Reaction Of Metmyoglobin Research Articles

Direct Detection of the Oxygen Rebound Intermediates, Ferryl Mb and NO2, in the Reaction of metMyoglobin with Peroxynitrite

Oxygenated hemoproteins are known to react rapidly with nitric oxide (NO) to produce peroxynitrite (PN) at the heme site. This process could lead either to attenuation of the effects of NO or to nitrosative protein damage. Peroxynitrite is a powerful nitrating and oxidizing agent that has been implicated in a variety of cell injuries. Accordingly, it is important to delineate the nature and variety of reaction mechanisms of PN reactions with heme proteins. Here, we present direct evidence that ferrylMb and NO(2) are both produced during the reaction of PN and metmyoglobin (metMb). Kinetic evidence indicates that these products evolve from initial formation of a caged radical intermediate [Fe(IV)=O *NO(2)]. This caged pair reacts mainly via internal return with a rate constant k(r) to form metMb and nitrate in an oxygen rebound scenario. Detectable amounts of ferrylMb are observed by stopped-flow spectrophotometry, appearing at a rate consistent with the rate, k(obs), of heme-mediated PN decomposition. Freely diffusing NO(2), which is liberated concomitantly from the radical pair (k(e)), preferentially nitrates Tyr103 in horse heart myoglobin. The ratio of the rates of in-cage rebound and cage escape, k(r)/k(e), was found to be approximately 10 by examining the nitration yields of fluorescein, an external NO(2) trap. This rebound/escape model for the metMb/PN interaction is analogous to the behavior of alkyl hyponitrites and the well-studied geminate recombination processes of deoxymyoglobin with O(2), CO, and NO. The scenario is also similar to the stepwise events of substrate hydroxylation by cytochrome P450 and other oxygenases. It is likely, therefore, that the reaction of metMb with ONOO(-) and that of oxyMb with NO proceed through the same [Fe(IV)=O *NO(2)] caged radical intermediate and lead to similar outcomes. The results indicate that while oxyMb may reduce the concentration of intracellular NO, it would not eliminate the formation of NO(2) as a decomposition product of peroxynitrite.

Inhibition of the metmyoglobin-induced peroxidation of linoleic acid by dietary antioxidants: Action in the aqueous vs. lipid phase

The gastric digestion of food containing oxidizable lipids and iron catalysts for peroxide decomposition such as (met)myoglobin from muscle meat can be accompanied by an extensive formation of potentially toxic lipid hydroperoxides. An early protective action by dietary antioxidants in the gastro-intestinal tract is plausible, especially for poorly bioavailable antioxidants such as polyphenols. Hence, the ability of antioxidants to inhibit lipid peroxidation initiated by dietary iron in mildly acidic emulsions is a valuable and general model. In this work, the ability of some ubiquitous dietary antioxidants representative of the main antioxidant classes (α-tocopherol, the flavonol quercetin, β-carotene) to inhibit the metmyoglobin-induced peroxidation of linoleic acid is investigated by UV–visible spectroscopy and HPLC in mildly acidic emulsions. The phenolic antioxidants quercetin and α-tocopherol come up as the most efficient peroxidation inhibitors. Inhibition by quercetin essentially proceeds in the aqueous phase via a fast reduction of an unidentified activated iron species (with a partially degraded heme) produced by reaction of metmyoglobin with the lipid hydroperoxides. This reaction is faster by, at least, a factor 40 than the reduction of ferrylmyoglobin (independently prepared by reacting metmyoglobin with hydrogen peroxide) by quercetin. By contrast, α-tocopherol mainly acts in the lipid phase by reducing the propagating lipid peroxyl radicals. The poorer inhibition afforded by β-carotene may be related to both its slower reaction with the lipid peroxyl radicals and its competitive degradation by autoxidation and/or photo-oxidation.

