Superoxide modulates the activity of myeloperoxidase and optimizes the production of hypochlorous acid.

Myeloperoxidase catalyses the conversion of H2O2 and Cl- to hypochlorous acid (HOCl). It also reacts with O2- to form the oxy adduct (compound III). To determine how O2- affects the formation of HOCl, chlorination of monochlorodimedon by myeloperoxidase was investigated using xanthine oxidase and hypoxanthine as a source of O2- and H2O2. Myeloperoxidase was mostly converted to compound III, and H2O2 was essential for chlorination. At pH 5.4, superoxide dismutase (SOD) enhanced chlorination and prevented formation of compound III. However, at pH 7.8, SOD inhibited chlorination and promoted formation of the ferrous peroxide adduct (compound II) instead of compound III. We present spectral evidence for a direct reaction between compound III and H2O2 to form compound II, and for the reduction of compound II by O2- to regenerate native myeloperoxidase. These reactions enable compound III and compound II to participate in the chlorination reaction. Myeloperoxidase catalytically inhibited O2- -dependent reduction of Nitro Blue Tetrazolium. This inhibition is explained by myeloperoxidase undergoing a cycle of reactions with O2-, H2O2 and O2-, with compounds III and II as intermediates, i.e., by myeloperoxidase acting as a combined SOD/catalase enzyme. By preventing the accumulation of inactive compound II, O2- enhances the activity of myeloperoxidase. We propose that, under physiological conditions, this optimizes the production of HOCl and may potentiate oxidant damage by stimulated neutrophils.     

Oxides of Nitrogen (NO· and NO2 –) as Cofactors of the Myeloperoxidase System

Myeloperoxidase is the main peroxisomal protein of neutrophils, monocytes, and a subpopulation of tissue macrophages; it plays the key role in protective and inflammatory responses of the organism. This role is mediated by various diffusible radicals formed during oxidative reactions catalyzed by the enzyme heme. Myeloperoxidase and nitric oxide synthase are stored in peroxisomes. Nitric oxide reacts with the heme of myeloperoxidase. Low nitric oxide concentrations increase peroxidase activity through reduction of Compound II to native myeloperoxidase. Conversely, high nitric oxide concentrations inhibit the catalytic activity of myeloperoxidase through formation of inactive nitrosyl–heme complexes. Such effect of nitric oxide on catalytic activity of myeloperoxidase has various consequences for infectious and local inflammatory processes. Another oxide of nitrogen, nitrite, is a good substrate for myeloperoxidase Compound I but slowly reacts with Compound II. Nitrogen dioxide is formed after nitrite oxidation by myeloperoxidase. Formation of nitrogen dioxide is another protective mechanism and nitration of microbial proteins by myeloperoxidase can represent an additional protective response of peroxisomes.     

Biomarkers of myeloperoxidase-derived hypochlorous acid

Hypochlorous acid is the major strong oxidant generated by neutrophils. The heme enzyme myeloperoxidase catalyzes the production of hypochlorous acid from hydrogen peroxide and chloride. Although myeloperoxidase has been implicated in the tissue damage that occurs in numerous diseases that involve inflammatory cells, it has proven difficult to categorically demonstrate that it plays a crucial role in any pathology. This situation should soon be rectified with the advent of sensitive biomarkers for hypochlorous acid. In this review, we outline the advantages and limitations of chlorinated tyrosines, chlorohydrins, 5-chlorocytosine, protein carbonyls, antibodies that recognize HOCl-treated proteins, and glutathione sulfonamide as potential biomarkers of hypochlorous acid. Levels of 3-chlorotyrosine and 3,5-dichlorotyrosine are increased in proteins after exposure to low concentrations of hypochlorous acid and we conclude that their analysis by gas chromatography and mass spectrometry is currently the best method available for probing the involvement of oxidation by myeloperoxidase in the pathology of particular diseases. The appropriate use of other biomarkers should provide complementary information.Keywords-Free radicals, Myeloperoxidase, Neutrophil oxidant, Hypochlorous acid, Chlorotyrosine, Chlorohydrin, Oxidant biomarker     

Antimicrobial Activity of Myeloperoxidase from Neutrophilic Peroxisome

Myeloperoxidase plays the key role in antimicrobial oxygen-dependent activity of neutrophils. This heme-containing enzyme catalyzes HOCl formation from H₂O₂ and Cl^–. HOCl is a strong oxidation agent produced at the significant level by neutrophils. Myeloperoxidase easily oxidizes thiocyanate to hypothiocyanate and Br^– to HOBr, which are involved in protective reactions. Myeloperoxidase reacts quickly with nitric oxide and peroxynitrite in inflammation foci. All these reactions affect neutrophil-induced oxidative stress.     

