), similar to Compound II of peroxidases, as well as a protein radical; this high oxidation state of myoglobin is known as ferrylmyoglobin. The interaction of Trolox, a water-soluble vitamin E analog, with ferrylmyoglobin entailed two sequential one-electron oxidations of the phenolic antioxidant with intermediate formation of a phenoxyl radical and accumulation of a quinone end product. These oxidation reactions were linked to individual reductions of ferrylmyoglobin to metmyoglobin, as indicated by the value of the relationship [metmyoglobin]formed/[Trolox]consumed: 1.92 +/- 0.28. The Trolox-mediated reduction of ferrylmyoglobin to metmyoglobin could proceed directly, i.e., electron transfer from the phenolic-OH group in Trolox to the oxoferryl moiety, or indirectly, i.e., sequential electron transfer from Trolox to a protein radical to the oxoferryl moiety. The former mechanism is supported by the finding that the high oxidation heme iron is reduced under conditions where the tyrosyl residues are blocked by o-acetylation and when hemin is substituted for myoglobin. The latter mechanism is consistent with the following observations: (a) the EPR signal ascribed to the protein radical is suppressed by Trolox, with the concomitant appearance of the EPR spectrum of the Trolox phenoxyl radical and (b) the rate of ferrylmyoglobin reduction by Trolox is decreased with increasing number of tyrosyl residues in the proteins of horse myoglobin (titrated by o-acetylation) and sperm whale myoglobin. The apparent discrepancy between these observations can be reconciled by considering that both electrophilic centers in ferrylmyoglobin--the oxoferryl heme moiety and the protein radical--function independently of each other and that recovery of ferrylmyoglobin by Trolox could be effected through the tyrosyl residues, albeit at slower rates. The mechanistic aspects of these results are discussed in terms of the two main redox transitions in the myoglobin molecule encompassing valence changes of the heme iron and electron transfer of the tyrosyl residue in the protein and linked to the two sequential one-electron oxidations of Trolox.     

In vitro nonenzymatic glycation enhances the role of myoglobin as a source of oxidative stress

Metmyoglobin (Mb) was glycated by glucose in a nonenzymatic in vitro reaction. Amount of iron release from the heme pocket of myoglobin was found to be directly related with the extent of glycation. After in vitro glycation, the unchanged Mb and glycated myoglobin (GMb) were separated by ion exchange (BioRex 70) chromatography, which eliminated free iron from the protein fractions. Separated fractions of Mb and GMb were converted to their oxy forms -MbO₂ and GMbO₂, respectively. H₂O₂-induced iron release was significantly higher from GMbO₂ than that from MbO₂. This free iron, acting as a Fenton reagent, might produce free radicals and degrade different cell constituents. To verify this possibility, degradation of different cell constituents catalyzed by these fractions in the presence of H₂O₂ was studied. GMbO₂ degraded arachidonic acid, deoxyribose and plasmid DNA more efficiently than MbO₂. Arachidonic acid peroxidation and deoxyribose degradation were significantly inhibited by desferrioxamine (DFO), mannitol and catalase. However, besides free iron-mediated free radical reactions, role of iron of higher oxidation states, formed during interaction of H₂O₂ with myoglobin might also be involved in oxidative degradation processes. Formation of carbonyl content, an index of oxidative stress, was higher by GMbO₂. Compared to MbO₂, GMbO₂ was rapidly auto-oxidized and co-oxidized with nitroblue tetrazolium, indicating increased rate of Mb and superoxide radical formation in GMbO₂. GMb exhibited more peroxidase activity than Mb, which was positively correlated with ferrylmyoglobin formation in the presence of H₂O₂. These findings correlate glycation-induced modification of myoglobin and a mechanism of increased formation of free radicals. Although myoglobin glycation is not significant within muscle cells, free myoglobin in circulation, if becomes glycated, may pose a serious threat by eliciting oxidative stress, particularly in diabetic patients.     

Myoglobin-catalyzed tyrosine nitration: no need for peroxynitrite.

