Thiols as myeloperoxidase-oxidase substrates.

Nine low-Mr thiols were compared with regard to their ability to function as myeloperoxidase-oxidase substrates under conditions where no auto-oxidation of the thiols could be observed. The methyl and ethyl esters of cysteine were found to be about twice as active as cysteamine at pH 7.0, in terms of increased O2 consumption. Cysteine itself was poorly active, whereas glutathione, N-acetylcysteine and penicillamine were completely inactive as myeloperoxidase-oxidase substrates under these conditions. The structure-activity relationships indicated that both a free thiol and free amino group were required for peroxidase-oxidase activity, and also that a free carboxy group abolished activity. In analogy with cysteamine, the activities of both cysteine esters were inhibited by superoxide dismutase (less than 5 micrograms/ml) and by catalase and not by the hydroxyl-radical scavenger mannitol. In contrast with cysteamine, the activities of both cysteine esters were stimulated more than 2-fold by high concentrations (greater than 5 micrograms/ml) of superoxide dismutase. The activities of both cysteine esters exhibited broad pH optima at pH 7. A mechanism for the myeloperoxidase-oxidase oxidation of the cysteine esters is proposed, which is partly different from that previously proposed for cysteamine.     

Synergism between substrate and non-substrate thiols in peroxidase-oxidase reactions

Cysteamine and reduced glutathione were shown to act synergistically as peroxidase-oxidase substrates as measured by oxygen consumption and Nitro Blue Tetrazolium reduction. Cysteine methyl ester could be substituted for cysteamine and N-acetylcysteine and penicillamine could be substituted for glutathione. The involvement of reduced oxygen species and the effects of pH and chloride were studied. A possible mechanism of peroxidase-oxidase oxidation of cysteamine and glutathione is proposed. These studies show that peroxidase oxidase reactions can occur with close to physiological concentrations of peroxidase and thiols.     

Involvement of cysteine, serotonin and their analogues in peroxidase-oxidase reactions

Myeloperoxidase-oxidase reactions with close to physiological concentrations of thiols and phenols were studied. Cysteine was shown to be a myeloperoxidase-oxidase substrate when catalytic amounts of serotonin were added as cosubstrate. Penicillamine could be substituted for cysteine and acetaminophen could be substituted for serotonin. The properties of these peroxidase-oxidase reactions, e.g. the dependence on substrate and myeloperoxidase concentration, reduced oxygen species, metal ions and pH, were studied. Also, eosinophil, lacto- and horseradish peroxidase could catalyse these reactions.     

The antioxidant action of taurine, hypotaurine and their metabolic precursors.

It has been suggested that taurine, hypotaurine and their metabolic precursors (cysteic acid, cysteamine and cysteinesulphinic acid) might act as antioxidants in vivo. The rates of their reactions with the biologically important oxidants hydroxyl radical (.OH), superoxide radical (O2.-), hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) were studied. Their ability to inhibit iron-ion-dependent formation of .OH from H2O2 by chelating iron ions was also tested. Taurine does not react rapidly with O2.-, H2O2 or .OH, and the product of its reaction with HOCl is still sufficiently oxidizing to inactivate alpha 1-antiproteinase. Thus it seems unlikely that taurine functions as an antioxidant in vivo. Cysteic acid is also poorly reactive to the above oxidizing species. By contrast, hypotaurine is an excellent scavenger of .OH and HOCl and can interfere with iron-ion-dependent formation of .OH, although no reaction with O2.- or H2O2 could be detected within the limits of our assay techniques. Cysteamine is an excellent scavenger of .OH and HOCl; it also reacts with H2O2, but no reaction with O2.- could be measured within the limits of our assay techniques. It is concluded that cysteamine and hypotaurine are far more likely to act as antioxidants in vivo than is taurine, provided that they are present in sufficient concentration at sites of oxidant generation.     

