Kinetics, subcellular localization, and contribution to parasite virulence of a <i>Trypanosoma cruzi</i> hybrid type A heme peroxidase ( <i>Tc</i> APx-CcP)

Abstract

The <i>Trypanosoma cruzi</i> ascorbate peroxidase is, by sequence analysis, a hybrid type A member of class I heme peroxidases [<i>Tc</i>APx-cytochrome <i>c</i> peroxidase (CcP)], suggesting both ascorbate (Asc) and cytochrome <i>c</i> (Cc) peroxidase activity. Here, we show that the enzyme reacts fast with H₂O₂ (<i>k</i> = 2.9 × 10⁷ M^-1⋅s^-1) and catalytically decomposes H₂O₂ using Cc as the reducing substrate with higher efficiency than Asc (<i>k</i>_cat/<i>K</i>_m = 2.1 × 10⁵ versus 3.5 × 10⁴ M^-1⋅s^-1, respectively). Visible-absorption spectra of purified recombinant <i>Tc</i>APx-CcP after H₂O₂ reaction denote the formation of a compound I-like product, characteristic of the generation of a tryptophanyl radical-cation (Trp^233•+). Mutation of Trp²³³ to phenylalanine (W233F) completely abolishes the Cc-dependent peroxidase activity. In addition to Trp^233•+, a Cys²²²-derived radical was identified by electron paramagnetic resonance spin trapping, immunospin trapping, and MS analysis after equimolar H₂O₂ addition, supporting an alternative electron transfer (ET) pathway from the heme. Molecular dynamics studies revealed that ET between Trp²³³ and Cys²²² is possible and likely to participate in the catalytic cycle. Recognizing the ability of <i>Tc</i>APx-CcP to use alternative reducing substrates, we searched for its subcellular localization in the infective parasite stages (intracellular amastigotes and extracellular trypomastigotes). <i>Tc</i>APx-CcP was found closely associated with mitochondrial membranes and, most interestingly, with the outer leaflet of the plasma membrane, suggesting a role at the host-parasite interface. <i>Tc</i>APx-CcP overexpressers were significantly more infective to macrophages and cardiomyocytes, as well as in the mouse model of Chagas disease, supporting the involvement of <i>Tc</i>APx-CcP in pathogen virulence as part of the parasite antioxidant armamentarium.