Peroxynitrite detoxification by Trypanosoma cruzi heme peroxidase supports parasite survival in macrophages

Trypanosoma cruzi, the causative agent of Chagas disease, must overcome a host's nitro-oxidative burst to establish the infection. Among the reactive species generated by immune cells, peroxynitrite stands out as a highly cytotoxic molecule for the parasite. Yet, the parasite's defense mechanisms against peroxynitrite remain incompletely understood. In this work, we demonstrate that the T. cruzi heme-containing ascorbate peroxidase cytochrome c peroxidase (APx-CcP), an antioxidant enzyme previously shown to increase parasite infectivity, acts as a key peroxynitrite-detoxifying enzyme. Using direct and competitive kinetic assays, we show that APx-CcP reacts rapidly with peroxynitrite (k = 3-4 × 10⁶ M^-1 s^-1 at pH 7.4 and 25 °C) via a two-electron reduction mechanism. This activity allows T. cruzi to neutralize peroxynitrite within the macrophage phagosome, increasing survival and infection capacity, particularly in strains with elevated APx-CcP expression. This is the first heme peroxidase of trypanosomatids in which a catalytic cycle of peroxynitrite decomposition is described. Moreover, the presence of physiological concentrations of ascorbate during infection enhances the enzyme's ability to scavenge both hydrogen peroxide and peroxynitrite, further highlighting its role in parasite survival.