Metmyoglobin Oxidation during Electron Transport Reactions in Mitochondria

Studies of the intracellular role of myoglobin were carried out by recording spectrophotometric changes in acid metmyoglobin and oxymyoglobin during electron transport reactions with mitochondria prepared from pigeon heart muscle by the method of Chance and Hagihara. The absorption peak of metmyoglobin at 409 mµ disappeared when substrate was added to normal or antimycin-inhibited preparations, and was replaced by a new maximum at 423 to 424 mµ, identified as due to the oxidation to ferrylmyoglobin. Further investigation revealed that the oxidation of metmyoglobin took place with the simultaneous oxidation of reduced flavoprotein. Hydrogen peroxide, formed by the reaction of reduced flavoprotein with oxygen, was considered to be the probable intermediate for the oxidation of metmyoglobin in experiments in which catalase was added as a competitor for the oxidant. When DPNH was added to the reaction mixture, the reductant acted to resynthesize the ferri-derivative by reaction with ferrylmyoglobin. Oxymyoglobin could not be used in place of metmyoglobin in these systems. Under the experimental conditions, oxymyoglobin dissociated when dissolved oxygen was depleted from the medium by enzyme oxidations; the resultant ferromyoglobin underwent oxidation to metmyoglobin.     

Kinetic analysis of myoglobin autoxidation by isoelectric-focusing electrophoresis.

The autoxidation of horse myoglobin was studied in the presence or absence of catalase (EC 1.11.1.6) and/or superoxide dismutase (EC 1.15.1.1) at various pH values (6.6-7.8). Changes in the percentages of oxymyoglobin and metmyoglobin during the reaction were analysed by means of isoelectric focusing on Ampholine gel plates. Oxymyoglobin was decreased in a first-order manner, with an accompanying increase in metmyoglobin, under the various conditions studied. The observed reaction rate constants obtained under these conditions were pH-dependent; however, they were also greatly affected by the presence of the enzymes. The pH-dependence of the overall reaction was explained by the acid-base three-state model of myoglobin proposed by Shikama & Sugawara [(1978) Eur. J. Biochem. 91, 407-413]. The reaction process of myoglobin autoxidation was explained by the model suggested by Winterbourn, McGrath & Carrell [(1976) Biochem. J. 155, 493-502], indicating that superoxide anion and hydrogen peroxide are involved in the reaction mechanism.     

Reduction of methemerythrin by deoxymyoglobin: a protein-protein redox reaction not involving electron-transfer proteins.

The stoichiometry and kinetics of reaction of methemerythrin with the deoxy forms of myoglobin and hemoglobin have been examined at I = 0.2 M and 25 degrees C. One mole of methemerythrin (on the basis of the monomer unit containing two irons) reacts with 2 mol of deoxymyoglobin and with 0.5 mol of deoxyhemoglobin. All reactions are second order. Rate constants for reaction with deoxymyoglobin are 0.25 M-1s-1 (Phascolopsis gouldii) and 5.6 M-1s-1 (Themiste pyroides) at pH 6.3. There is little effect of raising the ionic strength to 1.35 M and only a small decrease in rate when the pH is adjusted to 8.2. The rate constant for reaction of deoxyhemoglobin with P. gouldii methemerythrin is approximately 0.1 M-1s-1 at pH 6.3. Metmyohemerythrin from T. pyroides reacts slightly slower than the octamer form (k = 2.0 M-1s-1 at pH 6.3 and 7.0). Oxymyoglobin is converted to metmyoglobin by methemerythrin. The electron-transfer path is discussed and a self-exchange rate constant for hemerythrin assessed as 10(-3) M-1s-1 on the basis of Marcus's theory.     

Encapsulation of myoglobin with a mesoporous silicate results in new capabilities

A metmyoglobin (Fe3+), an oxidized form of myoglobin (Fe2+), was confined in nanospaces of about 4 nm in diameter in mesoporous silica (FSM; folded-sheet mesoporous material), forming a metmyoglobin (Fe3+)-FSM nanoconjugate. The spectral characteristics of metmyoglobin (Fe3+)- and myoglobin (Fe2+)-FSM show an absorption curve quite similar to that of native metmyoglobin, indicating that myoglobin retains its higher-order structure in the pores of FSM. The metmyoglobin (Fe3+)-FSM conjugate had not only a peroxidase-like activity in the presence of hydrogen peroxide (a hydrogen acceptor) and 2,2-azino-bis(3-ethylbenzothiazoline)-6-sulfomic acid (ABTS) or guaiacol (a hydrogen donor) but also an advanced molecular recognition ability enabling it to distinguish between ABTS and guaiacol. Furthermore, the metmyoglobin (Fe3+)-FSM showed the peroxidase-like activity even in an organic media using benzoyl peroxide as the hydrogen acceptor and leucocrystal violet as the hydrogen donor. The simple immobilization of metmyoglobin (Fe3+) into FSM results in enhanced catalytic activity in organic media compared to that of native metmyoglobin (Fe3+).     