Characteristics of luminol chemiluminescence induced by the catalytic action of myeloperoxidase

The effects of pH, luminol myeloperoxidase and hydrogen peroxide concentrations on the intensity of luminol chemiluminescence induced by myeloperoxidase catalysis were investigated. It was found that the intensity of luminescence is proportional to the enzyme concentration (up to 8.10(-8) M) and reaches the saturation level at higher enzyme concentrations. The dependence of chemiluminescence intensity on [H2O2] is bell-shaped: at H2O2 concentrations above 1.10(-4) M the luminescence is inhibited with a maximum at neutral values of pH. Luminol at concentrations above 5.10(-5) M inhibits this process. It was demonstrated that the effects of singlet oxygen, superoxide and hydroxyl radicals on the chemiluminescence reaction are insignificant. Luminol oxidation in the course of the myeloperoxidase reaction is induced by hypochlorite.     

The mechanism of myeloperoxidase-dependent chlorination of monochlorodimedon

Chlorination of monochlorodimedon is routinely used to measure the production of hypochlorous acid catalysed by myeloperoxidase from H2O2 and Cl-. We have found that the myeloperoxidase/H2O2/Cl- system, at pH 7.8, catalysed the loss of monochlorodimedon with a rapid burst phase followed by a much slower steady-state phase. The loss of monochlorodimedon in the absence of Cl- was only 10% of the steady-state rate in the presence of Cl-, which indicates that the major reaction of monochlorodimedon was with hypochlorous acid. During the steady-state reaction, myeloperoxidase was present as 100% compound II, which cannot participate directly in hypochlorous acid formation. Monochlorodimedon was necessary for formation of compound II, since it was not formed in the presence of methionine. Both the amount of hypochlorous acid formed during the burst phase, and the steady-state rate of hypochlorous acid production, increased with increasing concentrations of myeloperoxidase and with decreasing concentrations of monochlorodimedon. Inhibition by monochlorodimedon was competitive with Cl-. From these results, and the ability of myeloperoxidase to slowly peroxidase monochlorodimedon in the absence of Cl-, we propose that the reaction of monochlorodimedon with the myeloperoxidase/H2O2/Cl- system involves a major pathway due to hypochlorous acid-dependent chlorination and a minor peroxidative pathway. Only a small fraction of compound I needs to react with monochlorodimedon instead of Cl- at each enzyme cycle, for compound II to rapidly accumulate. Monochlorodimedon, therefore, cannot be regarded as an inert detector of hypochlorous acid production by myeloperoxidase, but acts to limit the chlorinating activity of the enzyme. In the presence of reducing species that act like monochlorodimedon, the activity of myeloperoxidase would depend on the rate of turnover of compound II. Components of human serum promoted the conversion of ferric-myeloperoxidase to compound II in the presence of H2O2. We suggest, therefore, that in vivo the rate of turnover of compound II may determine the rate of myeloperoxidase-dependent production of hypochlorous acid by stimulated neutrophils.     

Kinetic mechanism and specificity of bovine milk sulphydryl oxidase

Sulphydryl oxidase is known to catalyse the synthesis de novo of disulphide bonds in a variety of thiol-containing compounds. Reduced glutathione is the best thiol substrate; however, D- and L-cysteine, cysteamine and N-acetyl-L-cysteine, as well as cysteine-containing peptides and proteins, are also effectively oxidized. In contrast, oxidation of the thiol groups of mercaptoethanol, mercaptopyridine, dithiothreitol, dithioerythritol, mercaptoacetate, mercaptopropionate or lipoic acid is not detectably catalysed. In bovine milk, sulphydryl oxidase is closely associated with another glutathione-metabolizing enzyme, gamma-glutamyltransferase. Covalent chromatography of crude preparations on cysteinylsuccinamidopropyl-glass resolves the oxidase from the transferase, thus permitting the kinetic characterization of glutathione oxidation. Initial-rate data imply a Ter Bi substituted-enzyme mechanism, and the observed substrate inhibition by thiols suggest that O2 binds first. Independent, non-kinetic, data, namely the immobilization of sulphydryl oxidase on cysteinyl-matrices, support formation of a mixed-disulphide intermediate between the thiol and enzyme, as predicted by the proposed mechanism. The enzyme-catalysed reaction appears not to be mediated via a superoxide intermediate, since O2 consumption is not affected by the presence of Nitro Blue Tetrazolium. FAD, NAD+, NADP+ and Nitro Blue Tetrazolium are all inactive as electron acceptors for sulphydryl oxidase catalysis.     

