Trypanosoma brucei and Trypanosoma cruzi tryparedoxin peroxidases catalytically detoxify peroxynitrite via oxidation of fast reacting thiols.
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 degrees 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 10(5) and 7.2 x 10(5) m(-1) s(-1) at pH 7.4 and 37 degrees 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 Mn(3+)-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.
Trujillo
Madia
M
Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Avda Gral. Flores 2125, 11800 Montevideo, Uruguay.
Budde
Heike
H
Piñeyro
María Dolores
MD
Stehr
Matthias
M
Robello
Carlos
C
Flohé
Leopold
L
Radi
Rafael
R
D000818:Animals; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004583:Electrons; D005978:Glutathione / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008264:Macrophages / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008956:Models, Chemical; D010100:Oxygen / Q000378:metabolism; D010544:Peroxidases / Q000737:chemistry*; D030421:Peroxynitrous Acid / Q000737:chemistry / Q000378:metabolism; D011166:Porphyrins / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry*; D013095:Spermidine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D014346:Trypanosoma brucei brucei / Q000201:enzymology*; D014349:Trypanosoma cruzi / Q000201:enzymology*
D011166:Porphyrins; D015800:Protozoan Proteins; D013438:Sulfhydryl Compounds; D030421:Peroxynitrous Acid; D008345:Manganese; C044809:trypanothione; D010544:Peroxidases; C110959:tryparedoxin peroxidase; D005978:Glutathione; D003545:Cysteine; D010100:Oxygen; D013095:Spermidine