Reduction Of Peroxynitrite Research Articles

The Scavenging Effect of Myoglobin from Meat Extracts toward Peroxynitrite Studied with a Flow Injection System Based on Electrochemical Reduction over a Screen-Printed Carbon Electrode Modified with Cobalt Phthalocyanine: Quantification and Kinetics.

The scavenging activity of myoglobin toward peroxynitrite (PON) was studied in meat extracts, using a new developed electrochemical method (based on cobalt phthalocyanine-modified screen-printed carbon electrode, SPCE/CoPc) and calculating kinetic parameters of PON decay (such as half-time and apparent rate constants). As reactive oxygen/nitrogen species (ROS/RNS) affect the food quality, the consumers can be negatively influenced. The discoloration, rancidity, and flavor of meat are altered in the presence of these species, such as PON. Our new highly thermically stable, cost-effective, rapid, and simple electrocatalytical method was combined with a flow injection analysis system to achieve high sensitivity (10.843 nA µM−1) at a nanomolar level LoD (400 nM), within a linear range of 3–180 µM. The proposed biosensor was fully characterized using SEM, FTIR, Raman spectroscopy, Cyclic Voltammetry (CV), Differential Pulse Voltammetry (DPV), and Linear Sweep Voltammetry (LSV). These achievements were obtained due to the CoPc-mediated reduction of PON at very low potentials (around 0.1 V vs. Ag/AgCl pseudoreference). We also proposed a redox mechanism involving two electrons in the reduction of peroxynitrite to nitrite and studied some important interfering species (nitrite, nitrate, hydrogen peroxide, dopamine, ascorbic acid), which showed that our method is highly selective. These features make our work relevant, as it could be further applied to study the kinetics of important oxidative processes in vivo or in vitro, as PON is usually present in the nanomolar or micromolar range in physiological conditions, and our method is sensitive enough to be applied.

Reduction of peroxynitrite by some manganoporphyrins of AEOL series: DFT approach with dispersion correction and NBO analysis

The AEOL series of manganoporphyrins (MnP; AEOL compounds were named by US Aeolus pharmaceuticals) designed as superoxide dismutase mimic are well-known for their powerful catalytic activity to neutralize reactive oxygen and nitrogen species. Reductive oxygen atom cleavage from peroxynitrite (ONOO−) to form NO2 in aqueous solution by some AEOL compounds (AEOL-10113, AEOL-10150, AEOL-11114 and AEOL-11203) was studied by DFT/M06-2X computations with D3 dispersion correction and gCP (geometrical counterpoise correction) for basis set superposition error. DFT computation showed that AEOL-10150 can form the most stable association complex {MnP…OONO} among four AEOL models. AEOL-10150 complex with ONOO− has the lowest deformation energy. In AEOL compounds and their association complexes with ONOO−, Mn atom prefered the high spin state (S = 2) to the intermediate spin state (S = 1). Natural bond orbital analysis showed that electron transfer from the most negative oxygen atom in ONOO− to Mn atom in MnP has the biggest interaction energy among all kinds of donor-acceptor interactions between ONOO− and MnP.

Rapid peroxynitrite reduction by human peroxiredoxin 3: Implications for the fate of oxidants in mitochondria

Mitochondria are main sites of peroxynitrite formation. While at low concentrations mitochondrial peroxynitrite has been associated with redox signaling actions, increased levels can disrupt mitochondrial homeostasis and lead to pathology. Peroxiredoxin 3 is exclusively located in mitochondria, where it has been previously shown to play a major role in hydrogen peroxide reduction. In turn, reduction of peroxynitrite by peroxiredoxin 3 has been inferred from its protective actions against tyrosine nitration and neurotoxicity in animal models, but was not experimentally addressed so far. Herein, we demonstrate the human peroxiredoxin 3 reduces peroxynitrite with a rate constant of 1 × 107 M−1 s−1 at pH 7.8 and 25 °C. Reaction with hydroperoxides caused biphasic changes in the intrinsic fluorescence of peroxiredoxin 3: the first phase corresponded to the peroxidatic cysteine oxidation to sulfenic acid. Peroxynitrite in excess led to peroxiredoxin 3 hyperoxidation and tyrosine nitration, oxidative post-translational modifications that had been previously identified in vivo. A significant fraction of the oxidant is expected to react with CO2 and generate secondary radicals, which participate in further oxidation and nitration reactions, particularly under metabolic conditions of active oxidative decarboxylations or increased hydroperoxide formation. Our results indicate that both peroxiredoxin 3 and 5 should be regarded as main targets for peroxynitrite in mitochondria.