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.     

Studies on the NADPH oxidation by subcellular particles from phagocytosing polymorphonuclear leucocytes: evidence for the involvement of three mechanisms

1. The NADPH-oxidizing activity of a 100 000 X g particulate fraction of the postnuclear supernatant obtained frm guinea-pig phagocytosing poymorphonuclear leucocytes has been assayed by simultaneous determination of oxygen consumption, NADPH oxidation and O2- generation at pH 5.5 and 7.0 and with 0.15 mM and 1 mM NADPH. 2. The measurements of oxygen consumption and NADPH oxidation gave comparable results. The stoichiometry between the oxygen consumed and the NADPH oxidized was 1:1. 3. A markedly lower enzymatic activity was observed, under all the experimental conditions used, when the O2- generation assay was employed as compared to the assays of oxygen uptake and NADPH oxidation. 4. The explanation of this difference came from the analysis of the effect of superoxide dismutase and of cytochrome c which removes O2- formed during the oxidation of NADPH. 5. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reactin at pH 5.5. The inhibition was higher with 1 mM NADPH than with 0.15 mM NADPH. 6. Both superoxide dismutase and cytochrome c inhibited the NADPH-oxidizing reaction at pH 7.0 with 1 mM NADPH but less than at pH 5.5 with 1 mM NADPH. 7. The effect of superoxide dismutase at pH 7.0 with 0.15 mM NADPH was negligible. 8. In all instances the inhibitory effect of cytochrome c was greater than that of superoxide dismutase. 9. It was concluded that the NADPH-oxidizing reaction studied here is made up of three components: an enzymatic univalent reduction of O2; an enzymatic, apparently non-univalent, O2 reduction and a non-enzymatic chain reaction. 10. These three components are variably and independently affected by the experimental conditions used. For example, the chain reaction is freely operative at pH 5.5 with 1 mM NADPH but is almost absent at pH 7.0 with 0.15 mM NADPH, whereas the univalent reduction of O2 is optimal at pH 7.0 with 1 mM NADPH.     

Catalase-peroxidases (KatG) exhibit NADH oxidase activity

Catalase-peroxidases (KatG) produced by Burkholderia pseudomallei, Escherichia coli, and Mycobacterium tuberculosis catalyze the oxidation of NADH to form NAD+ and either H2O2 or superoxide radical depending on pH. The NADH oxidase reaction requires molecular oxygen, does not require hydrogen peroxide, is not inhibited by superoxide dismutase or catalase, and has a pH optimum of 8.75, clearly differentiating it from the peroxidase and catalase reactions with pH optima of 5.5 and 6.5, respectively, and from the NADH peroxidase-oxidase reaction of horseradish peroxidase. B. pseudomallei KatG has a relatively high affinity for NADH (Km=12 microm), but the oxidase reaction is slow (kcat=0.54 min(-1)) compared with the peroxidase and catalase reactions. The catalase-peroxidases also catalyze the hydrazinolysis of isonicotinic acid hydrazide (INH) in an oxygen- and H2O2-independent reaction, and KatG-dependent radical generation from a mixture of NADH and INH is two to three times faster than the combined rates of separate reactions with NADH and INH alone. The major products from the coupled reaction, identified by high pressure liquid chromatography fractionation and mass spectrometry, are NAD+ and isonicotinoyl-NAD, the activated form of isoniazid that inhibits mycolic acid synthesis in M. tuberculosis. Isonicotinoyl-NAD synthesis from a mixture of NAD+ and INH is KatG-dependent and is activated by manganese ion. M. tuberculosis KatG catalyzes isonicotinoyl-NAD formation from NAD+ and INH more efficiently than B. pseudomallei KatG.     

