title: Structural and molecular basis of the peroxynitrite-mediated nitration and inactivation of Trypanosoma cruzi iron-superoxide dismutases (Fe-SODs) A and B: disparate susceptibilities due to the repair of Tyr35 radical by Cys83 in Fe-SODB through intramolecular electron transfer.
abstract: Trypanosoma cruzi, the causative agent of Chagas disease, contains exclusively iron-dependent superoxide dismutases (Fe-SODs) located in different subcellular compartments. Peroxynitrite, a key cytotoxic and oxidizing effector biomolecule, reacted with T. cruzi mitochondrial (Fe-SODA) and cytosolic (Fe-SODB) SODs with second order rate constants of 4.6 ± 0.2 × 10(4) M(-1) s(-1) and 4.3 ± 0.4 × 10(4) M(-1) s(-1) at pH 7.4 and 37 °C, respectively. Both isoforms are dose-dependently nitrated and inactivated by peroxynitrite. Susceptibility of T. cruzi Fe-SODA toward peroxynitrite was similar to that reported previously for Escherichia coli Mn- and Fe-SODs and mammalian Mn-SOD, whereas Fe-SODB was exceptionally resistant to oxidant-mediated inactivation. We report mass spectrometry analysis indicating that peroxynitrite-mediated inactivation of T. cruzi Fe-SODs is due to the site-specific nitration of the critical and universally conserved Tyr(35). Searching for structural differences, the crystal structure of Fe-SODA was solved at 2.2 Å resolution. Structural analysis comparing both Fe-SOD isoforms reveals differences in key cysteines and tryptophan residues. Thiol alkylation of Fe-SODB cysteines made the enzyme more susceptible to peroxynitrite. In particular, Cys(83) mutation (C83S, absent in Fe-SODA) increased the Fe-SODB sensitivity toward peroxynitrite. Molecular dynamics, electron paramagnetic resonance, and immunospin trapping analysis revealed that Cys(83) present in Fe-SODB acts as an electron donor that repairs Tyr(35) radical via intramolecular electron transfer, preventing peroxynitrite-dependent nitration and consequent inactivation of Fe-SODB. Parasites exposed to exogenous or endogenous sources of peroxynitrite resulted in nitration and inactivation of Fe-SODA but not Fe-SODB, suggesting that these enzymes play distinctive biological roles during parasite infection of mammalian cells.
authors.affiliation: From the Departamento de Bioquímica and Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República, Montevideo 11800, Uruguay.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Martinez
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Peluffo
authors.forename: Ariel A
authors.initials: AA
authors.lastname: Petruk
authors.forename: Martín
authors.initials: M
authors.lastname: Hugo
authors.forename: Dolores
authors.initials: D
authors.lastname: Piñeyro
authors.forename: Verónica
authors.initials: V
authors.lastname: Demicheli
authors.forename: Diego M
authors.initials: DM
authors.lastname: Moreno
authors.forename: Analía
authors.initials: A
authors.lastname: Lima
authors.forename: Carlos
authors.initials: C
authors.lastname: Batthyány
authors.forename: Rosario
authors.initials: R
authors.lastname: Durán
authors.forename: Carlos
authors.initials: C
authors.lastname: Robello
authors.forename: Marcelo A
authors.initials: MA
authors.lastname: Martí
authors.forename: Nicole
authors.initials: N
authors.lastname: Larrieux
authors.forename: Alejandro
authors.initials: A
authors.lastname: Buschiazzo
authors.forename: Madia
authors.initials: M
authors.lastname: Trujillo
authors.forename: Rafael
authors.initials: R
authors.lastname: Radi
authors.forename: Lucía
authors.initials: L
authors.lastname: Piacenza
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D020134:Catalytic Domain; D014355:Chagas Disease / Q000469:parasitology; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D004789:Enzyme Activation / Q000187:drug effects; D006790:Host-Parasite Interactions; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D009566:Nitrates / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D017433:Protein Structure, Secondary; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013482:Superoxide Dismutase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D009566:Nitrates; D015800:Protozoan Proteins; D017382:Reactive Oxygen Species; D030421:Peroxynitrous Acid; D014443:Tyrosine; D013482:Superoxide Dismutase; D003545:Cysteine
keywords: Free Radicals; Nitration; Nitric Oxide; Oxidation-Reduction; Peroxynitrite; Superoxide; Superoxide Dismutase (SOD); Trypanosoma cruzi; Trypanosome