111 - Application of resonance raman spectroscopy on manganese porphyrin detection and redox state in endothelial cells: implications on cytoprotection towards peroxynitrite

Manganese(III) ortho N-substituted pyridylporphyrins (MnP) act as efficient antioxidants catalyzing superoxide dismutation and accelerating peroxynitrite reduction. Importantly, MnP can reach mitochondria offering protection against reactive species in different animal models of disease. In this scenario, we applied Resonance Raman (RR) spectroscopy as a direct method to detect both the uptake and the redox state of MnP in living cells. Using RR we were able to analyze the intracellular redox state changes of two potent MnP (MnTnBuOE-2-PyP5+ and MnTnHex-2-PyP5+) within endothelial cells exposed to SIN-1. RR experiments with cells and isolated mitochondria revealed that the reduction of Mn(III)P was affected by inhibitors of the electron transport chain, supporting the action of MnP as efficient redox active compounds in mitochondria. Indeed, RR spectra confirmed that MnP added in the Mn(III) state can be incorporated into the cells, readily reduced by intracellular components to the Mn(II) state and oxidized by peroxynitrite. We also evaluated the cytoprotective capacity of MnP by exposing the endothelial cells to nitro-oxidative stress induced by peroxynitrite. We observed a preservation of normal mitochondrial function, attenuation of cell damage and prevention of apoptotic cell death. These data introduce a novel application of RR spectroscopy for the direct detection of MnP and their redox states inside living cells, and helps to rationalize their antioxidant capacity in biological systems.

Manganese porphyrin redox state in endothelial cells: Resonance Raman studies and implications for antioxidant protection towards peroxynitrite

Cationic manganese(III) ortho N-substituted pyridylporphyrins (MnP) act as efficient antioxidants catalyzing superoxide dismutation and accelerating peroxynitrite reduction. Importantly, MnP can reach mitochondria offering protection against reactive species in different animal models of disease. Although an LC-MS/MS-based method for MnP quantitation and subcellular distribution has been reported, a direct method capable of evaluating both the uptake and the redox state of MnP in living cells has not yet been developed. In the present work we applied resonance Raman (RR) spectroscopy to analyze the intracellular accumulation of two potent MnP-based lipophilic SOD mimics, MnTnBuOE-2-PyP5+ and MnTnHex-2-PyP5+ within endothelial cells. RR experiments with isolated mitochondria revealed that the reduction of Mn(III)P was affected by inhibitors of the electron transport chain, supporting the action of MnP as efficient redox active compounds in mitochondria. Indeed, RR spectra confirmed that MnP added in the Mn(III) state can be incorporated into the cells, readily reduced by intracellular components to the Mn(II) state and oxidized by peroxynitrite. To assess the combined impact of reactivity and bioavailability, we studied the kinetics of Mn(III)TnBuOE-2-PyP5+ with peroxynitrite and evaluated the cytoprotective capacity of MnP by exposing the endothelial cells to nitro-oxidative stress induced by peroxynitrite. We observed a preservation of normal mitochondrial function, attenuation of cell damage and prevention of apoptotic cell death. These data introduce a novel application of RR spectroscopy for the direct detection of MnP and their redox states inside living cells, and helps to rationalize their antioxidant capacity in biological systems.

The labile iron pool attenuates peroxynitrite-dependent damage and can no longer be considered solely a pro-oxidative cellular iron source

The ubiquitous cellular labile iron pool (LIP) is often associated with the production of the highly reactive hydroxyl radical, which forms through a redox reaction with hydrogen peroxide. Peroxynitrite is a biologically relevant peroxide produced by the recombination of nitric oxide and superoxide. It is a strong oxidant that may be involved in multiple pathological conditions, but whether and how it interacts with the LIP are unclear. Here, using fluorescence spectroscopy, we investigated the interaction between the LIP and peroxynitrite by monitoring peroxynitrite-dependent accumulation of nitrosated and oxidized fluorescent intracellular indicators. We found that, in murine macrophages, removal of the LIP with membrane-permeable iron chelators sustainably accelerates the peroxynitrite-dependent oxidation and nitrosation of these indicators. These observations could not be reproduced in cell-free assays, indicating that the chelator-enhancing effect on peroxynitrite-dependent modifications of the indicators depended on cell constituents, presumably including LIP, that react with these chelators. Moreover, neither free nor ferrous-complexed chelators stimulated intracellular or extracellular oxidative and nitrosative chemistries. On the basis of these results, LIP appears to be a relevant and competitive cellular target of peroxynitrite or its derived oxidants, and thereby it reduces oxidative processes, an observation that may change the conventional notion that the LIP is simply a cellular source of pro-oxidant iron.