Transient and steady-state kinetics of the oxidation of substituted benzoic acid hydrazides by myeloperoxidase

Myeloperoxidase is the most abundant protein in neutrophils and catalyzes the production of hypochlorous acid. This potent oxidant plays a central role in microbial killing and inflammatory tissue damage. 4-Aminobenzoic acid hydrazide (ABAH) is a mechanism-based inhibitor of myeloperoxidase that is oxidized to radical intermediates that cause enzyme inactivation. We have investigated the mechanism by which benzoic acid hydrazides (BAH) are oxidized by myeloperoxidase, and we have determined the features that enable them to inactivate the enzyme. BAHs readily reduced compound I of myeloperoxidase. The rate constants for these reactions ranged from 1 to 3 x 10(6) M-1 s-1 (15 degrees C, pH 7.0) and were relatively insensitive to the substituents on the aromatic ring. Rate constants for reduction of compound II varied between 6.5 x 10(5) M-1 s-1 for ABAH and 1.3 x 10(3) M-1 s-1 for 4-nitrobenzoic acid hydrazide (15 degrees C, pH 7.0). Reduction of both compound I and compound II by BAHs adhered to the Hammett rule, and there were significant correlations with Brown-Okamoto substituent constants. This indicates that the rates of these reactions were simply determined by the ease of oxidation of the substrates and that the incipient free radical carried a positive charge. ABAH was oxidized by myeloperoxidase without added hydrogen peroxide because it underwent auto-oxidation. Although BAHs generally reacted rapidly with compound II, they should be poor peroxidase substrates because the free radicals formed during peroxidation converted myeloperoxidase to compound III. We found that the reduction of ferric myeloperoxidase by BAH radicals was strongly influenced by Hansch's hydrophobicity constants. BAHs containing more hydrophilic substituents were more effective at converting the enzyme to compound III. This implies that BAH radicals must hydrogen bond to residues in the distal heme pocket before they can reduce the ferric enzyme. Inactivation of myeloperoxidase by BAHs was related to how readily they were oxidized, but there was no correlation with their rate constants for reduction of compounds I or II. We propose that BAHs destroy the heme prosthetic groups of the enzyme by reducing a ferrous myeloperoxidase-hydrogen peroxide complex.     

Nitrite as a substrate and inhibitor of myeloperoxidase. Implications for nitration and hypochlorous acid production at sites of inflammation

Myeloperoxidase is a heme enzyme of neutrophils that uses hydrogen peroxide to oxidize chloride to hypochlorous acid. Recently, it has been shown to catalyze nitration of tyrosine. In this study we have investigated the mechanism by which it oxidizes nitrite and promotes nitration of tyrosyl residues. Nitrite was found to be a poor substrate for myeloperoxidase but an excellent inhibitor of its chlorination activity. Nitrite slowed chlorination by univalently reducing the enzyme to an inactive form and as a consequence was oxidized to nitrogen dioxide. In the presence of physiological concentrations of nitrite and chloride, myeloperoxidase catalyzed little nitration of tyrosyl residues in a heptapeptide. However, the efficiency of nitration was enhanced at least 4-fold by free tyrosine. Our data are consistent with a mechanism in which myeloperoxidase oxidizes free tyrosine to tyrosyl radicals that exchange with tyrosyl residues in peptides. These peptide radicals then couple with nitrogen dioxide to form 3-nitrotyrosyl residues. With neutrophils, myeloperoxidase-dependent nitration required a high concentration of nitrite (1 mM), was doubled by tyrosine, and increased 4-fold by superoxide dismutase. Superoxide is likely to inhibit nitration by reacting with nitrogen dioxide and/or tyrosyl radicals. We propose that at sites of inflammation myeloperoxidase will nitrate proteins, even though nitrite is a poor substrate, because the co-substrate tyrosine will be available to facilitate the reaction. Also, production of 3-nitrotyrosine will be most favorable when the concentration of superoxide is low.     