Autoxidation of oxymyoglobin. An overall stoichiometry including subsequent side reactions

Oxymyoglobin (MbO2) is oxidized easily to metmyoglobin (metMb) with generation of the superoxide anion, which can be converted by the spontaneous dismutation into H2O2, this being also a potent oxidant of MbO2. In the presence of sodium azide in stoichiometric amounts, however, the rate of autoxidation of MbO2 increased rapidly with increasing concentration of the anion, but soon reached a saturating level, the extent of which was about twice that of the normal autoxidation in buffer alone. Quantitative analysis has revealed that this enhancement is not due to the nucleophilic displacement of O2- from MbO2 by the anion (Satoh, Y., and Shikama, K. (1981) J. Biol. Chem. 256, 10272-10275), but is due to the additional oxidation of MbO2 by H2O2 freed from the metMb being occupied by the anion at the sixth coordination position. Based on these novel results and stoichiometric considerations, it is possible to propose a new view that H2O2 produced from O2- can be eliminated or decomposed mostly, if not completely, by the metMb resulting from the normal autoxidation reaction of MbO2, presumably via the formation of the ferryl species.     

Free fatty acids enhance the oxidation of oxymyoglobin and inhibit the peroxidase activity of metmyoglobin

The effect of low concentrations of sodium oleate on the oxidation of oxymyoglobin to metmyoglobin has been examined. This long chain fatty acid results in a tripling of the initial rate (1.5-4.3 h-1) at which oxymyoglobin is converted to metmyoglobin and more than doubling of the rate of the long-term reaction (0.12-0.33 h-1). Examination of rate constant enhancement over a range of oleate concentrations (0-0.215 mM) has allowed an estimate of association constants for both phases of the reaction system. The peroxidase activity expressed by metmyoglobin towards hydrogen peroxide is inhibited by the presence of sodium oleate by a fivefold increase in the apparent Km value (0.33-1.77 mM). The observed changes in oxymyoglobin concentration over time are discussed in terms of competition between metmyoglobin, which acts as a peroxidase decreasing in situ concentrations of H2O2, and oxymyoglobin, which also is oxidized by the peroxide. It is shown that oleate can bind to metmyoglobin and azidometmyoglobin, but not oxymyoglobin. Catalase reduces the oxidation rates of oxymyoglobin in the presence or in the absence of oleate, substantiating the involvement of H2O2. The results are discussed in relation to the potential increase in tissue peroxidations in the presence of ischaemically elevated fatty acid concentrations.     

Reaction of imidazole and hydroquinone with oxymyoglobin

Oxymyoglobin reacts with imidazole, substituted imidazoles, and hydroquinone to give metmyoglobin. The kinetics of these reactions have been studied. The rates are first order in both reactants, and second-order rate constants are reported. At pH 8.2, k₁ for imidazole is 2.5 ± 0.3 × 10^?3 M^?1 sec^?1 and for hydroquinone is 4 ± 0.4 × 10^?1 M^?1 sec^?1. The rates are independent of pH for imidazole but increase rapidly with pH for hydroquinone. The mechanism for all these reactions is thought to involve the two-electron reduction of molecular oxygen to peroxide with concurrent oxidation of both the protein and the reactant. An analogous mechanism has been suggested previously [1] for the reaction of oxyhemoglobin with hydroquinone. It has previously been shown [6] that imidazole can mediate the transfer of electrons to heme proteins by forming a transient reduced radical. The present results indicate that it can also form a transient oxidized radical under mild conditions. This dual capability may be important in biological electron-transfer processes.     

Metmyoglobin reductase. Identification and purification of a reduced nicotinamide adenine dinucleotide-dependent enzyme from bovine heart which reduces metmyoglobin.