Proton transfer reactions associated with the reaction of the fully reduced,purified cytochrome C oxidase with molecular oxygen and ferricyanide

A study is presented on proton transfer associated with the reaction of the fully reduced, purified bovine heart cytochrome c oxidase with molecular oxygen or ferricyanide. The proton consumption associated with aerobic oxidation of the four metal centers changed significantly with pH going from approximately 3.0 H(+)/COX at pH 6.2-6.3 to approximately 1.2 H(+)/COX at pH 8.0-8.5. Rereduction of the metal centers was associated with further proton uptake which increased with pH from approximately 1.0 H(+)/COX at pH 6.2-6.3 to approximately 2.8 H(+)/COX at pH 8.0-8.5. Anaerobic oxidation of the four metal centers by ferricyanide resulted in the net release of 1.3-1.6 H(+)/COX in the pH range 6.2-8.2, which were taken up by the enzyme on rereduction of the metal centers. The proton transfer elicited by ferricyanide represents the net result of deprotonation/protonation reactions linked to anaerobic oxidoreduction of the metal centers. Correction for the ferricyanide-induced pH changes of the proton uptake observed in the oxidation and rereduction phase of the reaction of the reduced oxidase with oxygen gave a measure of the proton consumption in the reduction of O(2) to 2H(2)O. The results show that the expected stoichiometric proton consumption of 4H(+) in the reduction of O(2) to 2H(2)O is differently associated, depending on the actual pH, with the oxidation and reduction phase of COX. Two H(+)/COX are initially taken up in the reduction of O(2) to two OH(-) groups bound to the binuclear Fe a(3)-Cu(B) center. At acidic pHs the third and fourth protons are also taken up in the oxidative phase with formation of 2H(2)O. At alkaline pHs the third and fourth protons are taken up with formation of 2H(2)O only upon rereduction of COX.     

Transient-kinetic studies of pig muscle lactate dehydrogenase

1. The very fast pre-steady-state formation of NADH catalysed by pig M(4) lactate dehydrogenase was equivalent to the enzyme-site concentration at pH values greater than 8.0 and to one-half the site concentration at pH6.8. 2. The rate of dissociation of NADH from the enzyme at pH8.0 (450s(-1)) in the absence of other substrates is faster than the steady-state oxidation of lactate (80s(-1)). The latter process is therefore controlled by a step before NADH dissociation but subsequent to the hydride transfer. 3. The oxidation of enzyme-NADH by excess of pyruvate was studied as a first-order process at pH9.0. There was no effect of NADD on this reaction and it was concluded that the ternary complex undergoes a rate-limiting change before the hydride-transfer step. 4. Some conclusions about the reactions catalysed by the M(4) isoenzyme were drawn from a comparison of these results with those obtained with the H(4) isoenzyme and liver alcohol dehydrogenase.     

Superoxide-dependent oxidation of extracellular reducing agents by isolated neutrophils

