Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP)
The Trypanosoma cruzi ascorbate peroxidase is, by sequence analysis, a hybrid type A member of class I heme peroxidases [TcAPx-cytochrome c peroxidase (CcP)], suggesting both ascorbate (Asc) and cytochrome c (Cc) peroxidase activity. Here, we show that the enzyme reacts fast with H2O2 (k = 2.9 × 107...
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todo:paper_00278424_v114_n8_pE1326_Hugo2023-10-03T14:38:16Z Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP) Hugo, M. Martínez, A. Trujillo, M. Estrada, D. Mastrogiovanni, M. Linares, E. Augusto, O. Issoglio, F. Zeida, A. Estrín, D.A. Heijnen, H.F.G. Piacenza, L. Radi, R. Heme peroxidase Kinetics Oxidants Trypanosoma cruzi Virulence ascorbate peroxidase cytochrome c peroxidase heme hydrogen peroxide phenylalanine cytochrome c peroxidase tryptophan absorption spectroscopy amastigote animal cell animal experiment Article cell kinetics cell membrane cellular distribution controlled study electron spin resonance electron transport enzyme activity enzyme mechanism enzyme substrate host parasite interaction mass spectrometry mitochondrial membrane molecular dynamics mouse nonhuman parasite virulence priority journal protein expression spin trapping Trypanosoma cruzi animal Bagg albino mouse C57BL mouse Chagas disease female kinetics male metabolism oxidation reduction reaction parasite parasitology pathogenicity physiology procedures site directed mutagenesis Trypanosoma cruzi virulence Animals Chagas Disease Cytochrome c Group Electron Spin Resonance Spectroscopy Electron Transport Female Heme Hydrogen Peroxide Kinetics Male Mice Mice, Inbred BALB C Mice, Inbred C57BL Mutagenesis, Site-Directed Oxidation-Reduction Parasites Peroxidase Phenylalanine Trypanosoma cruzi Tryptophan Virulence The Trypanosoma cruzi ascorbate peroxidase is, by sequence analysis, a hybrid type A member of class I heme peroxidases [TcAPx-cytochrome c peroxidase (CcP)], suggesting both ascorbate (Asc) and cytochrome c (Cc) peroxidase activity. Here, we show that the enzyme reacts fast with H2O2 (k = 2.9 × 107 M-1·s-1) and catalytically decomposes H2O2 using Cc as the reducing substrate with higher efficiency than Asc (kcat/Km = 2.1 × 105 versus 3.5 × 104 M-1·s-1, respectively). Visible-absorption spectra of purified recombinant TcAPx-CcP after H2O2 reaction denote the formation of a compound I-like product, characteristic of the generation of a tryptophanyl radical-cation (Trp233•+). Mutation of Trp233 to 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 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 TcAPx-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 of TcAPx-CcP in pathogen virulence as part of the parasite antioxidant armamentarium. © 2017, National Academy of Sciences. All rights reserved. Fil:Estrín, D.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_00278424_v114_n8_pE1326_Hugo |
| institution |
Universidad de Buenos Aires |
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I-28 |
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R-134 |
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Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
Heme peroxidase Kinetics Oxidants Trypanosoma cruzi Virulence ascorbate peroxidase cytochrome c peroxidase heme hydrogen peroxide phenylalanine cytochrome c peroxidase tryptophan absorption spectroscopy amastigote animal cell animal experiment Article cell kinetics cell membrane cellular distribution controlled study electron spin resonance electron transport enzyme activity enzyme mechanism enzyme substrate host parasite interaction mass spectrometry mitochondrial membrane molecular dynamics mouse nonhuman parasite virulence priority journal protein expression spin trapping Trypanosoma cruzi animal Bagg albino mouse C57BL mouse Chagas disease female kinetics male metabolism oxidation reduction reaction parasite parasitology pathogenicity physiology procedures site directed mutagenesis Trypanosoma cruzi virulence Animals Chagas Disease Cytochrome c Group Electron Spin Resonance Spectroscopy Electron Transport Female Heme Hydrogen Peroxide Kinetics Male Mice Mice, Inbred BALB C Mice, Inbred C57BL Mutagenesis, Site-Directed Oxidation-Reduction Parasites Peroxidase Phenylalanine Trypanosoma cruzi Tryptophan Virulence |
| spellingShingle |
