Factors Affecting the Ascorbate- and Phenolic-dependent Generation of Hydrogen Peroxide in Dulbecco's Modified Eagles Medium

Ascorbate and several polyphenolic compounds have been reported to undergo oxidation in cell culture media to generate hydrogen peroxide (H?0?), but the mechanism underlying this has not been established. We therefore investigated the parameters affecting H?0? production. H?0? gene ration from ascorbate, gallic acid and other phenolic compounds in Dulbecco's Modified Eagles' Medium (DMEM) at 37°C under 95% air - 5% C0? was not significantly inhibited by high (5-10 mM) concentration of EGTA, o-phenanthroline or desferrioxamine, but partial inhibition by EDTA and diethylenetriaminepentaacetic acid (DTPA) was observed. Incubation of DMEM alone at 37°C led to an upward drift of pH, even under an atmosphere of 95% air - 5% C0?. Prevention of this pH rise by increasing the concentration of N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (Hepes) buffer lowered the levels of H?0? generated by ascorbate and phenolic compounds, but there was still substantial H?0? generated at pH 7.4. Mixtures of ascorbate and phenolic compounds led to less H?0? generation than would be expected from the rates observed with ascorbate or phenolic compounds alone. Ascorbate prevented the loss of gallic acid incubated in DMEM. The role of metal ions and other constituents of the culture medium in promoting H?0? generation is discussed.     

Irradiation of cells with ultraviolet-A (320-400 nm) in the presence of cell culture medium elicits biological effects due to extracellular generation of hydrogen peroxide

Biological effects of ultraviolet A (UVA) irradiation have been ascribed to the photochemical generation of singlet oxygen. Not all effects described in the literature, however, are explicable solely by the generation of singlet oxygen, but rather resemble effects elicited by hydrogen peroxide (H 2 O 2 ). Here, we show that when cells are kept in cell culture media during exposure to UVA, stress kinases, including ERK 1 and ERK 2 as well as Akt (protein kinase B), are activated, whereas there is no or only minor activation when cells are kept in phosphate-buffered saline during irradiation. Indeed, the exposure of cell culture media to UVA (30 J/cm 2 ) results in the generation of significant amounts of H 2 O 2 , with concentrations of about 100 μM. H 2 O 2 concentrations are at least three-fold higher in HEPES-buffered culture media after UVA irradiation. From experiments with solutions of riboflavin, tryptophan or HEPES, as well as combinations thereof, it is concluded that riboflavin mediates the photooxidation of either tryptophan or HEPES, resulting in the generation of H 2 O 2 . Thus, if signaling effects of UVA radiation are to be investigated in cell culture systems, riboflavin and HEPES/tryptophan should be avoided during irradiation because of artificial H 2 O 2 generation. It should be taken into account, however, that in vivo tryptophan and riboflavin might play an important role in the generation of reactive oxygen species by UVA as both substances are abundant in living tissues.     

N-dealkylation of aminopyrine catalyzed by soybean lipoxygenase in the presence of hydrogen peroxide

A hypothesis that lipoxygenase may mediate N-dealkylation of xenobiotics was investigated using the prototype drug aminopyrine and soybean lipoxygenase as a model enzyme in the presence of hydrogen peroxide. Formaldehyde production as a result of N-demethylation of aminopyrine exhibited pH optimum of 6.5. The reaction was dependent on the incubation time, amount of enzyme, and concentration of aminopyrine and hydrogen peroxide. Under the experimental conditions employed, the specific activity for N-demethylation of aminopyrine was found to be 823 ± 93 nmoles per min/mg protein or 89 ± 10 nmoles per min/nmole of enzyme. The reaction was significantly inhibited by nordihydroguaiaretic acid and gossypol, the classical inhibitors of lipoxygenase. Spectrophotometric analyses indicated the generation of a nitrogen-centered free-radical cation as the initial oxidation product of aminopyrine. The rate of accumulation of this radical species was also dependent on pH, the amount of enzyme, and concentration of aminopyrine and hydrogen peroxide. The radical production was markedly suppressed by ascorbate, glutathione, and dithiothreitol in a concentration-dependent manner. Preliminary data gathered for the oxidation of other chemicals indicated that the lipoxygenase exhibits a unique substrate specificity. Collectively, the evidence presented suggests for the first time that lipoxygenase pathway may be involved in N-demethylation of aminopyrine and other chemicals. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 175–183, 1998     