Beef heart muscle has been found to contain an enzyme which will rapidly and directly reduce metmyoglobin in vitro. Reduction rates are far greater than any previously reported for nonspecific or nonenzymatic systems. The enzyme is NADH-dependent and requires the presence of ferrocyanide ion for in vitro assay. The artificial electron carriers, dichlorophenolindophenol and methylene blue, are not required. Nonenzymatic reduction of metmyoglobin, which has previously been reported, was not encountered under the assay conditions described herein. Demonstration of enzymatic activity is dependent on a suitable myoglobin substrate, NADH, and ferrocyanide. An equimolar amount of cytochrome b5 was more effective than ferrocyanide in the enzymatic reduction of metmyoglobin. The methods for preparation of beef heart myoglobin and for purification of the enzyme are presented. The enzyme has been purified over 2000-fold. The enzyme has a pH optimum about 6.5 and a Km of 5.0 x 10(-5) M, and is unaffected by the absence of O2. Sodium dodecyl sulfate-gel electrophoresis revealed a molecular weight around 30,000. Purified enzyme does not react with lipoamide. The reaction is markedly influenced by the composition of the buffering milieu. Enzyme activity is inhibited by p-chloromercuriphenyl sulfonic acid, quinacrine dihydrochloride, and N-ethyl-maleimide. Activity was slightly stimulated by FMN. The characteristics of the enzymatic activity and the assay system are similar to those reported by Hegesh et al. (J. Lab. Clin. Med. 72, 339-344, 1968) for erythrocyte methemoglobin reductase.     

Conformational changes in sperm-whale metmyoglobin due to combination with antibodies to apomyoglobin

1. No ferrihaem was detected in the precipitate formed by metmyoglobin with an antiserum to apomyoglobin and the extinction at 410mmu of metmyoglobin, due to ferrihaem, was decreased by the univalent fragments of apomyoglobin antibodies. It was concluded that the combination of apomyoglobin antibodies with metmyoglobin caused the release of ferrihaem. As the removal of ferrihaem from metmyoglobin is accompanied by a conformational change, it was concluded that the conformation of metmyoglobin was altered by the apomyoglobin antibodies. 2. Antisera to metmyoglobin were divided into two groups; antisera of the first group revealed differences between the immunological reactivities of metmyoglobin and apomyoglobin, whereas no differences were detected with antisera of the second group. 3. Metmyoglobin was only partially re-formed by adding haematin to the precipitate produced by apomyoglobin with an antiserum of the first group, whereas complete re-formation of metmyoglobin was achieved in the presence of antisera of the second group. No metmyoglobin was formed on the addition of haematin to the precipitates produced by either metmyoglobin or apomyoglobin with the anti-apomyoglobin serum. 4. Immune precipitates formed by antisera to metmyoglobin dissociated at pH1.8, whereas those formed by the anti-apomyoglobin serum did not dissociate. 5. These results suggest that apomyoglobin possessed different conformations when combined with metmyoglobin antibodies and apomyoglobin antibodies.     

Secondary structural changes of metmyoglobin and apomyoglobin in anionic and cationic surfactant solutions: Effect of the hydrophobic chain length of the surfactants on the structural changes

Secondary structural changes of metmyoglobin and apomyoglobin were examined in solutions of sodium alkylsulfates with hydrocarbon numbers of 8 and 12, and alkyltrimethylammonium bromides with hydrocarbon numbers of 10, 12, 14, and 16. The relative proportion ofa-helical structure was estimated by the curve-fitting method of circular dichroic spectrum. The helical proportions of metmyoglobin and apomyoglobin were 82 and 63%, respectively. The shorter the hydrocarbon chain the surfactant had, the higher the concentration necessary to disrupt the secondary structures of these proteins. However, the helical proportion had a tendency to decrease down to lower values in solutions of the cationic surfactants with short hydrophobic groups. On the other hand, thea-helical structure of apomyoglobin was disrupted in lower concentrations of each cationic surfactant than that of metmyoglobin, although the disruptions of the same structures in both the proteins occurred in the same concentration range of each anionic surfactant. It appeared likely that the removal of the heme group unstabilized the myoglobin conformation only in the cationic surfactant solutions.     

Horseradish peroxidase. XXXVI. On the difference between peroxidase and metmyoglobin.

A mechanism for the reaction of hydrogen peroxide with horseradish peroxidase is proposed which involves the catalytic activity of the carboxylate side chain of aspartate residue 43. The corresponding residue in the active site of metmyoglobin is glycine E8, which explains the inability of metmyoglobin to form compound I. Certain aspects of the proposed peroxidase mechanism may be relevant to the catalytic triad for the serine proteases.     

