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. 2013 Jan 1;529(1):45-54.
doi: 10.1016/j.abb.2012.10.012. Epub 2012 Nov 8.

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

Valeria Valez  1 , Adriana Cassina, Ines Batinic-Haberle, Balaraman Kalyanaraman, Gerardo Ferrer-Sueta, Rafael Radi
Affiliations

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

Valeria Valez et al. Arch Biochem Biophys. .

Erratum in

  • Arch Biochem Biophys. 2014 Apr 1;547:44

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.

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Figures

Fig. 1
Fig. 1. Luminol chemiluminescence induced by SMP and the effect of nitric oxide
A) SMP (0.5 mg/mL) were incubated with luminol (400 μM) in isotonic PBS at 37°C. The arrows show the time when (succ, 6 mM), antimycin A (AA, 2 μM), •NO (6 μM), and superoxide dismutase (SOD, 2 μM) were added to the reaction mixture. This experiment was performed in the Thorn Emi photon counter four independent times with similar results; herein we show a representative curve. B) SMP (0.5 mg/mL) were incubated in PBS with 400 μM luminol and different compounds added as indicated: 6 mM succinate, 2 μM antimycin A, and various concentrations of NOC-7; 10 μM methionine (met) was added in the presence of 10 μM NOC-7. Bars represent the maximum chemiluminescence reached in each condition in the LUMIstar Galaxy BMG Labtechnologies.
Fig. 2
Fig. 2. Coelenterazine chemiluminescence (CCL) induced by nitric oxide-exposed SMP
A) SMP (0.5 mg/mL) were incubated in PBS with coelenterazine (1 μM) at 37°C. The arrows indicate the order of addition to the reaction mixture of substrate (succ, 6 mM), antimycin A (2μM), the bolus of •NO (6μM) and SOD (2μM). This assay was performed in the same Thorn Emi photon counter used with luminol, four times with similar results. B) We evaluated the pre-incubation of SMP with methionine (5 mM) before the addition of substrates.
Fig. 3
Fig. 3. Dihydrorhodamine-123 oxidation by nitric oxide-exposed SMP
SMP (0.75 mg/mL) were incubated in PBS with dihydrorhodamine-123 (DHR, 50 μM). Rhodamine-123 fluorescence was followed at 37°C, under continuous agitation for 20 min. A) Fluorescence upon addition of succinate (6 mM), antimycin A (2 μM) as a function of NOC-7 concentrations. Inset: time course of RH accumulation B) The effect of methionine (10 mM) in fluorescence yields at 10 μM NOC-7-exposed SMP. Control conditions in the absence of either O2•− or •NO were performed as indicated.
Fig. 4
Fig. 4. Coumarin -7-boronic acid oxidation by SMP
SMP (0.75 mg/mL) were incubated in PBS with coumarin -7-boronic acid (CBA, 50 μM). Fluorescence of the oxidized form, 7-hydroxycoumarin (COH), was followed at 37°C under continuous agitation for 20 min. A) Fluorescence upon addition of succinate (6 mM), antimycin A (2 μM) as a function of NOC-7 concentrations. Inset: time course of COH accumulation. B) Controls were performed at 500 μM NOC-7 and the effect of cysteine (20 and 40 mM) on CBA oxidation was tested as well.
Fig. 5
Fig. 5. Detection of protein radical formation in SMP by DMPO immunospintrapping
A) SMP (1 mg/mL) were incubated in isotonic buffer at 37°C for 20 min with DMPO (100 mM) plus different reagents. Succinate (3 mM), antimycin A (2 μM), SOD (20 μM), methionine (10 mM), tiron (5 mM) and NOC-7 (10, 20 and 40 μM) were added as indicated. B) Controls were performed and band densities were represented in bars. Increasing concentrations of NOC-7 (5, 25 and 50 μM) produce augmented immunodetection with anti-DMPO. The inhibitory effect of methionine (10 mM) was tested on protein radical formation.
Fig. 6
Fig. 6. MnTE-2-PyP and MnTnHex-2-PyP reduction by SMP as a function of oxygen concentration
