Peroxynitrite formation in nitric oxide-exposed submitochondrial particles: Detection, oxidative damage and catalytic removal by Mn-porphyrins

Center for Free Radical and Biomedical Research, Universidad de la República, Avda. General Flores 2125, Montevideo 11800, Uruguay.
Archives of Biochemistry and Biophysics (Impact Factor: 3.02). 11/2012; 529(1). DOI: 10.1016/
Source: PubMed


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 M); 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.

Download full-text


Available from: Gerardo Ferrer-Sueta, Jan 29, 2014
42 Reads
  • Source
    • "However, the ALO-based mechanism contributes only to about a half of the Sups' toxicity as it follows from the effect of ALO inhibition by cholesterol (Popova et al., 2011). The partial protective effect of the iron porphyirin derivative, FeTPPS, used in previous studies to catalyze decomposition of peroxynitrite (Belik et al., 2010; Valez et al., 2012) indicates that this RNS may also play role in the toxicity of Sups. Due to the transient nature of peroxynitrite, it is difficult to observe it directly within the cells. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Bacillus anthracis is a dangerous pathogen of humans and many animal species. Its virulence has been mainly attributed to the production of Lethal and Edema toxins as well as the antiphagocytic capsule. Recent data indicate that the nitric oxide (NO) synthase (baNOS) plays an important pathogenic role at the early stage of disease by protecting bacteria from the host reactive species and S-nytrosylating the mitochondrial proteins in macrophages. In this study we for the first time present evidence that bacteria-derived NO participates in the generation of highly reactive oxidizing species which could be abolished by the NOS inhibitor L - NAME, free thiols, and superoxide dismutase but not catalase. The formation of toxicants is likely a result of the simultaneous formation of NO and superoxide leading to a labile peroxynitrite and its stable decomposition product, nitrogen dioxide. The toxicity of bacteria could be potentiated in the presence of bovine serum albumin. This effect is consistent with the property of serum albumin to serves as a trap of a volatile NO accelerating its reactions. Our data suggest that during infection in the hypoxic environment of pre-mortal host the accumulated NO is expected to have a broad toxic impact on host cell functions.
    Frontiers in Cellular and Infection Microbiology 05/2013; 3:16. DOI:10.3389/fcimb.2013.00016 · 3.72 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The loss of dopaminergic neurons induced by the parkinsonian toxins paraquat, rotenone and 1-methyl-4-phenylpyridinium (MPP(+)) is associated with oxidative stress. However, controversial reports exist regarding the source/compartmentalization of reactive oxygen species (ROS) generation and its exact role in cell death. We aimed to determine in detail the role of superoxide anion (O2(•-)), oxidative stress and their subcellular compartmentalization in dopaminergic cell death induced by parkinsonian toxins. Oxidative stress and ROS formation was determined in the cytosol, intermembrane (IMS) and mitochondrial matrix compartments, using dihydroethidine derivatives, the redox sensor roGFP, as well as electron paramagnetic resonance spectroscopy. Paraquat induced an increase in ROS and oxidative stress in both the cytosol and mitochondrial matrix prior to cell death. MPP(+) and rotenone primarily induced an increase in ROS and oxidative stress in the mitochondrial matrix. No oxidative stress was detected at the level of the IMS. In contrast to previous studies, overexpression of manganese superoxide dismutase (MnSOD) or copper/zinc SOD (CuZnSOD) had no effect on ROS steady state levels, lipid peroxidation, loss of mitochondrial membrane potential (ΔΨm) and dopaminergic cell death induced by MPP(+) or rotenone. In contrast, paraquat-induced oxidative stress and cell death were selectively reduced by MnSOD overexpression, but not by CuZnSOD or manganese-porphyrins. However, MnSOD also failed to prevent ΔΨm loss. Finally, paraquat, but not MPP(+) or rotenone, induced the transcriptional activation the redox-sensitive antioxidant response elements (ARE) and nuclear factor kappa-B (NF-κB). These results demonstrate a selective role of mitochondrial O2(•-) in dopaminergic cell death induced by paraquat, and show that toxicity induced by the complex I inhibitors rotenone and MPP(+) does not depend directly on mitochondrial O2(•-) formation.
    Free Radical Biology and Medicine 04/2013; 61. DOI:10.1016/j.freeradbiomed.2013.04.021 · 5.74 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Peroxynitrite is the product of the diffusion-controlled reaction of nitric oxide and superoxide radicals. Peroxynitrite, a reactive short-lived peroxide with a pKa = 6.8, is a good oxidant and nucleophile. It also yields secondary free radical intermediates such as nitrogen dioxide and carbonate radicals. Much of nitric oxide- and superoxide-dependent cytotoxicity resides on peroxynitrite, which affects mitochondrial function and triggers cell death via oxidation and nitration reactions. Peroxynitrite is an endogenous toxicant, but also a cytotoxic effector against invading pathogens. The biological chemistry of peroxynitrite is modulated by endogenous antioxidant mechanisms and neutralized by synthetic compounds with peroxynitrite scavenging capacity.
    Journal of Biological Chemistry 07/2013; 288(37). DOI:10.1074/jbc.R113.472936 · 4.57 Impact Factor
Show more