Mechanism of the Reaction of Human Manganese Superoxide Dismutase with Peroxynitrite: Nitration of Critical Tyrosine 34
Human Mn-containing superoxide dismutase (hMnSOD) is amitochondrial enzyme that metabolizes superoxide radical (O2?−). O2?− reacts atdiffusional rates with nitric oxide to yield a potent nitrating species, peroxynitriteanion (ONOO−). MnSOD is nitrated and inactivated in vivo, with active siteTyr34 a...
| Autores: | , , , , , , , , , , , |
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| Tipo de recurso: | artículo |
| Estado: | Versión publicada |
| Fecha de publicación: | 2016 |
| País: | Argentina |
| Institución: | Consejo Nacional de Investigaciones Científicas y Técnicas |
| Repositorio: | CONICET Digital (CONICET) |
| Idioma: | inglés |
| OAI Identifier: | oai:ri.conicet.gov.ar:11336/54248 |
| Acceso en línea: | http://hdl.handle.net/11336/54248 |
| Access Level: | acceso abierto |
| Palabra clave: | Nitration Sod Qm-Mm https://purl.org/becyt/ford/1.6 https://purl.org/becyt/ford/1 |
| Sumario: | Human Mn-containing superoxide dismutase (hMnSOD) is amitochondrial enzyme that metabolizes superoxide radical (O2?−). O2?− reacts atdiffusional rates with nitric oxide to yield a potent nitrating species, peroxynitriteanion (ONOO−). MnSOD is nitrated and inactivated in vivo, with active siteTyr34 as the key oxidatively modified residue. We previously reported a k of ∼1.0× 105 M−1 s−1 for the reaction of hMnSOD with ONOO− by direct stopped-flowspectroscopy and the critical role of Mn in the nitration process. In this study, wefurther established the mechanism of the reaction of hMnSOD with ONOO−,including the necessary re-examination of the second-order rate constant by anindependent method and the delineation of the microscopic steps that lead to theregio-specific nitration of Tyr34. The redetermination of k was performed bycompetition kinetics utilizing coumarin boronic acid, which reacts with ONOO−at a rate of ∼1 × 106 M−1 s−1 to yield the fluorescence product, 7-hydroxycoumarin. Time-resolved fluorescence studies in the presence of increasing concentrations of hMnSOD provided a kof ∼1.0 × 105 M−1 s−1, fully consistent with the direct method. Proteomic analysis indicated that ONOO−, but not othernitrating agents, mediates the selective modification of active site Tyr34. Hybrid quantum-classical (quantum mechanics/molecular mechanics) simulations supported a series of steps that involve the initial reaction of ONOO− with MnIII to yield MnIVand intermediates that ultimately culminate in 3-nitroTyr34. The data reported herein provide a kinetic and mechanistic basis forrationalizing how MnSOD constitutes an intramitochondrial target for ONOO− and the microscopic events, with atomic levelresolution, that lead to selective and efficient nitration of critical Tyr34. |
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