Nitroxides protect horseradish peroxidase from H2O2-induced inactivation and modulate its catalase-like activity

BackgroundHorseradish peroxidase (HRP) catalyzes H₂O₂ dismutation while undergoing heme inactivation. The mechanism underlying this process has not been fully elucidated. The effects of nitroxides, which protect metmyoglobin and methemoglobin against H₂O₂-induced inactivation, have been investigated.MethodsHRP reaction with H₂O₂ was studied by following H₂O₂ depletion, O₂ evolution and heme spectral changes. Nitroxide concentration was followed by EPR spectroscopy, and its reactions with the oxidized heme species were studied using stopped-flow.ResultsNitroxide protects HRP against H₂O₂-induced inactivation. The rate of H₂O₂ dismutation in the presence of nitroxide obeys zero-order kinetics and increases as [nitroxide] increases. Nitroxide acts catalytically since its oxidized form is readily reduced to the nitroxide mainly by H₂O₂. The nitroxide efficacy follows the order 2,2,6,6-tetramethyl-piperidine-N-oxyl (TPO) > 4-OH-TPO > 3-carbamoyl proxyl > 4-oxo-TPO, which correlates with the order of the rate constants of nitroxide reactions with compounds I, II, and III.ConclusionsNitroxide catalytically protects HRP against inactivation induced by H₂O₂ while modulating its catalase-like activity. The protective role of nitroxide at μM concentrations is attributed to its efficient oxidation by P940, which is the precursor of the inactivated form P670. Modeling the dismutation kinetics in the presence of nitroxide adequately fits the experimental data. In the absence of nitroxide the simulation fits the observed kinetics only if it does not include the formation of a Michaelis-Menten complex.General SignificanceNitroxides catalytically protect heme proteins against inactivation induced by H₂O₂ revealing an additional role played by nitroxide antioxidants in vivo.     

Catalase catalyzes nitrotyrosine formation from sodium azide and hydrogen peroxide

Sodium azide (NaN₃) is known as an inhibitor of catalase, and a nitric oxide (NO) donor in the presence of catalase and H₂O₂. We showed here that catalase-catalyzed oxidation of NaN₃ can generate reactive nitrogen species which contribute to tyrosine nitration in the presence of H₂O₂. The formation of free-tyrosine nitration and protein-bound tyrosine nitration by the NaN₃/catalase/H₂O₂ system showed a maximum level at pH 6.0. Free-tyrosine nitration induced by peroxynitrite was inhibited by ethanol and dimethyl-sulfoxide (DMSO), and augmented by superoxide dismutase (SOD). However, free-tyrosine nitration induced by the NaN₃/catalase/H₂O₂ system was not affected by ethanol, DMSO and SOD. NO^-₂ and NO donating agents did not affect free-tyrosine nitration by the NaN₃/catalase/H₂O₂ system. The reaction of NaN₃ with hydroxyl radical generating system showed free-tyrosine nitration, but no formation of nitrite and nitrate. The generation of nitrite (NO^-₂) and nitrate (NO^-₃) by the NaN₃/catalase/H₂O₂ system was maximal at pH 5.0. These results suggested that the oxidation of NaN₃ by the catalase/H₂O₂ system generates unknown peroxynitrite-like reactive nitrogen intermediates, which contribute to tyrosine nitration.     

The effect of neighboring methionine residue on tyrosine nitration and oxidation in peptides treated with MPO, H2O2, and NO2 or peroxynitrite and bicarbonate: Role of intramolecular electron transfer mechanism?