Superoxide-dependent oxidation of melatonin by myeloperoxidase

Myeloperoxidase uses hydrogen peroxide to oxidize numerous substrates to hypohalous acids or reactive free radicals. Here we show that neutrophils oxidize melatonin to N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) in a reaction that is catalyzed by myeloperoxidase. Production of AFMK was highly dependent on superoxide but not hydrogen peroxide. It did not require hypochlorous acid, singlet oxygen, or hydroxyl radical. Purified myeloperoxidase and a superoxide-generating system oxidized melatonin to AFMK and a dimer. The dimer would result from coupling of melatonin radicals. Oxidation of melatonin was partially inhibited by catalase or superoxide dismutase. Formation of AFMK was almost completely eliminated by superoxide dismutase but weakly inhibited by catalase. In contrast, production of melatonin dimer was enhanced by superoxide dismutase and blocked by catalase. We propose that myeloperoxidase uses superoxide to oxidize melatonin by two distinct pathways. One pathway involves the classical peroxidation mechanism in which hydrogen peroxide is used to oxidize melatonin to radicals. Superoxide adds to these radicals to form an unstable peroxide that decays to AFMK. In the other pathway, myeloperoxidase uses superoxide to insert dioxygen into melatonin to form AFMK. This novel activity expands the types of oxidative reactions myeloperoxidase can catalyze. It should be relevant to the way neutrophils use superoxide to kill bacteria and how they metabolize xenobiotics.     

The Mechanism of Bactericidal Activity in Phagosomes of Neutrophils

Myeloperoxidase plays the key role in antimicrobial of phagocytes. This enzyme uses hydrogen peroxide and chloride to catalyze hypochlorous acid formation. HOCl is the most probable agent in the oxygen-dependent bactericidal activity in the phagocyte phagosome. Chlorination markers indicate HOCl generation in the quantities lethal for bacteria. Enzymatic assay for myeloperoxidase indicates proceeding of other reactions involved in bactericidal activity. Superoxide integrates many activities of this kind and is important for physiological function of myeloperoxidase. Elucidation of phagosomes biochemistry can help us to understand why certain pathogens survive in such unfavorable environment.     

Myeloperoxidase-catalyzed chlorination: the quest for the active species

Myeloperoxidase (MPO) is a dominating enzyme of circulating polymorphonuclear neutrophils that catalyzes the two-electron oxidation of chloride, thereby producing the strong halogenating agent hypochlorous acid (ClO⁻/HOCl). In absence of MPO the tripeptide Pro-Gly-Gly reacts with HOCl faster than the amino acid taurine (2-aminoethanesulfonic acid, Tau), while the MPO-mediated chlorination shows reverse order. A comparative study of the enzymatic oxidation of both substrates at pH 4.0–6.0, varying H₂O₂ concentration is presented. Initial and equilibrium rates studies have been carried on, reaction rates in the latter being slower due to the chemical equilibrium between MPO-I and MPO-II–HO₂. A maximum of chlorination rate is observed for Pro-Gly-Gly and Tau when [H₂O₂] ≈ 0.3–0.7 mM and pH ≈ 4.5–5.0. Several mechanistic possibilities are considered, the proposed one implies that chlorination takes place via two pathways. One, for bulkier substrates, involves chlorination by free HOCl outside the heme cavity; ClO⁻ is released from the active center, diffuses away the heme cavity, and undergoes protonation to HOCl. The other implies the existence of compound I–Cl⁻ complex (MPO-I–Cl), capable of chlorinating smaller substrates in the heme pocket. Electronic structure calculations show the size of Pro-Gly-Gly comparable to the available gap in the substrate channel, this tripeptide being unable to reach the active site, and its chlorination is only possible by free HOCl outside the enzyme.     

