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Trypanosoma brucei and Trypanosoma cruzi Tryparedoxin Peroxidases Catalytically Detoxify Peroxynitrite via Oxidation of Fast Reacting Thiols

2004; Elsevier BV; Volume: 279; Issue: 33 Linguagem: Inglês

10.1074/jbc.m404317200

1083-351X

Madia Trujillo, Heike Budde, Marı́a Dolores Piñeyro, Matthias Stehr, Carlos Robello, Leopold Flohé, Rafael Radí,

Redox biology and oxidative stress

Macrophage activation is one of the hallmarks observed in trypanosomiasis, and the parasites must cope with the resulting oxidative burden, which includes the production of peroxynitrite, an unusual peroxo-acid that acts as a strong oxidant and trypanocidal molecule. Cytosolic tryparedoxin peroxidase (cTXNPx) has been recently identified as essential for oxidative defense in trypanosomatids. This peroxiredoxin decomposes peroxides using tryparedoxin (TXN) as electron donor, which in turn is reduced by dihydrotrypanothione. In this work, we studied the kinetics of the reaction of peroxynitrite with the different thiol-containing components of the cytosolic tryparedoxin peroxidase system in T. brucei (Tb) and T. cruzi (Tc), namely trypanothione, TXN, and cTXNPx. We found that whereas peroxynitrite reacted with dihydrotrypanothione and TbTXN at moderate rates (7200 and 3500 m–1 s–1, respectively, at pH 7.4 and 37 °C) and within the range of typical thiols, the second order rate constants for the reaction of peroxynitrite with reduced TbcTXNPx and TccTXNPx were 9 × 105 and 7.2 × 105m–1 s–1 at pH 7.4 and 37 °C, respectively. This reactivity was dependent on a highly reactive cTXNPx thiol group identified as cysteine 52. Competition experiments showed that TbcTXNPx inhibited other fast peroxynitrite-mediated processes, such as the oxidation of Mn3+-porphyrins. Moreover, steady-state kinetic studies indicate that peroxynitrite-dependent TbcTXNPx and TccTXNPx oxidation is readily reverted by TXN, supporting that these peroxiredoxins would be not only a preferential target for peroxynitrite reactivity but also be able to act catalytically in peroxynitrite decomposition in vivo. Macrophage activation is one of the hallmarks observed in trypanosomiasis, and the parasites must cope with the resulting oxidative burden, which includes the production of peroxynitrite, an unusual peroxo-acid that acts as a strong oxidant and trypanocidal molecule. Cytosolic tryparedoxin peroxidase (cTXNPx) has been recently identified as essential for oxidative defense in trypanosomatids. This peroxiredoxin decomposes peroxides using tryparedoxin (TXN) as electron donor, which in turn is reduced by dihydrotrypanothione. In this work, we studied the kinetics of the reaction of peroxynitrite with the different thiol-containing components of the cytosolic tryparedoxin peroxidase system in T. brucei (Tb) and T. cruzi (Tc), namely trypanothione, TXN, and cTXNPx. We found that whereas peroxynitrite reacted with dihydrotrypanothione and TbTXN at moderate rates (7200 and 3500 m–1 s–1, respectively, at pH 7.4 and 37 °C) and within the range of typical thiols, the second order rate constants for the reaction of peroxynitrite with reduced TbcTXNPx and TccTXNPx were 9 × 105 and 7.2 × 105m–1 s–1 at pH 7.4 and 37 °C, respectively. This reactivity was dependent on a highly reactive cTXNPx thiol group identified as cysteine 52. Competition experiments showed that TbcTXNPx inhibited other fast peroxynitrite-mediated processes, such as the oxidation of Mn3+-porphyrins. Moreover, steady-state kinetic