Irradiation of Cells with Ultraviolet-A (320-400 nm) in the Presence of Cell Culture Medium Elicits Biological Effects Due to Extracellular Generation of Hydrogen Peroxide

Biological effects of ultraviolet A (UVA) irradiation have been ascribed to the photochemical generation of singlet oxygen. Not all effects described in the literature, however, are explicable solely by the generation of singlet oxygen, but rather resemble effects elicited by hydrogen peroxide (H 2 O 2 ). Here, we show that when cells are kept in cell culture media during exposure to UVA, stress kinases, including ERK 1 and ERK 2 as well as Akt (protein kinase B), are activated, whereas there is no or only minor activation when cells are kept in phosphate-buffered saline during irradiation. Indeed, the exposure of cell culture media to UVA (30 J/cm 2 ) results in the generation of significant amounts of H 2 O 2 , with concentrations of about 100 &#119 M. H 2 O 2 concentrations are at least three-fold higher in HEPES-buffered culture media after UVA irradiation. From experiments with solutions of riboflavin, tryptophan or HEPES, as well as combinations thereof, it is concluded that riboflavin mediates the photooxidation of either tryptophan or HEPES, resulting in the generation of H 2 O 2 . Thus, if signaling effects of UVA radiation are to be investigated in cell culture systems, riboflavin and HEPES/tryptophan should be avoided during irradiation because of artificial H 2 O 2 generation. It should be taken into account, however, that in vivo tryptophan and riboflavin might play an important role in the generation of reactive oxygen species by UVA as both substances are abundant in living tissues.     

Composition of the redox environment of the endoplasmic reticulum and sources of hydrogen peroxide

The endoplasmic reticulum (ER) is a metabolically active organelle, which has a central role in proteostasis by translating, modifying, folding, and occasionally degrading secretory and membrane proteins. The lumen of the ER represents a separate compartment of the eukaryotic cell, with a characteristic proteome and metabolome. Although the redox metabolome and proteome of the compartment have not been holistically explored, it is evident that proper redox conditions are necessary for the functioning of many luminal pathways. These redox conditions are defined by local oxidoreductases and the membrane transport of electron donors and acceptors. The main electron carriers of the compartment are identical with those of the other organelles: glutathione, pyridine and flavin nucleotides, ascorbate, and others. However, their composition, concentration, and redox state in the ER lumen can be different from those observed in other compartments. The terminal oxidases of oxidative protein folding generate and maintain an “oxidative environment” by oxidizing protein thiols and producing hydrogen peroxide. ER-specific mechanisms reutilize hydrogen peroxide as an electron acceptor of oxidative folding. These mechanisms, together with membrane and kinetic barriers, guarantee that redox systems in the reduced or oxidized state can be present simultaneously in the lumen. The present knowledge on the in vivo conditions of ER redox is rather limited; development of new genetically encoded targetable sensors for the measurement of the luminal state of redox systems other than thiol/disulfide will contribute to a better understanding of ER redox homeostasis.     

Instability of, and generation of hydrogen peroxide by, phenolic compounds in cell culture media

Many papers in the literature have described complex effects of flavonoids and other polyphenols on cells in culture. In this paper we show that hydroxytyrosol, delphinidin chloride and rosmarinic acid are unstable in three commonly-used cell culture media (Dulbecco’s modified Eagle’s medium (DMEM), RPMI 1640 (RPMI) and Minimal Essential Medium Eagle (MEM)) and undergo rapid oxidation to generate H₂O₂. This may have confounded some previous studies on the cellular effects of these compounds. By contrast, apigenin, curcumin, hesperetin, naringenin, resveratrol and tyrosol did not generate significant H₂O₂ levels in these media. Nevertheless, curcumin and, to a lesser extent, resveratrol (but not tyrosol) were also unstable in DMEM, so the absence of detectable H₂O₂ production by a compound in cell culture media should not be equated to stability of that compound. Compound instability and generation of H₂O₂ must be taken into account in interpreting effects of phenolic compounds on cells in culture.     

