title: Mechanism of the Reaction of Human Manganese Superoxide Dismutase with Peroxynitrite: Nitration of Critical Tyrosine 34.
abstract: 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 × 10(5) 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 × 10(6) 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 × 10(5) 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.
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República , Avda. General Flores 2125, Montevideo, Uruguay.|Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República , Avda. General Flores 2125, Montevideo, Uruguay.
authors.forename: Verónica
authors.initials: V
authors.lastname: Demicheli
authors.affiliation: Instituto de Química de Rosario (IQUIR-CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario , Suipacha 531, S2002LRK Rosario, Argentina.
authors.forename: Diego M
authors.initials: DM
authors.lastname: Moreno
authors.affiliation: Departamento de Química Inorgánica, Analítica y Química-Física (INQUIMAE-CONICET), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria , Intendente Güiraldes 2160, C1428EGA Ciudad Autónoma de Buenos Aires, Argentina.
authors.forename: Gabriel E
authors.initials: GE
authors.lastname: Jara
authors.affiliation: Unidad Bioquímica y Proteómica Analíticas, Institut Pasteur de Montevideo , Montevideo, Uruguay.
authors.forename: Analía
authors.initials: A
authors.lastname: Lima
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República , Avda. General Flores 2125, Montevideo, Uruguay.|Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República , Avda. General Flores 2125, Montevideo, Uruguay.
authors.forename: Sebastián
authors.initials: S
authors.lastname: Carballal
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República , Avda. General Flores 2125, Montevideo, Uruguay.|Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República , Avda. General Flores 2125, Montevideo, Uruguay.|Departamento de Química Orgánica, Facultad de Química, Universidad de la República , Avda. General Flores 2124, Montevideo, Uruguay.
authors.forename: Natalia
authors.initials: N
authors.lastname: Ríos
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República , Avda. General Flores 2125, Montevideo, Uruguay.|Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República , Avda. General Flores 2125, Montevideo, Uruguay.|Unidad Bioquímica y Proteómica Analíticas, Institut Pasteur de Montevideo , Montevideo, Uruguay.
authors.forename: Carlos
authors.initials: C
authors.lastname: Batthyany
authors.affiliation: Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República , Avda. General Flores 2125, Montevideo, Uruguay.|Laboratorio de Fisicoquímica Biológica, Instituto de Química Biológica, Facultad de Ciencias, Universidad de la Repúbica , Igua 4225, Montevideo, Uruguay.
authors.forename: Gerardo
authors.initials: G
authors.lastname: Ferrer-Sueta
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República , Avda. General Flores 2125, Montevideo, Uruguay.|Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República , Avda. General Flores 2125, Montevideo, Uruguay.
authors.forename: Celia
authors.initials: C
authors.lastname: Quijano
authors.affiliation: Departamento de Química Inorgánica, Analítica y Química-Física (INQUIMAE-CONICET), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria , Intendente Güiraldes 2160, C1428EGA Ciudad Autónoma de Buenos Aires, Argentina.
authors.forename: Darío A
authors.initials: DA
authors.lastname: Estrı́n
authors.affiliation: Departamento de Química Biológica e IQUIBICEN-CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria , Intendente Güiraldes 2160, C1428EGA Ciudad Autónoma de Buenos Aires, Argentina.
authors.forename: Marcelo A
authors.initials: MA
authors.lastname: Martí
authors.affiliation: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República , Avda. General Flores 2125, Montevideo, Uruguay.|Center for Free Radical and Biomedical Research, Facultad de Medicina, Universidad de la República , Avda. General Flores 2125, Montevideo, Uruguay.
authors.forename: Rafael
authors.initials: R
authors.lastname: Radi
mesh_terms: D015153:Blotting, Western; D020134:Catalytic Domain; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009566:Nitrates / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid / Q000378:metabolism*; D040901:Proteomics; D013482:Superoxide Dismutase / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*; D014468:Umbelliferones / Q000378:metabolism
chemical_list: D009566:Nitrates; D014468:Umbelliferones; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D014443:Tyrosine; C031477:7-hydroxycoumarin; D013482:Superoxide Dismutase