Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP).

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