Resonance Raman characterization of a high‐spin six‐coordinate iron(III) intermediate in metmyoglobin–azido complex formation trapped by microsecond freeze‐hyperquenching (MHQ)

AbstractThe reaction of metmyoglobin with azide was used to characterize a novel freeze‐quench instrument with a mixing and freezing time resolution in the microsecond time range. Samples quenched within 95 and 245 µs after mixing of metmyoglobin with 1 M azide were characterized by low‐temperature UV–visible and resonance Raman spectroscopy with excitation into the Soret band. Comparison of these data with control samples where azide is absent or metmyoglobin is preincubated with azide demonstrates the formation of an intermediate complex in the first 95 µs after mixing that has fully decayed after 245 µs. Porphyrin skeletal modes displayed by this transient species identify it as a six‐coordinate high‐spin species. Minor low‐spin components are also observed and suggest that the hexacoordinated intermediate exists as a spin equilibrium. This preliminary study demonstrates the feasibility of this approach to detect new intermediates and to characterize early reaction intermediates in other metalloproteins. Copyright © 2005 John Wiley & Sons, Ltd.

Kinetic and Spectroscopic Characterization of a Hydroperoxy Compound in the Reaction of Native Myoglobin with Hydrogen Peroxide

The reaction of metmyoglobin with H2O2 was investigated in a pH range between 8.5 and 6.0 with the aid of stopped flow-rapid scan and rapid freezing-EPR techniques. Singular value decomposition analyses of the stopped flow data at pH 8.5 revealed that a spectral species previously unknown accumulated during the reaction and exhibited a Soret absorption maximum at >/=423 nm. In the EPR experiments, the new species exhibited a set of g values at 2.32, 2.19, and 1.94, indicating that the species was assignable to a ferric hydroperoxy (Fe(III)[O-O-H]-) compound. In contrast, the hydroperoxy compound scarcely accumulated in the reaction at pH 6.0, and the dominant intermediate species accumulated was compound I, which was derived from the oxygen-oxygen bond cleavage of the hydroperoxy compound. The accumulated amount of the hydroperoxy compound relative to compound I showed a pH dependence with an apparent pKa (pKaapp) from 6.95 to 7.27 depending on the metmyoglobins examined. This variation in pKaapp paralleled that in pKa of the acid-alkaline transition (pKaAB) of metmyoglobins, suggesting that the accumulation of hydroperoxy compound is controlled by the distal histidine. We propose that the H2O2 activation by metmyoglobin is promoted at the acidic condition due to the imidazolium form of the distal histidine, and we further propose that the controlled protonation state of the distal histidine is important for the facile O-O bond cleavage in heme peroxidases.

The Interaction of Resveratrol with Ferrylmyoglobin and Peroxynitrite; Protection Against LDL Oxidation

Resveratrol (3,4',5-trihydroxystilbene) is a natural phytoalexin synthesized in response to injury or fungal attack, found in the grape skin and wine, specially red wine. A large number of studies have demonstrated that resveratrol regulates many biological activities, namely protection against atherosclerosis by a set of pharmacological properties, including the antioxidant activity. In this study, we explored the capacity of resveratrol in protecting low density lipoproteins (LDL) against either ferrylmyoglobin- or peroxynitrite-mediated oxidation and the underlying mechanisms of its antioxidant potential. Resveratrol efficiently decreases the accumulation of hydroperoxides in LDL promoted by ferrylmyoglobin, a potent oxidant formed by the reaction of metmyoglobin with hydrogen peroxide, in a concentration-dependent manner, promptly reducing the oxoferryl complex to metmyoglobin. Simultaneously, resveratrol is consumed as detected by the rapid decrease in the characteristic peak at 310 nm, in a similar way to that observed upon its reaction with peroxidase/H 2 O 2 , pointing to a mechanism of one-electron oxidation and subsequent resveratrol dimer formation. On the other hand, resveratrol inhibits LDL apoprotein modifications induced by peroxynitrite, another potent oxidant formed by the reaction between superoxide and nitric oxide, as assessed by the decrease in apo-B net charge alterations and in carbonyl groups formation mediated by that oxidant. Resveratrol also interacts with peroxynitrite in a similar way to that observed with laccases, suggesting a mechanism of resveratrol oxidation rather than a nitration one. These mechanisms are discussed. Considering that either ferrylmyoglobin or peroxynitrite are physiologically relevant oxidants implicated in several pathologies, including atherosclerosis, our results certainly contribute to the understanding of the antioxidant action of resveratrol and consequently provide a new approach for the cardiovascular benefits associated with moderate consumption of red wine.

