Kinetic analysis of reactive oxygen species generated by the in vitro reconstituted NADPH oxidase and xanthine oxidase systems

The nicotinamide adenine dinucleotide (NADH)/nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and the xanthine oxidase (XOD) systems generate reactive oxygen species (ROS). In the present study, to characterize the difference between the two systems, the kinetics of ROS generated by both the NADH oxidase and XOD systems were analysed by an electron spin resonance (ESR) spin trapping method using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 5-(diethoxyphosphoryl)-5-methyl-pyrroline N-oxide (DEPMPO) and 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO). As a result, two major differences in ROS kinetics were found between the two systems: (i) the kinetics of (?)OH and (ii) the kinetics of hydrogen peroxide. In the NADH oxidase system, the interaction of hydrogen peroxide with each component of the enzyme system (NADPH, NADH oxidase and FAD) was found to generate (?)OH. In contrast, (?)OH generation was found to be independent of hydrogen peroxide in the XOD system. In addition, the hydrogen peroxide level in the NADPH-NADH oxidase system was much lower than measured in the XOD system. This lower level of free hydrogen peroxide is most likely due to the interaction between hydrogen peroxide and NADPH, because the hydrogen peroxide level was reduced by ~90% in the presence of NADPH.     

Hydrogen peroxide: a potent activator of dioxygenase activity of soybean lipoxygenase

Hydrogen peroxide, an ubiquitous biologically occurring peroxide, was found to stimulate the dioxygenase activity of soybean lipoxygenase at the physiologically attainable concentration. The increase in enzyme specific activity was directly proportional to hydrogen peroxide concentration up to 0.5 nM. A decrease in the stimulation of dioxygenase activity was observed at higher concentrations. At low enzyme concentration up to 28-fold stimulation was noted when the formation of lipid hydroperoxide was monitored spectrophotometrically. The stimulation was further confirmed by increased oxygen uptake. It is proposed that the mechanism for in vivo activation involves hydrogen peroxide.     

A kinetic analysis of the catalase activity of myeloperoxidase

The predominant physiological activity of myeloperoxidase is to convert hydrogen peroxide and chloride to hypochlorous acid. However, this neutrophil enzyme also degrades hydrogen peroxide to oxygen and water. We have undertaken a kinetic analysis of this reaction to clarify its mechanism. When myeloperoxidase was added to hydrogen peroxide in the absence of reducing substrates, there was an initial burst phase of hydrogen peroxide consumption followed by a slow steady state loss. The kinetics of hydrogen peroxide loss were precisely mirrored by the kinetics of oxygen production. Two mols of hydrogen peroxide gave rise to 1 mol of oxygen. With 100 microM hydrogen peroxide and 6 mM chloride, half of the hydrogen peroxide was converted to hypochlorous acid and the remainder to oxygen. Superoxide and tyrosine enhanced the steady-state loss of hydrogen peroxide in the absence of chloride. We propose that hydrogen peroxide reacts with the ferric enzyme to form compound I, which in turn reacts with another molecule of hydrogen peroxide to regenerate the native enzyme and liberate oxygen. The rate constant for the two-electron reduction of compound I by hydrogen peroxide was determined to be 2 x 10(6) M(-1) s(-1). The burst phase occurs because hydrogen peroxide and endogenous donors are able to slowly reduce compound I to compound II, which accumulates and retards the loss of hydrogen peroxide. Superoxide and tyrosine drive the catalase activity because they reduce compound II back to the native enzyme. The two-electron oxidation of hydrogen peroxide by compound I should be considered when interpreting mechanistic studies of myeloperoxidase and may influence the physiological activity of the enzyme.     