Recent reports suggest that intramolecular electron transfer reactions can profoundly affect the site and specificity of tyrosyl nitration and oxidation in peptides and proteins. Here we investigated the effects of methionine on tyrosyl nitration and oxidation induced by myeloperoxidase (MPO), H₂O₂ and NO₂⁻ and peroxynitrite (ONOO⁻) or ONOO⁻ and bicarbonate (HCO₃⁻) in model peptides, tyrosylmethionine (YM), tyrosylphenylalanine (YF) and tyrosine. Nitration and oxidation products of these peptides were analyzed by HPLC with UV/Vis and fluorescence detection, and mass spectrometry; radical intermediates were identified by electron paramagnetic resonance (EPR)-spin-trapping. We have previously shown (Zhang et al., J. Biol. Chem. 280 (2005) 40684-40698) that oxidation and nitration of tyrosyl residue was inhibited in tyrosylcysteine(YC)-type peptides as compared to free tyrosine. Here we show that methionine, another sulfur-containing amino acid, does not inhibit nitration and oxidation of a neighboring tyrosine residue in the presence of ONOO⁻ (or ONOOCO₂⁻) or MPO/H₂O₂/NO₂⁻ system. Nitration of tyrosyl residue in YM was actually stimulated under the conditions of in situ generation of ONOO⁻ (formed by reaction of superoxide with nitric oxide during SIN-1 decomposition), as compared to YF, YC and tyrosine. The dramatic variations in tyrosyl nitration profiles caused by methionine and cysteine residues have been attributed to differences in the direction of intramolecular electron transfer in these peptides. Further support for the interpretation was obtained by steady-state radiolysis and photolysis experiments. Potential implications of the intramolecular electron transfer mechanism in mediating selective nitration of protein tyrosyl groups are discussed.     

Consequences of MnSOD interactions with nitric oxide: Nitric oxide dismutation and the generation of peroxynitrite and hydrogen peroxide

The present study demonstrates that manganese superoxide dismutase (MnSOD) (Escherichia coli), binds nitric oxide (^√NO) and stimulates its decay under both anaerobic and aerobic conditions. The results indicate that previously observed MnSOD-catalyzed ^√NO disproportionation (dismutation) into nitrosonium (NO⁺) and nitroxyl (NO^? ) species under anaerobic conditions is also operative in the presence of molecular oxygen. Upon sustained aerobic exposure to ^√NO, MnSOD-derived NO^? species initiate the formation of peroxynitrite (ONOO^? ) leading to enzyme tyrosine nitration, oxidation and (partial) inactivation. The results suggest that both ONOO^? decomposition and ONOO^? -dependent tyrosine residue nitration and oxidation are enhanced by metal centre-mediated catalysis. We show that the generation of ONOO^? is accompanied by the formation of substantial amounts of H₂O₂. MnSOD is a critical mitochondrial antioxidant enzyme, which has been found to undergo tyrosine nitration and inactivation in various pathologies associated with the overproduction of ^√NO. The results of the present study can account for the molecular specificity of MnSOD nitration in vivo. The interaction of ^√NO with MnSOD may represent a novel mechanism by which MnSOD protects the cell from deleterious effects associated with overproduction of ^√NO.     

Triosephosphate isomerase tyrosine nitration induced by heme–NaNO2–H2O2 or peroxynitrite: Effects of different natural phenolic compounds

Peroxynitrite and heme peroxidases (or heme)–H₂O₂–NaNO₂ system are the two common ways to cause protein tyrosine nitration in vitro, but the effects of antioxidants on reducing these two pathways‐induced protein nitration and oxidation are controversial. Both nitrating systems can dose‐dependently induce triosephosphate isomerase (TIM) nitration, however, heme–H₂O₂–NaNO₂ was less destructive to protein secondary structures and led to more nitrated tyrosine residue than 3‐morpholinosydnonimine hydrochloride (SIN‐1, a peroxynitrite donor). Both of desferrioxamine and catechin could inhibit TIM nitration induced by heme–H₂O₂–NaNO₂ and SIN‐1 and protein oxidation induced by SIN‐1, but promoted heme–H₂O₂–NaNO₂‐induced protein oxidation. Moreover, the antagonism of natural phenolic compounds on SIN‐1‐induced tyrosine nitration was consistent with their radical scavenging ability, but no similar consensus was found in heme–H₂O₂–NaNO₂‐induced nitration. Our results indicated that peroxynitrite and heme–H₂O₂–NaNO₂‐induced protein nitration was different, and the later one could be a better model for anti‐nitration compounds screening.     

