In Kinetoplastida, comprising the medically important parasites Trypanosoma brucei, T. cruzi, and Leishmania species, 2-Cys peroxiredoxins described to date have been shown to catalyze reduction of peroxides by the specific thiol trypanothione using tryparedoxin, a thioredoxin-related protein, as an immediate electron donor. Here we show that a mitochondrial peroxiredoxin from L. infantum (LimTXNPx) is also a tryparedoxin peroxidase. In an heterologous system constituted by nicotinamide adenine dinucleotide phosphate (NADPH), T. cruzi trypanothione reductase, trypanothione and Crithidia fasciculata tryparedoxin (CfTXN1 and CfTXN2), the recombinant enzyme purified from Escherichia coli as an N-terminally His-tagged protein preferentially reduces H(2)O(2) and tert-butyl hydroperoxide and less actively cumene hydroperoxide. Linoleic acid hydroperoxide and phosphatidyl choline hydroperoxide are poor substrates in the sense that they are reduced weakly and inhibit the enzyme in a concentration- and time-dependent way. Kinetic parameters deduced for LimTXNPx are a k(cat) of 37.0 s(-1) and K(m) values of 31.9 and 9.1 microM for CfTXN2 and tert-butyl hydroperoxide, respectively. Kinetic analysis indicates that LimTXNPx does not follow the classic ping-pong mechanism described for other TXNPx (Phi(1,2) = 0.8 s x microM(2)). Although the molecular mechanism underlying this finding is unknown, we propose that cooperativity between the redox centers of subunits may explain the unusual kinetic behavior observed. This hypothesis is corroborated by high-resolution electron microscopy and gel chromatography that reveal the native enzyme to preferentially exist as a homodecameric ring structure composed of five dimers.