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.     

Phagocytes produce 5-chlorouracil and 5-bromouracil,two mutagenic products of myeloperoxidase,in human inflammatory tissue

Oxidative damage to DNA has been implicated in carcinogenesis during chronic inflammation. Epidemiological and biochemical studies suggest that one potential mechanism involves myeloperoxidase, a hemeprotein secreted by human phagocytes. In this study, we demonstrate that human neutrophils use myeloperoxidase to oxidize uracil to 5-chlorouracil in vitro. Uracil chlorination by myeloperoxidase or reagent HOCl exhibited an unusual pH dependence, being minimal at pH approximately 5, but increasing markedly under either acidic or mildly basic conditions. This bimodal curve suggests that myeloperoxidase initially produces HOCl, which subsequently chlorinates uracil by acid- or base-catalyzed reactions. Human neutrophils use myeloperoxidase and H2O2 to chlorinate uracil, suggesting that nucleobase halogenation reactions may be physiologically relevant. Using a sensitive and specific mass spectrometric method, we detected two products of myeloperoxidase, 5-chlorouracil and 5-bromouracil, in neutrophil-rich human inflammatory tissue. Myeloperoxidase is the most likely source of 5-chlorouracil in vivo because halogenated uracil is a specific product of the myeloperoxidase system in vitro. In contrast, previous studies have demonstrated that 5-bromouracil could be generated by either eosinophil peroxidase or myeloperoxidase, which preferentially brominates uracil at plasma concentrations of halide and under moderately acidic conditions. These observations indicate that the myeloperoxidase system promotes nucleobase halogenation in vivo. Because 5-chlorouracil and 5-bromouracil can be incorporated into nuclear DNA, and these thymine analogs are well known mutagens, our observations raise the possibility that halogenation reactions initiated by phagocytes provide one pathway for mutagenesis and cytotoxicity at sites of inflammation.     

Propofol inhibits the myeloperoxidase activity by acting as substrate through a redox process

BackgroundPropofol (2,6-diisopropylphenol) is frequently used as intravenous anesthetic agent, especially in its injectable form (Diprivan), to initiate and maintain sedative state during surgery or in intensive care units. Numerous studies have reported the antioxidant and anti-inflammatory effect of propofol. The oxidant enzyme myeloperoxidase (MPO), released from activated neutrophils, plays a key role in host defense. An increase of the circulating MPO concentration has been observed in patients admitted in intensive care unit and presenting a systemic inflammatory response related to septic shock or trauma.MethodsThis study investigates the immunomodulatory action of propofol and Diprivan as inhibitor of the oxidant activity of MPO. The understanding of the redox action mechanism of propofol and Diprivan on the myeloperoxidase chlorination and peroxidase activities has been refined using the combination of fluorescence and absorption spectroscopies with docking and cyclic voltammetry.ResultsPropofol acts as a reversible MPO inhibitor. The molecule interacts as a reducing substrate in the peroxidase cycle and promotes the accumulation of compound II. At acidic pH (5.5), propofol and Diprivan do not inhibit the chlorination activity, but their action increases at physiological pH (7.4). The main inhibitory action of Diprivan could be attributed to its HOCl scavenging property.General significancePropofol can act as a reversible MPO inhibitor at clinical concentrations. This property could, in addition to other previously proven anti-inflammatory actions, induce an immunomodulatory action, beneficial during clinical use, particularly in the treatment of systemic inflammation response syndrome.     

