Ferric Myeloperoxidase Research Articles

Myeloperoxidase-catalyzed oxidation of cyanide to cyanate: A potential carbamylation route involved in the formation of atherosclerotic plaques?

Protein carbamylation by cyanate is a post-translational modification associated with several (patho)physiological conditions, including cardiovascular disorders. However, the biochemical pathways leading to protein carbamylation are incompletely characterized. This work demonstrates that the heme protein myeloperoxidase (MPO), which is secreted at high concentrations at inflammatory sites from stimulated neutrophils and monocytes, is able to catalyze the two-electron oxidation of cyanide to cyanate and promote the carbamylation of taurine, lysine, and low-density lipoproteins. We probed the role of cyanide as both electron donor and low-spin ligand by pre-steady-state and steady-state kinetic analyses and analyzed reaction products by MS. Moreover, we present two further pathways of carbamylation that involve reaction products of MPO, namely oxidation of cyanide by hypochlorous acid and reaction of thiocyanate with chloramines. Finally, using an in vivo approach with mice on a high-fat diet and carrying the human MPO gene, we found that during chronic exposure to cyanide, mimicking exposure to pollution and smoking, MPO promotes protein-bound accumulation of carbamyllysine (homocitrulline) in atheroma plaque, demonstrating a link between cyanide exposure and atheroma. In summary, our findings indicate that cyanide is a substrate for MPO and suggest an additional pathway for in vivo cyanate formation and protein carbamylation that involves MPO either directly or via its reaction products hypochlorous acid or chloramines. They also suggest that chronic cyanide exposure could promote the accumulation of carbamylated proteins in atherosclerotic plaques.

Mechanisms of myeloperoxidase catalyzed oxidation of H2S by H2O2 or O2 to produce potent protein Cys-polysulfide-inducing species

The interaction of heme proteins with hydrogen sulfide is gaining attention as an important element in sulfide-mediated protection against oxidative stress and in regulation of redox signaling. In our previous study we reported the efficient reversible inhibition of myeloperoxidase (MPO) activity by sulfide and the kinetics of the reactions of sulfide with ferric MPO, Compound I and Compound II. Here we provide several lines of evidence that a central intermediate species in the turnover of MPO by sulfide is the Compound III state. Compound III is formed in the reactions of sulfide with ferric or ferrous MPO in the presence of oxygen or via the reductions of Compound I or Compound II by sulfide. The regeneration of active ferric MPO from Compound III is slow - representing the rate-limiting step during turnover - but facilitated by ascorbate or superoxide dismutase. These catalytic cycles produce inorganic sulfane sulfur species, which were shown to promote protein Cys persulfidation. Based on compiling experimental data we propose that in contrast to hemoglobin, myoglobin, catalase or lactoperoxidase the formation of a sulfheme derivative in the oxidative interactions of sulfide with MPO is not a major pathway. Using the Met243Val mutant we demonstrated that the sulfonium ion linkage of the Met243 sulfur to the heme pyrrole ring A, which is a unique feature of MPO, is pivotal in the catalytic oxidation of sulfide via Compound III. The proposed novel MPO-catalyzed sulfide oxidation model does not require the initial presence of hydrogen peroxide, only oxygen to provide a slow flux of sulfane sulfur species generation, which could be important in sulfide-mediated endogenous signaling. Furthermore, peroxide-induced formation of sulfane sulfur species by MPO may have a role in protection of regulatory or functional Cys residues during (for example neutrophil induced) inflammatory oxidative stress.