Stabilization of Snail by HuR in the process of hydrogen peroxide induced cell migration

Snail functions as a key regulator in the induction of a phenotypic change called epithelial to mesenchymal transition (EMT). Aberrant expression of Snail prevails in the onset and development of tumor. Here, we have observed increased expression of Snail under the treatment of hydrogen peroxide (H(2)O(2)). Investigation into the underlying mechanisms revealed that stabilization of Snail mRNA contributes partially to this process. H(2)O(2)-induced the luciferase activity of the reporter construct contains the 3'UTR of Snail. Deletion of the AU-rich elements in the UTR eliminated the response of the reporter to H(2)O(2), suggesting the potential role of HuR in the process. Lowering of endogenous HuR levels through knockdown of HuR by siRNA greatly reduced the inducability and half-life of Snail mRNA, which consequently inhibited the downregulation of E-cadherin by H(2)O(2). Our findings indicate that HuR plays a major role in regulating H(2)O(2)-induced Snail expression by enhancing Snail mRNA stability, which in turn enhances cell migrating ability through repressing expression of E-cadherin.     

Lignin synthesis: The generation of hydrogen peroxide and superoxide by horseradish peroxidase and its stimulation by manganese (II) and phenols

The enzyme horseradish peroxidase (EC 1.11.1.7) catalyses oxidation of NADH. NADH oxidation is prevented by addition of the enzyme superoxide dismutase (EC 1.15.1.1) to the reaction mixture before adding peroxidase but addition of dismutase after peroxidase has little inhibitory effect. Catalase (EC 1.11.1.6) inhibits peroxidase-catalysed NADH oxidation when added at any time during the reaction. Apparently the peroxidase uses hydrogen peroxide (H₂O₂) generated by non-enzymic breakdown of NADH to catalyse oxidation of NADH to a free-radical, NAD., which reduces oxygen to the superoxide free-radical ion, O₂ ^.-. Some of the O₂ ^.- reacts with peroxidase to give peroxidase compound III, which is catalytically inactive in NADH oxidation. The remaining O₂ ^.- undergoes dismutation to O₂ and H₂O₂. O₂ ^.- does not react with NADH at significant rates. Mn²⁺ or lactate dehydrogenase stimulate NADH oxidation by peroxidase because they mediate a reaction between O₂ ^.- and NADH. 2,4-Dichlorophenol, p-cresol and 4-hydroxycinnamic acid stimulate NADH oxidation by peroxidase, probably by breaking down compound III and so increasing the amount of active peroxidase in the reaction mixture. Oxidation in the presence of these phenols is greatly increased by adding H₂O₂. The rate of NADH oxidation by peroxidase is greatest in the presence of both Mn²⁺ and those phenols which interact with compound III. Both O₂ ^.- and H₂O₂ are involved in this oxidation, which plays an important role in lignin synthesis.     

Melatonin inhibits the contractile effect of vanadate in the isolated pulmonary arterial rings of rats: possible role of hydrogen peroxide

The effect and possible mechanism of action of vanadate on the isolated pulmonary arterial rings of normal rats were studied. Pulmonary arterial rings contracted in response to vanadate (0.1-1 mM) in a concentration-dependent manner. Preincubation of the pulmonary arterial rings with 1 mM melatonin significantly reduced the contractile effect of vanadate by more than 60%. Furthermore, addition of hydrogen peroxide (50 microM) or enzymatic generation of hydrogen peroxide by the addition of glucose oxidase (10 U/mL) to the medium containing glucose produced remarkable increases in the pulmonary arterial tension, 46.2 +/- 7.3 and 78.7 +/- 9.7 g tension/g tissue, respectively. Similarly, incubation of the pulmonary arterial rings with 1 mM melatonin significantly reduced the contractile responses of the arterial rings to hydrogen peroxide and glucose/glucose oxidase to 25.7 +/- 2.9 and 24.7 +/- 4.4 g tension/g tissue, respectively. Vanadate, in vitro, significantly stimulated the oxidation of NADH by xanthine oxidase, and the rate of oxidation was increased by increasing either time or vanadate concentration. Similarly, addition of melatonin to a reaction mixture containing xanthine oxidase and vanadate significantly inhibited the rate of NADH oxidation in a concentration-dependent fashion. The results of the present study indicated that vanadate induced contraction in the isolated pulmonary arterial rings, which was significantly reduced by melatonin. Furthermore, the contractile effect of vanadate on the pulmonary arterial rings may be attributed to the intracellular generation of hydrogen peroxide.     