studies indicate that peroxynitrite-dependent TbcTXNPx and TccTXNPx oxidation is readily reverted by TXN, supporting that these peroxiredoxins would be not only a preferential target for peroxynitrite reactivity but also be able to act catalytically in peroxynitrite decomposition in vivo. Trypanosoma brucei and Trypanosoma cruzi are the causative agents of African trypanosomiasis and Chagas disease, respectively, major public health problems affecting millions of people in Africa and Latin America. Both diseases are characterized by an increase in the number of macrophages and the presence of macrophage activation markers (1Vincendeau P. Jauberteau-Marchan M.O. Daulouede S. Ayed Z. Dumas M. Buguet A. Progress in Human African Trypanosomiasis. Springer-Verlag, Berlin1999: 137-156Google Scholar, 2Celentano A.M. Gonzalez Cappa S.M. Parasite Immunol. 1992; 14: 155-167Crossref PubMed Scopus (25) Google Scholar). However, T. brucei is an extracellular parasite, whereas T. cruzi proliferates inside the macrophages and in the cytoplasm of other nucleated cells. Macrophages from T. brucei- and T. cruzi-infected mice produce high levels of nitric oxide (•NO), which has antiparasitic effects in vitro and in vivo (3Vincendeau P. Daulouede S. Veyret B. Darde M.L. Bouteille B. Lemesre J.L. Exp. Parasitol. 1992; 75: 353-360Crossref PubMed Scopus (125) Google Scholar, 4Muñoz-Fernandez M.A. Fernandez M.A. Fresno M. Immunol. Lett. 1992; 33: 35-40Crossref PubMed Scopus (169) Google Scholar, 5Machado F.S. Martins G.A. Aliberti J.C. Mestriner F.L. Cunha F.Q. Silva J.S. Circulation. 2000; 102: 3003-3008Crossref PubMed Scopus (193) Google Scholar). In addition, reactive oxygen intermediates such as superoxide radical (O2−˙) and hydrogen peroxide (H2O2) are synthesized as a result of the oxidative burst by inflammatory cells from T. brucei- and T. cruzi-infected animals (6Grosskinsky C.M. Ezekowitz R.A. Berton G. Gordon S. Askonas B.A. Infect. Immun. 1983; 39: 1080-1086Crossref PubMed Google Scholar, 7Cardoni R.L. Rottenberg M.E. Segura E.L. Cell. Immunol. 1990; 128: 11-21Crossref PubMed Scopus (25) Google Scholar, 8Cardoni R.L. Antunez M.I. Morales C. Nantes I.R. Am. J. Trop. Med. Hyg. 1997; 56: 329-334Crossref PubMed Scopus (66) Google Scholar). Superoxide can be also formed by the parasite itself (e.g. during the generation of the irontyrosyl radical center in the small subunit of ribonucleotide reductase (9Fontecave M. Graslund A. Reichard P. J. Biol. Chem. 1987; 262: 12332-12336Abstract Full Text PDF PubMed Google Scholar), by mitochondrial respiration (10Boveris A. Stoppani A.O. Experientia. 1977; 33: 1306-1308Crossref PubMed Scopus (77) Google Scholar), or by redox cycling of antichagasic drugs (11Docampo R. Moreno S.N. Fed. Proc. 1986; 45: 2471-2476PubMed Google Scholar)). The diffusion-controlled reaction between •NO and O2−˙ leads to the formation of peroxynitrite anion (12Radi R. Denicola A. Alvarez B. Ferrer-Sueta G. Rubbo H. Ignarro L. Nitric Oxide Biology and Pathobiology. Academic Press, Inc., San Diego, CA2000: 57-82Google Scholar), a strong oxidizing and cytotoxic effector molecule against T. cruzi (13Denicola A. Rubbo H. Rodriguez D. Radi R. Arch. Biochem. Biophys. 1993; 304: 279-286Crossref PubMed Scopus (187) Google Scholar, 14Thomson L. Denicola A. Radi R. Arch. Biochem. Biophys. 