Purification and Some Properties of “Methemoglobin Reductase”

A procedure for obtaining the electrophoretically and ultracentrifugally homogenous preparation of “methemoglobin reductase” from erythrocytes of blue-white dolphin was developed. Method consists of DEAE-cellulose adsorption, fractionation with ammonium sulfate, Sephadex G-75 gel filtration and DEAE-Sephadex A-50 column chromatography. There were obtained three preparations of enzyme. All these preparations strongly reduced methemoglobin, metmyoglobin and cytochrome c in the presence of methyleneblue when NADPH or NADH was used as the cofactor. The activity of NADPH as the cofactor was higher than that of NADH. The enzyme contained neither flavin nor heme, and molecular weight was 23,000 ~ 28,000.     

Pro-oxidant and antioxidant potential of catecholestrogens against ferrylmyoglobin-induced oxidative stress

Ferryl heme proteins may play a major role in vivo under certain pathological conditions. Catecholestrogens, the estradiol-derived metabolites, can act either as antioxidants or pro-oxidants in iron-dependent systems. The aim of the present work was (1) to determine the effects of ferrylmyoglobin on hepatocyte cytotoxicity, and (2) to assess the pro/antioxidant potential of a series of estrogens (phenolic, catecholic and stilbene-derived) against ferrylmyoglobin induced lipid peroxidation in rat hepatocytes. Cells were exposed to metmyoglobin plus hydrogen peroxide to form ferrylmyoglobin in the presence of the transition metal chelator diethylentriaminepentaacetic acid. Results showed that ferrylmyoglobin induced an initial oxidative stress, mainly reflected in an early lipid peroxidation and further decrease in GSH and ATP. However, cells gradually adapted to this situation, by recovering the endogenous ATP and GSH levels at longer incubation times. Phenolic and stilbene-derived estrogens inhibited ferrylmyoglobin-induced lipid peroxidation to different degrees: diethylstilbestrol>estradiol>resveratrol. Catecholestrogens at concentrations higher than 1 microM also inhibited lipid peroxidation with similar efficacy. The ability of estrogens to reduce ferrylmyoglobin to metmyoglobin may account for their antioxidant activity. In contrast, physiological concentrations (100 pM-100 nM) of the catecholestrogens exerted pro-oxidant activities, 4-hydroxyestradiol being more potent than 2-hydroxyestradiol. The implications of these interactions should be considered in situations where local myoglobin or hemoglobin microbleeding takes place.     

Conformation of biological macromolecules. Circular dichroism and magnetic circular dichroism studies of metmyoglobin and its derivatives.

The circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of horse heart metmyoglobin and the following derivatives were measured in the Soret and near ultraviolet regions: metmyoglobin and its peroxide compound, and hydroxide, cyanide, azide, and fluoride derivatives. The heme-related CD bands in the Soret and near ultraviolet wavelength regions were altered by ligand substitution, though their relationships to the magnetic moment were quite different. In the Soret region, the CD peak had no definite relation to the magnetic moment, while in the near ultraviolet region the magnitude of the CD peak decreased with the magnetic moment. The MCD peak in the Soret and near Ultraviolet regions also varied with ligand substitution. The magnetic ellipticity decreased with the magnetic moment in both wavelength regions. There was a more quantitative correlation between the magnetic ellipticity and the magnetic moment in the near ultraviolet region than in the Soret region. Metmyoglobin peroxide compound exhibited slightly different behavior in the MCD spectrum from other derivatives. It is suggested that the heme iron of the metmyoglobin peroxide compound is in an oxidation state other than the ferric state and that the porphyrin structure of metmyoglobin may be modified by the reaction with hydrogen peroxide.     

The myoglobin protein radical. Coupling of Tyr-103 to Tyr-151 in the H2O2-mediated cross-linking of sperm whale myoglobin

Sperm whale metmyoglobin, which has tyrosine residues at positions 103, 146, and 151, dimerizes in the presence of H2O2. Equine metmyoglobin, which lacks Tyr-151, and red kangaroo metmyoglobin, which lacks Tyr-103 and Tyr-151, do not dimerize in the presence of H2O2. The dityrosine content of the sperm whale myoglobin dimer shows that it is primarily held together by dityrosine cross-links, although more tyrosine residues are lost than are accounted for by dityrosine formation. Digestion of the myoglobin dimer with chymotrypsin yields a peptide with the fluorescence spectrum of dityrosine. The amino acid composition, amino acid sequence, and mass spectrum of the peptide show that cross-linking involves covalent bond formation between Tyr-103 of one myoglobin chain and Tyr-151 of the other. Replacement of the prosthetic group of sperm whale myoglobin with zinc protoporphyrin IX prevents H2O2-induced dimerization even when intact horse metmyoglobin is present in the incubation. This suggests that the tyrosine radicals required for the dimerization reaction are generated by intra- rather than intermolecular electron transfer to the ferryl heme. Rapid electron transfer from Tyr-103 to the ferryl heme followed by slower electron transfer from Tyr-151 to Tyr-103 is most consistent with the present results.     