Metal-catalyzed protein tyrosine nitration in biological systems

Abstract

Protein tyrosine nitration is an oxidative postranslational modification that can affect protein structure and function. It is mediated in vivo by the production of nitric oxide-derived reactive nitrogen species (RNS), including peroxynitrite (ONOO^?) and nitrogen dioxide (^?NO₂). Redox-active transition metals such as iron (Fe), copper (Cu), and manganese (Mn) can actively participate in the processes of tyrosine nitration in biological systems, as they catalyze the production of both reactive oxygen species and RNS, enhance nitration yields and provide site-specificity to this process. Early after the discovery that protein tyrosine nitration can occur under biologically relevant conditions, it was shown that some low molecular weight transition-metal centers and metalloproteins could promote peroxynitrite-dependent nitration. Later studies showed that nitration could be achieved by peroxynitrite-independent routes as well, depending on the transition metal-catalyzed oxidation of nitrite (NO₂^?) to ^?NO₂ in the presence of hydrogen peroxide. Processes like these can be achieved either by hemeperoxidase-dependent reactions or by ferrous and cuprous ions through Fenton-type chemistry. Besides the in vitro evidence, there are now several in vivo studies that support the close relationship between transition metal levels and protein tyrosine nitration. So, the contribution of transition metals to the levels of tyrosine nitrated proteins observed under basal conditions and, specially, in disease states related with high levels of these metal ions, seems to be quite clear. Altogether, current evidence unambiguously supports a central role of transition metals in determining the extent and selectivity of protein tyrosine nitration mediated both by peroxynitrite-dependent and independent mechanisms.     

Nitric oxide diffusion to red blood cells limits extracellular,but not intraphagosomal,peroxynitrite formation by macrophages

Macrophage-derived nitric oxide (^•NO) participates in cytotoxic mechanisms against diverse microorganisms and tumor cells. These effects can be mediated by ^•NO itself or ^•NO-derived species such as peroxynitrite formed by its diffusion-controlled reaction with NADPH oxidase-derived superoxide radical anion (O₂^•−). In vivo, the facile extracellular diffusion of ^•NO as well as different competing consumption routes limit its bioavailability for the reaction with O₂^•− and, hence, peroxynitrite formation. In this work, we evaluated the extent by which ^•NO diffusion to red blood cells (RBC) can compete with activated macrophages-derived O₂^•− and affect peroxynitrite formation yields. Macrophage-dependent peroxynitrite production was determined by boron-based probes that react directly with peroxynitrite, namely, coumarin-7-boronic acid (CBA) and fluorescein-boronate (Fl-B). The influence of ^•NO diffusion to RBC on peroxynitrite formation was experimentally analyzed in co-incubations of ^•NO and O₂^•−-forming macrophages with erythrocytes. Additionally, we evaluated the permeation of ^•NO to RBC by measuring the intracellular oxidation of oxyhemoglobin to methemoglobin. Our results indicate that diluted RBC suspensions dose-dependently inhibit peroxynitrite formation, outcompeting the O₂^•− reaction. Computer-assisted kinetic studies evaluating peroxynitrite formation by its precursor radicals in the presence of RBC are in accordance with experimental results. Moreover, the presence of erythrocytes in the proximity of ^•NO and O₂^•--forming macrophages prevented intracellular Fl-B oxidation pre-loaded in L1210 cells co-cultured with activated macrophages. On the other hand, Fl-B-coated latex beads incorporated in the macrophage phagocytic vacuole indicated that intraphagosomal probe oxidation by peroxynitrite was not affected by nearby RBC. Our data support that in the proximity of a blood vessel, ^•NO consumption by RBC will limit the extracellular formation (and subsequent cytotoxic effects) of peroxynitrite by activated macrophages, while the intraphagosomal yield of peroxynitrite will remain unaffected.     