Artefacts in cell culture: Pyruvate as a scavenger of hydrogen peroxide generated by ascorbate or epigallocatechin gallate in cell culture media

Ascorbate and several phenolic compounds readily oxidise in cell culture media to generate hydrogen peroxide. However, media containing pyruvate showed much less H₂O₂ production, apparently because pyruvate can scavenge H₂O₂ in the medium. Researchers must be aware that compounds under test can sometimes readily oxidise in cell culture media, that this might not be detected by measurement of H₂O₂ if the media contain pyruvate, and that pyruvate can be substantially depleted in the media as a result.     

The involvement of hydrogen peroxide in abscisic acid-induced activities of ascorbate peroxidase and glutathione reductase in rice roots

We have monitored the changes in antioxidant enzyme activities and H₂O₂ concentrations in roots of rice (Oryza sativa L., cv. Taichung Native 1) seedlings treated with exogenous abscisic acid(ABA). Decrease in superoxide dismutase (SOD) and catalase (CAT) activities was observed in rice roots in the presence of ABA. However, ascorbate peroxide (APX) and glutathione reductase (GR) activities were increased after the ABA treatment. ABA treatment resulted in an increase in H₂O₂ concentrations in rice roots. Pre-treatment with dimethylthiourea, a chemical trap for H₂O₂, and diphenyleneiodonium chloride (DPI), a well known inhibitor of NADPH oxidase, inhibited ABA-induced accumulation of H₂O₂ and ABA-induced activities of APX and GR. ABA-induced accumulation of H₂O₂ was found to be prior to ABA-induced activities of APX and GR. Our results suggest that H₂O₂ is involved in ABA-induced APX and GR activities in rice roots.     

Involvement of malate,monophenols, and the superoxide radical in hydrogen peroxide formation by isolated cell walls from horseradish (Armoracia lapathifolia Gilib.)

Peroxidase associated with isolated horseradish cell walls catalyzes the formation of H₂O₂ in the presence of NADH. The reaction is stimulated by various monophenols, especially of coniferyl alcohol. NADH can be provided by a bound malate dehydrogenase. This system is capable of polymerizing coniferyl alcohol yielding an insoluble dehydrogenation polymer. NADH was found to be oxidized by two different mechanisms, one involving Mn²⁺, monophenol, and the superoxide radical O₂ ^·- in a reaction that is not affected by superoxide dismutase, and another one depending on the presence of free O₂ ^·- and probably of an enzyme-NADH complex. A scheme of these reaction chains, which are thought to be involved in the lignification process, is presented.Abbreviations DHP dehydrogenation polymer - GOT glutamate oxaloacetate transaminase (EC 2.6.1.1) - LDH lactate dehydrogenase (pig heart, EC 1.1.1.27) - MDH malate dehydrogenase (EC 1.1.1.37) - pCA p-coumaric acid - SOD superoxide dismutase (EC 1.15.1.1) - TLC thin-layer chromatography - XOD xanthine oxidase (EC 1.2.3.2)     

Accelerated CuZn-SOD-mediated oxidation and reduction in the presence of hydrogen peroxide

Copper, zinc-superoxide dismutase (CuZn-SOD) is a cytosolic, antioxidant enzyme that scavenges potentially damaging superoxide radical (()O(2)(-)). Under the proper conditions, CuZn-SOD also catalyzes the oxidation and reduction of certain small molecules. Here, we demonstrate that increased exposure to hydrogen peroxide (H(2)O(2)), a by-product of the ()O(2)(-) scavenging reaction, dramatically increases the ability of CuZn-SOD to oxidize melatonin and reduce S-nitrosoglutathione (GSNO). After a 15min in vitro incubation with CuZn-SOD and 1mM H(2)O(2), 76% of the melatonin was oxidized, compared to 52% with 0.25mM H(2)O(2), and just 9% without H(2)O(2). Pre-incubation with 1mM H(2)O(2) resulted in a 100% increase in the rate of GSNO breakdown by CuZn-SOD in the presence of glutathione (GSH) compared to untreated CuZn-SOD. Collectively, these data suggest that even small increases in intracellular H(2)O(2) levels may result in the oxidation and/or reduction of small molecules critical for proper cellular function.     