Antioxidant Activity of Quercetin against Metmyoglobin-induced Oxidation of Fish Oil-bile Salt Emulsion

The antioxidative effect of quercetin was examined in metmyoglobin-induced oxidation of a fish oil-bile salt emulsion (average diameter of particles; 2.0 μm) to evaluate its effectiveness during the digestion of highly oxidizabile oils. The activity of quercetin increased with the lowering of the initial peroxide value (PV) of the oil and its effectiveness was superior to that of α-tocopherol. A synergistic antioxidant effect was observed upon the addition of quercetin and α-tocopherol irrespective of the initial PV of the oils, and quercetin was consumed faster than α-tocopherol. The loss of quercetin was larger than that of α-tocopherol when cumene hydroperoxide and metmyoglobin were mixed in a trimyristin-bile salt emulsion. In an ultrafiltration experiment on emulsified oil with a membrane filter of 100 nm pore size, the recovery of quercetin in the filtrate was higher than that of α-tocopherol. These data suggest that quercetin was an antioxidant in the digestion of fish oil. The effectiveness seems to come from its distribution in the emulsified oil, different from that of α-tocopherol, and its ability to scavenge radicals generated from the reaction of lipid hydroperoxides with metmyoglobin.     

Occurrence of a New Enzyme Reducing Metmyoglobin in Dolphin Muscle

A new enzyme has been obtained in a crystalline state from the muscle of blue white dolphin. This enzyme resembles to methemoglobin reductase from erythrocyte with respect to (a) elution pattern of DEAE-Sephadex column chromatography, (b) absorption spectra, (c) molecular weight and (d) activity of reducing methemoglobin, metmyoglobin and ferric cytochrome c. However, distinct differences can be observed between two enzymes with regard to (a) sedimentation coefficient, (b) diffusion coefficient, (c) frictional ratio, (d) pH-mobility curve and (e) specific activity of reducing the above three substrates. It is advocated that enzyme is termed metmyoglobin reductase.     

Co-oxidation of salicylate and cholesterol during the oxidation of metmyoglobin by H2O2

The reaction between metmyoglobin and H2O2 proceeds with oxidation of the hemo-protein iron to a higher valence state and consumption of the peroxide. This reaction is further associated with (a) O2 evolution; (b) hydroxylation of the aromatic compound salicylate to yield a set of dihydroxybenzoic acid derivatives (analyzed by HPLC with electrochemical detection); (c) autoxidation of cholesterol with formation of 3 beta-hydroxy-5-alpha-cholest-6-ene-5-hydroperoxide; and (d) formation of electronically excited states detected by low-level chemiluminescence. The heterolytic scission of the O-O bond of hydroperoxides by metmyoglobin causes the formation of an oxidizing equivalent capable of promoting peroxidation of linoleate and arachidonate (as indicated by the parallel formation of thiobarbituric acid-reactive material and an enhancement of chemiluminescence intensity). The identity of the oxidizing equivalent(s) is discussed in terms of the formation of a relatively stable higher state of oxidation of heme Fe (FeIV-OH or FeV = O) as well as on possible intermediate species derived during the decomposition of H2O2 by metmyoglobin, such as HO.and 1O2. These species might be involved either simultaneously or sequentially in the peroxidation of fatty acids as well as in the tissue damage associated with the formation of H2O2 in ischemic-reperfusion states.     

Kinetic analysis of metsulphmyoglobin and metmyoglobin reduction by Fe(EDTA)2-.

Metsulphmyoglobin prepared from horse heart myoglobin was purified by ion-exchange chromatography to yield a product that on reduction with Fe(EDTA)2- has an A617/A561 ratio greater than 3.5:1. The kinetics of reduction of this purified metsulphmyoglobin and of native metmyoglobin by Fe(EDTA)2- were studied under various conditions of pH, ionic strength and temperature to compare the relative electron-transfer reactivities of a metallochlorin and a metalloporphyrin in identical protein environments. Although the rate of metsulphmyoglobin reduction is 2-7 times that of metmyoglobin under a variety of conditions, this difference can be more than compensated for by the reported difference in mid-point reduction potential between the two forms of the protein. The electrostatic and activation parameters observed for native metmyoglobin and metsulphmyoglobin are essentially identical, and small differences are found in the pH-dependence of the reduction reaction. These findings lead us to conclude that conversion of the porphyrin prosthetic group into a chlorin has relatively little effect on the electron-transfer reactivity of the central metal atom.