Stereoselective Electron-Transfer Reactions of Myoglobin and Cytochrome c with Chiral Viologen-Radical Cations

Abstract Electron-transfer (ET) reactions of metmyoglobin (metMb) and the oxidized form of cytochrome c (cyt c(III)) with chiral viologen-radical cations containing (1-phenyl- and 1-cyclohexylethyl)carbamoylmethyl groups were examined at pH 7.0 (10 mM phosphate buffer), ionic strengths (I) of 0.040–0.50 M, and temperatures of 10–35 °C. In the metMb system, the pseudo first-order rate constant of the decay of the viologen-radical cation was saturated with increasing concentrations of metMb, indicating that metMb interacts with the viologen-radical cation to form a complex followed by an intramolecular ET reaction. The stereoselectivity was observed in both the complex formation and the intramolecular ET processes for the (S,S)-isomers of chiral viologens. The ratios of the association constants and the intramolecular ET rate constants of the (S,S)-isomer with those of the (R,R)-isomer were 1.1–1.3 and 1.1–1.5, respectively. The saturation kinetics was not observed for the ET reaction between metMb and the achiral benzylviologen-radical cation, which has no CONH bond, suggesting that the hydrogen bonding of the CONH group of the chiral viologen with the side chain of the polypeptide in metMb plays an important role in the stereoselectivity. No appreciable complex formation was observed in the ET reaction between cyt c(III) and viologen-radical cations, although the stereoselectivity was observed. The ratios of the second-order rate constants of the (S,S)-isomer with those of the (R,R)-isomer were 1.1–1.4. The stereoselectivity is discussed based on the activation parameters.

Electrochemical generation of ferrylmyoglobin during oxidation of styrene with films of DNA and a poly(ester sulfonic acid) ionomer

The chemistry of electrochemically-driven myoglobin-catalyzed oxidation of styrene was investigated in films of DNA or Eastman AQ ionomer on optically transparent electrodes. Conversion of styrene to styrene oxide proceeded via a ferrylmyoglobin radical intermediate. Ferrylmyoglobins were clearly detected by spectroelectrochemistry in films of 1–4 mm thick. The ferrylmyoglobin radical is produced by reaction of metmyoglobin (Mb) in the films with hydrogen peroxide formed by electrochemical catalytic reduction of oxygen catalyzed by Mb. Thus, electrochemically-driven styrene oxidation with these films proceeds by a ‘doubly catalytic’ electrode-driven reduction–oxidation pathway. Ferrylmyoglobin formation during electrolysis of Mb–DNA films in aerobic solutions was much faster, and styrene oxidation occurred with less Mb decomposition compared to the Mb–AQ films. The better performance of Mb–DNA films is correlated with a larger fraction of electroactive Mb and better stability than for the Mb–AQ films.

Oxidation of Adrenaline by Ferrylmyoglobin

The oxidation of adrenaline by ferrylmyoglobin, the product formed by the oxidation of myoglobin with H 2O 2, was examined by absorption, fluorescence, and EPR spectroscopy in terms of the formation of intermediate free radicals and stable molecular products and the binding of adrenaline oxidation products to the apoprotein. The reaction of adrenaline with ferrylmyoglobin resulted in reduction of the hemoprotein to metmyoglobin and consumption of adrenaline. Quantification of metmyoglobin formed per adrenaline yielded a ratio of 1.66. The reaction was found first order on adrenaline concentration and second order on ferrylmyoglobin concentration. This, together with the above ratio, suggested a mechanism by which two oxoferryl moieties (ferrylmyoglobin) were reduced by adrenaline yielding metmyoglobin and the o-semiquinone state of adrenaline. The decay of the o-semiquinone to adrenochrome was confirmed by an increase in absorbance at 485 nm. The product was nonfluorescent; alkalinization of the reaction mixture resulted in a strong fluorescence at 540 nm ascribed to 3,5,6-trihydroxyindol or adrenolutin. Hence, adrenochrome and its alkali-catalyzed product, adrenolutin, are the major molecular products formed during the oxidation of adrenaline by ferrylmyoglobin. Semiquinones formed during the adrenaline/ferrylmyoglobin interaction were detected by EPR, spin stabilizing these species with Mg 2+. The six-line EPR spectrum observed (a N = 4.5 G, a N(CH 3) = 5.1, and a 2H = 0.91; g = 2.0040) may be assigned to the semiquinone forms of adrenochrome and/or adrenolutin or a composite of these species. The intensity of the EPR signal increased with time and its subsequent decay followed a second-order kinetics as inferred by the proportionality of the square of the EPR line intensity with H 2O 2 concentration. Heme destruction and lysine loss, inherent in the reaction of metmyoglobin with H 2O 2, were prevented 80 and 34% by adrenaline, respectively. The low protection exerted by adrenaline against lysine loss was possibly due to the formation of Schiff bases between the ϵ-NH 2 group of lysine and the o-quinone oxidation product(s) of adrenaline. The yield of Schiff base formation was 20–25%. The autoxidation of adrenaline at physiological pH is extremely slow or nonexistent. These data provide a rationale for the primary oxidation of adrenaline by the pseudoperoxidatic activity of ferrylmyoglobin and suggest implications of the free radicals thereby formed for the oxidative damage in reperfusion injury.

