Oxidation of oxymyoglobin to metmyoglobin with hydrogen peroxide: involvement of ferryl intermediate

Hydrogen peroxide, one of the potent oxidants in muscle tissues, can induce very rapid oxidation of oxymyoglobin (MbO2) to metmyoglobin (metMb) with an apparent rate constant of 7.5 X 10(4) h-1 M-1 (i.e., 20.8 s-1 M-1) over the wide pH range of 5.5-10.2 in 0.1 M buffer at 25 degrees C. Its molecular mechanism, however, is quite different from that of the autoxidation of MbO2 to metMb. Kinetic analysis has revealed that the hydrogen peroxide oxidation proceeds through the formation of ferryl-Mb(IV) from deoxy-Mb(II), which is in equilibrium with MbO2, by a two-equivalent oxidation with H2O2. Once the ferryl species is formed, it reacts rapidly with another deoxy-Mb(II) in a bimolecular fashion so as to yield 2 mol of metMb(III). Under physiological conditions, the rate-determining step was the oxidation of the deoxy species by H2O2, its rate constant being estimated to be on the order of 3.6 X 10(3) s-1 M-1 at 25 degrees C. These findings leads us to the view that a good supply of dioxygen provides rather an important defense against the oxidation of myoglobin with hydrogen peroxide in cardiac and skeletal muscle tissues.     

Hydrogen peroxide plays a key role in the oxidation reaction of myoglobin by molecular oxygen. A computer simulation.

The stability properties of the iron(II)-dioxygen bond in myoglobin and hemoglobin are of particular importance, because both proteins are oxidized easily to the ferric met-form, which cannot be oxygenated and is therefore physiologically inactive. In this paper, we have formulated all the possible pathways leading to the oxidation of myoglobin to metmyoglobin with each required rate constant in 0.1 M buffer (pH 7.0) at 25 degrees C, and have set up six rate equations for the elementary processes going on in a simultaneous way. By using the Runge-Kutta method to solve these differential equations, the concentration progress curves were then displayed for all the reactive species involved. In this complex reaction, the primary event was the autoxidation of MbO2 to metMb with generation of the superoxide anion, this anion being converted immediately and almost completely into H2O2 by the spontaneous dismutation. Under air-saturated conditions (PO2 = 150 Torr), the H2O2 produced was decomposed mostly by the metMb resulting from the autoxidation of MbO2. At lower pressures of O2, however, H2O2 can act as the most potent oxidant of the deoxyMb, which increases with decreasing O2 pressures, so that there appeared a well defined maximum rate in the formation of metMb at approximately 5 Torr of oxygen. Such examinations with the aid of a computer provide us, for the first time, with a full picture of the oxidation reaction of myoglobin as a function of oxygen pressures. These results also seem to be of primary importance from a point of view of clinical biochemistry of the oxygen supply, as well as of pathophysiology of ischemia, in red muscles such as cardiac and skeletal muscle tissues.     

Autoxidation of myoglobin from bigeye tuna fish (Thunnus obesus)

Native oxymyoglobin (MbO2) was isolated directly from the skeletal muscle of bigeye tuna (Thunnus obesus) with complete separation from metmyoglobin (metMb) on a CM-cellulose column. It was examined for its stability properties over a wide range of pH values (pH 5-12) in 0.1 M buffer at 25 degrees C. When compared with sperm whale MbO2 as a reference, the tuna MbO2 was found to be much more susceptible to autoxidation. Kinetic analysis has revealed that the rate constant for a nucleophilic displacement of O2- from MbO2 by an entering water molecule is 10-times higher than the corresponding value for sperm whale MbO2. The magnitude of the circular dichroism of bigeye tuna myoglobin at 222 nm was comparable to that of sperm whale myoglobin, but its hydropathy profile revealed the region corresponding to the distal side of the heme iron to be apparently less hydrophobic. The kinetic simulation also demonstrated that accessibility of the solvent water molecule to the heme pocket is clearly a key factor in the stability properties of the bound dioxygen.     

Role of globin moiety in the autoxidation reaction of oxymyoglobin: effect of 8 M urea.