Ohr plays a central role in bacterial responses against fatty acid hydroperoxides and peroxynitrite

Organic hydroperoxide resistance (Ohr) enzymes are unique Cys-based, lipoyl-dependent peroxidases. Here, we investigated the involvement of Ohr in bacterial responses toward distinct hydroperoxides. In silico results indicated that fatty acid (but not cholesterol) hydroperoxides docked well into the active site of Ohr from Xylella fastidiosa and were efficiently reduced by the recombinant enzyme as assessed by a lipoamide-lipoamide dehydrogenase-coupled assay. Indeed, the rate constants between Ohr and several fatty acid hydroperoxides were in the 107-108 M-1⋅s-1 range as determined by a competition assay developed here. Reduction of peroxynitrite by Ohr was also determined to be in the order of 107 M-1⋅s-1 at pH 7.4 through two independent competition assays. A similar trend was observed when studying the sensitivities of a ∆ohr mutant of Pseudomonas aeruginosa toward different hydroperoxides. Fatty acid hydroperoxides, which are readily solubilized by bacterial surfactants, killed the ∆ohr strain most efficiently. In contrast, both wild-type and mutant strains deficient for peroxiredoxins and glutathione peroxidases were equally sensitive to fatty acid hydroperoxides. Ohr also appeared to play a central role in the peroxynitrite response, because the ∆ohr mutant was more sensitive than wild type to 3-morpholinosydnonimine hydrochloride (SIN-1 , a peroxynitrite generator). In the case of H2O2 insult, cells treated with 3-amino-1,2,4-triazole (a catalase inhibitor) were the most sensitive. Furthermore, fatty acid hydroperoxide and SIN-1 both induced Ohr expression in the wild-type strain. In conclusion, Ohr plays a central role in modulating the levels of fatty acid hydroperoxides and peroxynitrite, both of which are involved in host-pathogen interactions.

37 - Resonance Raman Studies on Manganese Porphyrin Detection and Redox State in Endothelial Cells

Manganese(III) cationic ortho N-substituted pyridylporhyrins (MnPs) act as efficient antioxidants catalyzing superoxide dismutation and accelerating peroxynitrite reduction. Importantly, MnPs reach mitochondria and protect them in different animal models of disease, furthermore, MnTnBuOE-2-PyP 5+ is presently in Phase I/II clinical trial as a normal tissue radioprotectant. An LC-MS/MS -based method for MnPs quantitation and subcellular distribution has been reported; however, a direct method to evaluate the uptake and redox state of MnPs in vivo has not been developed. Here, we applied a spectroscopic technique, based on Resonance Raman (RR) confocal microscopy, in order to observe the intracellular accumulation of two potent MnP-based lipophilic SOD mimics, MnTnBuOE-2-PyP 5+ and MnTnHex-2-PyP 5+ within endothelial cells. RR spectra confirmed that MnPs were incorporated into endothelial cells where the reduced Mn(II)P is the predominant oxidation state, confirming that the Mn(III)P added can be reduced by intracellular components, in particular those of the mitochondrial electron transport chain. Thus, the incubation of Mn(III)P with isolated mitochondria or submitochondrial particles (SMP), in the presence of succinate as substrate, revealed the reduction to Mn(II)P, which was in part affected by the inhibitors of electron transport chain. Such data support the action of MnPs as efficient redox active compounds in mitochondria. Additional studies were performed in order to evaluate the cytoprotective capacity of MnPs by exposing the endothelial cells to nitro-oxidative stress induced by the peroxynitrite donor, SIN-1. We observed a preservation of normal mitochondrial function, attenuation of cell damage and prevention of apoptotic cell death. The data presented herein introduce a novel application of the RR spectroscopy for the direct detection of MnPs and their redox state in vivo , and helps to rationalize its antioxidant capacity in biological systems.

