Mechanism of the Reaction of Human Manganese Superoxide Dismutase with Peroxynitrite: Nitration of Critical Tyrosine 34

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• 作者：Verónica Demicheli ; Diego M. Moreno ; Gabriel E. Jara ; Analía Lima ; Sebastián Carballal ; Natalia Ríos ; Carlos Batthyany ; Gerardo Ferrer-Sueta ; Celia Quijano ; Darío A. Estrı́n ; Marcelo A. Martí ; Rafael Radi
• 刊名：Biochemistry
• 出版年：2016
• 出版时间：June 21, 2016
• 年：2016
• 卷：55
• 期：24
• 页码：3403-3417
• 全文大小：665K
• 年卷期：0
• ISSN：1520-4995

文摘

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 of ∼1.0 × 10⁵ M^–1 s^–1 for the reaction of hMnSOD with ONOO^– by direct stopped-flow spectroscopy and the critical role of Mn in the nitration process. In this study, we further established the mechanism of the reaction of hMnSOD with ONOO^–, including the necessary re-examination 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 redetermination of k was performed by competition 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 k of ∼1.0 × 10⁵ M^–1 s^–1, fully consistent with the direct method. Proteomic analysis indicated that ONOO^–, but not other nitrating 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 Mn^III to yield Mn^IV and intermediates that ultimately culminate in 3-nitroTyr34. The data reported herein provide a kinetic and mechanistic basis for rationalizing 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.