Functional characterization of the mitochondrial peroxiredoxin I from Leishmania braziliensis evaluating the peroxidase and chaperone activity

Abstract

Peroxiredoxins (Prxs) belonging to the subfamily Prx1, are typical 2-Cys peroxidases responsible for controlling the intracellular level of H2O2 and seem to assume a chaperone function under oxidative stress conditions. Members of this subfamily have two catalytic cysteines and constitute two homodimers with identical active sites located on opposite sides. In most cases, these dimers can associate non-covalently forming a decamer. The dimer-to-decamer interconversion seems to play an important role in the catalytic cycle, influencing the enzymatic efficiency. Recent studies show that the decamer can form larger complexes that have the chaperone activity. The redox state is traditionally described as the main factor that regulates the oligomeric state of Prxs, however the concentration, ionic strength and pH can also affect the oligomerization of these proteins. In the PhD project (FAPESP 2012/24134-3), in progress, we intend to investigate the oligomerization mechanisms of the mitochondrial tryparedoxin peroxidase Leishmania braziliensis (mLbTXNPx), belonging to the subfamily Prx1. Studies show that this enzyme is essential for the survival of Leishmania sp. during oxidative stress generated by macrophages and its overexpression results in an increased growth rate, less duplication and increased resistance to oxidative stress in the parasite. Interestingly, our results show that in this enzyme, the decamerization can be regulated by divalent cations. Through structural analysis and site-directed mutations it is possible to propose a divalent cation binding site. Furthermore, the molecular mechanisms of regulation by the pH are also investigated by biophysical analyses and site-directed mutagenesis. Importantly, the possible regulation by divalent cations has been seen for the first time, and, although the influence of pH has already been described, the molecular mechanisms concerning this regulation remained unknown. For this period abroad, we intend to evaluate as divalent cations and pH influence the peroxidase and chaperone activities for the mLbTXNPx wild-type and mutants, providing essential information for the understanding of the regulatory mechanisms of Prxs1 from trypanosomatids. (AU)