Reactions of superoxide with the myoglobin tyrosyl radical

The contribution of superoxide-mediated injury to oxidative stress is not fully understood. A potential mechanism is the reaction of superoxide with tyrosyl radicals, which either results in repair of the tyrosine or formation of tyrosine hydroperoxide by addition. Whether these reactions occur with protein tyrosyl radicals is of interest because they could alter protein structure or modulate enzyme activity. Here, we have used a xanthine oxidase/acetaldehyde system to generate tyrosyl radicals on sperm whale myoglobin in the presence of superoxide. Using mass spectrometry we found that superoxide prevented myoglobin dimer formation by repairing the protein tyrosyl radical. An addition product of superoxide at Tyr151 was also identified, and exogenous lysine promoted the formation of this product. In our system, reaction of tyrosyl radicals with superoxide was favored over dimer formation with the ratio of repair to addition being approximately 10:1. Our results demonstrate that reaction of superoxide with protein tyrosyl radicals occurs and may play a role in free radical-mediated protein injury.     

Spectral and kinetic evidence for reaction of superoxide with compound I of myeloperoxidase

Superoxide and myeloperoxidase (MPO) are essential for the oxidative killing of bacteria by neutrophils. Previously, we developed a kinetic model to demonstrate that within the confines of neutrophil phagosomes, superoxide should react exclusively with MPO and be converted to hypochlorous acid. The model consists of all known reactions and rate constants for reactions of superoxide, hydrogen peroxide, and chloride ions with MPO, except for the reaction of superoxide with compound I, which could only be estimated. Compound I is a transitory redox intermediate of MPO that is responsible for oxidizing chloride ions to hypochlorous acid. To tackle the challenge of observing the reaction between two transient species, we combined stopped-flow spectrophotometry with pulse radiolysis. Using this technique, we directly observed the reduction of compound I by superoxide. The rate constant for the reaction was determined to be 5.6±0.3×10(6)M(-1)s(-1). This value establishes superoxide as one of the best substrates for compound I. Based on this value, the rate constant for reduction of compound II by superoxide was determined to be 1.2±0.1×10(6)M(-1)s(-1). Within phagosomes, the reduction of compound I by superoxide will compete with the oxidation of chloride ions so that the relative concentrations of these two substrates will affect the yield of hypochlorous acid. Characterization of this reaction confirms that superoxide is a physiological substrate for MPO and that their interactions are central to an important host defense mechanism.     

A pulse radiolysis investigation of the reactions of myeloperoxidase with superoxide and hydrogen peroxide

Using pulse radiolysis, the rate constant for the reaction of ferric myeloperoxidase with O2- to give compound III was measured at pH 7.8, and values of 2.1.10(6) M-1.s-1 for equine ferric myeloperoxidase and 1.1.10(6) M-1.s-1 for human ferric myeloperoxidase were obtained. Under the same conditions, the rate constant for the reaction of human ferric myeloperoxidase with H2O2 to give compound I was 3.1.10(7) M-1.s-1. Our results indicate that although the reaction of ferric myeloperoxidase with O2- is an order of magnitude slower than with H2O2, the former reaction is sufficiently rapid to influence myeloperoxidase-dependent production of hypochlorous acid by stimulated neutrophils.     

The reactions of horseradish peroxidase, lactoperoxidase, and myeloperoxidase with enzymatically generated superoxide

The formation and decay of intermediate compounds of horseradish peroxidase, lactoperoxidase, and myeloperoxidase formed in the presence of the superoxide/hydrogen peroxide-generating xanthine/xanthine oxidase system has been studied by observation of spectral changes in both the Soret and visible spectral regions and both on millisecond and second time scales. It is tentatively concluded that in all cases compound III is formed in a two-step reaction of native enzyme with superoxide. The presence of superoxide dismutase completely inhibited compound III formation; the presence of catalase had no effect on the process. Spectral data which indicate differences in the decay of horseradish peroxidase compound III back to the native state in comparison with compounds III of lactoperoxidase and myeloperoxidase are also presented.     