2003; 412: 55-64Crossref PubMed Scopus (66) Google Scholar). Moreover, inflammatory lesions in the central nervous system of mice chronically infected with T. brucei brucei and in the myocardium of acute chagasic rats express type II nitric-oxide synthase and show protein 3-nitrotyrosine immunoreactivity, which has been ascribed to peroxynitrite 1The term peroxynitrite is used to refer to both peroxynitrite anion (ONOO–) and peroxynitrous acid (ONOOH). IUPAC-recommended names are oxoperoxinitrate (1–) and hydrogen oxoperoxonitrate, respectively. 1The term peroxynitrite is used to refer to both peroxynitrite anion (ONOO–) and peroxynitrous acid (ONOOH). IUPAC-recommended names are oxoperoxinitrate (1–) and hydrogen oxoperoxonitrate, respectively. and/or nitrogen dioxide (•NO2) formation (15Keita M. Vincendeau P. Buguet A. Cespuglio R. Vallat J.M. Dumas M. Bouteille B. Exp. Parasitol. 2000; 95: 19-27Crossref PubMed Scopus (42) Google Scholar, 16Chandrasekar B. Melby P.C. Troyer D.A. Freeman G.L. Clin. Exp. Immunol. 2000; 121: 112-119Crossref PubMed Scopus (36) Google Scholar, 17Radi R. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4003-4008Crossref PubMed Scopus (1207) Google Scholar).Peroxynitrous acid is an unusual peroxo-acid, since it has a low pKa value (6.8 versus 11.6 of the first proton dissociation in hydrogen peroxide, H2O2) and a weak O–O bond (bond strength of 90 kJ mol–1versus 170 kJ mol–1 H2O2) (18Hughes M.N. Biochim. Biophys. Acta. 1999; 1411: 263-272Crossref PubMed Scopus (242) Google Scholar) that makes it an unstable species that decomposes by homolysis (k = 0.9 s–1 in phosphate buffer, pH 7.4 and 37 °C) to yield hydroxyl radical (•OH) and •NO2, which either recombine to form nitrate or react with substrates. The short lifetime of peroxynitrous acid and its fast reaction with carbon dioxide (CO2) frequently present in buffers and in biological systems (12Radi R. Denicola A. Alvarez B. Ferrer-Sueta G. Rubbo H. Ignarro L. Nitric Oxide Biology and Pathobiology. Academic Press, Inc., San Diego, CA2000: 57-82Google Scholar) makes biochemical studies more difficult to perform than with H2O2 and organic hydroperoxides. Preferential targets for peroxynitrite in vivo are thiols that can be oxidized both by direct bimolecular reaction and by the reactions with peroxynitrite-derived radicals (12Radi R. Denicola A. Alvarez B. Ferrer-Sueta G. Rubbo H. Ignarro L. Nitric Oxide Biology and Pathobiology. Academic Press, Inc., San Diego, CA2000: 57-82Google Scholar). The direct peroxynitrite-mediated thiol oxidation is a twoelectron oxidation process that leads to the formation of nitrite and the thiol-derived sulfenic acid, which, in the presence of an accessible thiol group, forms a disulfide, resulting in an overall stoichiometry of two thiols oxidized per peroxynitrite (19Radi R. Beckman J.S. Bush K.M. Freeman B.A. J. Biol. Chem. 1991; 266: 4244-4250Abstract Full Text PDF PubMed Google Scholar). The reaction involves peroxynitrous acid and the deprotonated form of the thiol (thiolate, RS–). The second order rate constants for the reactions between peroxynitrous acid and low molecular weight thiols at pH 7.4 (∼102 to 104m–1 s–1) are inversely related to the thiol pKSH (20Trujillo M. Radi R. Arch. Biochem. Biophys. 2002; 397: 91-98Crossref PubMed Scopus (161) Google Scholar). However, there is an increasing number of highly reactive protein thiols that react with peroxynitrite at rates of 105 to 107m–1 s–1 at pH 7.4 and 37 °C (21Souza J.M. Radi R. Arch. Biochem. Biophys. 1998; 360: 187-194Crossref PubMed Scopus (144) Google Scholar, 22Konorev E.A. Hogg N. Kalyanaraman B. FEBS Lett. 1998; 427: 171-174Crossref PubMed Scopus (135) Google Scholar). Among them, the bacterial peroxiredoxin alkylhydroperoxide reductase subunit C serves to catalytically detoxify peroxynitrite (23Bryk R. Griffin P. Nathan C. Nature. 