The tyrosinase activity of melanosomes from the harding-passey melanoma: The absence of a peroxidase component in vitro

Melanosomes were isolated from the Harding-Passey melanoma with a density gradient technique. Using the Pomerantz radioassay for tyrosinase activity it was found that these isolated melanosomes could hydroxylate tyrosine in the presence of catalase sufficient to deny the enzyme any hydrogen peroxide. It was further found that the rate of hydroxylation was unaffected by the presence of exogenous hydrogen peroxide. Tyrosinase activity could be suppressed by preincubation in diethyldithiocarbamate followed by removal of this inhibitor before enzyme assay. Attempts to regain enzymatic activity, however, by addition of copper II ions were unsuccessful. No peroxidase activity could be detected on the isolated granules, and indeed evidence for a peroxidase inhibitor on the granules was found. It was also found that the peroxidase activity present in a 20% homogenate of mouse muscle did not demonstrate any tyrosinase activity with the Pomerantz assay even in the presence of hydrogen peroxide. It is concluded from these studies that there is tyrosinase on these melanosomes which is capable in vitro of hydroxylating tyrosine without any contribution from an active peroxidase.     

Product of extracellular-superoxide dismutase catalysis

Extracellular-superoxide dismutase is a tetrameric enzyme containing four copper atoms. It has previously been shown to catalyse the decay of the superoxide radical, but the resulting product was not determined. In a xanthine oxidase-xanthine system in which about 30% of the electron flux resulted in superoxide radical formation, accumulation of hydrogen peroxide was determined. Catalysis of superoxide radical decay by extracellular-superoxide dismutase was found to result in hydrogen peroxide formation. The catalysed reaction is thus identical to those of previously investigated superoxide dismutases. Human manganese superoxide dismutase was also found to dismute the superoxide radical to hydrogen peroxide and water.     

Effect of Asp-235-->Asn substitution on the absorption spectrum and hydrogen peroxide reactivity of cytochrome c peroxidase.

The spectroscopic properties of a mutant cytochrome c peroxidase, in which Asp-235 has been replaced by an asparagine residue, were examined in both nitrate and phosphate buffers between pH 4 and 10.5. The spin state of the enzyme is pH dependent, and four distinct spectroscopic species are observed in each buffer system: a predominantly high-spin Fe(III) species at pH 4, two distinct low-spin forms between pH 5 and 9, and the denatured enzyme above pH 9.3. The spectrum of the mutant enzyme at pH 4 is dependent upon specific ion effects. Increasing the pH above 5 converts the mutant enzyme to a predominantly low-spin hydroxy complex. Subsequent conversion to a second low-spin form is essentially complete at pH 7.5. The second low-spin form has the distal histidine, His-52, coordinated to the heme iron. To evaluate the effect of the changes in coordination state upon the reactivity of the enzyme, the reaction between hydrogen peroxide and the mutant enzyme was also examined as a function of pH. The reaction of CcP(MI,D235N) with peroxide is biphasic. At pH 6, the rapid phase of the reaction can be attributed to the bimolecular reaction between hydrogen peroxide and the hydroxy-ligated form of the mutant enzyme. Despite the hexacoordination of the heme iron in this form, the bimolecular rate constant is approximately 22% that of pentacoordinate wild-type yeast cytochrome c peroxidase. The bimolecular reaction of the mutant enzyme with peroxide exhibits the same pH dependence in nitrate-containing buffers that has been described for the wild-type enzyme, indicating a loss of reactivity with the protonation of a group with an apparent pKa of 5.4. This observation eliminates Asp-235 as the source for this heme-linked ionization and strengthens the hypothesis that the pKa of 5.4 is associated with His-52. The slower phase of the reaction between peroxide and the mutant enzyme saturates at high peroxide concentration and is attributed to conversion of unreactive to reactive forms of the enzyme. The fraction of enzyme which reacts via the slow phase is dependent upon both pH and specific ion effects.     