Reactions of superoxide with myeloperoxidase

When neutrophils ingest bacteria, they discharge superoxide and myeloperoxidase into phagosomes. Both are essential for killing of the phagocytosed micro-organisms. It is generally accepted that superoxide is a precursor of hydrogen peroxide which myeloperoxidase uses to oxidize chloride to hypochlorous acid. Previously, we demonstrated that superoxide modulates the chlorination activity of myeloperoxidase by reacting with its ferric and compound II redox states. In this investigation we used pulse radiolysis to determine kinetic parameters of superoxide reacting with redox forms of myeloperoxidase and used these data in a steady-state kinetic analysis. We provide evidence that superoxide reacts with compound I and compound III. Our estimates of the rate constants for the reaction of superoxide with compound I, compound II, and compound III are 5 x 10(6) M-1 s-1, 5.5 +/- 0.4 x 10(6) M-1 s-1, and 1.3 +/- 0.2 x 10(5) M-1 s-1, respectively. These reactions define new activities for myeloperoxidase. It will act as a superoxide dismutase when superoxide reacts consecutively with ferric myeloperoxidase and compound III. It will also act as a superoxidase by using hydrogen peroxide to oxidize superoxide via compound I and compound II. The favorable kinetics of these reactions indicate that, within the confines of a phagosome, superoxide will react with myeloperoxidase and affect the reactions it will catalyze. These interactions of superoxide and myeloperoxidase will have a major influence on the way neutrophils use oxygen to kill bacteria. Consequently, superoxide should be viewed as a cosubstrate that myeloperoxidase uses to elicit bacterial killing.     

Identification of proteins susceptible to thiol oxidation in endothelial cells exposed to hypochlorous acid and N-chloramines

Hypochlorous acid (HOCl) is a potent oxidant produced by the enzyme myeloperoxidase, which is released by neutrophils under inflammatory conditions. Although important in the immune system, HOCl can also damage host tissue, which contributes to the development of disease. HOCl reacts readily with free amino groups to form N-chloramines, which also cause damage in vivo, owing to the extracellular release of myeloperoxidase and production of HOCl. HOCl and N-chloramines react readily with cellular thiols, which causes dysfunction via enzyme inactivation and modulation of redox signaling processes. In this study, the ability of HOCl and model N-chloramines produced on histamine and ammonia at inflammatory sites, to oxidize specific thiol-containing proteins in human coronary artery endothelial cells was investigated. Using a proteomics approach with the thiol-specific probe, 5-iodoacetamidofluorescein, we show that several proteins including peptidylprolyl isomerase A (cyclophilin A), protein disulfide isomerase, glyceraldehyde-3-phosphate dehydrogenase and galectin-1 are particularly sensitive to oxidation by HOCl and N-chloramines formed at inflammatory sites. This will contribute to cellular dysfunction and may play a role in inflammatory disease pathogenesis.     

Molecular chlorine generated by the myeloperoxidase-hydrogen peroxide-chloride system of phagocytes produces 5-chlorocytosine in bacterial RNA

Myeloperoxidase, a heme enzyme secreted by activated phagocytes, uses H(2)O(2) and Cl(-) to generate the chlorinating intermediate hypochlorous acid (HOCl). This potent cytotoxic oxidant plays a critical role in host defenses against invading pathogens. In this study, we explore the possibility that myeloperoxidase-derived HOCl might oxidize nucleic acids. When we exposed 2'-deoxycytidine to the myeloperoxidase-H(2)O(2)-Cl(-) system, we obtained a single major product that was identified as 5-chloro-2'-deoxycytidine using mass spectrometry, high performance liquid chromatography, UV-visible spectroscopy, and NMR spectroscopy. 5-Chloro-2'-deoxycytidine production by myeloperoxidase required H(2)O(2) and Cl(-), suggesting that HOCl is an intermediate in the reaction. However, reagent HOCl failed to generate 5-chloro-2'-deoxycytidine in the absence of Cl(-). Moreover, chlorination of 2'-deoxycytidine was optimal under acidic conditions in the presence of Cl(-). These results implicate molecular chlorine (Cl(2)), which is in equilibrium with HOCl through a reaction requiring Cl(-) and H(+), in the generation of 5-chloro-2'-deoxycytidine. Activated human neutrophils were able to generate 5-chloro-2'-deoxycytidine. Cellular chlorination was blocked by catalase and heme poisons, consistent with a myeloperoxidase-catalyzed reaction. The myeloperoxidase-H(2)O(2)-Cl(-) system generated similar levels of 5-chlorocytosine in RNA and DNA in vitro. In striking contrast, only cell-associated RNA acquired detectable levels of 5-chlorocytosine when intact Escherichia coli was exposed to the myeloperoxidase system. This observation suggests that oxidizing intermediates generated by myeloperoxidase selectively target intracellular RNA for chlorination. Collectively, these results indicate that Cl(2) derived from HOCl generates 5-chloro-2'-deoxycytidine during the myeloperoxidase-catalyzed oxidation of 2'-deoxycytidine. Phagocytic generation of Cl(2) therefore may constitute one mechanism for oxidizing nucleic acids at sites of inflammation.     