It is in the ferrous form that myoglobin or hemoglobin can bind molecular oxygen reversibly and carry out its function. To understand the possible role of the globin moiety in stabilizing the FeO2 bond in these proteins, we examined the autoxidation rate of bovine heart oxymyoglobin (MbO2) to its ferric met-form (metMb) in the presence of 8 M urea at 25 degrees C and found that the rate was markedly enhanced above the normal autoxidation in buffer alone over the whole range of pH 5-13. Taking into account the concomitant process of unfolding of the protein in 8 M urea, we then formulated a kinetic procedure to estimate the autoxidation rate of the unfolded form of MbO2 that might appear transiently in the possible pathway of denaturation. As a result, the fully denatured MbO2 was disclosed to be extremely susceptible to autoxidation with an almost constant rate over a wide range of pH 5-11. At pH 8.5, for instance, its rate was nearly 1000 times higher than the corresponding value of native MbO2. These findings lead us to conclude that the unfolding of the globin moiety allows much easier attack of the solvent water molecule or hydroxyl ion on the FeO2 center and causes a very rapid formation of the ferric met-species by the nucleophilic displacement mechanism. In the molecular evolution from simple ferrous complexes to myoglobin and hemoglobin molecules, therefore, the protein matrix can be depicted as a breakwater of the FeO2 bonding against protic, aqueous solvents.     

Dual nature of the distal histidine residue in the autoxidation reaction of myoglobin and hemoglobin comparison of the H64 mutants.

The oxygenated form of myoglobin or hemoglobin is oxidized easily to the ferric met-form with generation of the superoxide anion. To make clear the possible role(s) of the distal histidine (H64) residue in the reaction, we have carried out detailed pH-dependence studies of the autoxidation rate, using some typical H64 mutants of sperm whale myoglobin, over the wide range of pH 5-12 in 0.1 M buffer at 25 degrees C. Each mutation caused a dramatic increase in the autoxidation rate with the trend H64V >/= H64G >/= H64L > H64Q > H64 (wild-type) at pH 7.0, whereas each mutant protein showed a characteristic pH-profile which is essentially different from that of the wild-type or native sperm whale MbO2. In particular, all the mutants have lost the acid-catalyzed process that can play a dominant role in the autoxidation reaction of most mammalian myoglobins or hemoglobins. Kinetic analyses of various types of pH-profiles lead us to conclude that the distal histidine residue can play a dual role in the nucleophilic displacement of O2- from MbO2 or HbO2 in protic, aqueous solution. One is in a proton-relay mechanism via its imidazole ring, and the other is in the maximum protection of the FeO2 center against a water molecule or an hydroxyl ion that can enter the heme pocket from the surrounding solvent.     

Shark myoglobins. II. Isolation, characterization and amino acid sequence of myoglobin from Galeorhinus japonicus

Native oxymyoglobin (MbO2) was isolated from red muscle of G. japonicus by chromatographic separation from metmyoglobin (metMb) on DEAE-cellulose and the amino acid sequence of the major chain was determined with the aid of sequence homology with that of G. australis. It was shown to differ in amino acid sequence from that of G. australis by 10 replacements, to be acetylated at the amino terminus and to contain glutamine at the distal (E7) residue. It was also shown to have a spectrum very similar to that of mammalian MbO2. However, the pH-dependence for the autoxidation of MbO2 was seen to be quite different from that of sperm whale (Physeter catodon) MbO2. Although the sequence homology between sperm whale and G. japonicus myoglobins is about 40%, their hydropathy profiles were very similar, indicating that they have a similar geometry in their globin folding.     