PrxQ B from Mycobacterium tuberculosis is a monomeric, thioredoxin-dependent and highly efficient fatty acid hydroperoxide reductase

Mycobacterium tuberculosis (M. tuberculosis) is the intracellular bacterium responsible for tuberculosis disease (TD). Inside the phagosomes of activated macrophages, M. tuberculosis is exposed to cytotoxic hydroperoxides such as hydrogen peroxide, fatty acid hydroperoxides and peroxynitrite. Thus, the characterization of the bacterial antioxidant systems could facilitate novel drug developments. In this work, we characterized the product of the gene Rv1608c from M. tuberculosis, which according to sequence homology had been annotated as a putative peroxiredoxin of the peroxiredoxin Q subfamily (PrxQ B from M. tuberculosis or MtPrxQ B). The protein has been reported to be essential for M. tuberculosis growth in cholesterol-rich medium. We demonstrated the M. tuberculosis thioredoxin B/C-dependent peroxidase activity of MtPrxQ B, which acted as a two-cysteine peroxiredoxin that could function, although less efficiently, using a one-cysteine mechanism. Through steady-state and competition kinetic analysis, we proved that the net forward rate constant of MtPrxQ B reaction was 3 orders of magnitude faster for fatty acid hydroperoxides than for hydrogen peroxide (3×106vs 6×103M−1s−1, respectively), while the rate constant of peroxynitrite reduction was (0.6−1.4) ×106M−1s−1 at pH 7.4. The enzyme lacked activity towards cholesterol hydroperoxides solubilized in sodium deoxycholate. Both thioredoxin B and C rapidly reduced the oxidized form of MtPrxQ B, with rates constants of 0.5×106 and 1×106M−1s−1, respectively. Our data indicated that MtPrxQ B is monomeric in solution both under reduced and oxidized states. In spite of the similar hydrodynamic behavior the reduced and oxidized forms of the protein showed important structural differences that were reflected in the protein circular dichroism spectra.

Peroxynitrite: From interception to signaling

Peroxynitrite is a strong oxidant and nitrating species that mediates certain biological effects of superoxide and nitrogen monoxide. These biological effects include oxidative damage to proteins as well as the formation of 3-nitrotyrosyl moieties in proteins. As a consequence, such proteins may lose their activity, gain altered function, or become prone to proteolytic degradation – resulting in modulation of cellular protein turnover and in the modulation of signaling cascades. In analogy to hydrogen peroxide, peroxynitrite may be scavenged by selenoproteins like glutathione peroxidase-1 (GPx-1) or by selenocompounds with a GPx-like activity, such as ebselen; in further analogy to H2O2, peroxiredoxins have also been established as contributors to peroxynitrite reduction. This review covers three aspects of peroxynitrite biochemistry, (i) the interaction of selenocompounds/-proteins with peroxynitrite, (ii) peroxynitrite-induced modulation of cellular proteolysis, and (iii) peroxynitrite-induced modulation of cellular signaling.

Degradation of Peroxynitrite by Simple, Recyclable Selenolanes

Abstract Two cyclic organoselenium compounds, DHS (trans-3,4-dihydroxy-1-selenolane) and MAS (3-amino-1-selenolane) were examined for their reaction with peroxynitrite. Both DHS and MAS scavenge peroxynitrite with rate constants of the order of 103 M−1 s−1. Additionally the two compounds efficiently react with carbonate (CO3•−) radical (formed by reaction of peroxynitrite with carbon dioxide) by one-electron oxidation and their selenoxides convert the nitrite formed by peroxynitrite reduction to nitrate. Even though the reactivity of DHS/MAS was fairly the same with peroxynitrite and its derived radicals, they differ in their ability to protect plasmid pBR322 DNA and CHO cells from peroxynitrite induced toxicity. To understand this, analysis of crucial products formed during peroxynitrite reaction was carried out. On reaction with peroxynitrite, both the compounds were converted to their corresponding selenoxide, identified and estimated by NMR and HPLC. Comparing the relative yields of the selenoxides, it has been confirmed that DHS produced significantly higher amount of selenoxide than MAS. The selenoxide can be regenerated back to the parent compound, either by thiols or by nitrite. Based on these results it is concluded that formation of stable and recyclable selenoxide makes selenolanes, a novel class of selenium compounds as antioxidants for peroxynitrite degradation.