Nitroxide radicals as research tools: Elucidating the kinetics and mechanisms of catalase-like and “suicide inactivation” of metmyoglobin

BackgroundMetmyoglobin (MbFe^III) reaction with H₂O₂ has been a subject of study over many years. H₂O₂ alone promotes heme destruction frequently denoted “suicide inactivation,” yet the mechanism underlying H₂O₂ dismutation associated with MbFe^III inactivation remains obscure.MethodsMbFe^III reaction with excess H₂O₂ in the absence and presence of the nitroxide was studied at pH 5.3–8.1 and 25 °C by direct determination of reaction rate constants using rapid-mixing stopped-flow technique, by following H₂O₂ depletion, O₂ evolution, spectral changes of the heme protein, and the fate of the nitroxide by EPR spectroscopy.ResultsThe rates of both H₂O₂ dismutation and heme inactivation processes depend on [MbFe^III], [H₂O₂] and pH. Yet the inactivation stoichiometry is independent of these variables and each MbFe^III molecule catalyzes the dismutation of 50 ± 10 H₂O₂ molecules until it is inactivated. The nitroxide catalytically enhances the catalase-like activity of MbFe^III while protecting the heme against inactivation. The rate-determining step in the absence and presence of the nitroxide is the reduction of MbFe^IVO by H₂O₂ and by nitroxide, respectively.ConclusionsThe nitroxide effects on H₂O₂ dismutation catalyzed by MbFe^III demonstrate that MbFe^IVO reduction by H₂O₂ is the rate-determining step of this process. The proposed mechanism, which adequately fits the pro-catalytic and protective effects of the nitroxide, implies the intermediacy of a compound I–H₂O₂ adduct, which decomposes to a MbFe^IVO and an inactivated heme at a ratio of 25:1.General significanceThe effects of nitroxides are instrumental in elucidating the mechanism underlying the catalysis and inactivation routes of heme proteins.     

Efficiency of methemoglobin, hemin and ferric citrate in catalyzing protein tyrosine nitration, protein oxidation and lipid peroxidation in a bovine serum albumin-liposome system: Influence of pH

Protein tyrosine nitration, protein oxidation and lipid peroxidation are nitrative/oxidative modification of protein and lipids. In this paper, a BSA (bovine serum albumin)-lecithin liposome system was used to study the nature of different forms of iron, including methemoglobin, hemin and ferric citrate, in catalyzing H₂O₂-nitrite system to oxidize protein and lipid as well as nitrate protein. It was found that in pH range of 5.0-9.0, in pure BSA solution or pure liposome solution, hemin and methemoglobin catalyzed protein tyrosine nitration and lipid peroxidation were decreased with the increasing of pH, while hemin and methemoglobin catalyzed protein oxidation was significantly and moderately increased, respectively. Lipid completely inhibited hemin catalyzed protein tyrosine nitration but only partially inhibited methemoglobin catalyzed protein tyrosine nitration, and its inhibitory effect on hemin induced protein oxidation was also more pronounced. In addition, BSA showed more efficient in inhibiting hemin and ferric citrate induced lipid peroxidation. At the same condition, ferric citrate was relatively ineffective in all tests. Considering protein tyrosine nitration, protein oxidation and lipid oxidation as overall oxidative damage, these results indicated that methemoglobin is more toxic than hemin and ferric citrate, the degradation procedure of heme containing macromolecules, e.g. hemoglobin to hemin and finally to low molecular weight bounded iron, is step by step detoxification. These results provide fundamental knowledge on oxidative/nitrative of biomolecules in lipid-protein coexistence system.     