2000; 407: 211-215Crossref PubMed Scopus (562) Google Scholar). It has been postulated that highly reactive cysteines in proteins are located close to positively charged amino acids (24Flohé L. Budde H. Bruns K. Castro H. Clos J. Hofmann B. Kansal-Kalavar S. Krumme D. Menge U. Plank-Schumacher K. Sztajer H. Wissing J. Wylegalla C. Hecht H.J. Arch. Biochem. Biophys. 2002; 397: 324-335Crossref PubMed Scopus (109) Google Scholar) or at the positive edges of aromatic rings, which promote dissociation of the thiol (pKSH as low as 18 megaohms·cm–1) to minimize trace metal contamination.Expression and Purification of T. brucei Tryparedoxin and T. brucei and T. cruzi Cytosolic Tryparedoxin Peroxidases—Proteins were obtained by heterologous expression of the respective genes in Escherichia coli. The gene of TXN of T. brucei brucei, as identified by Lüdemann et al. (40Lüdemann H. Dormeyer M. Sticherling C. Stallmann D. Follmann H. Krauth-Siegel R.L. FEBS Lett. 1998; 431: 381-385Crossref PubMed Scopus (93) Google Scholar), was expressed as an N-terminally His-tagged protein that was purified as described previously (32Budde H. Flohé L. Hecht H.J. Hofmann B. Stehr M. Wissing J. Lünsdorf H. Biol. Chem. 2003; 384: 619-633Crossref PubMed Scopus (76) Google Scholar). Molecular mutants of TbTXN were obtained according to Ref. 32Budde H. Flohé L. Hecht H.J. Hofmann B. Stehr M. Wissing J. Lünsdorf H. Biol. Chem. 2003; 384: 619-633Crossref PubMed Scopus (76) Google Scholar. T. brucei brucei cytosolic TXNPx and mutated forms of the enzyme were also prepared as N-terminally His-tagged proteins as described previously (32Budde H. Flohé L. Hecht H.J. Hofmann B. Stehr M. Wissing J. Lünsdorf H. Biol. Chem. 2003; 384: 619-633Crossref PubMed Scopus (76) Google Scholar). T. cruzi cytosolic TXNPx was purified as in Ref. 41Guerrero S.A. Lopez J.A. Steinert P. Montemartini M. Kalisz H.M. Colli W. Singh M. Alves M.J. Flohé L. Appl. Microbiol. Biotechnol. 2000; 53: 410-414Crossref PubMed Scopus (41) Google Scholar with the following modifications. The amplified T. cruzi TXNPx gene was cloned into the pQE-30 vector (Qiagen) between SacI and HindIII. pQE30-TcH6TXNPx in E. coli M15 cells was grown at 30 °C with vigorous aeration in LB broth containing 100 μg/ml ampicillin and 25 μg/ml kanamycin. Expression of recombinant TcH6TXNPx was induced with 0.5 mm isopropyl-β-d-thiogalactopyranoside when the culture reached A600 = 0.6. The purification was performed in a 5-ml HiTrap affinity column (Amersham Biosciences) charged with Ni2+ and equilibrated with binding buffer (50 mm sodium phosphate, pH 7.6, containing 10 mm imidazole, 500 mm NaCl) at a flow rate of 3 ml/min. The His-tagged TXNPx was eluted in 50 mm sodium phosphate, pH 7.6, containing 300 mm imidazole, 500 mm NaCl.Peroxynitrite Synthesis—Peroxynitrite was synthesized in a quenched flow reactor from sodium nitrite and H2O2 under acidic conditions and quantitated as described previously (19Radi R. Beckman J.S. Bush K.M. Freeman B.A. J. Biol. Chem. 1991; 266: 4244-4250Abstract Full Text PDF PubMed Google Scholar). Treating a stock solution of peroxynitrite with granular manganese dioxide eliminated H2O2 remaining from the synthesis. Nitrite (NO–2) present in samples of peroxynitrite decomposed at acidic pH was typically 10-fold excess of dithiothreitol. Excess reductant was removed immediately before use by passing proteins through a high