The nitration of tyrosine residues in protein to yield 3-nitrotyrosine derivatives has been suggested to represent a specific footprint for peroxynitrite formation in vivo. However, recent studies suggest that certain hemoproteins such as peroxidases catalyze the H(2)O(2)-dependent nitration of tyrosine to yield 3-nitrotyrosine in a peroxynitrite-independent reaction. Because 3-nitrotyrosine has been shown to be present in the postischemic myocardium, we wished to assess the ability of myoglobin to catalyze the nitration of tyrosine in vitro. We found that myoglobin catalyzed the oxidation of nitrite and promoted the nitration of tyrosine. Both nitrite oxidation and tyrosine nitration were H(2)O(2)-dependent and required the formation of ferryl (Fe(+4)) myoglobin. In addition, nitrite oxidation and tyrosine nitration were pH-dependent with a pH optimum of approximately 6.0. Taken together, these data suggest that the acidic pH and low oxygen tension produced during myocardial ischemia will facilitate myoglobin-catalyzed, peroxyntrite-independent formation of 3-nitrotyrosine.     

Oxidation of deoxy myoglobin by [Fe(CN)6]3-.

The ability of myoglobin (Mb) to reversibly bind O2 and other ligands has been well characterized. Mb also participates with a variety of redox metals to form metmyoglobin (metMb). By using an anaerobic stopped-flow device we have measured outer-sphere oxidation by [Fe(CN)6]3 of native sperm whale myoglobin, recombinant wild-type Mb, and a series of mutant Mb proteins in which the distal His-64 was changed to Gly, Phe, Leu or Val. Second-order rate constants for oxidation of mutant proteins are 10-15 times greater than for recombinant or native (kox approximately 10(6) M-1 s-1). We attribute the reduced rate of oxidation of wild-type protein to a higher reorganization energy imposed by the presence of the unique water/His-64/heme interaction, which is absent in the mutant proteins.     

The Effect of Myoglobin on the Stability of the Hydroxyl-Radical Adducts of 5, 5 Dimethyl-1-Pyrolline-N-Oxide (DMPO), 3, 3,5, 5 Tetramethyl-1-pyrolline-N-Oxide(TMPO) and 1-Alpha-Phenyl-Tert-Butyl Nitrone (PBN) in the Presence of Hydrogen Peroxide

The hydroxyl radical adducts of 5, 5 dimethyl-1-pyrolline-N-oxide (DMPO) and 3, 3,5, 5 tetramethyl-1-pyrolline-N-oxide (TMPO) formed in the presence of hydrogen peroxide and Fe are normally quite stable, but in the presence of 5-20 micromolar myoglobin their ESR signals decay rapidly. This decay probably reflects further oxidation of the adduct to nonparamgnetic products.

The ESR signal of the hydroxyl radical adduct of 1-alpha-phenyl-tert-butyl nitrone (PBN) formed under similar conditions is subject to non-heme dependent attenuation, possibly via hydroxyl radical scavenging, but not to heme dependent decay. Hydrogen peroxide readily converts myoglobin to its ferryl (Fe^IV) derivative, and this centre may be responsible for the oxidation of the DMPO and TMPO adducts. The different behaviour of PBN may be due to differences in susceptibility to ferrylmyoglobin mediated oxidation, or to steric differences controlling access to the heme pocket of myoglobin, and is relevant to the choice of spin trap for biological experiments aimed at detecting hydroxyl radicals in the presence of myoglobin or other heme proteins.     

The dimer-monomer conversion of Cerithidea myoglobin coupled with the heme iron oxidation

With regard to the distal (E7) residue, gastropod sea mollusc contains both types of myoglobin, one with and the other lacking the distal histidine. We have isolated a myoglobin from the radular muscle of Cerithidea rhizophorarum, a small whelk found on the Japanese coast. Unlike Aplysia myoglobin having a single histidine residue at position 95, Cerithidea myoglobin contains three histidines at positions 48, 66 and 98. Moreover, Cerithidea MbO₂ exists as homodimers and is oxidized, not to the usual form of metMb but to the hemichrome monomers. It was also found that the hemichrome monomers thus produced can easily be converted back to the dimerized oxy-form, if the ferric protein was reduced carefully with a slight excess of sodium hydrosulfite. This dimer-monomer conversion coupled with the heme-iron oxidation in Cerithidea myoglobin is very unique, and the distal histidine at position 66 is probably responsible for its reversible formation of hemichrome from the ferric met-form that occurred transiently in due course of the oxidation reaction of Cerithidea MbO₂     

Respiratory chain defect of myocardial mitochondria in idiopathic dilated cardiomyopathy of Doberman pinscher dogs.