New insights into thiocyanate oxidation by human myeloperoxidase

Human myeloperoxidase (MPO) uses chloride and thiocyanate as physiological substrates at neutral pH. Oxidation of thiocyanate to hypothiocyanite mediated by the redox intermediate Compound I rapidly restores the ferric state of MPO. At low thiocyanate concentration and in the presence of hydrogen peroxide the observed reaction sequence is Compound I→ferric MPO→Compound II→MPO-cyanide complex, whereas at high thiocyanate concentrations and in the absence of H2O2 the only observed transition is Compound I→ferric MPO. The reaction of ferric MPO with hypothiocyanite directly forms the MPO-cyanide complex, whereas a transient product derived from the reaction between hypothiocyanite and hydrogen peroxide is demonstrated to mediate the conversion of ferric MPO to Compound II. Mechanisms for those reactions are discussed and proposed.

Interactions of hydrogen sulfide with myeloperoxidase.

The actions of hydrogen sulfide in human physiology have been extensively studied and, although it is an essential mediator of many biological functions, the underlying molecular mechanisms of its actions are ill-defined. To elucidate the roles of sulfide in inflammation, we have investigated its interactions with human myeloperoxidase (MPO), a major contributor to inflammatory oxidative stress. The interactions of sulfide and MPO were investigated using electron paramagnetic resonance, electronic circular dichroism, UV-vis and stopped-flow spectroscopies. We found favourable reactions between sulfide and the native-ferric enzyme as well as the MPO redox intermediates, ferrous MPO, compound I and compound II. Sulfide was a potent reversible inhibitor of MPO enzymic activity with an IC50 of 1 µM. In addition, the measured second-order rate constants for the reactions of sulfide with compound I [k = (1.1 ± 0.06) × 10(6) M(-1) s(-1)] and compound II [k = (2.0 ± 0.03) × 10(5) M(-1) s(-1)] suggest that sulfide is a potential substrate for MPO in vivo. Endogenous levels of sulfide are likely to inhibit the activity of circulating and endothelium-bound MPO. The fully reversible inhibition suggests a mediatory role of sulfide on the oxidant-producing function of the enzyme. Furthermore, the efficient HOCl oxidation of sulfide to give polysulfides (recently recognized as important components of sulfide biology) together with MPO-catalysed sulfide oxidation and the lack of interaction between MPO and sulfide oxidation products, predict a modulatory role of MPO in sulfide signalling.

CO Binding and Ligand Discrimination in Human Myeloperoxidase

Despite the fact that ferrous myeloperoxidase (MPO) can bind both O(2) and NO, its ability to bind CO has been questioned. UV/visible spectroscopy was used to confirm that CO induces small spectral shifts in ferrous MPO, and Fourier transform infrared difference spectroscopy showed definitively that these arose from formation of a heme ferrous-CO compound. Recombination rates after CO photolysis were monitored at 618 and 645 nm as a function of CO concentration and pH. At pH 6.3, k(on) and k(off) were 0.14 mM(-1) x s(-1) and 0.23 s(-1), respectively, yielding an unusually high K(D) of 1.6 mM. This affinity of MPO for CO is 10 times weaker than its affinity for O(2). The observed rate constant for CO binding increased with increasing pH and was governed by a single protonatable group with a pK(a) of 7.8. Fourier transform infrared spectroscopy revealed two different conformations of bound CO with frequencies at 1927 and 1942 cm(-1). Their recombination rate constants were identical, indicative of two forms of bound CO that are in rapid thermal equilibrium rather than two distinct protein populations with different binding sites. The ratio of bound states was pH-dependent (pK(a) approximately 7.4) with the 1927 cm(-1) form favored at high pH. Structural factors that account for the ligand-binding properties of MPO are identified by comparisons with published data on a range of other ligand-binding heme proteins, and support is given to the recent suggestion that the proximal His336 in MPO is in a true imidazolate state.

Myeloperoxidase: a target for new drug development?