pressure liquid chromatographyconnected Hitrap column (Amersham Biosciences) with a UV-visible detector at 280 nm and collected manually in rubber-capped tubes. The elution buffer was 100 mm potassium phosphate, pH 7, plus 100 μm DTPA, which was extensively degassed before use. Once collected, samples were bubbled for 5 min with argon at 4 °C. Protein concentration was measured by the Bradford method, as well as by their absorbance at 280 nm as previously described (31Tetaud E. Giroud C. Prescott A.R. Parkin D.W. Baltz D. Biteau N. Baltz T. Fairlamb A.H. Mol. Biochem. Parasitol. 2001; 116: 171-183Crossref PubMed Scopus (73) Google Scholar). Alkylation of cTXNPx thiol groups by NEM was performed by incubation of the enzyme with a 10–20-fold excess of NEM for 5 min.Thiol Measurements—Low molecular weight thiols as well as protein thiols were quantitated using the DTNB assay (42Ellman G.L. Arch. Biochem. Biophys. 1959; 82: 70-77Crossref PubMed Scopus (21069) Google Scholar).Dihydrorhodamine Oxidation—Stock solutions of dihydrorhodamine (DHR) (28.9 mm) in dimethyl sulfoxide were purged with argon and stored at –20 °C. Dihydrorhodamine oxidation to rhodamine was followed spectrophotometrically at 500 nm (ϵ500 = 78.8 mm–1 cm–1) (43Crow J.P. Nitric Oxide. 1997; 1: 145-157Crossref PubMed Scopus (551) Google Scholar).Direct Kinetic Studies—The kinetics of peroxynitrite decomposition were studied in a stopped-flow spectrophotometer (SF17MV; Applied Photophysics) with a mixing time of <2 ms. Although peroxynitrite decomposition is usually measured at 302 nm (ϵ = 1670 m–1 cm–1 302) (44Hughes M.N. Nicklin H.G. J. Chem. Soc. A. 1968; : 450-452Crossref Google Scholar), we monitored it at 310 nm (ϵ300 = 1600 m–1 cm–1) in order to avoid interferences by background protein absorption at 302 nm. When the initial rate approach was used (45Alvarez B. Ferrer-Sueta G. Freeman B.A. Radi R. J. Biol. Chem. 1999; 274: 842-848Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar), the peroxynitrite decomposition at 2–10 ms was fitted to a linear plot. To calculate initial rates of peroxynitrite decomposition, the slopes were divided by the molar extinction coefficient of peroxynitrite at 310 nm and multiplied by 1.2 (since the absorption at 310 nm derives from peroxynitrite anion, which represents 80% total peroxynitrite concentration at pH 7.4). In pseudo-first-order analysis, apparent rate constants for peroxynitrite decomposition, kobs (s–1) values were determined by fitting stopped-flow data to single exponential decays with floating end point. Reported values are the average of at least seven separate determinations. Temperature was maintained at 37 ± 0.1 °C, and the pH was measured at the outlet.Competition Kinetic Studies—Mn3+ porphyrins are rapidly oxidized by peroxynitrite (k = 105 to 107m–1 s–1 at pH 7.4 and 37 °C) to the O=Mn4+ derivative, in a process that can be conveniently monitored at the Soret band as the decay of absorbance at 462 nm (46Ferrer-Sueta G. Vitturi D. Batinic-Haberle I. Fridovich I. Goldstein S. Czapski G. Radi R. J. Biol. Chem. 2003; 278: 27432-27438Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). In the case of Mn3+-TM-4-PyP, the second order rate constant for its reaction with peroxynitrite was determined previously as 3.7 × 106m–1 s–1 at pH 7.4 and 37 °C (46Ferrer-Sueta G. Vitturi D. Batinic-Haberle I. Fridovich I. Goldstein S. Czapski G. Radi R. J. Biol. Chem. 2003; 278: 27432-27438Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). The effect of increasing concentrations of TbcTXNPX on peroxynitrite-mediated Mn3+-TM-4-PyP oxidation was determined by stopped flow (47Jaeger T. Budde H. Flohé L. Menge U. Singh M. Trujillo M. Radi R. Arch. Biochem. Biophys. 