Quantification of superoxide radical production in thylakoid membrane using cyclic hydroxylamines

Applicability of two lipophilic cyclic hydroxylamines (CHAs), CM-H and TMT-H, and two hydrophilic CHAs, CAT1-H and DCP-H, for detection of superoxide anion radical (O₂^∙−) produced by the thylakoid photosynthetic electron transfer chain (PETC) of higher plants under illumination has been studied. ESR spectrometry was applied for detection of the nitroxide radical originating due to CHAs oxidation by O₂^∙−. CHAs and corresponding nitroxide radicals were shown to be involved in side reactions with PETC which could cause miscalculation of O₂^∙− production rate. Lipophilic CM-H was oxidized by PETC components, reducing the oxidized donor of Photosystem I, P₇₀₀⁺, while at the same concentration another lipophilic CHA, TMT-H, did not reduce P₇₀₀⁺. The nitroxide radical was able to accept electrons from components of the photosynthetic chain. Electrostatic interaction of stable cation CAT1-H with the membrane surface was suggested. Water-soluble superoxide dismutase (SOD) was added in order to suppress the reaction of CHA with O₂^∙− outside the membrane. SOD almost completely inhibited light-induced accumulation of DCP^∙, nitroxide radical derivative of hydrophilic DCP-H, in contrast to TMT^∙ accumulation. Based on the results showing that change in the thylakoid lumen pH and volume had minor effect on TMT^∙ accumulation, the reaction of TMT-H with O₂^∙− in the lumen was excluded. Addition of TMT-H to thylakoid suspension in the presence of SOD resulted in the increase in light-induced O₂ uptake rate, that argued in favor of TMT-H ability to detect O₂^∙− produced within the membrane core. Thus, hydrophilic DCP-H and lipophilic TMT-H were shown to be usable for detection of O₂^∙− produced outside and within thylakoid membranes.     

Inhibition of myeloperoxidase- and neutrophil-mediated oxidant production by tetraethyl and tetramethyl nitroxides

The powerful oxidant HOCl (hypochlorous acid and its corresponding anion, ⁻OCl) generated by the myeloperoxidase (MPO)–H₂O₂–Cl⁻ system of activated leukocytes is strongly associated with multiple human inflammatory diseases; consequently there is considerable interest in inhibition of this enzyme. Nitroxides are established antioxidants of low toxicity that can attenuate oxidation in animal models, with this ascribed to superoxide dismutase or radical-scavenging activities. We have shown (M.D. Rees et al., Biochem. J. 421, 79–86, 2009) that nitroxides, including 4-amino-TEMPO (4-amino-2,2,6,6-tetramethylpiperidin-1-yloxyl radical), are potent inhibitors of HOCl formation by isolated MPO and activated neutrophils, with IC₅₀ values of ~1 and ~6 µM respectively. The utility of tetramethyl-substituted nitroxides is, however, limited by their rapid reduction by biological reductants. The corresponding tetraethyl-substituted nitroxides have, however, been reported to be less susceptible to reduction. In this study we show that the tetraethyl species were reduced less rapidly than the tetramethyl species by both human plasma (89–99% decreased rate of reduction) and activated human neutrophils (62–75% decreased rate). The tetraethyl-substituted nitroxides retained their ability to inhibit HOCl production by MPO and activated neutrophils with IC₅₀ values in the low-micromolar range; in some cases inhibition was enhanced compared to tetramethyl substitution. Nitroxides with rigid structures (fused oxaspiro rings) were, however, inactive. Overall, these data indicate that tetraethyl-substituted nitroxides are potent inhibitors of oxidant formation by MPO, with longer plasma and cellular half-lives compared to the tetramethyl species, potentially allowing lower doses to be employed.     

Involvement of Molecular Oxygen in the Enzyme-Catalyzed NADH Oxidation and Ferric Leghemoglobin Reduction