Oxidation of Phe454 in the Gating Segment Inactivates Trametes multicolor Pyranose Oxidase during Substrate Turnover

The flavin-dependent enzyme pyranose oxidase catalyses the oxidation of several pyranose sugars at position C-2. In a second reaction step, oxygen is reduced to hydrogen peroxide. POx is of interest for biocatalytic carbohydrate oxidations, yet it was found that the enzyme is rapidly inactivated under turnover conditions. We studied pyranose oxidase from Trametes multicolor (TmPOx) inactivated either during glucose oxidation or by exogenous hydrogen peroxide using mass spectrometry. MALDI-MS experiments of proteolytic fragments of inactivated TmPOx showed several peptides with a mass increase of 16 or 32 Da indicating oxidation of certain amino acids. Most of these fragments contain at least one methionine residue, which most likely is oxidised by hydrogen peroxide. One peptide fragment that did not contain any amino acid residue that is likely to be oxidised by hydrogen peroxide (DAFSYGAVQQSIDSR) was studied in detail by LC-ESI-MS/MS, which showed a +16 Da mass increase for Phe454. We propose that oxidation of Phe454, which is located at the flexible active-site loop of TmPOx, is the first and main step in the inactivation of TmPOx by hydrogen peroxide. Oxidation of methionine residues might then further contribute to the complete inactivation of the enzyme.     

Biosynthesis of ethylene. Enzymes involved in its formation from methional

1. Two enzymes were shown to be necessary for the production of ethylene from methional; they were separated from extracts of cauliflower florets by fractionation on Sephadex and other methods. 2. The first enzyme, generating hydrogen peroxide, appears to be similar to the fungal glucose oxidase, for like the latter it is highly specific for its substrate d-glucose. 3. The second enzyme, in the presence of cofactors and peroxide generated by the first enzyme, cleaves methional to ethylene. 4. It was also found that hydrogen peroxide in these reactions may be replaced by hydroperoxide generated from linolenic acid by lipoxidase enzymes. 5. Dihydroxyphenols were shown to have a marked inhibitory effect on these reactions and to account for the initial phase of low activity that is always observed in aqueous extracts prepared from the floret tissue.     

Electrochemical study of the effect of nano-zinc oxide on microperoxidase and its application to more sensitive hydrogen peroxide biosensor preparation

Direct electron transfer reactions of microperoxidase were achieved with the help of semiconductive zinc oxide nanoparticles on a pyrolytic graphite electrode. The enzyme could also exhibit fine electrocatalytic activity towards the reduction of hydrogen peroxide. Thereby, a hydrogen peroxide biosensor was constructed based on the electrocatalysis of microperoxidase. Further studies revealed that after irradiating the microperoxidase/zinc oxide nanoparticles co-modified electrode with UV light for 4h, the catalytic ability of microperoxidase could be greatly promoted, which could be beneficial to developing more sensitive hydrogen peroxide biosensors. As comparison, it was found that the catalytic activity of the enzyme would be depressed if microperoxidase/agarose co-modified electrode was irradiated. We supposed it was the photovoltaic effect of the zinc oxide nanoparticles that improved the catalytic ability of microperoxidase.     

The mechanism of ferrocytochrome C oxidation by a horseradish isoperoxidase.

The oxidation of ferrocytochrome c catalysed by highly purified horse-radish isoperoxidase P2 was studied kinetically. To take into account the low turnover number of the enzyme and the tendency to autocatalytic oxidation of ferrocytochrome c, experimental conditions were used which prevented us from using the steady-state treatment. According to kinetic results reported by several authors, a kinetic scheme involving a ternary complex between the enzyme and the substrates was postulated and simulated on a hybrid computer. By assuming that the interaction of peroxidase with hydrogen peroxide is much faster than the interaction with ferrocytochrome c, one can verify that this scheme explains the fact that initial velocity does not vary in relation to the hydrogen peroxide concentration and that a sudden change of slope occurs in the kinetic curve for an initial hydrogen peroxide/ferrocytochrome c ratio lower than 0.5.     

The reaction mechanism of the novel vanadium-bromoperoxidase. A steady-state kinetic analysis

