Kinetics, subcellular localization, and contribution to parasite virulence of aTrypanosoma cruzihybrid type A heme peroxidase (TcAPx-CcP).
TheTrypanosoma cruziascorbate peroxidase is, by sequence analysis, a hybrid type A member of class I heme peroxidases [TcAPx-cytochromecperoxidase (CcP)], suggesting both ascorbate (Asc) and cytochromec(Cc) peroxidase activity. Here, we show that the enzyme reacts fast with H2O2(k= 2.9 × 107M-1⋅s-1) and catalytically decomposes H2O2using Cc as the reducing substrate with higher efficiency than Asc (kcat/Km= 2.1 × 105versus 3.5 × 104M-1⋅s-1, respectively). Visible-absorption spectra of purified recombinantTcAPx-CcP after H2O2reaction denote the formation of a compound I-like product, characteristic of the generation of a tryptophanyl radical-cation (Trp233•+). Mutation of Trp233to phenylalanine (W233F) completely abolishes the Cc-dependent peroxidase activity. In addition to Trp233•+, a Cys222-derived radical was identified by electron paramagnetic resonance spin trapping, immunospin trapping, and MS analysis after equimolar H2O2addition, supporting an alternative electron transfer (ET) pathway from the heme. Molecular dynamics studies revealed that ET between Trp233and Cys222is possible and likely to participate in the catalytic cycle. Recognizing the ability ofTcAPx-CcP to use alternative reducing substrates, we searched for its subcellular localization in the infective parasite stages (intracellular amastigotes and extracellular trypomastigotes).TcAPx-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.TcAPx-CcP overexpressers were significantly more infective to macrophages and cardiomyocytes, as well as in the mouse model of Chagas disease, supporting the involvement ofTcAPx-CcP in pathogen virulence as part of the parasite antioxidant armamentarium.
Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo 11800, Uruguay.|Center for Free Radical and Biomedical Research, Universidad de la República, Montevideo 11800, Uruguay.
Martín
M
Hugo
Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo 11800, Uruguay.|Center for Free Radical and Biomedical Research, Universidad de la República, Montevideo 11800, Uruguay.
Alejandra
A
Martínez
Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo 11800, Uruguay.|Center for Free Radical and Biomedical Research, Universidad de la República, Montevideo 11800, Uruguay.
Madia
M
Trujillo
Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo 11800, Uruguay.|Center for Free Radical and Biomedical Research, Universidad de la República, Montevideo 11800, Uruguay.
Damián
D
Estrada
Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo 11800, Uruguay.|Center for Free Radical and Biomedical Research, Universidad de la República, Montevideo 11800, Uruguay.
Mauricio
M
Mastrogiovanni
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, 05508-000, Sao Paulo, Brazil.
Edlaine
E
Linares
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, 05508-000, Sao Paulo, Brazil.
Ohara
O
Augusto
Departamento de Química Inorgánica, Analítica y Química-Física and Instituto de Química Física de Materiales, Medio Ambiente y Energía (INQUIMAE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C1428EHA Buenos Aires, Argentina.
Federico
F
Issoglio
Departamento de Química Inorgánica, Analítica y Química-Física and Instituto de Química Física de Materiales, Medio Ambiente y Energía (INQUIMAE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C1428EHA Buenos Aires, Argentina.
Ari
A
Zeida
Departamento de Química Inorgánica, Analítica y Química-Física and Instituto de Química Física de Materiales, Medio Ambiente y Energía (INQUIMAE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, C1428EHA Buenos Aires, Argentina.
Darío A
DA
Estrín
Department of Clinical Chemistry and Hematology, Cell Microscopy Core, University Medical Center, 3584CX Utrecht, The Netherlands.
Harry F G
HF
Heijnen
Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo 11800, Uruguay; rradi@fmed.edu.uy lpiacenza@fmed.edu.uy.|Center for Free Radical and Biomedical Research, Universidad de la República, Montevideo 11800, Uruguay.
Lucía
L
Piacenza
Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo 11800, Uruguay; rradi@fmed.edu.uy lpiacenza@fmed.edu.uy.|Center for Free Radical and Biomedical Research, Universidad de la República, Montevideo 11800, Uruguay.
Rafael
R
Radi
D000818:Animals; D014355:Chagas Disease / Q000378:metabolism / Q000469:parasitology; D003574:Cytochrome c Group / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004579:Electron Transport / Q000502:physiology; D005260:Female; D006418:Heme / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D016297:Mutagenesis, Site-Directed / Q000379:methods; D010084:Oxidation-Reduction; D010271:Parasites / Q000378:metabolism* / Q000472:pathogenicity*; D009195:Peroxidase / Q000378:metabolism*; D010649:Phenylalanine / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000378:metabolism* / Q000472:pathogenicity*; D014364:Tryptophan / Q000378:metabolism; D014774:Virulence / Q000502:physiology*
D003574:Cytochrome c Group; D006418:Heme; D010649:Phenylalanine; D014364:Tryptophan; D006861:Hydrogen Peroxide; D009195:Peroxidase
Trypanosoma cruzi; heme peroxidase; kinetics; oxidants; virulence