Ferric leghemoglobin reductase (FLbR) from soybean (Glycine max [L.] Merr) nodules catalyzed oxidation of NADH, reduction of ferric leghemoglobin (Lb⁺³), and reduction of dichloroindophenol (diaphorase activity). None of these reactions was detectable when O₂ was removed from the reaction system, but all were restored upon readdition of O₂. In the absence of exogenous electron carriers and in the presence of O₂ and excess NADH, FLbR catalyzed NADH oxidation with the generation of H₂O₂ functioning as an NADH oxidase. The possible involvement of peroxide-like intermediates in the FLbR-catalyzed reactions was analyzed by measuring the effects of peroxidase and catalase on FLbR activities; both enzymes at low concentrations (about 2 μg/mL) stimulated the FLbR-catalyzed NADH oxidation and Lb⁺³ reduction. The formation of H₂O₂ during the FLbR-catalyzed NADH oxidation was confirmed using a sensitive assay based on the fluorescence emitted by dichlorofluorescin upon reaction with H₂O₂. The stoichiometry ratios between the FLbR-catalyzed NADH oxidation and Lb⁺³ reduction were not constant but changed with time and with concentrations of NADH and O₂ in the reaction solution, indicating that the reactions were not directly coupled and electrons from NADH oxidation were transferred to Lb⁺³ by reaction intermediates. A study of the affinity of FLbR for O₂ showed that the enzyme required at least micromolar levels of dissolved O₂ for optimal activities. A mechanism for the FLbR-catalyzed reactions is proposed by analogy with related oxidoreductase systems.     

Angiotensin II Inhibits Insulin-Stimulated GLUT4 Translocation and Akt Activation through Tyrosine Nitration-Dependent Mechanisms

Angiotensin II (Ang II) plays a major role in the pathogenesis of insulin resistance and diabetes by inhibiting insulin''s metabolic and potentiating its trophic effects. Whereas the precise mechanisms involved remain ill-defined, they appear to be associated with and dependent upon increased oxidative stress. We found Ang II to block insulin-dependent GLUT4 translocation in L6 myotubes in an NO- and O₂ ^.−-dependent fashion suggesting the involvement of peroxynitrite. This hypothesis was confirmed by the ability of Ang II to induce tyrosine nitration of the MAP kinases ERK1/2 and of protein kinase B/Akt (Akt). Tyrosine nitration of ERK1/2 was required for their phosphorylation on Thr and Tyr and their subsequent activation, whereas it completely inhibited Akt phosphorylation on Ser⁴⁷³ and Thr³⁰⁸ as well as its activity. The inhibitory effect of nitration on Akt activity was confirmed by the ability of SIN-1 to completely block GSK3α phosphorylation in vitro. Inhibition of nitric oxide synthase and NAD(P)Hoxidase and scavenging of free radicals with myricetin restored insulin-stimulated Akt phosphorylation and GLUT4 translocation in the presence of Ang II. Similar restoration was obtained by inhibiting the ERK activating kinase MEK, indicating that these kinases regulate Akt activation. We found a conserved nitration site of ERK1/2 to be located in their kinase domain on Tyr^156/139, close to their active site Asp^166/149, in agreement with a permissive function of nitration for their activation. Taken together, our data show that Ang II inhibits insulin-mediated GLUT4 translocation in this skeletal muscle model through at least two pathways: first through the transient activation of ERK1/2 which inhibit IRS-1/2 and second through a direct inhibitory nitration of Akt. These observations indicate that not only oxidative but also nitrative stress play a key role in the pathogenesis of insulin resistance. They underline the role of protein nitration as a major mechanism in the regulation of Ang II and insulin signaling pathways and more particularly as a key regulator of protein kinase activity.     

Potent methyl oxidation of 5-methyl-2′-deoxycytidine by halogenated quinoid carcinogens and hydrogen peroxide via a metal-independent mechanism