Transformed target cell-derived superoxide anions drive apoptosis induction by myeloperoxidase

Myeloperoxidase induces apoptosis in src- or raxs-transformed fibroblasts, but not in parental nontransformed fibroblasts. This selectivity seems to be based on superoxide anion production by transformed cells, a recently described characteristic feature of transformed cells. Myeloperoxidase-mediated apoptosis induction is inhibited by SOD, catalase, 4-aminobenzoyl hydrazide, taurine and DMSO. This pattern of inhibition allows us to conclude that transformed cell derived superoxide anions dismutate to hydrogen peroxide, which fosters HOCl formation by myeloperoxidase. Hydrogen peroxide formation thereby is the rate-limiting step and depends on the cell density. In a second step, HOCl interacts with superoxide anions to yield the highly reactive apoptosis inducing hydroxyl radical. This conclusion was verified through selective apoptosis induction in transformed cells by direct addition of HOCl, which was also inhibited by SOD and DMSO. Our findings demonstrate a specific interplay between target cell derived superoxide anions and MPO during selective apoptosis induction.     

The superoxide dismutase activity of myeloperoxidase; formation of compound III

The reaction of superoxide anions with myeloperoxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7), which results in the formation of Compound III of myeloperoxidase, was investigated. It is shown that myeloperoxidase has a high affinity for superoxide anions because formation of Compound III was only partially inhibited by high concentrations of superoxide dismutase. Furthermore, when superoxide anions were generated in a mixture of both cytochrome c and myeloperoxidase in the absence of Cl-, only Compound III was formed and reduction of cytochrome c was not observed. In the presence of Cl-, Compound III was also formed and reduction of cytochrome c was inhibited. From the results described in this paper we conclude that Compound III is able to react with superoxide anions, probably resulting in formation of an intermediate (Compound I) which is catalytically active in the oxidation of Cl- to yield hypochlorous acid (HOCl). Because Compound III of myeloperoxidase is formed in phagocytosing neutrophils (Winterbourn, C.C., Garcia, R.C. and Segal, A.W. (1985) Biochem. J. 228, 583-592) we propose that, in vivo, myeloperoxidase also acts as a superoxide dismutase, and via formation of Compound I uses superoxide anions in the formation of HOCl.     

Subcellular distribution of superoxide dismutases in human neutrophils. Influence of myeloperoxidase on the measurement of superoxide dismutase activity

We have identified two distinct pools of superoxide dismutase in fractions of human peripheral neutrophils obtained by the isopycnic fractionation of homogenates of the latter with linear sucrose gradients. Superoxide dismutase activity, observed with polyacrylamide gels impregnated with Nitro Blue Tetrazolium, was present in: (1) the mitochondrial fraction [density (rho) 1.169g/ml], containing the high-molecular-weight KCN-resistant enzyme, and (2) the cytoplasm fraction, containing the low-molecular-weight KCN-sensitive enzyme. Superoxide dismutase activity, observed with a quantitative assay involving cytochrome c, was present in: (1) the mitochondria, (2) the cytoplasm, and (3) the azurophil-granule fractions (rho=1.206 and 1.222g/ml). No substantial enzyme activity was observed in specific-granule fractions (rho=1.187g/ml) or in the membranous fraction (rho=1.136g/ml) in either assay. The apparent superoxide dismutase activity observed in the azurophil granules with the cytochrome c assay was attributable not to true superoxide dismutase but to myeloperoxidase, an enzyme found solely in the azurophil granules. In the presence of H(2)O(2), human neutrophil myeloperoxidase oxidized ferrocytochrome c. Thus, in the cytochrome c assay for superoxide dismutase, the oxidation of ferrocytochrome c by myeloperoxidase mimicked the inhibition of reduction of ferricytochrome c by superoxide dismutase. When myeloperoxidase was removed from azurophilgranule fractions by specific immuno-affinity chromatography, both myeloperoxidase and apparent superoxide dismutase activities were removed. It is concluded that there is no detectable superoxide dismutase in either the azurophil or specific granules of human neutrophils. Mitochondrial superoxide dismutase, 15% of the total dismutase activity of the cells, occurred only in fractions of density 1.160g/ml, where isocitrate dehydrogenase and cytochrome oxidase were also observed.     

Reactions involving superoxide and normal and unstable haemoglobins.