Aplysia oxymyoglobin with an unusual stability property: involvement of two kinds of carboxyl groups

Unlike mammalian oxymyoglobins, Aplysia MbO2 is extremely susceptible to autoxidation, and its pH dependence is also unusual. Kinetic formulation has revealed that two kinds of dissociable group with pK1 = 4.3 and pK2 = 6.1, respectively, at 25 degrees C are involved in the stability property of Aplysia MbO2. In order to characterize thermodynamically these dissociation processes involved, the effect of temperature on K1 and K2 was studied by analyzing the pH dependence for the autoxidation rate of Aplysia MbO2 in 0.1 M buffer over the pH range of 4-11, and at 15, 25 and 35 degrees C. The resulting thermodynamic parameters for each group were both those to be expected for the ionization of a carboxyl group; the delta H degrees value being numerically much less than 1 kcal.mol-1, or zero in practice, but being associated with a large negative value of delta S degrees of the order of -20 cal.mol-1.K-1. Taking into account the fact that Aplysia myoglobin contains only a single histidine residue corresponding to the heme-binding proximal one, we can unequivocally conclude that the two kinds of the dissociable group involved in the unusual stability of Aplysia MbO2 must both be carboxyl groups, the protonation of these groups being responsible for an increase in its autoxidation rate in the acidic pH range.     

Kinetic analysis and mechanistic aspects of autoxidation of catechins

A peroxidase-based bioelectrochemical sensor of hydrogen peroxide (H(2)O(2)) and a Clark-type oxygen electrode were applied to continuous monitoring and kinetic analysis of the autoxidation of catechins. Four major catechins in green tea, (-)-epicatechin, (-)-epicatechin gallate, (-)-epigallocatechin, and (-)-epigallocatechin gallate, were used as model compounds. It was found that dioxygen (O(2)) is quantitatively reduced to H(2)O(2). The initial rate of autoxidation is suppressed by superoxide dismutase and H(+), but is independent of buffer capacity. Based on these results, a mechanism of autoxidation is proposed; the initial step is the one-electron oxidation of the B ring of catechins by O(2) to generate a superoxide anion (O(2)(*-)) and a semiquinone radical, as supported in part by electron spin resonance measurements. O(2)(*-) works as a stronger one-electron oxidant than O(2) against catechins and is reduced to H(2)O(2). The semiquinone radical is more susceptible to oxidation with O(2) than fully reduced catechins. The autoxidation rate increases with pH. This behavior can be interpreted in terms of the increase in the stability of O(2)(*-) and the semiquinone radical with increasing pH, rather than the acid dissociation of phenolic groups. Cupric ion enhances autoxidation; most probably it functions as a catalyst of the initial oxidation step of catechins. The product cuprous ion can trigger a Fenton reaction to generate hydroxyl radical. On the other hand, borate ion suppresses autoxidation drastically, due to the strong complex formation with catechins. The biological significance of autoxidation and its effectors are also discussed.     

Role of myoglobin as a scavenger of cellular NO in myocardium

Recent studies have detected a (1)H nuclear magnetic resonance (NMR) reporter signal of metmyoglobin (metMb) during bradykinin stimulation of an isolated mouse heart. The observation has led to the hypothesis that Mb reacts with cellular nitric oxide (NO). However, the hypothesis depends on an unequivocal detection of metMb signals in vivo. In solution, nitrite oxidization of Mb produces a characteristic set of paramagnetically shifted (1)H NMR signals. In the upfield spectral region, MbO(2) and MbCO exhibit the gammaCH(3) Val E11 signals at -2.8 and -2.4 ppm, respectively. In the same spectral region, nitrite oxidation of Mb produces a set of signals at -3.7 and -4.7 ppm at 35 degrees C. Previous studies have confirmed the visibility of metMb signals in perfused rat myocardium. With bradykinin infusion, perfusion pressure and rate-pressure product decrease, consistent with endogenous NO formation. However, neither myocardial O(2) consumption nor high-energy phosphate levels, as reflected in the (31)P NMR signals, show any significant change. Bradykinin still triggers a similar physiological response even in the presence of CO that is sufficient to inhibit 86% Mb. In all cases, the (1)H NMR spectra from perfused rat myocardium reveal no metMb signals. The results suggest that bradykinin-induced NO does not interact significantly with cellular Mb to produce an NMR-detectable quantity of metMb in the perfused rat myocardium. As a consequence, the experiments cannot confirm the intriguing proposal that Mb acts as a cellular NO scavenger.     