The reaction of vanadium-bromoperoxidase from the brown alga Ascophyllum nodosum with hydrogen peroxide, bromide, and 2-chlorodimedone has been subjected to an extensive steady-state kinetic analysis. Systematic variation of pH and the concentrations of these three components demonstrate that the reaction model includes four enzyme species: native bromoperoxidase, a bromoperoxidase-bromide inhibitory complex, a bromoperoxidase-hydrogen peroxide intermediate, and a bromoperoxidase-HOBr species. This latter intermediate did not display any direct interaction with the nucleophilic reagent as oxidized bromine species (Br-3, Br2, and/or HOBr) were the primary reaction products. The generation of oxidized bromine species was as fast as the bromination of 2-chlorodimedone. The enzyme did not show any specificity with regard to bromination of various organic compounds. Formation of the bromoperoxidase-bromide inhibitory complex was competitive with the reaction between hydrogen peroxide and enzyme. From the steady-state kinetic data lower limits for the second-order rate constants at various pH values were calculated for individual steps in the catalytic cycle. This pH study showed that native enzyme must be unprotonated prior to binding of hydrogen peroxide (second-order association rate constant of 2.5.10(6) M-1.s-1 at pH greater than 6). The pKa for the functional group controlling the binding of hydrogen peroxide was 5.7 and is ascribed to a histidine residue. The reaction rate between bromide and enzyme-hydrogen peroxide intermediate also depended on pH (second-order association rate constant of 1.7.10(5) M-1.s-1 at pH 4.0).     

A quantitative study of peroxidase activity in unfixed tissue sections of the guinea-pig thyroid gland

Summary A technique for the cytochemical demonstration of peroxidase activity in unfixed guinea-pig thyroid tissue is described in this paper. The substrate 3,3-diaminobenzidine tetrahydrochloride (DAB) is oxidized by the peroxidase to form an insoluble reaction product. Optimal results were obtained after 20 min incubation at 37° C in reaction medium containing 1.4mm DAB (in 0.1m Tris-HCl) and 0.15mm hydrogen peroxide at pH 8.0. Peroxidase activity was seen in the thyroid follicle cells as a diffuse brown reaction product (which was more dense and granular in erythrocytes). The enzyme activity was quantified using a scanning-integrating microdensitometer, and the effects of two specific peroxidase inhibitors were evaluated. Both 3-amino-1,2,4-triazole and methimazole inhibited peroxidase activity in the follicle cells (enzyme activity was still seen in the erythrocytes), maximal inhibition occurring at 10mm. Stimulation of peroxidase in the thyroid was observedin vivo (1 I.U. TSH administered every 8 h for two days), with the maximal stimulation occurring after 1 day.     

Characterization of an iron- and manganese-containing superoxide dismutase from Methylobacillus sp. strain SK1 DSM 8269

A superoxide dismutase was purified 62-fold in seven steps to homogeneity from Methylobacillus sp. strain SK1, an obligate methanol-oxidizing bacterium, with a yield of 9.6%. The final specific activity was 4,831 units per milligram protein as determined by an assay based on a 50% decrease in the rate of cytochrome c reduction. The molecular weight of the native enzyme was estimated to be 44,000. Sodium dodecyl sulfate gel electrophoresis revealed two identical subunits of molecular weight 23,100. The isoelectric point of the purified enzyme was found to be 4.4. Maximum activity of the enzyme was measured at pH 8. The enzyme was stable at pH range from 6 to 8 and at high temperature. The enzyme showed an absorption peak at 280 nm with a shoulder at 292 nm. Hydrogen peroxide and sodium azide, but not sodium cyanide, was found to inhibit the purified enzyme. The enzyme activity in cell-free extracts prepared from cells grown in manganese-rich medium, however, was not inhibited by hydrogen peroxide but inhibited by sodium azide. The activity in cell extracts from cells grown in iron-rich medium was found to be highly sensitive to hydrogen peroxide and sodium azide. One mol of native enzyme was found to contain 1.1 g-atom of iron and 0.7 g-atom of manganese. The N-terminal amino acid sequence of the purified enzyme was Ala-Tyr-Thr-Leu-Pro-Pro-Leu-Asn-Tyr-Ala-Tyr. The superoxide dismutase of Methylobacillus sp. strain SK1 was found to have antigenic sites identical to those of Methylobacillus glycogenes enzyme. The enzyme, however, shared no antigenic sites with Mycobacterium sp. strain JC1, Methylovorus sp. strain SS1, Methylobacterium sp. strain SY1, and Methylosinus trichosproium enzymes.     