Induction of inducible nitric oxide synthase (iNOS) expression by oxLDL inhibits macrophage derived foam cell migration

ObjectiveDeletion of inducible nitric oxide synthase (iNOS) in apolipoprotein E knockout mice was shown to mitigate the extent of arteriosclerosis. Oxidized low density lipoprotein (oxLDL) inhibits macrophage migration and traps foam cells, possibly through a mechanism involving oxidative stress. Here, we addressed whether a reduction of iNOS-mediated oxidative stress remobilizes macrophage-derived foam cells and may reverse plaque formation. MethodsMigration of RAW264.7 cells and bone marrow cells was quantified using a modified Boyden chamber. iNOS expression, phalloidin staining, focal adhesion kinase phosphorylation, lipid peroxides, nitric oxide (NO) and reactive oxygen species (ROS) production were assessed. ResultsoxLDL treatment significantly reduced cell migration compared to unstimulated cells (p < 0.05). This migratory arrest was reversed by co-incubation with a pharmacologic iNOS inhibitor 1400W (p < 0.05) and iNOS-siRNA (p > 0.05). Furthermore, apoE/iNOS double knockout macrophages do not show migratory arrest in response to oxLDL uptake, compared to apoE knockout controls (p > 0.05). We documented significantly increased iNOS expression following oxLDL treatment and downregulation using 1400W and small inhibitory RNA (siRNA). iNOS inhibition was associated with a reduction in NO and peroxynitrite (ONOO–)– and increased superoxide generation. Trolox treatment of RAW264.7 cells restored migration indicating that peroxynitrite mediated lipid peroxide formation is involved in the signaling pathway mediating cell arrest.. ConclusionsHere, we provide pharmacologic and genetic evidence that oxLDL induced iNOS expression inhibits macrophage-derived foam cell migration. Therefore, reduction of peroxynitrite and possibly lipid hydroperoxide levels in plaques represents a valuable therapeutic approach to reverse migratory arrest of macrophage-derived foam cells and to impair plaque formation.

Hemin‐Functionalized Reduced Graphene Oxide Nanosheets Reveal Peroxynitrite Reduction and Isomerization Activity

Facile and efficient reduction of graphene oxide (GO) and novel applications of the reduced graphene oxide (RGO) based materials are of current interest. Herein, we report a novel and facile method for the reduction of GO by using a biocompatible reducing agent dithiothreitol (DTT). Stabilization of DTT by the formation of a six-membered ring with internal disulfide linkage upon oxidation is responsible for the reduction of GO. The reduced graphene oxide is characterized by several spectroscopic and microscopic techniques. Dispersion of RGO in DMF remained stable for several weeks suggesting that the RGO obtained by DTT-mediated reduction is hydrophobic in nature. This method can be considered for large scale production of good quality RGO. Treatment of RGO with hemin afforded a functional hemin-reduced graphene oxide (H-RGO) hybrid material that exhibited remarkable protective effects against the potentially harmful peroxynitrite (PN). A detailed inhibition study on PN-mediated oxidation and nitration reactions indicate that the interaction between hemin and RGO results in a synergistic effect, which leads to an efficient reduction of PN to nitrate. The RGO also catalyzes the isomerization of PN to nitrate as the RGO layers facilitate the rapid recombination of (·)NO(2) with Fe(IV)=O species. In the presence of reducing agents such as ascorbic acid, the Fe(IV)=O species can be reduced to Fe(III), thus helping to maintain the PN reductase cycle.

A Comparison of Thiol Peroxidase Mechanisms

Thiol peroxidases comprise glutathione peroxidases (GPx) and peroxiredoxins (Prx). The enzymes of both families reduce hydroperoxides with thiols by enzyme-substitution mechanisms. H(2)O(2) and organic hydroperoxides are reduced by all thiol peroxidases, most efficiently by SecGPxs, whereas fast peroxynitrite reduction is more common in Prxs. Reduction of lipid hydroperoxides is the domain of monomeric GPx4-type enzymes and of some Prxs. The catalysis starts with oxidation of an active-site selenocysteine (U(P)) or cysteine (C(P)). Activation of Cys (Sec) for hydroperoxide reduction in the GPx family is achieved by a typical tetrad composed of Cys (Sec), Asn, Gln, and Trp, whereas a triad of Cys Thr (or Ser) and Arg is the signature of Prx. In many of the CysGPxs and Prxs, a second Cys (C(R)) is required. In these 2-CysGPxs and 2-CysPrxs, the C(P) oxidized to a sulfenic acid forms an intra- or intermolecular disulfide (typical 2-CysPrx) with C(R), before a stepwise regeneration of ground-state enzyme by redoxin-type proteins can proceed. In SecGPxs and sporadically in Prxs, GSH is used as the reductant. Diversity combined with structural variability predestines thiol peroxidases for redox regulation via ROOH sensing and direct or indirect transduction of oxidant signals to specific protein targets.