The interaction of oxymyoglobin with hydrogen peroxide: a kinetic anomaly at large excesses of hydrogen peroxide

The reaction of oxymyoglobin (MbO2) with H2O2 has been examined at pH 7.2 and 20(+/- 2) degrees C for reactant ratios of [H2O2]:[MbO2] greater than approximately 15:1. Under the conditions of large excesses of H2O2, the reaction is characterized by an increase in the rate of loss of MbO2 as [H2O2] is increased, for which a value of k(MbO2 + H2O2) approximately 3 M-1 s-1 is obtained. This kinetic behavior contrasts the saturation kinetics observed previously at lower values of [H2O2]. The change in kinetics at increasing excesses of H2O2 is accompanied by a progressive tendency toward the direct formation of ferrimyoglobin at the expense of ferrylmyoglobin formation. A mechanism is proposed in which an initially formed intermediate produces the ferryl derivative in competition with the formation of ferrimyoglobin through the interaction of further H2O2. Overall, the H2O2 is catalytically decomposed by the MbO2. This mechanism is integrated with that determined previously at low excesses of H2O2 into a complex general scheme that applies over the entire studied range of [H2O2]:[MbO2]. No evidence is obtained for the conversion of ferrylmyoglobin to oxymyoglobin by the large excesses of H2O2, regardless of whether the ferryl derivative is the product of the reaction of H2O2 with the oxy or ferri derivative of myoglobin.     

Nature of the FeO2 bonding in myoglobin and hemoglobin: A new molecular paradigm

The iron(II)-dioxygen bond in myoglobin and hemoglobin is a subject of wide interest. Studies range from examinations of physical-chemical properties dependent on its electronic structure, to investigations of the stability as a function of oxygen supply. Among these, stability properties are of particular importance in vivo. Like all known dioxygen carriers synthesized so far with transition metals, the oxygenated forms of myoglobin and hemoglobin are known to be oxidized easily to their ferric met-forms, which cannot bind molecular oxygen and are therefore physiologically inactive. The mechanistic details of this autoxidation reaction, which are of clinical, as well as of physical-chemical, interest, have long been investigated by a number of authors, but a full understanding of the heme oxidation has not been reached so far. Recent kinetic and thermodynamic studies of the stability of oxymyoglobin (MbO2) and oxyhemoglobin (HbO2) have revealed new features in the FeO2 bonding. In vivo, the iron center is always subject to a nucleophilic attack of the water molecule or hydroxyl ion, which can enter the heme pocket from the surrounding solvent and thereby irreversibly displace the bound dioxygen from MbO2 or HbO2 in the form of O2- so that the iron is converted to the ferric met-form. Since the autoxidation reaction of MbO2 or HbO2 proceeds through a nucleophilic displacement following one-electron transfer from iron(II) to the bound O2, this reaction may be viewed as a meeting point of the stabilization and the activation of molecular oxygen performed by hemoproteins. Along with these lines of evidence, we finally discuss the stability property of human HbO2 and provide with the most recent state of hemoglobin research. The HbA molecule contains two types of alphabeta contacts and seems to differentiate them quite properly for its functional properties. The alpha1beta2 or alpha2beta1 contact is associated with the cooperative oxygen binding, whereas the alpha1beta1 or alpha2beta2 contact is used for controlling the stability of the bound O2. We can thus form a unified picture for hemoglobin function by closely integrating the cooperative and the stable binding of molecular oxygen with iron(II) in aqueous solvent. These new views on the nature of FeO2 bonding and the possible role of globin moiety in stabilizing MbO2 and HbO2 are of primary importance, not only for a full understanding of various hemoprotein reactions with O2, but also for planning new molecular designs for synthetic oxygen carriers which may be able to function in aqueous solvent and at physiological temperature.     