Effect of dietary phenolic compounds on apoptosis of human cultured endothelial cells induced by oxidized LDL.

1. Oxidized low density lipoproteins (LDL) are toxic to cultured endothelial cells. Mildly oxidized LDL, characterized by relatively low levels of TBARS and only minor modifications of apoB, were obtained by using 2 experimental model systems of oxidation, namely oxidation by u.v. radiation or ferrylmyoglobin (a two electron oxidation product from the reaction of metmyoglobin with H2O2). 2. Toxic concentrations of mildly oxidized LDL induce apoptosis (programmed cell death) of cultured endothelial cells, as shown by typical morphological features, by the in situ TUNEL procedure and by DNA fragmentation revealed on gel electrophoresis. This apoptosis is calcium-dependent and subsequent to the intense and sustained cytosolic [Ca2+]i peak elicited by oxidized LDL. 3. Five naturally occurring phenolic compounds present in food and beverages were able to prevent, in a concentration-dependent manner, the apoptosis of endothelial cells induced by oxidized LDL. Among the compounds tested, caffeic acid was the most effective. Under the conditions used, the protective effect of caffeic acid (IC50 8.3+/-2.1 micromol l[-1]) in the prevention of apoptosis induced by oxidized LDL was significantly higher than that of the other compounds tested (IC50s were 12.4+/-3.2, 14.1+/-4.1, 20.4+/-4.4 and 72.6+/-9.2 micromol l(-1) for ferulic, protocatechuic, ellagic and p-coumaric acids, respectively). 4. The anti-apoptotic effect of caffeic acid results from the addition of two effects, (i) the antioxidant effect which prevents LDL oxidation and subsequent toxicity ('indirect' protective effect); (ii) a 'direct' cytoprotective effect, acting at the cellular level. 5. Effective concentrations of caffeic acid acted at the cellular level by blocking the intense and sustained cytosolic [Ca2+]i rise elicited by oxidized LDL. 6. In conclusion, phenolic acids (caffeic and ferulic acids being the most potent of the compounds tested under the conditions used) exhibit a potent cytoprotective effect of cultured endothelial cells against oxidized LDL. In addition to antioxidant effect delaying LDL oxidation, caffeic acid acts as a cytoprotective agent, probably by blocking the intracellular signalling triggered by oxidized LDL and culminating in the sustained calcium rise which is involved in oxidized LDL-induced apoptosis.

Cholesteryl Ester Hydroperoxide Formation in Myoglobin-Catalyzed Low Density Lipoprotein Oxidation: Concerted Antioxidant Activity of Caffeic and p-Coumaric Acids with Ascorbate