Halogenated quinones are a class of carcinogenic intermediates and are newly identified chlorination disinfection by-products in drinking water. We found recently that the highly reactive and biologically important hydroxyl radical (^•OH) can be produced by halogenated quinones and H₂O₂ independent of transition metal ions. However, it is not clear whether these quinoid carcinogens and H₂O₂ can oxidize the nucleoside 5-methyl-2′-deoxycytidine (5mdC) to its methyl oxidation products and, if so, what the underlying molecular mechanism is. Here we show that three methyl oxidation products, 5-(hydroperoxymethyl)-, 5-(hydroxymethyl)-, and 5-formyl-2′-deoxycytidine, could be produced when 5mdC was treated with tetrachloro-1,4-benzoquinone (TCBQ) and H₂O₂. The formation of the oxidation products was markedly inhibited by typical ^•OH scavengers and under anaerobic conditions. Analogous effects were observed with other halogenated quinones and the classic Fenton system. Based on these data, we propose that the oxidation of 5mdC by TCBQ/H₂O₂ might be through the following mechanism: ^•OH produced by TCBQ/H₂O₂ may first abstract hydrogen from the methyl group of 5mdC, leading to the formation of 5-(2′-deoxycytidylyl)methyl radical, which may combine with O₂ to form the peroxyl radical. The unstable peroxyl radical transforms into the corresponding hydroperoxide 5-(hydroperoxymethyl)-2′-deoxycytidine, which reacts with TCBQ and results in the formation of 5-(hydroxymethyl)-2′-deoxycytidine and 5-formyl-2′-deoxycytidine. This is the first report that halogenated quinoid carcinogens and H₂O₂ can induce potent methyl oxidation of 5mdC via a metal-independent mechanism, which may partly explain their potential carcinogenicity.     

Electrochemical Characteristics of Nitro-Heterocyclic Compounds of Biological Interest IV. Lifetime of the Metronidazole Radical Anion

Electrochemical studies on metronidazole using mixed aqueous/dimethylformamide (DMF) solvents have allowed us to generate the one-electron addition product, the nitro radical anion, RNO^?₂. Cyclic volt-ammetric techniques have been employed to study the tendency of RNO^?₂ to undergo further chemical reaction. The return-to-forward peak current ratio. ip/ip_f. was found to increase towards unity with increasing DMF content of the medium, indicating the extended lifetime of RNO^?₂. Second order kinetics for the decay of RNO^?₂ were established at all DMF concentrations examined. Extrapolation has allowed the rate constant and a first half-life of 8.4 × 10⁴dm²/mol-sec and 0.059 seconds respectively, to be determined for the decay of RNO^?₂ in a purely aqueous media. This is impossible by direct electrochemical measurement in water. due to a different reduction mechanism, giving the hydroxylamine derivative in a single 4-electron step. The application of the technique to other nitro-aromatic compounds is discussed.     

Nitration Transforms a Sensitive Peroxiredoxin 2 into a More Active and Robust Peroxidase

Peroxiredoxins (Prx) are efficient thiol-dependent peroxidases and key players in the mechanism of H₂O₂-induced redox signaling. Any structural change that could affect their redox state, oligomeric structure, and/or interaction with other proteins could have a significant impact on the cascade of signaling events. Several post-translational modifications have been reported to modulate Prx activity. One of these, overoxidation of the peroxidatic cysteine to the sulfinic derivative, inactivates the enzyme and has been proposed as a mechanism of H₂O₂ accumulation in redox signaling (the floodgate hypothesis). Nitration of Prx has been reported in vitro as well as in vivo; in particular, nitrated Prx2 was identified in brains of Alzheimer disease patients. In this work we characterize Prx2 tyrosine nitration, a post-translational modification on a noncatalytic residue that increases its peroxidase activity and its resistance to overoxidation. Mass spectrometry analysis revealed that treatment of disulfide-oxidized Prx2 with excess peroxynitrite renders mainly mononitrated and dinitrated species. Tyrosine 193 of the YF motif at the C terminus, associated with the susceptibility toward overoxidation of eukaryotic Prx, was identified as nitrated and is most likely responsible for the protection of the peroxidatic cysteine against oxidative inactivation. Kinetic analyses suggest that tyrosine nitration facilitates the intermolecular disulfide formation, transforming a sensitive Prx into a robust one. Thus, tyrosine nitration appears as another mechanism to modulate these enzymes in the complex network of redox signaling.     