Incubation of stimulated neutrophils with sulfhydryl (RSH) compounds or ascorbic acid (ascorbate) results in rapid superoxide (O2-)-dependent oxidation of these reducing agents. Oxidation of RSH compounds to disulfides (RSSR) is faster than the rate of O2- production by the neutrophil NADPH-oxidase, whereas about one ascorbate is oxidized per O2-. Ascorbate is oxidized to dehydroascorbate, which is also oxidized but at a slower rate. Oxidation is accompanied by a large increase in oxygen (O2) uptake that is blocked by superoxide dismutase. Lactoferrin does not inhibit, indicating that ferric (Fe3+) ions are not required, and Fe3+-lactoferrin does not catalyze RSH or ascorbate oxidation. Two mechanisms contribute to oxidation: 1) O2- oxidizes ascorbate or reduced glutathione and is reduced to hydrogen peroxide (H2O2), which also oxidizes the reductants. O2- reacts directly with ascorbate, but reduced glutathione oxidation is mediated by the reaction of O2- with manganese (Mn2+). The H2O2-dependent portion of oxidation is mediated by myeloperoxidase-catalyzed oxidation of chloride to hypochlorous acid (HOCl) and oxidation of the reductants by HOCl. 2) O2- initiates Mn2+-dependent auto-oxidation reactions in which RSH compounds are oxidized and O2 is reduced. Part of this oxidation is due to the RSH-oxidase activity of myeloperoxidase. This activity is blocked by superoxide dismutase but does not require O2- production by the NADPH-oxidase, indicating that myeloperoxidase produces O2- when incubated with RSH compounds. It is proposed that an important role for O2- in the cytotoxic activities of phagocytic leukocytes is to participate in oxidation of reducing agents in phagolysosomes and the extracellular medium. Elimination of these protective agents allows H2O2 and products of peroxidase/H2O2/halide systems to exert cytotoxic effects.     

A study of the kinetics and mechanism of rabbit muscle l-glycerol 3-phosphate dehydrogenase

1. The kinetics of oxidation of l-glycerol 3-phosphate by NAD(+) and of reduction of dihydroxyacetone phosphate by NADH catalysed by rabbit muscle glycerol 3-phosphate dehydrogenase were studied over the range pH6-9. 2. The enzyme was found to catalyse the oxidation of glyoxylate by NAD(+) at pH8.0 and the kinetics of this reaction were also studied. 3. The results are consistent with a compulsory mechanism of catalysis for glycerol 3-phosphate oxidation and dihydroxyacetone phosphate reduction in the intermediate regions of pH, but modifications to the basic mechanism are required to fully explain results at the extremes of the pH range, with these substrates and for glyoxylate oxidation at pH8.0.     

Ascorbic acid, metal ions and the superoxide radical.

1. No evidence could be found for production of the superoxide radical, O2-, during autoxidation of ascorbic acid at alkaline pH values. Indeed, ascorbate may be important in protection against O2- genat-d in vivo. 2. Oxidation of ascorbate at pH 10.2 was stimulated by metal ions. Stimulation by Fe2+ was abolished by superoxide dismutase, probably because of generation of O2-- during reduction of O2 by Fe2+, followed by reaction of O2-- with ascorbate. EDTA changed the mechanism of Fe2+-stimulated ascorbate oxidation. 3. Stimulation of ascorbate oxidation by Cu2+ was also decreased by superoxide dismutase, but this appears to be an artifact, since apoenzyme or bovine serum albumin showed similar effects.     

Mechanism of melatonin-induced oscillations in the peroxidase-oxidase reaction

Melatonin induces oscillations in the peroxidase-oxidase (PO) reaction catalyzed by horseradish peroxidase. We present here studies of the effect of pH, enzyme concentration, and concentration of melatonin on the oscillation frequency. We also present a mechanistic model to explain the experimentally observed changes in oscillation frequency. Using the data obtained here we are able to predict that oscillations will also occur in the PO reaction catalyzed by myeloperoxidase. Myeloperoxidase is an important protein in activated neutrophils and we provide evidence that the oscillations of NAD(P)H, superoxide and hydrogen peroxide in these cells may involve this enzyme. Thus, our experimental system can be considered a model system for the nonrespiratory oxygen metabolism in activated neutrophils and other similar cells participating in the defence against invading pathogens.     