8-Nitro-2'-deoxyguanosine, a specific marker of oxidation by reactive nitrogen species, is generated by the myeloperoxidase-hydrogen peroxide-nitrite system of activated human phagocytes

Reactive intermediates generated by phagocytes damage DNA and may contribute to the link between chronic inflammation and cancer. Myeloperoxidase, a heme protein secreted by activated phagocytes, is a potential catalyst for such reactions. Recent studies demonstrate that this enzyme uses hydrogen peroxide (H2O2) and nitrite (NO2-) to generate reactive nitrogen species which convert tyrosine to 3-nitrotyrosine. We now report that activated human neutrophils use myeloperoxidase, H2O2, and NO2- to nitrate 2'-deoxyguanosine, one of the nucleosides of DNA. Through HPLC, UV/vis spectroscopy, and mass spectrometry, the two major products of this reaction were identified as 8-nitroguanine and 8-nitro-2'-deoxyguanosine. Nitration required each component of the complete enzymatic system and was inhibited by catalase and heme poisons. However, it was independent of chloride ion and little affected by scavengers of hypochlorous acid, suggesting that the reactive agent is a nitrogen dioxide-like species that results from the one-electron oxidation of NO2- by myeloperoxidase. Alternatively, 2'-deoxyguanosine might be oxidized directly by the enzyme to yield a radical species which subsequently reacts with NO2- or NO2* to generate the observed products. Human neutrophils stimulated with phorbol ester also generated 8-nitroguanine and 8-nitro-2'-deoxyguanosine. The reaction required NO2- and was inhibited by catalase and heme poisons, implicating myeloperoxidase in the cell-mediated pathway. These results indicate that human neutrophils use the myeloperoxidase-H2O2-NO2- system to generate reactive species that can nitrate the C-8 position of 2'-deoxyguanosine. Our observations raise the possibility that reactive nitrogen species generated by myeloperoxidase and other peroxidases contribute to nucleobase oxidation and tissue injury at sites of inflammation.     

Formation of myeloperoxidase compound II during aerobic stimulation of rat neutrophils

We have made simultaneous spectrophotometric and O₂ measurements on suspensions of rat neutrophils during activation of the respiratory burst. Under aerobic conditions an absorption increase attributable to myeloperoxidase compound II was observed in parallel with the rapid phase of O₂ uptake. Identification of this compound was confirmed by analysis of a spectrum obtained with purified myeloperoxidase and H₂O₂. Whereas a second addition of stimulus did not increase O₂ uptake any further, a second phase of myeloperoxidase release and compound II formation was observed. These results suggest thatin vivo myeloperoxidase reacts with H₂O₂ generatedvia the respiratory burst to form compound II under conditions in which the chlorination reaction would be the expected major pathway.Abbreviations FMLP N-formylmethionylleucyl phenylalanine - MPO²⁺.H₂O₂ Myeloperoxidase compound II - MPO³⁺.H₂O₂ Myeloperoxidase compound I - {ei275-1} superoxide     

Oxidation of hydroquinone by myeloperoxidase. Mechanism of stimulation by benzoquinone.