Superoxide ions (O2-) oxidized oxyhaemoglobin to methaemoglobin and reduced methaemoglobin to oxyhaemoglobin. The reactions of superoxide and H2O2 with oxyhaemoglobin or methaemoglobin and their inhibition by superoxide dismutase or catalase were used to detect the formation of superoxide or H2O2 on autoxidation of oxyhaemoglobin. The rate of autoxidation was decreased at about 35% in the presence of both enzymes. The copper-catalysed autoxidation of Hb (haemoglobin) was also shown to involve superoxide production. Superoxide was released on autoxidation of three unstable haemoglobins and isolated alpha and beta chains, at rates faster than with Hb A. Reactions of superoxide with Hb Christchurch and Hb Belfast were identical with those with Hb A, and occurred at the same rate. Hb Koln contrasted with the other haemoglobins in that the thiol groups of residue beta-93 as well as the haem groups reacted with superoxide. Haemichrome formation from methaemoglobin occurred very rapidly with Hb Christchurch and Hb Belfast, as well as the isolated chains, compared with Hb A. The process did not involve superoxide production or utilization. The relative importance of autoxidation and superoxide production compared with haemichrome formation in the haemolytic process associated with these abnormal haemoglobins and thalassaemia is considered.     

Reactions of superoxide radicals with curcumin: probable mechanisms by optical spectroscopy and EPR

Reactions of superoxide-crown ether complex with curcumin have been studied in acetonitrile. Optical absorption spectra showed that curcumin on reaction with superoxide forms a blue color intermediate absorbing at 560 nm, which subsequently decayed in a few hours with the development of the absorption band corresponding to the parent curcumin. The regeneration was 100% at low superoxide concentrations (1:1, or 1:2 or 1:3 of curcumin:superoxide) but reduced to 60% at high superoxide concentration (>1:5). The regeneration of curcumin is confirmed by HPLC analysis. Stopped-flow studies in acetonitrile following either the decay of parent curcumin at 420 nm or formation of 560 nm absorption have been used to determine the rate constant for the reaction of superoxide with curcumin. EPR studies confirmed the disappearance of characteristic superoxide signal in presence of curcumin with the formation of new featureless signal with g = 2.0067. Based on these studies it is concluded that at low superoxide concentrations curcumin effectively causes superoxide dismutation without itself undergoing any chemical change. At higher concentrations of superoxide, curcumin inhibits superoxide activity by reacting with it.     

Modeling the reactions of superoxide and myeloperoxidase in the neutrophil phagosome: implications for microbial killing

Neutrophils kill bacteria by ingesting them into phagosomes where superoxide and cytoplasmic granule constituents, including myeloperoxidase, are released. Myeloperoxidase converts chloride and hydrogen peroxide to hypochlorous acid (HOCl), which is strongly microbicidal. However, the role of oxidants in killing and the species responsible are poorly understood and the subject of current debate. To assess what oxidative mechanisms are likely to operate in the narrow confines of the phagosome, we have used a kinetic model to examine the fate of superoxide and its interactions with myeloperoxidase. Known rate constants for reactions of myeloperoxidase have been used and substrate concentrations estimated from neutrophil morphology. In the model, superoxide is generated at several mm/s. Most react with myeloperoxidase, which is present at millimolar concentrations, and rapidly convert the enzyme to compound III. Compound III turnover by superoxide is essential to maintain enzyme activity. Superoxide stabilizes at approximately 25 microM and hydrogen peroxide in the low micromolar range. HOCl production is efficient if there is adequate chloride supply, but further knowledge on chloride concentrations and transport mechanisms is needed to assess whether this is the case. Low myeloperoxidase concentrations also limit HOCl production by allowing more hydrogen peroxide to escape from the phagosome. In the absence of myeloperoxidase, superoxide increases to >100 microM but hydrogen peroxide to only approximately 30 microM. Most of the HOCl reacts with released granule proteins before reaching the bacterium, and chloramine products may be effectors of its antimicrobial activity. Hydroxyl radicals should form only after all susceptible protein targets are consumed.     