Reversible inhibition of the calvin cycle and activation of oxidative pentose phosphate cycle in isolated intact chloroplasts by hydrogen peroxide

Hydrogen peroxide (6x10^-4 M) causes a 90% inhibition of CO₂-fixation in isolated intact chloroplasts. The inhibition is reversed by adding catalase (2500 U/ml) or DTT (10 mM). If hydrogen peroxide is added to a suspension of intact chloroplasts in the light, the incorporation of carbon into hexose- and heptulose bisphosphates and into pentose monophosphates is significantly increased, whereas; carbon incorporation into hexose monophosphates and ribulose 1,5-bisphosphate is decreased. At the same time formation of 6-phosphogluconate is dramatically stimulated, and the level of ATP is increased. All these changes induced by hydrogen peroxide are reversed by addition of catalase or DTT. Additionally, the conversion of [¹⁴C]glucose-6-phosphate into different metabolites by lysed chloroplasts in the dark has been studied. In presence of hydrogen peroxide, formation of ribulose-1,5-bisphosphate is inhibited, whereas formation of other bisphosphates,of triose phosphates, and pentose monophosphates is stimulated. Again, DTT has the opposite effect. The release of ¹⁴CO₂ from added [¹⁴C]glucose-6-phosphate by the soluble fraction of lysed chloroplasts via the reactions of oxidative pentose phosphate cycle is completely inhibited by DTT (0.5 mM) and re-activated by comparable concentrations of hydrogen peroxide. These results indicate that hydrogen peroxide interacts with reduced sulfhydryl groups which are involved in the light activation of enzymes of the Calvin cycle at the site of fructose- and sedoheptulose bisphophatase, of phosphoribulokinase, as well as in light-inactivation of oxidative pentose phosphate cycle at the site of glucose-6-phosphate dehydrogenase.Abbreviations ADPG ADP-glucose - DHAP dihydroxyacetone phosphate - DTT dithiothreitol - FBP fructose-1,6-bisphosphate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - HMP hexose monophosphates (fructose-6-phosphate, glucose-6-phosphate, glucose-1-phosphate) - 6-PGI 6-phosphogluconate - PMP pentose monophosphates (xylulose-5-phosphate, ribose-5-phosphate, ribulose-5-phosphate) - RuBP ribulose-1,5-bisphosphate - S7P sedoheptulose-7-phosphate - SBP sedoheptulose-1,7-bisphosphate Dedicated to Prof. Dr. W. Simonis on the occasion of his 70th birthday     

Generation of superoxide radicals in alkaline solutions of hydrogen peroxide and the effect of superoxide dismutase on this system

Superoxide radicals in high concentrations were generated from alkaline H₂O₂ without using catalysts or irradiation. The dependence of the intensity and parameters of the superoxide radical EPR spectrum on pH, temperature, viscosity and H₂O₂ concentration were studied. The observed changes are explained on the base of matrix effects. The addition of superoxide dismutase to alkaline H₂O₂ led initially to a drop in the EPR spectrum intensity, followed by an increase in the concentration of superoxide radicals.     

The kinetics of the complex formation between iron(III)-ethylenediaminetetraacetate and hydrogen peroxide in aqueous solution

The kinetics of the formation of the purple complex [Fe^III(EDTA)O₂]³⁻, between Fe^III-EDTA and hydrogen peroxide was studied as a function of pH (8.22-11.44) and temperature (10-40 °C) in aqueous solutions using a stopped-flow method. The reaction was first-order with respect to both reactants. The observed second-order rate constants decrease with an increase in pH and appear to be related to deprotonation of Fe^III-EDTA ([Fe(EDTA)H₂O]⁻ ⇔ Fe(EDTA)OH]²⁻ + H⁺). The rate law for the formation of the complex was found to be d[Fe^IIIEDTAO₂]³⁻/dt=[(k₄[H⁺]/([H⁺] + K₁)][Fe^III-EDTA][H₂O₂], where k₄=8.15±0.05×10⁴ M⁻¹ s⁻¹ and pK₁=7.3. The steps involved in the formation of [Fe(EDTA)O₂]³⁻ are briefly discussed.     