Myeloperoxidase (MPO) is a prime candidate for mediating the inflammatory tissue damage of neutrophils because it converts Cl- to the potent oxidant hypochlorous acid. It also oxidizes xenobiotics to reactive free radicals. We have found that the kinetics of oxidation of hydroquinone by myeloperoxidase are inadequately explained by the classical peroxidase mechanism. Peroxidation of hydroquinone displayed a distinct lag phase, which was practically abolished by excluding O2 and was eliminated by adding benzoquinone at the start of the reaction. Superoxide dismutase increased the rate of peroxidation by 40% but did not eliminate the lag phase. Spectral investigations revealed that during the initial phase of the reaction, MPO was converted to oxy-MPO, or compound III, by a mechanism that was not reliant on superoxide. Benzosemiquinone, however, was able to convert ferric-MPO to compound III. Both compound III and ferro-MPO reacted with benzoquinone to regenerate ferric-MPO. We propose that the lag phase occurs because benzosemiquinone reduces ferric-MPO to ferro-MPO, which rapidly binds O2 to form compound III. Since compound III is outside the peroxidation cycle, conversion of hydroquinone to benzoquinone is retarded. However, as benzoquinone accumulates, it oxidizes ferro-MPO and compound III to ferric-MPO, thereby increasing the rate of peroxidation. There is a minimal lag phase under an atmosphere of N2 because ferro-MPO would be rapidly oxidized by benzoquinone, without formation of compound III. We conclude that when substrates produce radicals capable of reducing ferric-MPO, they will be peroxidized efficiently only if oxy-MPO is readily recycled. Furthermore, these radicals will prevent MP3+ from reacting with H2O2, and thereby prevent the enzyme from oxidizing Cl- to hypochlorous acid. Thus, this mechanism could be exploited to prevent hypochlorous acid-mediated inflammatory tissue damage.     

Superoxide enhances hypochlorous acid production by stimulated human neutrophils

Stimulated neutrophils undergo a respiratory burst discharging large quantities of superoxide and hydrogen peroxide. They also release myeloperoxidase, which catalyses the conversion of hydrogen peroxide and Cl- to hypochlorous acid. Human neutrophils stimulated with opsonized zymosan promoted the loss of monochlorodimedon. This loss was entirely due to hypochlorous acid, since it did not occur in Cl(-)-free buffer, was inhibited by azide and cyanide, and was enhanced by adding exogenous myeloperoxidase. It was not inhibited by desferal, diethylenetriaminepentaacetic acid, mannitol or dimethylsulfoxide, which excluded involvement of the hydroxyl radical. Approx. 30% of the detectable superoxide generated was converted to hypochlorous acid. As expected, formation of hypochlorous acid was completely inhibited by catalase, but it was also inhibited by up to 70% by superoxide dismutase. Superoxide dismutase also inhibited the production of hypochlorous acid by neutrophils stimulated with phorbol myristate acetate. Our results indicate that generation of superoxide by neutrophils enables these cells to enhance their production of hypochlorous acid. Furthermore, inhibition of neutrophil processes by superoxide dismutase and catalase does not necessarily implicate the hydroxyl radical. It is proposed that superoxide may potentiate oxidant damage at inflammatory sites by optimizing the myeloperoxidase-dependent production of hypochlorous acid.     

Ceruloplasmin Is an Endogenous Inhibitor of Myeloperoxidase

Myeloperoxidase is a neutrophil enzyme that promotes oxidative stress in numerous inflammatory pathologies. It uses hydrogen peroxide to catalyze the production of strong oxidants including chlorine bleach and free radicals. A physiological defense against the inappropriate action of this enzyme has yet to be identified. We found that myeloperoxidase oxidized 75% of the ascorbate in plasma from ceruloplasmin knock-out mice, but there was no significant loss in plasma from wild type animals. When myeloperoxidase was added to human plasma it became bound to other proteins and was reversibly inhibited. Ceruloplasmin was the predominant protein associated with myeloperoxidase. When the purified proteins were mixed, they became strongly but reversibly associated. Ceruloplasmin was a potent inhibitor of purified myeloperoxidase, inhibiting production of hypochlorous acid by 50% at 25 nm. Ceruloplasmin rapidly reduced Compound I, the Fe^V redox intermediate of myeloperoxidase, to Compound II, which has Fe^IV in its heme prosthetic groups. It also prevented the fast reduction of Compound II by tyrosine. In the presence of chloride and hydrogen peroxide, ceruloplasmin converted myeloperoxidase to Compound II and slowed its conversion back to the ferric enzyme. Collectively, our results indicate that ceruloplasmin inhibits myeloperoxidase by reducing Compound I and then trapping the enzyme as inactive Compound II. We propose that ceruloplasmin should provide a protective shield against inadvertent oxidant production by myeloperoxidase during inflammation.