Amino acid sequence of myoglobin from the mollusc Dolabella auricularia

The complete amino acid sequence of the myoglobin from Dolabella auricularia, a common gastropodic mollusc on the Japanese coast, has been determined. The myoglobin is composed of 146 amino acid residues, is acetylated at the NH2 terminus, and contains a single histidine residue at position 95 which most likely corresponds to the heme-binding proximal histidine. The sequence of Dolabella myoglobin shows strong homology (72-77%) with those of Aplysia myoglobins. The autoxidation rate of Dolabella oxymyoglobin (MbO2) was examined in 0.1 M buffer at 25 degrees C over pH range 4.8-12. Dolabella MbO2 was extremely unstable between pH 7 and 11, and the pH dependence of the stability was quite different from that of sperm whale MbO2. This property may be partly due to the absence of a distal (E7) histidine in Dolabella myoglobin.     

Observation of an isotope-sensitive low-frequency Raman band specific to metmyoglobin

A resonance Raman band involving significantly the iron(III)-histidine stretching (upsilonFe-His) character is identified for metmyoglobin (metMb) through isotope sensitivity of its low-frequency resonance Raman bands, but the identification was not successful for methemoglobin (metHb) and its isolated alpha and beta subunits. A band at 218 cm-1 of natural abundance metMb exhibited a low-frequency shift for 15N-His-labeled metMb (-1.4 cm-1 shift), while the strong porphyrin bands at 248 and 271 cm-1 did not shift significantly. The frequency of the 218-cm-1 band of metMb decreased by 1.6 cm-1 in D2O, probably due to Ndelta-deuteration of the proximal His, in a similar manner to that of the upsilonFe-His band of deoxyMb in D2O. This 218-cm-1 band shifted slightly to a lower frequency in H2(18)O, whereas it did little upon 54Fe isotopic substitution (<0.3 cm-1), presumably because of the six-coordinate structure. The lack of the 54Fe-isotope shift shows that the 218-cm-1 band is specific to metMb and not due to the deoxy species. The intensity of this band decreased for hydroxymetMb and was indiscernible for cyanometMb. For metHb and its alpha and beta subunits, however, the frequencies of the band around 220 cm-1 were not D2O sensitive. These results suggest an assignment of the band around 220 cm-1 to a pyrrole tilting mode, which significantly contains the Fe-His stretching character for metMb but scarcely for metHb and its subunits. The differences in the isotope sensitivity of this band in different proteins are considered to reflect the heme distortion from the planarity and the Fe-His geometry specific to individual proteins.     

A long-lived o-semiquinone radical anion is formed from N-beta-alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), an insect-derived antibacterial substance

N-beta-Alanyl-5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD), an insect-derived antibacterial peptide, generates hydrogen peroxide (H(2)O(2)) that exerts antitumour activity. We have investigated the precise mechanism of H(2)O(2) production from 5-S-GAD by autoxidation aiming to understand its action toward tumour cells. Using the electron spin resonance (ESR) technique, we detected a strong signal due to radical formation from 5-S-GAD. Surprisingly, the ESR signal of the radical derived from 5-S-GAD appeared after incubation for 30 min at 37 degrees C in the buffer at pH 7.4; the signal was persistently detected for 10 h in the absence of catalytic metal ions. The computer simulation of the observed ESR spectrum together with the theoretical calculation of the spin density of the radical species indicates that an o-semiquinone radical anion was formed from 5-S-GAD. We demonstrated that H(2)O(2) is produced via the formation of superoxide anion O2(.-) by the electron-transfer reduction of molecular oxygen by the 5-S-GAD anion, which is in equilibrium with 5-S-GAD in the aqueous solution. The radical formation and the subsequent H(2)O(2) production were inhibited by superoxide dismutase (SOD), when the antitumour activity of 5-S-GAD was inhibited by SOD. Thus, the formation of the o-semiquinone radical anion would be necessary for the antitumour activity of 5-S-GAD as an intermediate in the production of cytotoxic H(2)O(2).     

Aplysia oxymyoglobin with an unusual stability property

Native oxymyoglobin was isolated directly from the radular muscle of Aplysia kurodai with complete separation from metmyoglobin on a DEAE-cellulose column. It was examined for its spectral and stability properties. The spectrum of Aplysia MbO2 , which lacks the distal histidine, is very similar to those of mammalian oxymyoglobins , the alpha-peak being higher than the beta-peak and the absorbance ratio being 1.03. Its stability, however, is quite different from those of the mammalian oxymyoglobins , and Aplysia MbO2 is found to be extremely susceptible to autoxidation. Its rate is one-hundred times higher at pH 9.0, and its pH dependence is unusual and much less steep, when compared with sperm whale MbO2 as reference.     