Two diet-derived phenolic acids, caffeic and p-coumaric acids, interplayed with ascorbate in the protection of low density lipoproteins (LDL) from oxidation promoted by ferrylmyoglobin. Ferrylmyoglobin, a two-electron oxidation product from the reaction of metmyoglobin and H 2O 2, was able to oxidize LDL, degrading free cholesterol and cholesteryl esters. Upon exposure to ferrylmyoglobin, LDL became rapidly depleted of cholesteryl arachidonate and linoleate, which turn into the corresponding hydroperoxides. Cholesteryl oleate and cholesterol were, comparatively, more resistant to oxidation. Caffeic (2 μM) and p-coumaric (12 μM) acids efficiently delayed oxidations, as reflected by an increase in the lag times required for linoleate hydroperoxide and 7-ketocholesterol formation as well as for cholesteryl linoleate consumption. At the same concentration, ascorbate, a standard water-soluble antioxidant, was less efficient than the phenolic acids. Additionally, phenolic acids afforded a protection to LDL that, conversely to ascorbate, extends along the time, as inferred from the high levels of cholesteryl linoleate and cholesteryl arachidonate left after 22 hr of oxidation challenging. Significantly, the coincubation of LDL with ascorbate and each of the phenolic acids resulted in a synergistic protection from oxidation. This was inferred from the lag phases of cholesteryl linoleate hydroperoxide (the major peroxide found in LDL) formation in the presence of mixtures of ascorbate with phenolic acids longer than the sum of individual lag phases of ascorbate and the phenolic acids. A similar description could be drawn for the accumulation of a late product of oxidation, 7-ketocholesterol. It is concluded that ferrylmyoglobin induces a typical pattern of LDL lipid peroxidation, the oxidation rate of cholesteryl esters being a function of unsaturation; furthermore, there is a synergistic antioxidant activity of diet-derived phenolic acids with ascorbate in the protection of LDL from oxidation, a finding of putative physiological relevance.

Stimulation by Nitroxides of Catalase-like Activity of Hemeproteins: KINETICS AND MECHANISM

The ability of stable nitroxide radicals to detoxify hypervalent heme proteins such as ferrylmyoglobin (MbFeIV) produced in the reaction of metmyoglobin (MbFeIII) and H2O2 was evaluated by monitoring O2 evolution, H2O2 depletion, and redox changes of the heme prosthetic group. The rate of H2O2 depletion and O2 evolution catalyzed by MbFeIII was enhanced by stable nitroxides such as 4-OH-2,2,6,6-tetramethyl-piperidinoxyl (TPL) in a catalytic fashion. The reduction of MbFeIV to MbFeIII was the rate-limiting step. Excess TPL over MbFeIII enhanced catalase-like activity more than 4-fold. During dismutation of H2O2, [TPL] and [MbFeIV] remained constant. NADH caused: (a) inhibition of H2O2 decay; (b) progressive reduction of TPL to its respective hydroxylamine TPL-H; and (c) arrest/inhibition of oxygen evolution or elicit consumption of O2. Following depletion of NADH the evolution of O2 resumed, and the initial concentration of TPL was restored. Kinetic analysis showed that two distinct forms of MbFeIV might be involved in the process. In summary, by shuttling between two oxidation states, namely nitroxide and oxoammonium cation, stable nitroxides enhance the catalase mimic activity of MbFeIII, thus facilitating H2O2 dismutation accompanied by O2 evolution and providing protection against hypervalent heme proteins.

Reduction of ferrylmyoglobin and ferrylhemoglobin by nitric oxide: a protective mechanism against ferryl hemoprotein-induced oxidations.

The reactions of metmyoglobin (metMb) and methemoglobin (metHb), oxidized to their respective oxoferryl free radical species (.Mb-FeIV = O/.Hb-4FeIV = O) by tert-butyl hydroperoxide (t-BuOOH), with nitric oxide (NO.) were studied by a combination of optical, electron spin resonance (ESR), ionspray mass (MS), fluorescence, and chemiluminescence spectrometries to gain insight into the mechanism by which NO. protects against oxidative injury produced by .Mb-FeIV = O/.Hb-4FeIV = O. Oxidation of metMb/metHb by t-BuOOH in a nitrogen atmosphere proceeded via the formation of two protein electrophilic centers, which were heme oxoferryl and the apoprotein radical centered at tyrosine (for the .Mb-FeIV = O form, the g value was calculated to be 2.0057), and was accompanied by the formation of t-BuOOH-derived tert-butyl(per)oxyl radicals. We hypothesized that NO. may reduce both oxoferryl and apoprotein free radical electrophilic centers of .Mb-FeIV = O/.Hb-4FeIV = O and eliminate tert-butyl(per)oxyl radicals, thus protecting against oxidative damage. We found that NO. reduced .Mb-FeIV = O/.Hb-4FeIV = O to their respective ferric (met) forms and prevented the following: (i) oxidation of cis-parinaric acid (PnA) in liposomes, (ii) oxidation of luminol, and (iii) formation of the tert-butyl(per)oxyl adduct with the spin trap DMPO. NO. eliminated the signals of tyrosyl radical detected by ESR and oxoferryl detected by MS in the reaction of t-BuOOH with metMb. As evidenced by MS of apomyoglobin, this effect was due to the two-electron reduction of .Mb-FeIV = O by NO. at the oxoferryl center rather than to nitrosylation of the tyrosine residues. Results of our in vitro experiments suggest that NO. exhibits a potent, targetable antioxidant effect against oxidative damage produced by oxoferryl Mb/Hb.