Myeloperoxidase (MPO), a member of the haem peroxidase-cyclooxygenase superfamily, is abundantly expressed in neutrophils and to a lesser extent in monocytes and certain type of macrophages. MPO participates in innate immune defence mechanism through formation of microbicidal reactive oxidants and diffusible radical species. A unique activity of MPO is its ability to use chloride as a cosubstrate with hydrogen peroxide to generate chlorinating oxidants such as hypochlorous acid, a potent antimicrobial agent. However, evidence has emerged that MPO-derived oxidants contribute to tissue damage and the initiation and propagation of acute and chronic vascular inflammatory disease. The fact that circulating levels of MPO have been shown to predict risks for major adverse cardiac events and that levels of MPO-derived chlorinated compounds are specific biomarkers for disease progression, has attracted considerable interest in the development of therapeutically useful MPO inhibitors. Today, detailed information on the structure of ferric MPO and its complexes with low- and high-spin ligands is available. This, together with a thorough understanding of reaction mechanisms including redox properties of intermediates, enables a rationale attempt in developing specific MPO inhibitors that still maintain MPO activity during host defence and bacterial killing but interfere with pathophysiologically persistent activation of MPO. The various approaches to inhibit enzyme activity of MPO and to ameliorate adverse effects of MPO-derived oxidants will be discussed. Emphasis will be put on mechanism-based inhibitors and high-throughput screening of compounds as well as the discussion of physiologically useful HOCl scavengers.

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.

Redox Thermodynamics of the Fe(III)/Fe(II) Couple of Human Myeloperoxidase in Its High-Spin and Low-Spin Forms

Myeloperoxidase (MPO) (donor, hydrogen peroxide oxidoreductase, EC 1.11.1.7) is the most abundant neutrophil enzyme and catalyzes predominantly the two-electron oxidation of ubiquitous chloride (Cl-), to generate the potent bleaching oxidant hypochlorous acid (HOCl), thus contributing to bacterial killing and inflammatory reactions of neutrophils. Here, the thermodynamics of the one-electron reduction of the ferric heme in its ferric high-spin and cyanide-bound low-spin forms were determined through spectroelectrochemical experiments. The E(o)' values for free and cyanide-bound MPO (5 and -37 mV, respectively, at 25 degrees C and pH 7.0) are significantly higher than those of other heme peroxidases. Variable-temperature experiments revealed that the enthalpic stabilization of ferric high-spin MPO is much weaker than in other heme peroxidases and is exactly compensated by the entropic change upon reduction. In contrast to those of other heme peroxidases, the stabilization of the ferric cyanide-bound MPO is also very weak and fully entropic. This peculiar behavior is discussed with respect to the MPO-typical covalent heme to protein linkages as well as to the published structures of ferric MPO and its cyanide complex and the recently published structure of lactoperoxidase as well as the physiological role of MPO in bacterial killing.

A novel multistep mechanism for oxygen binding to ferrous hemoproteins: rapid kinetic analysis of ferrous-dioxy myeloperoxidase (compound III) formation.

Myeloperoxidase (MPO), a hemoprotein that uses H(2)O(2) as the electron acceptor in the catalysis of oxidative reactions, is implicated as a participant in inflammatory injury and cardiovascular diseases. Mechanisms for turning off this enzyme once released, preventing unwanted tissue injury, are poorly understood. We recently demonstrated that MPO heme reduction causes collapse of the heme pocket, as monitored by significant reductions in the rates of diatomic ligand binding to the heme iron. Using spectral and rapid kinetic measurements, we now demonstrate that molecular oxygen (O(2)) binds to ferrous MPO (MPO-Fe(II)) in a distinct and novel mechanism. Rather than occurring through a simple, reversible, one-step mechanism, as is typical for O(2) binding to other ferrous hemoproteins, the reaction involves several kinetically and spectroscopically distinguishable intermediates. Diode array spectrophotometric and stopped-flow studies reveal that the formation of the MPO-Fe(II)-O(2) complex consists of at least three elementary steps and includes at least two sequential transient intermediates. The first step involves reversible formation of a transient intermediate via an O(2)-dependent mechanism, followed by two sequential O(2)-independent steps that appear to be conformational in origin. Insights into mechanisms for inactivating MPO and the novel mode of O(2) binding to the hemoprotein may provide important clues toward understanding the catalytic action of MPO.