Idiopathic dilated cardiomyopathy (IDCM) is a primary myocardial disease of unknown cause. We tested the hypothesis that IDCM was associated with a myocardial metabolic defect by determining a comprehensive biochemical profile of metabolite concentrations and enzyme activities for the major metabolic pathways of the myocardium. We used the Doberman pinscher breed as a naturally occurring canine model of IDCM and compared its myocardial profile with that of healthy adult mongrels. Compared with controls, myocardium in IDCM had markedly reduced mitochondrial electron transport activity and myoglobin concentration, in association with acidosis and energy depletion following anoxic challenge: 60% decreased NADH dehydrogenase and 50% decreased ATP synthetase activities; 90% decreased myoglobin concentration; and 30% reduced ATP and 50% increased lactate and proton concentrations. Sarcoplasmic reticulum Ca(2+)-transport ATPase was decreased by 42%. There was a 15% compensatory increase in fatty acid oxidation and Krebs cycle activity. Other biochemical changes were mild by comparison with the mitochondrial defects. We conclude that IDCM is associated with a marked impairment of mitochondrial production of ATP, arising from decreased activity of the mitochondrial electron transport system, including myoglobin. These changes may be secondary to an underlying genetic defect or may indicate a deficiency of the mitochondrial respiratory chain that predisposes this breed to heart failure.     

Catalytic effect of ferricyanide between myoglobin and luminol and effect of temperature.

Specific catalytic oxidation of oxymyoglobin (MbO(2)) and luminol by ferricyanide was studied in a flow-injection system. MbO(2) in different redox states (ferric and ferrous) was oxidized to Mb(Fe(III)) by ferricyanide, and then specific binding of the ferrocyanide anion to Mb(Fe(III)) to the His 119 (GH1) region accelerated the electron transfer between Mb(Fe(III)) and luminol, which produced a chemiluminescence (CL) signal at 425 nm. The increased CL emission was correlated with the myoglobin concentration in the range 0.16-7.5 microg/mL. Thermogravimetry and differential scanning calorimetry were used to investigate the temperature effects on this reaction. The results showed that the CL intensity in the presence of myoglobin changed considerably with heating in the range 15-50 degrees C, and the maximal CL intensity was observed at 40 degrees C, corresponding to the glass transition temperature of myoglobin. The effect of different ligands and interferences were also studied.     

Toxicity of myoglobin and haemoglobin: oxidative stress in patients with rhabdomyolysis and subarachnoid haemorrhage

Haemolytic events, such as those following rhabdomyolysis and subarachnoid haemorrhage, often result in pathological complications such as vasoconstriction. Haem-protein cross-linked myoglobin and haemoglobin are generated by ferric-ferryl redox cycling, and thus can be used as markers of oxidative stress. We have found haem-protein cross-linked myoglobin in the urine of patients suffering from rhabdomyolysis and haem-protein cross-linked haemoglobin in the cerebrospinal fluid of patients following subarachnoid haemorrhage. These findings provide strong evidence that these respiratory haem proteins can be involved in powerful oxidation processes in vivo. We have previously proposed that these oxidation processes in rhabdomyolysis include the formation of potent vasoconstrictor molecules, generated by the myoglobin-catalysed oxidation of membranes, inducing nephrotoxicity and renal failure. Haem-protein cross-linked haemoglobin in cerebrospinal fluid suggests that a similar mechanism of lipid oxidation is present and that this may provide a mechanistic basis for the delayed vasospasm that follows subarachnoid haemorrhage.