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Apocynin has been reported to require dimerization by myeloperoxidase (MPO) to inhibit leukocyte NADPH oxidase. (-)-Epicatechin, a dietary flavan-3-ol, has been identified as a 'prodrug' of apocynin-like metabolites that inhibit endothelial NADPH oxidase activity and elevate the cellular level of nitric oxide. Since (-)-epicatechin has tentatively been identified as substrate of MPO, we studied the one-electron oxidation of (-)-epicatechin by MPO. By using multi-mixing stopped-flow technique, we demonstrate that (-)-epicatechin is one of the most efficient electron donors for heme peroxidases investigated so far. Second order rate constants for the (-)-epicatechin-mediated conversion of MPO-compound I to compound II and compound II to resting enzyme were estimated to be 1.9 x 10{sup 7} and 4.5 x 10{sup 6} M{sup -1} s{sup -1}, respectively (pH 7, 25 deg. C). The data indicate that (-)-epicatechin is capable of undergoing fast MPO-mediated one-electron oxidation.

Catalase-peroxidases (KatGs) are multifunctional heme peroxidases exhibiting an overwhelming catalase activity and a substantial peroxidase activity of broad specificity. Here, we show that catalase-peroxidases are also haloperoxidases capable of oxidizing chloride, bromide, and iodide in a peroxide- and enzyme-dependent manner. Recombinant KatG and the variants R119A, W122F, and W122A from the cyanobacterium Synechocystis PCC 6803 have been tested for their halogenation activity. Halogenation of monochlorodimedon (MCD), formation of triiodide and tribromide, and bromide- and chloride-mediated oxidation of glutathione have been tested. Halogenation of MCD by chloride, bromide, and iodide was shown to be catalyzed by wild-type KatG and the variant R119A. Generally, rates of halogenation increased in the order Cl(-) < Br(-) < I(-) and/or by decreasing pH. The halogenation activity of R119A was about 7-9% that of the wild-type enzyme. Upon exchange of the distal Trp122 by Phe and Ala, both the catalase and halogenation activities were lost but the overall peroxidase activity was increased. The findings suggest that the same redox intermediate is involved in H(2)O(2) and halide oxidation and that distal Trp122 is involved in both two-electron reactions. That halides compete with H(2)O(2) for the same redox intermediate is also emphasized by the fact that the polarographically measured catalase activity is influenced by halides, with bromide being more effective than chloride.

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.

Photoautotrophically grown cyanobacterium Nostoc sp. strain Mac (PCC 8009) released up to about 10 nmol of a c-type cytochrome per ml packed cells after treatment with EDTA under conditions that left the plasma membrane absolutely intact as judged from the absence of cytosolic proteins in the supernatant. Spectra of the ascorbate reduced cytochrome revealed peaks at 553, 522 and 416 nm. The protein was purified to an A-553/A-275 ratio of 0.8. Midpoint potential (at pH 7), isoelectric point and apparent molecular weight of the cytochrome were +0.35 V, 8.6, and around 10,500, respectively. The cytochrome proved to be an excellent electron donor to the aa3-type cytochrome oxidase in both plasma and thylakoid membranes isolated and purified from Nostoc Mac. Chemoheterotrophic growth of the cells increased the level of periplasmic cytochrome c up to 10-fold and cytochrome oxidase activity of plasma membranes up to 90-fold. The periplasmic cytochrome also transferred electrons to photosystem I in illuminated thylakoid membranes. We conclude that cyanobacteria contain a periplasmic c-type cytochrome presumably identical to so-called cytochrome c6 or c-553 which has long been known as a photosynthetic (i.e. thylakoid-associated) redox protein in these organisms, and which is capable of donating electrons (from the periplasmic space) to the cytochrome oxidase in the plasma membrane and (from the thylakoid lumen) to both P700 and cytochrome oxidase in the thylakoid membrane.

In human myeloperoxidase (MPO) the heme is covalently attached to the protein via two ester linkages and a unique sulfonium ion linkage between the sulfur atom of Met243 and the {beta}-carbon of the vinyl ring on pyrrole ring A. Here, we have investigated the variant Met243Val produced in Chinese hamster ovary cells in order to elucidate the role of the electron withdrawing sulfonium bond in compound I formation and reduction. Disruption of this MPO-typical bond causes a blue-shifted UV-vis spectrum and an increase in the heme flexibility. This had no impact on compound I formation mediated by hydrogen peroxide (2.2 x 10{sup 7} M{sup -1} s{sup -1} at pH 7.0 and 25 {sup o}C). Compared with wild-type recombinant MPO the cyanide association rate with ferric Met243Val was significantly enhanced as were also the calculated apparent bimolecular compound I reduction rates by iodide (>10{sup 8} M{sup -1} s{sup -1}) and thiocyanate (>10{sup 8} M{sup -1} s{sup -1}). By contrast, the overall chlorination and bromination activities were decreased by 98.1% and 87.4%, respectively, compared with the wild-type protein. Compound I reduction by chloride was slower than compound I decay to a compound II-like species (0.4 s{sup -1}), whereas compound I reduction bymore » bromide was about 10-times slower (1.3 x 10{sup 4} M{sup -1} s{sup -1}) than the wild-type rate. These findings are discussed with respect to the known crystal structure of MPO and its bromide complex as well as the known redox chemistry of its intermediates and substrates.« less