Decay Studies of Dmpo-Spin Adducts of Free Radicals Produced by Reactions of Metmyoglobin and Methemoglobin with Hydrogen Peroxide

The 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) spin adduct of myoglobin (Mb) or hemoglobin (Hb) was formed when metmyoglobin (MetMb) or methemoglobin (MetHb) reacted with H2O2 in the presence of DMPO, and both decayed with half-life of a few minutes. The DMPO spin adduct of Mb decayed with biphasic kinetics with k1 = 0.645 min-1 and k2 = 0.012 min-1, indicating that the spin adduct consisted of two kinetically heterogeneous species, stable and unstable ones. The DPMO spin adduct of Hb, however, was homogeneous. Decay of both spin adducts was accelerated in the presence of tyrosine, tryptophan or cysteine, but not phenylalanine, methionine or histidine. The decay obeyed the first order kinetics at varying concentrations of the spin adducts. The decay was accelerated by denaturation and proteolysis of protein moiety. The decay rate was not affected by the extra addition of MetMb or MetHb to each spin adduct. The decay rate of the spin adduct of Mb was increased by hematin in the presence of H2O2 and decreased by catalase. Decay of stable spin adduct of Mb, however, was not significantly changed under any experimental conditions used. These results led us to conclude that instability of the DMPO-spin adducts of Mb and Hb is due to intramolecular redox reactions between the spin adducts and amino acid residues and/or products of the reaction between heme and H2O2.

Identification of initiating agents in myoglobin-induced lipid peroxidation

Reaction of metmyoglobin with peroxides is known to involve the formation, possibly via a porphyrin radical-cation, of a ferryl (iron(IV)-oxo) species and a protein radical; there is a little information available as to which of these species initiates the damage which is observed on incubating membrane fractions with such mixtures. It is shown in this study that the initial protein radical, which is a tyrosine phenoxyl radical centered at position 103 in the protein, does not react rapidly with erythrocyte membranes. However a tyrosine peroxyl radical, formed by addition of oxygen to this species, does react rapidly, and it is concluded that this radical, together with the ferryl species, is an important initiating species in myoglobin-induced lipid peroxidation.

Identification of a globin free radical in equine myoglobin treated with peroxides

Reaction of metmyoglobin (MetMb) with H 2O 2 generates a ferryl species which is analogous to Compound II of peroxidases and is one oxidising equivalent above the initial level. The second oxidising equivalent from the peroxide is rapidly transferred into the surrounding protein generating a protein radical. Previous studies have suggested that this radical is centered on an aromatic residue, probably a tyrosine, but have not allowed unambiguous assignment. The identity of this radical has been investigated using stopped flow EPR. Reaction of equine MetMb with equimolar H 2O 2 gives a 6-line signal with g 2.0044 and coupling constants approx. α H 2.26 and α 2H 0.75 mT, together with a second, broad, underlying signal. Both signals decay rapidly. The parameters of the multiplet signal are consistent with the presence of a sterically constrained tyrosine phenoxyl radical. This radical reacts further with O 2, H 2O 2 and a number of other substrates. This activity, together with the observation that such a species is not detected with MetMb which has been iodinated solely at the Tyr-103 residue, suggests that this species is being generated from the readily accessible Tyr-103 residue, which is near the haem centre, rather than the more distant, buried, Tyr-146 residue. Reaction of this phenoxyl radical with oxygen gives the tyrosine peroxyl radical which has been previously trapped with the spin trap DMPO.

