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J. Biol. Chem., Vol. 279, Issue 33, 34175-34182, August 13, 2004

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

Madia Trujillo, Heike Budde¶, María Dolores Piñeyro||, Matthias Stehr¶, Carlos Robello, Leopold Flohé¶**, and Rafael Radi

From the Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, 11800 Uruguay, ^¶Biochemistry Department, Technical University of Braunschweig, D-38124 Braunschweig, Germany, and ^**MOLISA GmbH, D-39106 Magdeburg, Germany

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 x 10⁵ and 7.2 x 10⁵ M^–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.

Received for publication, April 19, 2004 , and in revised form, May 14, 2004.

^* This work was supported by grants from the Howard Hughes Medical Institute and the John Simon Guggenheim Memorial Foundation (to R. R.), the Deutsche Forschungsgemeinschaft (to L. F.), and the Comisión Sectorial de Investigación Científica (Universidad de la República, Uruguay) (to C. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains an additional figure.

^|| Recipient of a fellowship from Programa de Desarrollo de Ciencias Básicas (PEDECIBA Biología, Uruguay).

To whom correspondence should be addressed: Dept. de Bioquímica, Facultad de Medicina, Avda. Gral. Flores 2125, 11800 Montevideo, Uruguay. Tel.: 5982-9249561; Fax: 5982-9249563; E-mail: rradi{at}fmed.edu.uy.

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