Pure MnTBAP selectively scavenges peroxynitrite over superoxide: Comparison of pure and commercial MnTBAP samples to MnTE-2-PyP in two models of oxidative stress injury, an SOD-specific Escherichia coli model and carrageenan-induced pleurisy

MnTBAP is often referred to as an SOD mimic in numerous models of oxidative stress. We have recently reported that pure MnTBAP does not dismute superoxide, but commercial or poorly purified samples are able to perform O 2 ·−dismutation with low-to-moderate efficacy via non-innocent Mn-containing impurities. Herein, we show that neither commercial nor pure MnTBAP could substitute for SOD enzyme in a SOD-deficient Escherichia coli model, whereas MnTE-2-PyP-treated SOD-deficient E. coli grew as well as a wild-type strain. This SOD-specific system indicates that MnTBAP does not act as an SOD mimic in vivo. In another model, carrageenan-induced pleurisy in mice, inflammation was evidenced by increased pleural fluid exudate and neutrophil infiltration and activation: these events were blocked by 0.3 mg/kg MnTE-2-PyP and, to a slightly lesser extent, by 10 mg/kg of either MnTBAP. Also, 3-nitrotyrosine formation, an indication of peroxynitrite existence in vivo, was blocked by both compounds; again MnTE-2-PyP was 33-fold more effective. Pleurisy model data indicate that MnTBAP exerts some protective actions in common with MnTE-2-PyP, which are not O 2 ·− related and can be fully rationalized if one considers that the common biological role shared by MnTBAP and MnTE-2-PyP is related to their reduction of peroxynitrite and carbonate radical, the latter arising from ONOOCO 2 adduct. The log k cat (O 2 ·−) value for MnTBAP is estimated to be about 3.16, which is ~ 5 and ~ 6 orders of magnitude smaller than the SOD activities of the potent SOD mimic MnTE-2-PyP and Cu,Zn-SOD, respectively. This very low value indicates that MnTBAP is too inefficient at dismuting superoxide to be of any biological impact, which was confirmed in the SOD-deficient E. coli model. The peroxynitrite scavenging ability of MnTBAP, however, is only ~ 2.5 orders of magnitude smaller than that of MnTE-2-PyP and is not significantly affected by the presence of the SOD-active impurities in the commercial MnTBAP sample (log k red (ONOO −) = 5.06 for pure and 4.97 for commercial sample). The reduction of carbonate radical is equally fast with MnTBAP and MnTE-2-PyP. The dose of MnTBAP required to yield oxidative stress protection and block nitrotyrosine formation in the pleurisy model is > 1.5 orders of magnitude higher than that of MnTE-2-PyP, which could be related to the lower ability of MnTBAP to scavenge peroxynitrite. The slightly better protection observed with the commercial MnTBAP sample (relative to the pure MnTBAP) could arise from its impurities, which, by scavenging O 2 ·−, reduce consequently the overall peroxynitrite and secondary ROS/RNS levels. These observations have profound biological repercussions as they may suggest that the effect of MnTBAP observed in numerous studies may conceivably relate to peroxynitrite scavenging. Moreover, provided that pure MnTBAP is unable to dismute superoxide at any significant extent, but is able to partially scavenge peroxynitrite and carbonate radical, this compound may prove valuable in distinguishing ONOO −/CO 3 ·− from O 2 ·− pathways.