On the mechanism of the Mn3(+)-induced neurotoxicity of dopamine:prevention of quinone-derived oxygen toxicity by DT diaphorase and superoxide dismutase

Dopamine (DA) is rapidly oxidized by Mn3(+)-pyrophosphate to its cyclized o-quinone (cDAoQ), a reaction which can be prevented by NADH, reduced glutathione (GSH) or ascorbic acid. The oxidation of DA by Mn3+, which appears to be irreversible, results in a decrease in the level of DA, but not in a formation of reactive oxygen species, since oxygen is neither consumed nor required in this reaction. The formation of cDAoQ can initiate the generation of superoxide radicals (O2-.) by reduction-oxidation cycling, i.e. one-electron reduction of the quinone by various NADH- or NADPH-dependent flavoproteins to the semiquinone (QH.), which is readily reoxidized by O2 with the concomitant formation of O2-.. This mechanism is believed to underly the cytotoxicity of many quinones. Two-electron reduction of cDAoQ to the hydroquinone can be catalyzed by the flavoprotein DT diaphorase (NAD(P)H:quinone oxidoreductase). This enzyme efficiently maintains DA quinone in its fully reduced state, although some reoxidation of the hydroquinone (QH2) is observed (QH2 + O2----QH. + O2-. + H+; QH. + O2----Q + O2-.). In the presence of Mn3+, generated from Mn2+ by O2-. (Mn2+ + 2H+ + O2-.----Mn3+ + H2O2) formed during the autoxidation of DA hydroquinone, the rate of autoxidation is increased dramatically as is the formation of H2O2. Furthermore, cDAoQ is no longer fully reduced and the steady-state ratio between the hydroquinone and the quinone is dependent on the amount of DT diaphorase present. The generation of Mn3+ is inhibited by superoxide dismutase (SOD), which catalyzes the disproportionation of O2-. to H2O2 and O2. It is noteworthy that addition of SOD does not only result in a decrease in the amount of H2O2 formed during the regeneration of Mn3+, but, in fact, prevents H2O2 formation. Furthermore, in the presence of this enzyme the consumption of O2 is low, as is the oxidation of NADH, due to autoxidation of the hydroquinone, and the cyclized DA o-quinone is found to be fully reduced. These observations can be explained by the newly-discovered role of SOD as a superoxide:semiquinone (QH.) oxidoreductase catalyzing the following reaction: O2-. + QH. + 2H+----QH2 + O2. Thus, the combination of DT diaphorase and SOD is an efficient system for maintaining cDAoQ in its fully reduced state, a prerequisite for detoxication of the quinone by conjugation with sulfate or glucuronic acid. In addition, only minute amounts of reactive oxygen species will be formed, i.e. by the generation of O2-., which through disproportionation to H2O2 and further reduction by ferrous ions can be converted to the hydroxyl radical (OH.). Absence or low levels of these enzymes may create an oxidative stress on the cell and thereby initiate events leading to cell death.     

Oxygen activation during oxidation of methoxyhydroquinones by laccase from Pleurotus eryngii