Electroenzymatic oxidation of veratryl alcohol by lignin peroxidase

This paper reports the formation of veratraldehyde by electroenzymatic oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) hybridizing both electrochemical and enzymatic reactions and using lignin peroxidase. The novel electroenzymatic method was found to be effective for replacement of hydrogen peroxide by an electrochemical reactor, which is essential for enzyme activity of lignin peroxidase. The effects of operating parameters such as enzyme dosage, pH, and electric potential were investigated. Further, the kinetics of veratryl alcohol oxidation in an electrochemical reactor were compared to oxidation when hydrogen peroxide was supplied externally.     

Cytosolic glutathione peroxidase from liver of pacu (Piaractus mesopotamicus), a hypoxia-tolerant fish of the Pantanal

Pacu (Piaractus mesopotamicus Holmberg, 1887, Characiformes) dwells in waters of Pantanal, in which it has adapted for alternate concentrations of dissolved oxygen. Intracellular antioxidant protection should be vital for such an adaptation. Accordingly, we found that cytosol from liver of pacu has the highest antioxidant glutathione peroxidase activity so far reported for fish and murine species. To clarify whether this activity was due to a selenium independent glutathione S-transferase or to a glutathione peroxidase, we purified it and studied its kinetics. The substrates cumene hydroperoxide and hydrogen peroxide were promptly reduced by the enzyme, but peroxidized phosphatidylcholine had to undergo previous fatty acid removal with phospholipase A(2). Augmenting concentrations (from 2 to 6 mM) of reduced glutathione activated the pure enzyme. Curves of velocity versus different micromolar concentrations of hydrogen peroxide in the presence of 2, 4 or 8 mM reduced glutathione indicated that at least 2.5 mM reduced glutathione should be available in vivo for an efficient continuous destruction of micromolar concentrations of hydrogen peroxide by this peroxidase. Molecular exclusion HPLC and SDS-polyacrylamide gel electrophoresis indicated that the purified peroxidase is a homotetramer. Data from internal sequences showed selenocysteine in its primary structure and that the enzyme was a homologue of the type-1 glutathione peroxidase found in rat, bull, trout, flounder and zebra fish. Altogether, our data establish that in liver cells of pacu, a hypoxia-tolerant fish from South America, there are high levels of a cytosolic GPX-1 capable of quenching hydrogen peroxide and fatty acid peroxides, providing an effective antioxidant action.     

Utilization of methanol by a catalase-negative mutant of Hansenula polymorpha

Summary In methanol-utilizing yeasts, catalase is an essential enzyme for the destruction of hydrogen peroxide generated by methanol oxidase (E.C. 1.1.3.13). It was found however that a catalase-negative mutant of Hansenula polymorpha is able to consume methanol in the presence of glucose in continuous cultures. At a dilution rate of 0.1 h^-1, stable continuous cultures could be obtained during growth on methanol/glucose mixtures with a molar ratio of methanol/glucose between 0 to 2.4. In these cultures methanol oxidase was induced up to a level of 40% of that obtained in the wild-type strain. The hydrogen peroxide-decomposition activity of the mutant was studied in more detail by pulsing methanol to samples of steady-state cultures. Only after the addition of excess methanol the hydrogen peroxide-decomposing system became saturated, and the cells excreted hydrogen peroxide. This was accompanied by excretion of formaldehyde and a rapid loss of viability. The presence of extracellular catalase during a methanol pulse prevented the loss of viability. The nature of the alternative hydrogen peroxide-decomposing enzyme system remains to be elucidated. Its capacity strongly depended on the cultivation conditions and pretreatment of the cells. Cells grown on formaldehyde/glucose mixtures showed a lower methanol tolerance than those grown on the methanol/glucose mixtures. Freeze-drying of cells drastically enhanced the excretion of hydrogen peroxide, probably as a result of an inactivation of the decomposing system.     

A kinetic study of the reaction between cytochrome c peroxidase and hydrogen peroxide. Dependence on pH and ionic strength.