Nitric oxide-derived oxidants (e.g., peroxynitrite) are believed to participate in antimicrobial activities as part of normal host defenses but also in oxidative tissue injury in inflammatory disorders. A similar role is ascribed to the heme enzyme myeloperoxidase (MPO), the most abundant protein of polymorphonuclear leukocytes, which are the terminal phagocytosing effector cells of the innate immune system. Concomitant production of peroxynitrite and release of millimolar MPO are characteristic events during phagocytosis. In order to understand the mode of interaction between MPO and peroxynitrite, we have performed a comprehensive stopped-flow investigation of the reaction between all physiological relevant redox intermediates of MPO and peroxynitrite. Both iron(III) MPO and iron(II) MPO are rapidly converted to compound II by peroxynitrite in monophasic reactions with calculated rate constants of (6.8+/-0.1) x 10(6) M(-1)s(-1) and (1.3+/-0.2) x 10(6) M(-1)s(-1), respectively (pH 7.0 and 25 degrees C). Besides these one- and two-electron reduction reactions of peroxynitrite, which produce nitrogen dioxide and nitrite, a one-electron oxidation to the oxoperoxonitrogen radical must occur in the fast monophasic transition of compound I to compound II mediated by peroxynitrite at pH 7.0 [(7.6+/-0.1) x 10(6) M(-1)s(-1)]. In addition, peroxynitrite induced a steady-state transition from compound III to compound II with a rate of (1.0+/-0.3) x 10(4) M(-1)s(-1). Thus, the interconversion among the various oxidation states of MPO that is prompted by peroxynitrite is remarkable. Reaction mechanisms are proposed and the physiological relevance is discussed.

Hypochlorous acid (HOCl) is the most powerful oxidant produced by human neutrophils and contributes to the damage caused by these inflammatory cells. It is produced from H2O2 and chloride by the heme enzyme myeloperoxidase (MPO). Based on findings that betalains provide antioxidant and anti-inflammatory effects, we performed the present kinetic study on the interaction between the betalains, betanin and indicaxanthin, with the redox intermediates, compound I and compound II of MPO, and its major cytotoxic product HOCl. It is shown that both betalains are good peroxidase substrates for MPO and function as one-electron reductants of its redox intermediates, compound I and compound II. Compound I is reduced to compound II with a second-order rate constant of (1.5+/-0.1) x 10(6) M(-1) s(-1) (betanin) and (1.1+/-0.2) x 10(6) M(-1) s(-1) (indicaxanthin), respectively, at pH 7.0 and 25 degrees C. Formation of ferric (native) MPO from compound II occurs with a second-order rate constant of (1.1+/-0.1) x 10(5) M(-1) s(-1) (betanin) and (2.9+/-0.1) x 10(5) M(-1) s(-1) (indicaxanthin), respectively. In addition, both betalains can effectively scavenge hypochlorous acid with determined rates of (1.8+/-0.2) x 10(4) M(-1) s(-1) (betanin) and (7.7+/-0.1) x 10(4) M(-1) s(-1) (indicaxanthin) at pH 7.0 and 25 degrees C. At neutral pH and depending on their concentration, both betalains can exhibit a stimulating and inhibitory effect on the chlorination activity of MPO, whereas at pH 5.0 only inhibitory effects were observed even at micromolar concentrations. These findings are discussed with respect to our knowledge of the enzymatic mechanisms of MPO.

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 2 O 2 compound II slowly decays to ferric MPO, whereas upon addition of millimolar H 2 O 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 2 O 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.

Myeloperoxidase (MPO) is an important component of the neutrophil's antimicrobial armory and has been implicated in promoting tissue damage in numerous inflammatory diseases. For the first time the standard reduction potential of the redox couple compound II/native enzyme has been determined to be (0.97+/-0.01)V at pH 7.0 and 25 degrees C. This was achieved by rapid mixing of preformed compound II with either tyrosine or nitrite by using the sequential-mixing stopped-flow technique and measuring spectrophotometrically the concentrations of the reacting species and products at equilibrium. Using the recently determined standard reduction potential for the couple compound I/native enzyme (1.16 V), the reduction potential of the couple compound I/compound II was calculated to be 1.35 V at pH 7 and 25 degrees C. These data reveal substantial differences between the two known heme peroxidase superfamilies and reflect the dramatic differences observed in the oxidisability of substrates by the MPO redox intermediates compound I and compound II.

Cytosolic extracts of the cyanobacterium Anacystis nidulans exhibit both catalase and o-dianisidine peroxidase activity. Native polyacrylamide gel electrophoresis demonstrates one distinct enzyme, which has been purified to essential homogeneity and found to be composed of two identical subunits of equal size (80.5 kDa). The isoelectric point is at pH 4.7. It is a very efficient catalase with a broad pH optimum between 6.5 and 7.5 and a Km for H2O2 of 4.3 mM, a calculated turnover number of 7200 s(-1), and an overall-rate constant of 3.5 x 10(6) M(-1) s(-1). The behaviour of this protoheme-enzyme is typical of the class of prokaryotic catalase-peroxidases, which is sensitive to cyanide (Ki = 27.2 microM) and insensitive to the eukaryotic catalase inhibitor 3-amino-1,2,4-triazole. The enzyme accepts electrons from o-dianisidine, but not from ascorbate, glutathione, and NADH. With hydrogen peroxide in steady-state conditions the enzyme is mainly in the ferric state indicating that Compound I is much faster reduced by H2O2 than it is formed. The native enzyme is in the high-spin state, which is transformed to low-spin upon addition of cyanide. With peroxoacetic acid Compound I is formed at a rate of 5.9 x 10(4) M(-1) s(-1) at pH 7.0 and 25 degrees C with about 50% hypochromicity, a Soret-maximum at 405 nm and isosbestic points at 354 and 427 nm.