Oxygen concentration and MnP reduction were followed simultaneously as indicated under Materials and Methods. A) SMP (0.1 mg/mL) were incubated in isotonic buffer with MnTE-2-PyP (5 μM) and NADH (1 mM) or B) succinate (2.5 mM) for complex I and II- dependent respiration, respectively. C) SMP (0.1 mg/mL) were incubated with MnTnHex-2-PyP (5 μM) and NADH (1 mM) or D) succinate (2.5 mM) for complex I and II, respectively. In the graphs, 10 μM oxygen is indicated with a line to facilitate data analysis. E) Redox potentials scheme of mitochondrial respiratory chain complexes, the MnP used in this work (MnTE-2-PyP and MnTnHex-2-PyP) and molecular oxygen.
Fig. 6
Fig. 6. MnTE-2-PyP and MnTnHex-2-PyP reduction by SMP as a function of oxygen concentration
Oxygen concentration and MnP reduction were followed simultaneously as indicated under Materials and Methods. A) SMP (0.1 mg/mL) were incubated in isotonic buffer with MnTE-2-PyP (5 μM) and NADH (1 mM) or B) succinate (2.5 mM) for complex I and II- dependent respiration, respectively. C) SMP (0.1 mg/mL) were incubated with MnTnHex-2-PyP (5 μM) and NADH (1 mM) or D) succinate (2.5 mM) for complex I and II, respectively. In the graphs, 10 μM oxygen is indicated with a line to facilitate data analysis. E) Redox potentials scheme of mitochondrial respiratory chain complexes, the MnP used in this work (MnTE-2-PyP and MnTnHex-2-PyP) and molecular oxygen.
Fig. 7
Fig. 7. Antioxidant effect of MnTE-2-PyP on peroxynitrite-dependent inactivation of mitochondrial complex I
A) Representative primary record of the oxygen concentration as a function of time, evaluating complex I (NADH dehydrogenase)-dependent respiration in SMP before and after a peroxynitrite flux for 3 minutes and the protective capacity of MnP. Once the system is equilibrated and calibrated (a) in PBS at 37 °C with continuous agitation, SMP (0.1 mg/mL) was injected. After that, respiratory substrates were added as indicated and the oxygen consumption starts. This slope (b) corresponds to the 100 % respiratory velocity of each experiment (100%). At near anoxic levels a peroxynitrite flux (50 μM/min) for 3 minutes was initiated (c) and after that the system was reoxygenated by simply opening the chamber to allow oxygen equilibration (d). When the oxygen level reached ca.100 μM, the chamber was closed and substrates were added again leading to a new, post-exposure, slope (e). The solid trace represents a record for SMP plus MnTE-2-PyP added at the beginning of the experiment and the dashed one without MnP just to evaluate the peroxynitrite-mediated inactivation. B) SMP (0.1 mg/mL) were incubated with or without MnTE-2-PyP (5 μM) and NADH (1 mM) is added in isotonic buffer. Bars represent the recovery of the respiratory rate (%) after infusion.
Fig. 8
Fig. 8. Mn-Porphyrins protect from peroxynitrite-dependent protein radical formation in SMP
SMP (2 mg/mL) were incubated in capped eppendorf tubes with DMPO (100 mM), succinate (6 mM) and MnTE-2-PyP (5 μM) (when indicated) in isotonic buffer at 37°C with continuous shaking for 5 minutes to allow MnP reduction. After that, NOC-7 (25 μM) were added as indicated for 20 minutes. Samples were ultra-centrifuged to concentrate them to 40 mg/mL and western blot analyses were performed using anti-DMPO antibody. Results were expressed as a function of fold increase relative to control samples.
Fig. 9
Fig. 9. Mn-Porphyrins protect from peroxynitrite-dependent protein tyrosine nitration in SMP
Protein tyrosine nitration was assessed in SMP (0.1 mg/mL) incubated with MnTE-2-PyP or MnTnHex-2-PyP (5 μM) and exposed to ONOO− fluxes (100 μM/min) during 3 minutes in isotonic buffer. Samples were collected and ultra-centrifuged at 100,000 g, resuspended to 40 mg/mL, and analyzed by Western blot using anti -nitro tyrosine antibody.
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