The rate of the reaction between cytochrome c peroxidase and hydrogen peroxide was investigated using the stopped-flow technique. The apparent bimolecular rate constant was determined between pH 3.3 and pH 11 as a function of ionic strength. The pH dependence of the apparent bimolecular rate constant can be explained by assuming that two ionizable groups on the enzyme strongly influence the rate of the reaction. At 0.1 M ionic strength, a group with a pKa of 5.5 must be unprotonated and a group with a pKa of 9.8 must be protonated for the enzyme to react rapidly with hydrogen peroxide. The apparent acid dissociation constants depend upon the ionic strength. The true bimolecular rate constant has a value of (4.5 +/- 0.3) X 10(7) M-1 sec-1 and is independent of ionic strength.     

Purification and properties of a glutathione peroxidase from Southern bluefin tuna (Thunnus maccoyii) liver

A glutathione peroxidase (GPX) protein was purified approximately 1000-fold from Southern bluefin tuna (Thunnus maccoyii) liver to a final specific activity of 256 micromol NADPH oxidised min(-1) mg(-1) protein. Gel filtration chromatography and denaturing protein gel electrophoresis of the purified preparation indicated that the protein has a native molecular mass of 85 kDa and is most likely a homotetramer with subunits of approximately 24 kDa. The Km values of the purified enzyme for hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide and glutathione were 12, 90, 90 and 5900 microM, respectively. The Km values for cumene hydroperoxide and t-butyl hydroperoxide were approximately 8-fold greater than the Km value for hydrogen peroxide. Thus, the SBT liver GPX has a considerably greater affinity for hydrogen peroxide than for the other two substrates. The pH optimum of the purified enzyme was pH 8.0. Immunoblotting experiments with polyclonal antibodies, raised against a recombinant human GPX, provided further evidence that the purified SBT enzyme is a genuine GPX.     

Characterization of DNA damage in yeast apoptosis induced by hydrogen peroxide, acetic acid, and hyperosmotic shock

Saccharomyces cerevisiae has been reported to die, under certain conditions, from programmed cell death with apoptotic markers. One of the most important markers is chromosomal DNA fragmentation as indicated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. We found TUNEL staining in S. cerevisiae to be a consequence of both single- and double-strand DNA breaks, whereas in situ ligation specifically stained double-strand DNA breaks. Cells treated with hydrogen peroxide or acetic acid staining positively for TUNEL assay stained negatively for in situ ligation, indicating that DNA damage in both cases mainly consists of single-strand DNA breaks. Pulsed field gel electrophoresis of chromosomal DNA from cells dying from hydrogen peroxide, acetic acid, or hyperosmotic shock revealed DNA breakdown into fragments of several hundred kilobases, consistent with the higher order chromatin degradation preceding DNA laddering in apoptotic mammalian cells. DNA fragmentation was associated with death by treatment with 10 mM hydrogen peroxide but not 150 mM and was absent if cells were fixed with formaldehyde to eliminate enzyme activity before hydrogen peroxide treatment. These observations are consistent with a process that, like mammalian apoptosis, is enzyme dependent, degrades chromosomal DNA, and is activated only at low intensity of death stimuli.     

The reaction of hydrogen peroxide with the dimethyl ester heme derivative of cytochrome c peroxidase

A modified cytochrome c peroxidase was prepared by reconstituting apocytochrome c peroxidase with protoheme in which both heme propionic acid groups were converted to the methyl ester derivatives. The modified enzyme reacted with hydrogen peroxide with a rate constant of (1.3 +/- 0.2) x 10(7) M-1 s-1, which is 28% that of the native enzyme. The reaction between the modified enzyme and hydrogen peroxide was pH-dependent with an apparent pK of 5.1 +/- 0.1 compared to a value of 5.4 +/- 0.1 for the native enzyme. These observations support the conclusion that the apparent ionization near pH 5.4, which influences the hydrogen peroxide-cytochrome c peroxidase reaction is not due to the ionization of the propionate side chains of the heme group in the native enzyme. A second apparent ionization, with pK of 6.1 +/- 0.1, influences the spectrum of the modified enzyme which changes from a high spin type at low pH to a low spin type at high pH.