INVESTIGADORES
MORENO Diego Martin
artículos
Título:
Mechanism of the Reaction of human Mn-Superoxide Dismutase with Peroxynitrite: Nitration of Critical Tyrosine-34.
Autor/es:
VERÓNICA DEMICHELI; DIEGO M. MORENO; GABRIEL E. JARA; ANALIA LIMA; SEBASTIAN CARBALLAL; NATALIA RIOS; CARLOS BATTHYANY; GERARDO FERRER-SUETA; CELIA QUIJANO; DARIO ESTRIN; MARCELO MARTI; RAFAEL RADI
Revista:
BIOCHEMISTRY
Editorial:
AMER CHEMICAL SOC
Referencias:
Lugar: Washington; Año: 2016 vol. 55 p. 3403 - 3417
ISSN:
0006-2960
Resumen:
Human Mn-containing superoxide dismutase (hMnSOD) is a mitochondrial enzyme that metabolizes superoxide radical (O2?−). O2?− reacts at diffusional rates with nitric oxide to yield a potent nitrating species, peroxynitrite anion (ONOO−). MnSOD is nitrated and inactivated in vivo, with active site Tyr34 as the key oxidatively-modified residue. We previously reported a k~1.0x105 M-1s-1 for the hMnSOD reaction with ONOO− by direct stopped-flow spectroscopy and the critical role of Mn in the nitration process. In the present study, we further established the mechanism of the reaction of hMnSOD with ONOO−, including the necessary reexamination of the second order rate constant by an independent method and the delineation of the microscopic steps that lead to the regio-specific nitration of Tyr34. The re-determination of k was performed by competition kinetics utilizing coumarin boronic acid which reacts with ONOO− at ~1x106 M-1s-1 to yield the fluorescence product, 7-hydroxycoumarin. Time-resolved fluorescence studies in the presence of increasing concentrations of hMnSOD provided a k~1.0x105 M-1s-1, fully consistent with the direct method. Proteomic analysis indicated that ONOO-, but not other nitrating agents, mediate the selective modification of active site Tyr34. Hybrid quantum-classical (QM/MM) simulations supported a series of steps that involve the initial reaction of ONOO- with MnIII to yield MnIV and intermediates that ultimately culminate in nitroTyr34. The data reported herein provide kinetic and mechanistic basis to rationalize how MnSOD constitutes an intramitochondrial target for ONOO- and the microscopic events, with atomic level resolution, that lead to selective and efficient nitration of critical Tyr34.