Kinetics of oxygen binding to ferrous myeloperoxidase

Myeloperoxidase (MPO), which is involved in host defence and inflammation, is a unique peroxidase in having a globin-like standard reduction potential of the ferric/ferrous couple. Intravacuolar and exogenous MPO released from stimulated neutrophils has been shown to exist in the oxyferrous form, called compound III. To investigate the reactivity of ferrous MPO with molecular oxygen, a stopped-flow kinetic analysis was performed. In the absence of dioxygen, ferrous MPO decays to ferric MPO (0.04 s −1 at pH 8 versus 1.4 s −1 at pH 5). At pH 7.0 and 25 °C, compound III formation (i.e., binding of dioxygen to ferrous MPO) occurs with a rate constant of (1.1 ± 0.1) × 10 4 M −1 s −1. The rate doubles at pH 5.0 and oxygen binding is reversible. At pH 7.0, the dissociation equilibrium constant of the oxyferrous form is (173 ± 12) μM. The rate constant of dioxygen dissociation from compound III is much higher than conversion of compound III to ferric MPO (which is not affected by the oxygen concentration). This allows an efficient transition of compound III to redox intermediates which actually participate in the peroxidase or halogenation cycle of MPO.

Direct conversion of ferrous myeloperoxidase to compound II by hydrogen peroxide: an anaerobic stopped-flow study

Myeloperoxidase (MPO) is one of the essential components of the antimicrobial systems of polymorphonuclear neutrophils. It is unique in having a globin-like standard reduction potential of the ferric/ferrous couple. Here, it is shown that ferrous MPO heterolytically cleaves hydrogen peroxide forming water and oxyferryl MPO (compound II). The two-electron oxidation reaction follows second-order kinetics with the apparent bimolecular rate constant being (6.8 ± 0.6) × 10 4 M −1 s −1 at pH 7.0. After depletion of (micromolar) H 2O 2 compound II slowly decays to ferric MPO, whereas upon addition of millimolar H 2O 2 to ferrous MPO, compound III (oxyperoxidase) is formed in a sequence of two reactions involving compound II formation and its direct reaction with H 2O 2, which also follows second-order kinetics [(78 ± 2) M −1 s −1 at pH 7.0]. It is discussed how these reactions contribute to the interconversion of compound II and compound III and could explain the catalase activity of MPO.

Two-electron reduction and one-electron oxidation of organic hydroperoxides by human myeloperoxidase

The reaction of native myeloperoxidase (MPO) and its redox intermediate compound I with hydrogen peroxide, ethyl hydroperoxide, peroxyacetic acid, t-butyl hydroperoxide, 3-chloroperoxybenzoic acid and cumene hydroperoxide was studied by multi-mixing stopped-flow techniques. Hydroperoxides are decomposed by MPO by two mechanisms. Firstly, the hydroperoxide undergoes a two-electron reduction to its corresponding alcohol and heme iron is oxidized to compound I. At pH 7 and 15°C, the rate constant of the reaction between 3-chloroperoxybenzoic acid and ferric MPO was similar to that with hydrogen peroxide (1.8×107 M−1 s−1 and 1.4×107 M−1 s−1, respectively). With the exception of t-butyl hydroperoxide, the rates of compound I formation varied between 5.2×105 M−1 s−1 and 2.7×106 M−1 s−1. Secondly, compound I can abstract hydrogen from these peroxides, producing peroxyl radicals and compound II. Compound I reduction is shown to be more than two orders of magnitude slower than compound I formation. Again, with 3-chloroperoxybenzoic acid this reaction is most effective (6.6×104 M−1 s−1 at pH 7 and 15°C). Both reactions are controlled by the same ionizable group (average pKa of about 4.0) which has to be in its conjugated base form for reaction.

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.