Peroxynitrite Detoxification and Its Biologic Implications

Peroxynitrite is a cytotoxic oxidant formed in vivo from the diffusional-controlled reaction between nitric oxide and superoxide radicals. Increased peroxynitrite formation has been related to the pathogenesis of multiple diseases, thus underlining the importance of understanding the mechanisms of its detoxification. In nature, different enzymatic routes for peroxynitrite decomposition have evolved. Among them, peroxiredoxins catalytically reduce peroxynitrite in vitro; modulation of their expression affects peroxynitrite-mediated cytotoxicity, and their content changes in pathologic conditions associated with increased peroxynitrite formation in vivo, thus indicating a physiologic role of these enzymes in peroxynitrite reduction. Selenium-containing glutathione peroxidase also catalyzes peroxynitrite reduction, but its role in vivo is still a matter of debate. In selected cellular systems, heme proteins also play a role in peroxynitrite detoxification, such as its isomerization by oxyhemoglobin in red blood cells. Moreover, different pharmacologic approaches have been used to decrease the toxicity related to peroxynitrite formation. Manganese or iron porphyrins catalyze peroxynitrite decomposition, and their protective role in vivo has been confirmed in biologic systems. Glutathione peroxidase mimetics also rapidly reduce peroxynitrite, but their biologic role is less well established. Flavonoids, nitroxides, and tyrosine-containing peptides decreased peroxynitrite-mediated toxicity under different conditions, but their mechanism of action is indirect.

Formation of Hydrogen Peroxide and Reduction of Peroxynitrite via Dismutation of Superoxide at Reperfusion Enhances Myocardial Blood Flow and Oxygen Consumption in Postischemic Mouse Heart

Reactive oxygen/nitrogen species suppress myocardial oxygen consumption. In this study, we determined that endogenous hydrogen peroxide through dismutation of superoxide enhances postischemic myocardial blood perfusion and oxygen consumption. Electron paramagnetic resonance oximetry was applied to monitor in vivo tissue Po2 in mouse heart subjected to regional ischemia reperfusion. Heart rate, arterial blood pressure, blood flow, infarction, and activities of mitochondrial NADH dehydrogenase and cytochrome c oxidase were measured in six groups of wild-type (WT) and endothelial nitricoxide synthase knock-out (eNOS(-/-)) mice treated with phosphate-buffered saline (PBS), superoxide dismutase mimetic (SOD(m)) M40403 [a manganese(II)-bis(cyclohexylpyridine)-substituted macrocyclic superoxide dismutase mimetic, C21H35Cl2MnN5], 10006329 EUK 134 [EUK134, manganese 3-methoxy N,N(1)-bis(salicyclidene)ethylenediamine chloride], and SOD(m) plus glibenclamide to study the protective effect of hydrogen peroxide via dismutation of superoxide on the activation of sarcolemmal potassium channels. In the PBS group, there was an overshoot of tissue Po2 after reperfusion. Treatment with SOD(m), EUK134, and SOD(m) + glibenclamide protected mitochondrial enzyme activities, reduced infarct size, and suppressed the postischemic hyperoxygenation. In particular, in the SOD(m)-treated group, there was a transient peak of tissue Po2 at 9 min after reperfusion, which was dependent on endogenous hydrogen peroxide but not nitric oxide formation as it appeared in both WT and eNOS(-/-) mice. Blood flow and rate pressure product were higher in the SOD(m) group than in other groups, which contributed to the transient oxygen peak. Thus, SOD mimetics protected mouse heart from superoxide-induced reperfusion injury. With treatment of different SOD mimetics, it is concluded that endogenous hydrogen peroxide via dismutation of superoxide at reperfusion enhances postischemic myocardial blood perfusion and mitochondrial oxygen consumption, possibly through activation of sarcolemmal ATP-sensitive potassium channels.

Cardiac capsaicin‐sensitive sensory nerves regulate myocardial relaxation via S‐nitrosylation of SERCA: role of peroxynitrite

Sensory neuropathy develops in the presence of cardiovascular risk factors (e.g. diabetes, dyslipidemia), but its pathological consequences in the heart are unclear. We have previously shown that systemic sensory chemodenervation by capsaicin leads to impaired myocardial relaxation and diminished cardiac nitric oxide (NO) content. Here we examined the mechanism of diminished NO formation and if it may lead to a reduction of peroxynitrite (ONOO(-))-induced S-nitrosylation of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA2a). Male Wistar rats were treated with capsaicin for 3 days to induce sensory chemodenervation. Seven days later, myocardial function and biochemical parameters were measured. Capsaicin pretreatment significantly increased left ventricular end-diastolic pressure (LVEDP) decreased cardiac NO level, Ca(2+)-dependent NO synthase (NOS) activity, and NOS-3 mRNA. Myocardial superoxide content, xanthine oxidoreductase and NADPH oxidase activities did not change, although superoxide dismutase (SOD) activity increased. Myocardial and serum ONOO(-) concentration and S-nitrosylation of SERCA2a were significantly decreased. Our results show that sensory chemodenervation decreases cardiac NO via decreased expression and activity of Ca(2+)-dependent NOS and increases SOD activity, thereby leading to decreased basal ONOO(-) formation and reduction of S-nitrosylation of SERCA2a, which causes impaired myocardial relaxation characterized by increased left ventricular end-diastolic pressure (LVEDP). This suggests that capsaicin sensitive sensory neurons regulate myocardial relaxation via maintaining basal ONOO(-) formation and SERCA S-nitrosylation.