Oxygen activation during oxidation of the lignin-derived hydroquinones 2-methoxy-1,4-benzohydroquinone (MBQH(2)) and 2, 6-dimethoxy-1,4-benzohydroquinone (DBQH(2)) by laccase from Pleurotus eryngii was examined. Laccase oxidized DBQH(2) more efficiently than it oxidized MBQH(2); both the affinity and maximal velocity of oxidation were higher for DBQH(2) than for MBQH(2). Autoxidation of the semiquinones produced by laccase led to the activation of oxygen, producing superoxide anion radicals (Q(*-) + O(2) <--> Q + O(2)(*-)). As this reaction is reversible, its existence was first noted in studies of the effect of systems consuming and producing O(2)(*-) on quinone formation rates. Then, the production of H(2)O(2) in laccase reactions, as a consequence of O(2)(*-) dismutation, confirmed that semiquinones autoxidized. The highest H(2)O(2) levels were obtained with DBQH(2), indicating that DBQ(*-) autoxidized to a greater extent than did MBQ(*-). Besides undergoing autoxidation, semiquinones were found to be transformed into quinones via dismutation and laccase oxidation. Two ways of favoring semiquinone autoxidation over dismutation and laccase oxidation were increasing the rate of O(2)(*-) consumption with superoxide dismutase (SOD) and recycling of quinones with diaphorase (a reductase catalyzing the divalent reduction of quinones). These two strategies made the laccase reaction conditions more natural, since O(2)(*-), besides undergoing dismutation, reacts with Mn(2+), Fe(3+), and aromatic radicals. In addition, quinones are continuously reduced by the mycelium of white-rot fungi. The presence of SOD in laccase reactions increased the extent of autoxidation of 100 microM concentrations of MBQ(*-) and DBQ(*-) from 4.5 to 30.6% and from 19.6 to 40.0%, respectively. With diaphorase, the extent of MBQ(*-) autoxidation rose to 13.8% and that of DBQ(*-) increased to 39.9%.     

Monoamine-dependent production of reactive oxygen species catalyzed by pseudoperoxidase activity of human hemoglobin

Hemoglobin (Hb) solution-based blood substitutes are being developed as oxygen-carrying agents for the prevention of ischemic tissue damage and low blood volume-shock. However, the cell-free Hb molecule has intrinsic toxicity to the tissue since harmful reactive oxygen species (ROS) are readily produced during autoxidation of Hb from the ferrous state to the ferric state, and the cell-free Hb also causes distortion in the oxidant/antioxidant balance in the tissues. There may be further hindering dangers in the use of free Hb as a blood substitute. It has been reported that Hb has peroxidase-like activity oxidizing peroxidase substrates such as aromatic amines. Here we observed the Hb-catalyzed ROS production coupled to oxidation of a neurotransmitter precursor, beta-phenylethylamine (PEA). Addition of PEA to Hb solution resulted in generation of superoxide anion (O2*-). We also observed that PEA increases the Hb-catalyzed monovalent oxidation of ascorbate to ascorbate free radicals (Asc'). The O2*- generation and Asc formation were detected by O2*--specific chemiluminescence of the Cypridina lucigenin analog and electron spin resonance spectroscopy, respectively. PEA-dependent O2*- production and monovalent oxidation of ascorbate in the Hb solution occurred without addition of H2O2, but a trace of H2O2 added to the system greatly increased the production of both O2*- and Asc*. Addition of GSH completely inhibited the PEA-dependent production of O2*- and Asc* in Hb solution. We propose that the O2*- generation and Asc* formation in the Hb solution are due to the pseudoperoxidase activity-dependent oxidation of PEA and resultant ROS may damage tissues rich in monoamines, if the Hb-based blood substitutes were circulated without addition of ROS scavengers such as thiols.     

Myoglobin as a model system for designing heme protein based blood substitutes

The ligand binding properties and resistances to denaturation of >300 different site-directed mutants of sperm whale, pig, and human myoglobin have been examined over the past 15 years. This library of recombinant proteins has been used to derive chemical mechanisms for ligand binding and to examine the factors governing holo- and apoglobin stability. We have also examined the effects of mutagenesis on the dioxygenation of NO by MbO(2) to form NO(3)(-) and metMb. This reaction rapidly detoxifies NO and is a key physiological function of both myoglobins and hemoglobins. The mechanisms derived for O(2) binding and NO dioxygenation have been used to design safer, more efficient, and more stable heme protein-prototypes for use as O(2) delivery pharmaceuticals in transfusion therapy (i.e. blood substitutes). An interactive database is being developed (http://olsonnt1.bioc.rice.edu/web/myoglobinhome.asp) to allow rapid access to the ligand binding parameters, stability properties, and crystal structures of the entire set of recombinant myoglobins. The long-range goal is to use this library for developing general protein engineering principles and for designing individual heme proteins for specific pharmacological and industrial uses.