Mechanism of reaction of myeloperoxidase with nitrite

Myeloperoxidase (MPO) is a major neutrophil protein and may be involved in the nitration of tyrosine residues observed in a wide range of inflammatory diseases that involve neutrophils and macrophage activation. In order to clarify if nitrite could be a physiological substrate of myeloperoxidase, we investigated the reactions of the ferric enzyme and its redox intermediates, compound I and compound II, with nitrite under pre-steady state conditions by using sequential mixing stopped-flow analysis in the pH range 4-8. At 15 degrees C the rate of formation of the low spin MPO-nitrite complex is (2.5 +/- 0.2) x 10(4) m(-1) s(-1) at pH 7 and (2.2 +/- 0.7) x 10(6) m(-1) s(-1) at pH 5. The dissociation constant of nitrite bound to the native enzyme is 2.3 +/- 0.1 mm at pH 7 and 31.3 +/- 0.5 micrometer at pH 5. Nitrite is oxidized by two one-electron steps in the MPO peroxidase cycle. The second-order rate constant of reduction of compound I to compound II at 15 degrees C is (2.0 +/- 0.2) x 10(6) m(-1) s(-1) at pH 7 and (1.1 +/- 0.2) x 10(7) m(-1) s(-1) at pH 5. The rate constant of reduction of compound II to the ferric native enzyme at 15 degrees C is (5.5 +/- 0.1) x 10(2) m(-1) s(-1) at pH 7 and (8.9 +/- 1.6) x 10(4) m(-1) s(-1) at pH 5. pH dependence studies suggest that both complex formation between the ferric enzyme and nitrite and nitrite oxidation by compounds I and II are controlled by a residue with a pK(a) of (4.3 +/- 0.3). Protonation of this group (which is most likely the distal histidine) is necessary for optimum nitrite binding and oxidation.     

Interaction of human myeloperoxidase with nitrite.

EPR (electron paramagnetic resonance) and optical spectroscopy show that human neutrophil myeloperoxidase is converted from ferric high-spin to low-spin by the addition of nitrite. The Soret peak shifts from 429 to 447 nm and new peaks appear in the visible region at 573 and 627 nm; the EPR g-values change from 6.84, 5.02, 1.95 to 2.55, 2.31, 1.82. Small differences are seen in the EPR (but, not optical) spectra of myeloperoxidase isoenzyme I compared to isoenzymes II and III. The reaction with nitrite is detectable by EPR in intact neutrophils and is thus a possible in vivo monitor of NO/nitrite production by these cells.     

Production of the superoxide adduct of myeloperoxidase (compound III) by stimulated human neutrophils and its reactivity with hydrogen peroxide and chloride.

Examination of the spectra of phagocytosing neutrophils and of myeloperoxidase present in the medium of neutrophils stimulated with phorbol myristate acetate has shown that superoxide generated by the cells converts both intravacuolar and exogenous myeloperoxidase into the superoxo-ferric or oxyferrous form (compound III or MPO2). A similar product was observed with myeloperoxidase in the presence of hypoxanthine, xanthine oxidase and Cl-. Both transformations were inhibited by superoxide dismutase. Thus it appears that myeloperoxidase in the neutrophil must function predominantly as this superoxide derivative. MPO2 autoxidized slowly (t 1/2 = 12 min at 25 degrees C) to the ferric enzyme. It did not react directly with H2O2 or Cl-, but did react with compound II (MP2+ X H2O2). MPO2 catalysed hypochlorite formation from H2O2 and Cl- at approximately the same rate as the ferric enzyme, and both reactions showed the same H2O2-dependence. This suggests that MPO2 can enter the main peroxidation pathway, possibly via its reaction with compound II. Both ferric myeloperoxidase and MPO2 showed catalase activity, in the presence or absence of Cl-, which predominated over chlorination at H2O2 concentrations above 200 microM. Thus, although the reaction of neutrophil myeloperoxidase with superoxide does not appear to impair its chlorinating ability, the H2O2 concentration in its environment will determine whether the enzyme acts primarily as a catalase or peroxidase.     

The photoprotein pholasin as a luminescence substrate for detection of superoxide anion radicals and myeloperoxidase activity in stimulated neutrophils

Pholasin, the photoprotein of the common piddock Pholas dactylus, emits an intense luminescence upon oxidation. The contribution of superoxide anion radicals and myeloperoxidase (MPO) to Pholasin luminescence in stimulated neutrophils was investigated. Data on Pholasin luminescence were compared with results of superoxide anion radical generation detected by the cytochrome c test as well as with the release of elastase and MPO. In N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated neutrophils, most of the luminescence is caused by superoxide anion radicals, whereas MPO shows only a small effect as shown by coincubation with superoxide dismutase (SOD) as well as potassium cyanide (KCN), an inhibitor of MPO. However, both, O₂^- and MPO contribute to light emission in fMLP/cytochalasin B and phorbol myristoyl acetate (PMA) stimulated cells. Thus, the kinetics of O₂^- generation and MPO release can be very well detected by Pholasin luminescence in stimulated neutrophils.

