title: Peroxynitrite formation in nitric oxide-exposed submitochondrial particles: detection, oxidative damage and catalytic removal by Mn-porphyrins.
abstract: Peroxynitrite (ONOO(-)) formation in mitochondria may be favored due to the constant supply of superoxide radical (O(2)(∙-)) by the electron transport chain plus the facile diffusion of nitric oxide ((∙)NO) to this organelle. Herein, a model system of submitochondrial particles (SMP) in the presence of succinate plus the respiratory inhibitor antimycin A (to increase O(2)(∙-) rates) and the (∙)NO-donor NOC-7 was studied to directly establish and quantitate peroxynitrite by a multiplicity of methods including chemiluminescence, fluorescence and immunochemical analysis. While all the tested probes revealed peroxynitrite at near stoichiometric levels with respect to its precursor radicals, coumarin boronic acid (a probe that directly reacts with peroxynitrite) had the more straightforward oxidation profile from O(2)(∙-)-forming SMP as a function of the (∙)NO flux. Interestingly, immunospintrapping studies verified protein radical generation in SMP by peroxynitrite. Substrate-supplemented SMP also reduced Mn(III)porphyrins (MnP) to Mn(II)P under physiologically-relevant oxygen levels (3-30 μM); then, Mn(II)P were capable to reduce peroxynitrite and protect SMP from the inhibition of complex I-dependent oxygen consumption and protein radical formation and nitration of membranes. The data directly support the formation of peroxynitrite in mitochondria and demonstrate that MnP can undergo a catalytic redox cycle to neutralize peroxynitrite-dependent mitochondrial oxidative damage.
authors.affiliation: Center for Free Radical and Biomedical Research, Universidad de la República, Avda. General Flores 2125, Montevideo 11800, Uruguay.
authors.forename: Valeria
authors.initials: V
authors.lastname: Valez
authors.forename: Adriana
authors.initials: A
authors.lastname: Cassina
authors.forename: Ines
authors.initials: I
authors.lastname: Batinic-Haberle
authors.forename: Balaraman
authors.initials: B
authors.lastname: Kalyanaraman
authors.forename: Gerardo
authors.initials: G
authors.lastname: Ferrer-Sueta
authors.forename: Rafael
authors.initials: R
authors.lastname: Radi
mesh_terms: D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D000975:Antioxidants / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D002384:Catalysis; D004579:Electron Transport / Q000187:drug effects; D006834:Hydrazines / Q000494:pharmacology; D008163:Luminescent Measurements; D008345:Manganese / Q000737:chemistry* / Q000378:metabolism; D008665:Metalloporphyrins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D008929:Mitochondria, Heart / Q000187:drug effects / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009569:Nitric Oxide / Q000737:chemistry* / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D013050:Spectrometry, Fluorescence; D013367:Submitochondrial Particles / Q000187:drug effects / Q000378:metabolism*; D019802:Succinic Acid / Q000378:metabolism; D013481:Superoxides / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: C115239:3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine; D000975:Antioxidants; D006834:Hydrazines; D008665:Metalloporphyrins; D020030:Nitric Oxide Donors; D009243:NAD; D013481:Superoxides; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D008345:Manganese; D000968:Antimycin A; D019802:Succinic Acid; D010100:Oxygen