Heme peroxidase Kinetics Oxidants Trypanosoma cruzi Virulence ascorbate peroxidase cytochrome c peroxidase heme hydrogen peroxide phenylalanine cytochrome c peroxidase tryptophan absorption spectroscopy amastigote animal cell animal experiment Article cell kinetics cell membrane cellular distribution controlled study electron spin resonance electron transport enzyme activity enzyme mechanism enzyme substrate host parasite interaction mass spectrometry mitochondrial membrane molecular dynamics mouse nonhuman parasite virulence priority journal protein expression spin trapping Trypanosoma cruzi animal Bagg albino mouse C57BL mouse Chagas disease female kinetics male metabolism oxidation reduction reaction parasite parasitology pathogenicity physiology procedures site directed mutagenesis Trypanosoma cruzi virulence Animals Chagas Disease Cytochrome c Group Electron Spin Resonance Spectroscopy Electron Transport Female Heme Hydrogen Peroxide Kinetics Male Mice Mice, Inbred BALB C Mice, Inbred C57BL Mutagenesis, Site-Directed Oxidation-Reduction Parasites Peroxidase Phenylalanine Trypanosoma cruzi Tryptophan Virulence Hugo, M. Martínez, A. Trujillo, M. Estrada, D. Mastrogiovanni, M. Linares, E. Augusto, O. Issoglio, F. Zeida, A. Estrín, D.A. Heijnen, H.F.G. Piacenza, L. Radi, R. Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP) |
| topic_facet |
Heme peroxidase Kinetics Oxidants Trypanosoma cruzi Virulence ascorbate peroxidase cytochrome c peroxidase heme hydrogen peroxide phenylalanine cytochrome c peroxidase tryptophan absorption spectroscopy amastigote animal cell animal experiment Article cell kinetics cell membrane cellular distribution controlled study electron spin resonance electron transport enzyme activity enzyme mechanism enzyme substrate host parasite interaction mass spectrometry mitochondrial membrane molecular dynamics mouse nonhuman parasite virulence priority journal protein expression spin trapping Trypanosoma cruzi animal Bagg albino mouse C57BL mouse Chagas disease female kinetics male metabolism oxidation reduction reaction parasite parasitology pathogenicity physiology procedures site directed mutagenesis Trypanosoma cruzi virulence Animals Chagas Disease Cytochrome c Group Electron Spin Resonance Spectroscopy Electron Transport Female Heme Hydrogen Peroxide Kinetics Male Mice Mice, Inbred BALB C Mice, Inbred C57BL Mutagenesis, Site-Directed Oxidation-Reduction Parasites Peroxidase Phenylalanine Trypanosoma cruzi Tryptophan Virulence |
| description |
The Trypanosoma cruzi ascorbate peroxidase is, by sequence analysis, a hybrid type A member of class I heme peroxidases [TcAPx-cytochrome c peroxidase (CcP)], suggesting both ascorbate (Asc) and cytochrome c (Cc) peroxidase activity. Here, we show that the enzyme reacts fast with H2O2 (k = 2.9 × 107 M-1·s-1) and catalytically decomposes H2O2 using Cc as the reducing substrate with higher efficiency than Asc (kcat/Km = 2.1 × 105 versus 3.5 × 104 M-1·s-1, respectively). Visible-absorption spectra of purified recombinant TcAPx-CcP after H2O2 reaction denote the formation of a compound I-like product, characteristic of the generation of a tryptophanyl radical-cation (Trp233•+). Mutation of Trp233 to 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 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 TcAPx-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 of TcAPx-CcP in pathogen virulence as part of the parasite antioxidant armamentarium. © 2017, National Academy of Sciences. All rights reserved. |
| format |
JOUR |
| author |
Hugo, M. Martínez, A. Trujillo, M. Estrada, D. Mastrogiovanni, M. Linares, E. Augusto, O. Issoglio, F. Zeida, A. Estrín, D.A. Heijnen, H.F.G. Piacenza, L. Radi, R. |
| author_facet |
Hugo, M. Martínez, A. Trujillo, M. Estrada, D. Mastrogiovanni, M. Linares, E. Augusto, O. Issoglio, F. Zeida, A. Estrín, D.A. Heijnen, H.F.G. Piacenza, L. Radi, R. |
| author_sort |
Hugo, M. |
| title |
Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP) |
| title_short |
Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP) |
| title_full |
Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP) |
| title_fullStr |
Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP) |
| title_full_unstemmed |
Kinetics, subcellular localization, and contribution to parasite virulence of a Trypanosoma cruzi hybrid type A heme peroxidase (TcAPx-CcP) |
| title_sort |
kinetics, subcellular localization, and contribution to parasite virulence of a trypanosoma cruzi hybrid type a heme peroxidase (tcapx-ccp) |
| url |
http://hdl.handle.net/20.500.12110/paper_00278424_v114_n8_pE1326_Hugo |
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