Nitrative and oxidative modifications of enolase are associated with iron in iron-overload rats and in vitro

Iron overload is one of the most common iron-related toxicities, and liver is the major organ that is injured. Although oxidative stress is well accepted in the pathological mechanism of iron overload, nitrative modification in liver and the role of iron are relatively unknown. In this work, the nitrative and oxidative stress in liver was investigated in an iron-overload rat model. It was found that after 15 weeks of iron dextran administration, consistent with the increase of iron content in rat liver, both protein tyrosine nitration and protein oxidation were clearly elevated. By means of immunoprecipitation analysis, it was found that enolase nitration and oxidation status were significantly increased in iron-overload liver, whereas both α-enolase expression and activity were clearly decreased. The effects of different forms of iron on NaNO₂–H₂O₂- and peroxynitrite (ONOO⁻)-dependent enolase nitration and oxidation were further investigated in vitro to elucidate the possible role of iron in enolase dysfunction in vivo. Compared with EDTA–Fe(III), ferric citrate, and ferritin, heme (hemin and hemoglobin) showed higher efficiency in catalyzing protein nitration in both models. Besides the major contribution of free iron (Fe²⁺ and Fe³⁺) to catalyze protein oxidation, Fe²⁺ also directly acted as a competitive inhibitor and produced a significant decrease in enzyme activity. These results suggest that the existence of various forms of iron is an important contributing factor to the elevated nitrative/oxidative modifications and diminished activity of α-enolase in the development and progress of iron-overload-associated syndromes.     

On the mechanism of H+ translocation by mitochondrial H+-ATPase. Studies with chemical modifier of tyrosine residues

In this paper a detailed study of the effect of nitration of tyrosine residues by tetranitromethane on H⁺ conduction and other reactions catalyzed by the H⁺-ATPase complex in phosphorylating submitochondrial particles, uncoupled particles, and the purified complex is presented. Tetranitromethane treatment of submitochondrial particles results in marked inhibition of ATP hydrolysis, ATP-³³P_i exchange, and proton conduction by the H⁺-ATPase complex. These effects are caused by nitration of tyrosine residues of H⁺-ATPase complex as shown by the appearance of the absorption peak at 360 nm (specific for nitrotyrosine formation) and inhibition of ATP hydrolysis and ATP-³³P_i exchange in the complex purified from tetranitromethane-treated particles. H⁺ conduction in phospholipid vesicles inlaid with F₀ is also inhibited by tetranitromethane treatment. These observations indicate that tyrosine residue(s) of F₀ are critically involved in energy-linked proton translocation in the ATP-ase complex.     

Nitro-Tyrosine as Promoter of Free Radical Damage in a DNA Model System

Nitro-tyrosine considerably promotes the degradation of DNA, when incubated with Cu²⁺ and ascorbate in oxygenated aqueous solution. This deleterious process requires oxygen and can be inhibited with catalase, indicating that H₂O₂ is involved, via the reduction of oxygen. Menadione and 2,4,6-trinitro-benzenesulfonate, known to catalyze particularly fast such reduction of oxygen, were only slightly more active than nitro-tyrosine. Degradation of DNA can be explained by a site-specific Fenton type reaction of H₂O₂ with the DNA-Cu⁺ complex.

DNA-Cu⁺ + H₂O₂ → DNA' ' 'OH + Cu²⁺ + OH^?

Copper-chelating agents (EDTA and penicillamine) prevent DNA degradation, whereas OH-scavengers (t-butanol) are ineffective. The deleterious activity of nitro-tyrosine (and of other nitroaromatics) in the DNA model system may indicate important toxicological implications, since aromatic nitration is a significant mode of action of nitrogen dioxide.     

Peroxynitrite formation and function in plants

Peroxynitrite (ONOO⁻) is a reactive nitrogen species formed when nitric oxide (NO) reacts with the superoxide anion (O₂⁻). It was first identified as a mediator of cell death in animals but was later shown to act as a positive regulator of cell signaling, mainly through the posttranslational modification of proteins by tyrosine nitration. In plants, peroxynitrite is not involved in NO-mediated cell death and its physiological function is poorly understood. However, it is emerging as a potential signaling molecule during the induction of defense responses against pathogens and this could be mediated by the selective nitration of tyrosine residues in a small number of proteins. In this review we discuss the general role of tyrosine nitration in plants and evaluate recent evidence suggesting that peroxynitrite is an effector of NO-mediated signaling following pathogen infection.