期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 289, 期 18, 页码 12760-12778出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M113.545590
关键词
Free Radicals; Nitric Oxide; Oxidation-Reduction; Superoxide Dismutase (SOD); Trypanosome; Trypanosoma cruzi; Nitration; Peroxynitrite; Superoxide
资金
- National Institutes of Health [1R01AI095173]
- Universidad de la Republica (CSIC, Uruguay)
- PEDECIBA (Progama de Desarrollo de Ciencias Basicas, Uruguay)
- CeBEM (Centro de Biolog a Estructural del Mercosur)
- Agencia Nacional de Investigacion e Innovacion (ANII, Uruguay)
- Biriden
- ASM
- Ridaline through Fundacion Manuel Perez, Facultad de Medicina, Universidad de la Republica
Background: Superoxide dismutases are inactivated by peroxynitrite. Results:T. cruzi cytosolic Fe-SODB is highly resistant toward peroxynitrite-mediated tyrosine nitration and inactivation as compared with mitochondrial Fe-SODA. Conclusion: Intramolecular electron transfer in Fe-SODB from Cys(83) to critical Tyr(35) prevents enzyme nitration and inactivation. Significance: Disparate susceptibilities of Fe-SODs to peroxynitrite can influence parasite virulence during T. cruzi infection of mammalian cells. 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 10(4) m(-1) s(-1) and 4.3 +/- 0.4 x 10(4) m(-1) s(-1) at pH 7.4 and 37 degrees 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 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.
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