INVESTIGADORES
MORENO Diego Martin
artículos
Título:
Structural and molecular basis of the peroxynitrite-mediated nitration and inactivation of Trypanosoma cruzi Fe-superoxide dismutases A and B: Disparate susceptibilities due to the repair of Tyr35 radical by Cys83 in Fe-SODB through intramolecular electro
Autor/es:
ALEJANDRA MARTINEZ; GONZALO PELUFFO; ARIEL A. PETRUK; MARTIN HUGO; DOLORES PINEYRO; VERÓNICA DEMICHELI; DIEGO M. MORENO; ANALIA LIMA; CARLOS BATTHYANY; ROSARIO DURÁN; CARLOS RUBELLO; MARCELO A. MARTI; NICOLE LARRIEUX; ALEJANDRO BUSCHIAZO; MADIA TRUJILLO; RAFAEL RADI; LUCIA PIACENZA
Revista:
JOURNAL OF BIOLOGICAL CHEMISTRY
Editorial:
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Referencias:
Lugar: Bethesda, Maryland; Año: 2014 vol. 289 p. 12760 - 12778
ISSN:
0021-9258
Resumen:
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 x 104 M-1s-1 and 4.3±0.4 x 104 M-1s-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 towards peroxynitrite was similar to that previously reported for E. 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 tyrosine-35 (Tyr35). Searching for structural differences, the crystal structure of Fe-SODA was solved at 2.2Åresolution. Structural analysis comparing both Fe-SODs isoforms reveals differences in key cysteines and tryptophan residues. Thiol alkylation of Fe-SODB cysteines made the enzyme more susceptible to peroxynitrite. In particular, Cys83 mutation (C83S, absent in Fe-SODA) increased the Fe-SODB sensitivity towards peroxynitrite. Molecular dynamics, electron paramagnetic resonance and immunospin-trapping analysis revealed that Cys83 present in Fe-SODB acts as an electron donor that repairs Tyr35 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.