The Mycobacterial Thioredoxin Peroxidase Can Act as a One-cysteine Peroxiredoxin

Thioredoxin peroxidase (TPx) has been reported to dominate the defense against H(2)O(2), other hydroperoxides, and peroxynitrite at the expense of thioredoxin (Trx) B and C in Mycobacterium tuberculosis (Mt). By homology, the enzyme has been classified as an atypical 2-C-peroxiredoxin (Prx), with Cys(60) as the "peroxidatic" cysteine (C(P)) forming a complex catalytic center with Cys(93) as the "resolving" cysteine (C(R)). Site-directed mutagenesis confirms Cys(60) to be C(P) and Cys(80) to be catalytically irrelevant. Replacing Cys(93) with serine leads to fast inactivation as seen by conventional activity determination, which is associated with oxidation of Cys(60) to a sulfinic acid derivative. However, in comparative stopped-flow analysis, WT-MtTPx and MtTPx C93S reduce peroxynitrite and react with TrxB and -C similarly fast. Reduction of pre-oxidized WT-MtTPx and MtTPx C93S by MtTrxB is demonstrated by monitoring the redox-dependent tryptophan fluorescence of MtTrxB. Furthermore, MtTPx C93S remains stable for 10 min at a morpholinosydnonimine hydrochloride-generated low flux of peroxynitrite and excess MtTrxB in a dihydrorhodamine oxidation model. Liquid chromatography-tandem mass spectrometry analysis revealed disulfide bridges between Cys(60) and Cys(93) and between Cys(60) and Cys(80) in oxidized WT-MtTPx. Reaction of pre-oxidized WT-MtTPx and MtTPx C93S with MtTrxB C34S or MtTrxC C40S yielded dead-end intermediates in which the Trx mutants are preferentially linked via disulfide bonds to Cys(60) and never to Cys(93) of the TPx. It is concluded that neither Cys(80) nor Cys(93) is required for the catalytic cycle of the peroxidase. Instead, MtTPx can react as a 1-C-Prx with Cys(60) being the site of attack for both the oxidizing and the reducing substrate. The role of Cys(93) is likely to conserve the oxidation equivalents of the sulfenic acid state of C(P) as a disulfide bond to prevent overoxidation of Cys(60) under a restricted supply of reducing substrate.

Reduction of manganese porphyrins by flavoenzymes and submitochondrial particles: A catalytic cycle for the reduction of peroxynitrite

The reduction of manganese(III) meso-tetrakis(( N-ethyl)pyridinium-2-yl)porphyrin (MnTE-2-PyP) to manganese(II) was catalyzed by flavoenzymes such as xanthine oxidase and glucose oxidase, and by Complex I and Complex II of the mitochondrial electron transport chain. The reduced manganese porphyrin has been previously shown to react rapidly with superoxide and carbonate radical anion. Herein, we describe the reaction of a reduced manganese porphyrin with peroxynitrite that proceeds as a two-electron process, has a rate constant greater than 7 × 10 6 M −1 s −1 (at pH 7.25 and 37°C), and produces nitrite and the Mn(IV)Porphyrin. The Mn(II)/Mn(IV) redox cycle was used to divert peroxynitrite from the inactivation of succinate dehydrogenase. In a typical experiment, 5 μM MnTE-2-PyP in the presence of excess succinate was able to protect the succinate dehydrogenase and succinate oxidase activities of submitochondrial particles challenged with a cumulative dose of 140 μM peroxynitrite infused in the course of 2 h. Other MnPorphyrins that are reduced more slowly do not provide as much protection underscoring the rate limiting character of the reduction step. The data presented here serve to rationalize the pharmacological action of MnPorphyrins as peroxynitrite reduction catalysts in vivo and opens avenues for the development of MnPorphyrins to protect mitochondria from oxidative damage.