Mechanism of inactivation of myeloperoxidase by 4-aminobenzoic acid hydrazide.

Hypochlorous acid is the most powerful oxidant generated by neutrophils and is likely to contribute to the damage mediated by these inflammatory cells. The haem enzyme myeloperoxidase catalyses its production from hydrogen peroxide and chloride. 4-Aminobenzoic acid hydrazide (ABAH) is a potent inhibitor of hypochlorous acid production. In this investigation we show that, in the presence of hydrogen peroxide, ABAH irreversibly inactivates myeloperoxidase. ABAH was oxidized by myeloperoxidase, and kinetic analysis of the inactivation conformed to that for a mechanism-based inhibitor. Inactivation was exacerbated by concentrations of hydrogen peroxide greater than 50 microM and by the absence of oxygen. Hydrogen peroxide alone caused minimal inactivation. Reduced glutathione inhibited the oxidation of ABAH as well as the irreversible inhibition of myeloperoxidase. In the presence of oxygen, ABAH and hydrogen peroxide initially converted myeloperoxidase into compound III, which subsequently lost haem absorbance. In the absence of oxygen, the enzyme was converted into ferrous myeloperoxidase and its haem groups were rapidly destroyed. We propose that myeloperoxidase oxidizes ABAH to a radical that reduces the enzyme to its ferrous intermediate. Ferrous myeloperoxidase reacts either with oxygen to allow enzyme turnover, or with hydrogen peroxide to give irreversible inactivation.

Aromatic hydroxylation during the myeloperoxidase-oxidase oxidation of hydrazines

Benzoic acid was found to be hydroxylated by a mixture of myeloperoxidase (MPO) and the mycobactericidal drug, isoniazid. Aromatic hydroxylation and formation of compound III (oxyper-oxidase) were coincident during the MPO-oxidase oxidation of isoniazid which proceeded without augmentation from the reagent hydrogen peroxide. An intermediate of isoniazid reduced ferric MPO to ferrous MPO which associated with dioxygen to form compound III. Aromatic hydroxylation also occurred in a mixture of isoniazid (or phenylhydrazine) and a ferric salt. Hydroxylations in both the enzymatic and nonenzymatic reaction systems were inhibited by the iron chelator, desferal, as well as by the specific hydroxyl radical scavenger, mannitol. To distinguish between the hydroxylating intermediates in the different reaction systems, the unique properties of the natural antioxidant, phytic acid, were exploited. Phytic acid inhibited aromatic hydroxylation in the Fe 3+INH system, which is in accordance with its known properties as a powerful inhibitor of iron-driven reactions ( żOH formation). By contrast, phytic acid stimulated hydroxylation in the enzymatic system which was accompanied by a concomitant stimulation in the rate of compound III formation. These events were, however, not directly related to each other. Phytic acid had a direct effect on the redox transformation of isoniazid by stimulating superoxide generation during auto-oxidation of the drug. In addition, phytic acid also facilitated compound III decay in the absence of isoniazid, suggesting that it may also regulate the oxygen affinity of MPO, similar to its effect on the oxygenation of haemoglobin. The data on aromatic hydroxylation in the MPO-isoniazid system do not support a role for żOH in the reaction and may fit the model for the P450 mixed oxidase system.

Influence of superoxide on myeloperoxidase kinetics measured with a hydrogen peroxide electrode