Degranulation of azurophilic granules was assessed using an ELISA test kit for released MPO or detection of elastase activity with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide in the supernatant of stimulated cells. Both approaches revealed concurrently similar results concerning the amount and kinetics of enzyme release with data of Pholasin luminescence. Both, cytochrome c measurements and Pholasin luminescence indicate that fMLP/cytochalasin B and PMA stimulated neutrophils produce more O₂^- than fMLP stimulated cells. Thus, Pholasin luminescence can be used to detect, sensitively and specifically, O₂^- production and MPO release from stimulated neutrophils.     

The photoprotein pholasin as a luminescence substrate for detection of superoxide anion radicals and myeloperoxidase activity in stimulated neutrophils

Pholasin, the photoprotein of the common piddock Pholas dactylus, emits an intense luminescence upon oxidation. The contribution of superoxide anion radicals and myeloperoxidase (MPO) to Pholasin luminescence in stimulated neutrophils was investigated. Data on Pholasin luminescence were compared with results of superoxide anion radical generation detected by the cytochrome c test as well as with the release of elastase and MPO. In N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated neutrophils, most of the luminescence is caused by superoxide anion radicals, whereas MPO shows only a small effect as shown by coincubation with superoxide dismutase (SOD) as well as potassium cyanide (KCN), an inhibitor of MPO. However, both, O₂^- and MPO contribute to light emission in fMLP/cytochalasin B and phorbol myristoyl acetate (PMA) stimulated cells. Thus, the kinetics of O₂^- generation and MPO release can be very well detected by Pholasin luminescence in stimulated neutrophils.

Degranulation of azurophilic granules was assessed using an ELISA test kit for released MPO or detection of elastase activity with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide in the supernatant of stimulated cells. Both approaches revealed concurrently similar results concerning the amount and kinetics of enzyme release with data of Pholasin luminescence. Both, cytochrome c measurements and Pholasin luminescence indicate that fMLP/cytochalasin B and PMA stimulated neutrophils produce more O₂^- than fMLP stimulated cells. Thus, Pholasin luminescence can be used to detect, sensitively and specifically, O₂^- production and MPO release from stimulated neutrophils.     

Mechanism of reaction of melatonin with human myeloperoxidase

Recently, it was suggested that melatonin (N-acetyl-5-methoxytryptamine) is oxidized by activated neutrophils in a reaction most probably involving myeloperoxidase (Biochem. Biophys. Res. Commun. (2000) 279, 657-662). Myeloperoxidase (MPO) is the most abundant protein of neutrophils and is involved in killing invading pathogens. To clarify if melatonin is a substrate of MPO, we investigated the oxidation of melatonin by its redox intermediates compounds I and II using transient-state spectral and kinetic measurements at 25 degrees C. Spectral and kinetic analysis revealed that both compound I and compound II oxidize melatonin via one-electron processes. The second-order rate constant measured for compound I reduction at pH 7 and pH 5 are (6.1 +/- 0.2) x 10(6) M(-1) s(-1) and (1.0 +/- 0.08) x 10(7) M(-1) s(-1), respectively. The rates for the one-electron reduction of compound II back to the ferric enzyme are (9.6 +/- 0.3) x 10(2) M(-1) s(-1) (pH 7) and (2.2 +/- 0.1) x 10(3) M(-1) s(-1) (pH 5). Thus, melatonin is a much better electron donor for compound I than for compound II. Steady-state experiments showed that the rate of oxidation of melatonin is dependent on the H(2)O(2) concentration, is not affected by superoxide dismutase, and is quickly terminated by sodium cyanide. Melatonin can markedly inhibit the chlorinating activity of MPO at both pH 7 and pH 5. The implication of these findings in the activated neutrophil is discussed.     

Reaction of autoxidation products of penicillamine with myeloperoxidase

Spectral evidence is presented which shows that penicillamine is able to initiate the formation of the oxidized intermediates of myeloperoxidase in the absence of exogenous hydrogen peroxide. The autoxidation of penicillamine presumably produces superoxide which dismutates spontaneously to form hydrogen peroxide. Thus, the formation of both compounds II and III of myeloperoxidase was observed. We also report that penicillamine can directly reduce cytochrome c and therefore, it could possibly act as a one-electron donor to myeloperoxidase.