2004; 423: 182-191Crossref PubMed Scopus (128) Google Scholar). Experiments were performed at 37 °C, and the pH was measured at the outlet. Kinetics of peroxynitrite-dependent TbcTXNPx oxidation in the presence of Mn3+-TM-4-PyP was estimated by computer-assisted simulation, varying the apparent second order rate constant for the reaction of the enzyme and peroxynitrite at pH 7.4 and 37 °C so as to get the best fit to experimental data. In this system, peroxynitrite can (a) decompose to nitrate after proton-catalyzed isomerization (k = 0.9 s–1) (12Radi R. Denicola A. Alvarez B. Ferrer-Sueta G. Rubbo H. Ignarro L. Nitric Oxide Biology and Pathobiology. Academic Press, Inc., San Diego, CA2000: 57-82Google Scholar) (Reaction 1), (b) react with the reduced enzyme (Reaction 2), or (c) react with Mn3+-TM-4-PyP (Reaction 3). ONOOH→NO3−+H+ ONOOH+TbcTXNPxred→NO2−+TbcTXNPxox ONOOH+Mn3+-TM-4-PyP→O=Mn4+-TM-4-PyP+·NO2 Reaction 1-3 Alternatively, the kinetics of the competition reaction was studied using a pseudo-first-order approach (48Espenson J.H. Speer J.B. Morris J.M. Chemical Kinetics and Reaction Mechanisms. McGraw-Hill, Inc., New York1995: 46-69Google Scholar), following the effect of increasing concentrations of the enzyme on the apparent first-order rate constants for peroxynitrite reduction as measured through the oxidation of Mn3+-TM-4-PyP at 462 nm. Although TbcTXNPx concentrations used were not much greater than peroxynitrite concentrations, computer-assisted simulations indicated that reduced enzyme concentrations would change minimally (∼10%) over the time course of the experiment under these conditions.Computer-assisted Simulations—Computer-assisted simulations were performed using the Gepasi program (49Mendes P. Trends Biochem. Sci. 1997; 22: 361-363Abstract Full Text PDF PubMed Scopus (493) Google Scholar).Statistics—All experiments reported here were repeated and reproduced at different days. Results are expressed as mean values with the corresponding S.D. values. Graphics and data analysis were performed using Sigma Plot.RESULTSThe Reaction of Peroxynitrite with Trypanothione and T. brucei Tryparedoxin—The addition of increasing excess dihydrotrypanothione concentrations to peroxynitrite led to an increase of the exponential decay of peroxynitrite at pH 7.4 and 37 °C. Dihydrotrypanothione reacted with peroxynitrite with an apparent (pH-dependent) second order rate constant of 7200 m–1 s–1 at pH 7.4 and 37 °C (Fig. 1). In contrast, similar concentrations of trypanothione disulfide did not lead to any increase on the rate of peroxynitrite decomposition, indicating that the thiol groups of the molecule are the responsible for the reactivity (Table I).Table ISecond order rate constants (k2) for the reaction of peroxynitrite and different thiol-containing compounds in T. brucei and T. cruziCompoundk2aAt pH 7.4 and 37 °C.m-1 s-1Dihydrotrypanothione7.2 ± 0.5 × 103Trypanothione disulfideNo reactionTbTXN∼3.5 × 103TbcTXNPx (wild type)9 ± 1 × 105TbcTXNPx (C52S)∼1 × 104TbcTXNPx (C173S)3.5 ± 0.5 × 105TccTXNPx7.2 ± 0.6 × 105a At pH 7.4 and 37 °C. Open table in a new tab Up to 150 μm reduced T. brucei tryparedoxin caused only a modest effect on peroxynitrite (24 μm) decomposition rate, from 0.89 ± 0.04 to 1.4 ± 0.05 s–1, indicating that the second order rate constant between peroxynitrite and the reduced