Stimulated neutrophils discharge large quantities of superoxide (O2.-), which dismutates to form H2O2. In combination with Cl-, H2O2 is converted into the potent oxidant hypochlorous acid (HOCl) by the haem enzyme myeloperoxidase. We have used an H2O2 electrode to monitor H2O2 uptake by myeloperoxidase, and have shown that in the presence of Cl- this accurately represents production of HOCl. Monochlorodimedon, which is routinely used to assay production of HOCl, inhibited H2O2 uptake by 95%. This result confirms that monochlorodimedon inhibits myeloperoxidase, and that the monochlorodimedon assay grossly underestimates the activity of myeloperoxidase. With 10 microM-H2O2 and 100 mM-Cl-, myeloperoxidase had a neutral pH optimum. Increasing the H2O2 concentration to 100 microM lowered the pH optimum to pH 6.5. Above the pH optimum there was a burst of H2O2 uptake that rapidly declined due to accumulation of Compound II. High concentrations of H2O2 inhibited myeloperoxidase and promoted the formation of Compound II. These effects of H2O2 were decreased at higher concentrations of Cl-. We propose that H2O2 competes with Cl- for Compound I and reduces it to Compound II, thereby inhibiting myeloperoxidase. Above pH 6.5, O2.- generated by xanthine oxidase and acetaldehyde prevented H2O2 from inhibiting myeloperoxidase, increasing the initial rate of H2O2 uptake. O2.- allowed myeloperoxidase to function optimally with 100 microM-H2O2 at pH 7.0. This occurred because, as previously demonstrated, O2.- prevents Compound II from accumulating by reducing it to ferric myeloperoxidase. In contrast, at pH 6.0, where Compound II did not accumulate, O2.- retarded the uptake of H2O2. We propose that by generating O2.- neutrophils prevent H2O2 and other one-electron donors from inhibiting myeloperoxidase, and ensure that this enzyme functions optimally at neutral pH.

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 O 2 − 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 H 2O 2 to give compound I was 3.1 · 10 7 M −1 · s −1. Our results indicate that although the reaction of ferric myeloperoxidase with O 2 − is an order of magnitude slower than with H 2O 2, the former reaction is sufficiently rapid to influence myeloperoxidase-dependent production of hypochlorous acid by stimulated neutrophils.

The effect of chloride on the redox and EPR properties of myeloperoxidase.

Myeloperoxidase was purified from human polymorphonuclear leukocytes and the effect of chloride upon the EPR and potentiometric properties was studied. The redox titration between the ferrous and ferric states of the enzyme yielded n = 1 Nernst plots between pH 9 and 4, with clear isosbestic points in the optical spectra during the redox change. The midpoint potential (Em) between the ferric and ferrous forms of the enzyme exhibited a pH-dependent change between pH 4 and 9, and the effect of added chloride ion indicated that Cl- competed with OH- for a binding site on the enzyme. Interestingly, the pH dependence of the Em indicated that the overall redox reactions of the enzyme was: ferric myeloperoxidase + 2e- + 1H+ = ferrous myeloperoxidase. Myeloperoxidase exhibited a rhombic high spin EPR signal which exhibited reduced rhombicity upon the binding of chloride. Our results strongly suggest that chloride binds to the sixth coordination position of the chlorin iron in myeloperoxidase by replacing the water which is the sixth ligand in the resting state. It is also concluded that the two iron centers are identical and that there is no interaction between them.

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 nitrosyl compounds of ferrous animal haloperoxidases

Human myeloperoxidase, human eosinophil peroxidase and bovine lactoperoxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) reduced with ascorbic acid form nitrosyl compounds which show rhombic EPR signals centered at g = 2. Using 14NO ( I N = 1), the central resonance signal exhibited a hyperfine structure of nine lines originating from a triplet with a small hyperfine splitting ( A II( z) = 0.69 mT for myeloperoxidase and 0.73 mT for eosinophil peroxidase and lactoperoxidase) superimposed upon a triplet with a larger hyperfine splitting ( A I ( z) = 2.34, 2.32 and 2.09 mT for myeloperoxidase, eosinophil peroxidase and lactoperoxidase, respectively). Using 15NO ( I N = 1 2 ), the nitrosyl compound of ferrous myeloperoxidase and ferrous lactoperoxidase showed a doublet of triplets superimposed upon the central resonance signal. These results demonstrate that a nitrogen nucleus is present at the fifth ligand position of the haem iron in these peroxidases.