Analysis of the lifetime and spatial localization of hydrogen peroxide generated in the cytosol using a reduced kinetic model

Hydrogen peroxide (H₂O₂) acts as a signaling molecule via its reactions with particular cysteine residues of certain proteins. Determining the roles of direct oxidation by H₂O₂ versus disulfide exchange reactions (i.e. relay reactions) between oxidized and reduced proteins of different identities is a current focus. Here, we use kinetic modeling to estimate the spatial and temporal localization of H₂O₂ and its most likely oxidation targets during a sudden increase in H₂O₂ above the basal level in the cytosol. We updated a previous redox kinetic model with recently measured parameters for HeLa cells and used the model to estimate the length and time scales of H₂O₂ diffusion through the cytosol before it is consumed by reaction. These estimates were on the order of one micron and one millisecond, respectively. We found oxidation of peroxiredoxin by H₂O₂ to be the dominant reaction in the network and that the overall concentration of reduced peroxiredoxin is not significantly affected by physiological increases in intracellular H₂O₂ concentration. We used this information to reduce the model from 22 parameters and reactions and 21 species to a single analytical equation with only one dependent variable, i.e. the concentration of H₂O₂, and reproduced results from the complete model. The reduced kinetic model will facilitate future efforts to progress beyond estimates and precisely quantify how reactions and diffusion jointly influence the distribution of H₂O₂ within cells.     

Antioxidant responses to enhanced generation of superoxide anion radical and hydrogen peroxide in the copper-stressed mulberry plants

The aim of the study was to implicate the generation of reactive oxygen species (ROS) and altered cellular redox environment with the effects of Cu-deficiency or Cu-excess in mulberry (Morus alba L.) cv. Kanva 2 plants. A study of antioxidative responses, indicators of oxidative damage and cellular redox environment in Cu-deficient or Cu-excess mulberry plants was undertaken. While the young leaves of plants supplied with nil Cu showed chlorosis and necrotic scorching of laminae, the older and middle leaves of plants supplied with nil or 0.1 μM Cu showed purplish-brown pigmented interveinal areas that later turned necrotic along the apices and margins of leaves. The Cu-excess plants showed accelerated senescence of the older leaves. The Cu-deficient plants showed accumulation of hydrogen peroxide and superoxide anion radical. The accumulation of hydrogen peroxide was strikingly intense in the middle portion of trichomes on Cu-deficient leaves. Though the concentration of total ascorbate increased with the increasing supply of Cu, the ratio of the redox couple (DHA/ascorbic acid) increased in Cu-deficient or Cu-excess plants. The activities of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6), peroxidase (EC 1.11.1.7), ascorbate peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2) increased in both Cu-deficient and Cu-excess plants. The results suggest that deficiency of Cu aggravates oxidative stress through enhanced generation of ROS and disturbed redox couple. Excess of Cu damaged roots, accelerated the rate of senescence in the older leaves, induced antioxidant responses and disturbed the cellular redox environment in the young leaves of mulberry plants.     

Tryparedoxin peroxidases from Trypanosoma cruzi: high efficiency in the catalytic elimination of hydrogen peroxide and peroxynitrite

During host cell infection, Trypanosoma cruzi parasites are exposed to reactive oxygen and nitrogen species. As part of their antioxidant defense systems, they express two tryparedoxin peroxidases (TXNPx), thiol-dependent peroxidases members of the peroxiredoxin family. In this work, we report a kinetic characterization of cytosolic (c-TXNPx) and mitochondrial (m-TXNPx) tryparedoxin peroxidases from T. cruzi. Both c-TXNPx and m-TXNPx rapidly reduced hydrogen peroxide (k = 3.0 × 10⁷ and 6 × 10⁶ M⁻¹ s⁻¹ at pH 7.4 and 25 °C, respectively) and peroxynitrite (k = 1.0 × 10⁶ and k = 1.8 × 10⁷ M⁻¹ s⁻¹ at pH 7.4 and 25 °C, respectively). The reductive part of the catalytic cycle was also studied, and the rate constant for the reduction of c-TXNPx by tryparedoxin I was 1.3 × 10⁶ M⁻¹ s⁻¹. The catalytic role of two conserved cysteine residues in both TXNPxs was confirmed with the identification of Cys52 and Cys173 (in c-TXNPX) and Cys81 and Cys204 (in m-TXNPx) as the peroxidatic and resolving cysteines, respectively. Our results indicate that mitochondrial and cytosolic TXNPxs from T. cruzi are highly efficient peroxidases that reduce hydrogen peroxide and peroxynitrite, and contribute to the understanding of their role as virulence factors reported in vivo.