The metabolism of cyclopentanol by Pseudomonas N.C.I.B. 9872

1. Pseudomonas N.C.I.B. 9872 grown on cyclopentanol as carbon source oxidized it at a rate of 228mul of O(2)/h per mg dry wt. and the overall consumption of 5.9mumol of O(2)/mumol of substrate. Cyclopentanone was oxidized at a similar rate with the overall consumption of 5.2mumol of O(2)mumol of substrate. Cells grown with sodium acetate as sole source of carbon were incapable of significant immediate oxidation of these two substrates. 2. Disrupted cells catalysed the oxidation of cyclopentanol to cyclopentanone by the action of an NAD(+)-linked dehydrogenase with an alkaline pH optimum. 3. A cyclopentanolinduced cyclopentanone oxygenase (specific activity 0.11mumol of NADPH oxidized/min per mg of protein) catalysed the consumption of 1mumol of NADPH and 0.9mumol of O(2) in the presence of 1mumol of cyclopentanone. NADPH oxidation did not occur under anaerobic conditions. The only detectable reaction product with 100000g supernatant was 5-hydroxyvalerate. 4. Extracts of cyclopentanol-grown cells contained a lactone hydrolase (specific activity 7.0mumol hydrolysed/min per mg of protein) that converted 5-valerolactone into 5-hydroxyvalerate. 5. Cyclopentanone oxygenase fractions obtained from a DEAE-cellulose column were almost devoid of 5-valerolactone hydrolase and catalysed the formation of 5-valerolactone in high yield from cyclopentanone in the presence of NADPH. 6. Incubation of 5-hydroxyvalerate with the 100000g supernatant, NAD(+) and NADP(+) under aerobic conditions resulted in the consumption of O(2) and the conversion of 5-hydroxyvalerate into glutarate. 7. The high activity of isocitrate lyase in cyclopentanol-grown cells suggests that the further oxidation of glutarate proceeds through as yet uncharacterized reactions to acetyl-CoA. 8. The reaction sequence for the oxidation of cyclopentanol by Pseudomonas N.C.I.B. 9872 is: cyclopentanol --> cyclopentanone --> 5-valerolactone --> 5-hydroxyvalerate --> glutarate --> --> acetyl-CoA.     

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.     

Studies on the mechanism of metabolic stimulation in polymorphonuclear leucocytes during phagocytosis. I. Evidence for superoxide anion involvement in the oxidation of NADPH2.

1. The oxidation of NADPH2 by leucocyte granules, as measured at acid pH in the presence of Mn-2+, was found to be inhibited by superoxide dismutase. 2. Omission of Mn-2+ markedly lowered the oxidase activity at acid pH, which was still inhibited by superoxide dismutase. 3. At alkaline pH the oxidase activity was lower than at acid pH. 4. During oxidation of NADPH2 by leucocyte granules, reduction of cytochrome c occurred which was partially inhibited by superoxide dismutase. 5. It was concluded that NADPH2 oxidation occurs through an enzymatic reaction and a nonenzymatic chain reaction. Superoxide anion (O-minus-2 and NADPH- free radical would be involved in the chain reaction. The differential sensitivity of NADPH2 oxidation to superoxide dismutase in different experimental conditions (see above 1, 2 and 3) was explained on the basis of changes in the properties of the chain reaction.     

Mechanism of protection of peroxidase activity by oscillatory dynamics.

The peroxidase-oxidase reaction is known to involve reactive oxygen species as intermediates. These intermediates inactivate many types of biomolecules, including peroxidase itself. Previously, we have shown that oscillatory dynamics in the peroxidase-oxidase reaction seem to protect the enzyme from inactivation. It was suggested that this is due to a lower average concentration of reactive oxygen species in the oscillatory state compared to the steady state. Here, we studied the peroxidase-oxidase reaction with either 4-hydroxybenzoic acid or melatonin as cofactors. We show that the protective effect of oscillatory dynamics is present in both cases. We also found that the enzyme degradation depends on the concentration of the cofactor and on the pH of the reaction mixture. We simulated the oscillatory behaviour, including the oscillation/steady state bistability observed experimentally, using a detailed reaction scheme. The computational results confirm the hypothesis that protection is due to lower average concentrations of superoxide radical during oscillations. They also show that the shape of the oscillations changes with increasing cofactor concentration resulting in a further decrease in the average concentration of radicals. We therefore hypothesize that the protective effect of oscillatory dynamics is a general effect in this system.