ESR spin-trapping studies of free radicals generated by hydrogen peroxide activation of metmyoglobin

The reaction of metmyoglobin (MetMb) with hydrogen peroxide (H 2 O 2 ) generated a free radical that was detectable by ESR at room temperature only in the presence of the spin trap 5,5-dimethyl-1-pyrroline-N-oxyl (DMPO). The formation of the free radical was favored at pH≥7 and was depressed at low pH. The free radical spin-trapped by DMPO may be a ferryl radical or an amino acid free radical originating from the myoglobin moiety

Detection of Myoglobin-Derived Radicals On Reaction of Metmyoglobin With Hydrogen Peroxide and Other Peroxidic Compounds

The reaction of metmyoglobin with equimolar concentrations of hydrogen peroxide has been studied using both electron spin resonance (e.s.r.) and optical spectroscopy. Using the former technique a strong anisotropic e.s.r. signal is observed, in the presence of the spin trap DMPO, which decays relatively rapidly. This previously unobserved signal, which is also observed on reaction of metmyoglobin with a number of other powerful oxidants (peracetic acid, 3-chloroperoxybenzoic acid, monoperoxyphthalic acid, iodosyl benzene, tBuOOH and cumene hydroperoxide) is assigned to a slowly-tumbling, metmyoglobin-derived, spin adduct. The parameters of this signal (aN 1.45, aH 0.83 mT) are consistent with the trapped radical having a heteroatom centre: this is believed to be oxygen. The concentration of this species is not affected by compounds such as 2-deoxyribose, mannitol and phenylalanine which are all efficient hydroxyl radical scavengers, demonstrating that the formation of this radical is not due to reaction of "free" HO. generated by breakdown of H2O2 by released iron ions. The concentration of this species is however decreased by desferal, ascorbate. Trolox C, salicylate and, to a lesser extent, linoleic acid; with the first three of these compounds further substrate-derived radicals are also observed. Examination of similar reaction systems (though in the absence of DMPO) by optical spectroscopy shows that the myoglobin (IV) species is formed and that this species behaves in a somewhat different manner with these added compounds. These results suggest that the radical trapped in the e.s.r. experiments is a myoglobin-derived species, probably a tyrosine peroxyl radical, arising from oxidative damage to the globin moiety. The diminution of both the e.s.r. signal of the spin adduct and the optical absorption of the myoglobin (IV) species in the presence of linoleic acid suggests that these myoglobin-derived species can initiate oxidative damage but that this process can be ameliorated by the presence of a number of water-soluble compounds such as ascorbate, Trolox C, desferal and salicylate.

Direct Detection of Peroxyl Radicals Formed in the Reactions of Metmyoglobin and Methaemoglobin with t-butyl Hydroperoxide

The reaction of metmyoglobin and methaemoglobin with t-butyl hydroperoxide has been investigated using stopped-flow e.s.r. spectroscopy. The major species observed immediately after mixing has been identified as the basis of its g value (2.014) and line width as being due to a peroxyl radical. The detection of this species under anaerobic conditions suggests that this species is the t-butyl peroxyl radical rather than a secondary species. The immediate observation of this species on mixing suggests that this species is arising via a reaction involving the intact haem moiety rather than via 'free iron' produced via oxidative damage. Detection of a second, oxygen-dependent, species, which is formed at a slower rate, in the metmyoglobin reaction suggests that the initial peroxyl radical subsequently reacts with the protein to give a protein-derived radical.

ChemInform Abstract: Metmyoglobin and Methemoglobin as Efficient Traps for Nitrosyl Hydride (Nitroxyl) in Neutral Aqueous Solution.

AbstractThe reactions of metmyoglobin and methemoglobin with trioxodinitrate monoanion HN2O3′ in neutral aqueous solution have been studied at 25°C under anaerobic conditions.

Reactivity differences between haemoglobins. Part XIV. The thermodynamics of the reaction of sperm whale metmyoglobin with azide and hydroxyl ions

The formation constants of the azide and hydroxyl ion complexes of sperm whale metmyoglobin have been measured as a function of pH and temperature. The observed behaviour is that of a typical methaemoglobin in that small variations with pH of the free energy of complex formation are accompanied by much larger compensating enthalpy and entropy changes, –ΔH° passing through a maximum. The behaviour of hydroxyl ion as a ligand is different from that for other ligands studied in this series, –ΔH° passing through a minimum at a pH close to that for which –ΔH° is a maximum for other ligands. The amount of hydrogen ion taken up upon reaction of metmyoglobin with azide ion has been measured as a function of pH and ionic strength. Using an expression for the activity coefficient of the metmyoglobin based on a dielectric cavity model for the protein the variation of log KL with pH at I= 0·01 is shown to be consistent with the amount of hydrogen ion taken up.