Reactions of desferrioxamine with peroxynitrite-derived carbonate and nitrogen dioxide radicals

The iron chelating agent desferrioxamine inhibits peroxynitrite-mediated oxidations and attenuates nitric oxide and oxygen radical-dependent oxidative damage both in vitro and in vivo. The mechanism of protection is independent of iron chelation and has remained elusive over the past decade. Herein, stopped-flow studies revealed that desferrioxamine does not react directly with peroxynitrite. However, addition of peroxynitrite to desferrioxamine in both the absence and the presence of physiological concentrations of CO2 and under excess nitrite led to the formation of a one-electron oxidation product, the desferrioxamine nitroxide radical, consistent with desferrioxamine reacting with the peroxynitrite-derived species carbonate (CO3*-) and nitrogen dioxide (*NO2) radicals. Desferrioxamine inhibited peroxynitrite-dependent free radical-mediated processes, including tyrosine dimerization and nitration, oxyhemoglobin oxidation in the presence of CO2, and peroxynitrite plus carbonate-dependent chemiluminescence. The direct two-electron oxidation of glutathione by peroxynitrite was unaffected by desferrioxamine. The reactions of desferrioxamine with CO3*- and *NO2 were unambiguously confirmed by pulse radiolysis studies, which yielded second-order rate constants of 1.7 x 10(9) and 7.6 x 10(6) M(-1) s(-1), respectively. Desferrioxamine also reacts with tyrosyl radicals with k = 6.3 x 10(6) M(-1) s(-1). However, radical/radical combination reactions between tyrosyl radicals or of tyrosyl radical with *NO2 outcompete the reaction with desferrioxamine and computer-assisted simulations indicate that the inhibition of tyrosine oxidation can be fully explained by scavenging of the peroxynitrite-derived radicals. The results shown herein provide an alternative mechanism to account for some of the biochemical and pharmacological actions of desferrioxamine via reactions with CO3*- and *NO2 radicals.     

The reaction of nitric oxide with ubiquinol: kinetic properties and biological significance

The reaction of nitric oxide (*NO) with ubiquinol-0 and ubiquinol-2, short-chain analogs of coenzyme Q, was examined in anaerobic and aerobic conditions in terms of formation of intermediates and stable molecular products. The chemical reactivity of ubiquinol-0 and ubiquinol-2 towards *NO differed only quantitatively, the reactions of ubiquinol-2 being slightly faster than those of ubiquinol-0. The ubiquinol/*NO reaction entailed oxidation of ubiquinol to ubiquinone and reduction of *NO to NO-, the latter identified by its reaction with metmyoglobin to form nitroxylmyoglobin and indirectly by measurement of nitrous oxide (N2O) by gas chromatography. Both the rate of ubiquinone accumulation and *NO consumption were linearly dependent on ubiquinol and *NO concentrations. The stoichiometry of *NO consumed per either ubiquinone formed or ubiquinol oxidized was 1.86 A 0.34. The reaction of *NO with ubiquinols proceeded with intermediate formation of ubisemiquinones that were detected by direct EPR. The second order rate constants of the reactions of ubiquinol-0 and ubiquinol-2 with *NO were 0.49 and 1.6 x 10(4) M(-1)s(-1), respectively. Studies in aerobic conditions revealed that the reaction of *NO with ubiquinols was associated with O2 consumption. The formation of oxyradicals - identified by spin trapping EPR- during ubiquinol autoxidation was inhibited by *NO, thus indicating that the O2 consumption triggered by *NO could not be directly accounted for in terms of oxyradical formation or H2O2 accumulation. It is suggested that oxyradical formation is inhibited by the rapid removal of superoxide anion by *NO to yield peroxynitrite, which subsequently may be involved in the propagation of ubiquinol oxidation. The biological significance of the reaction of ubiquinols with *NO is discussed in terms of the cellular O2 gradients, the steady-state levels of ubiquinols and *NO, and the distribution of ubiquinone (largely in its reduced form) in biological membranes with emphasis on the inner mitochondrial membrane.     

Kinetic and mechanistic studies of the NO*-mediated oxidation of oxymyoglobin and oxyhemoglobin

The second-order rate constants for the reactions between nitrogen monoxide and oxymyoglobin or oxyhemoglobin, determined by stopped-flow spectroscopy, increase with increasing pH. At pH 7.0 the rates are (43.6 +/- 0.5) x 10(6) M(-1) x s(-1) for oxymyoglobin and (89 +/- 3) x 10(6) M(-1) x s(-1) for oxyhemoglobin (per heme), whereas at pH 9.5 they are (97 +/- 3) x 10(6) M(-1) x s(-1) and (144 +/- 3) x 10(6) M(-1) x s(-1), respectively. The rate constants for the reaction between oxyhemoglobin and NO* depend neither on the association grade of the protein (dimer/tetramer) nor on the concentration of the phosphate buffer (100-1 mM). The nitrogen monoxide-mediated oxidations of oxymyoglobin and oxyhemoglobin proceed via intermediate peroxynitrito complexes which were characterized by rapid scan UV/vis spectroscopy. The two complexes MbFe(III)OONO and HbFe(III)OONO display very similar spectra with absorption maxima around 500 and 635 nm. These species can be observed at alkaline pH but rapidly decay to the met-form of the proteins under neutral or acidic conditions. The rate of decay of MbFe(III)OONO increases with decreasing pH and is significantly larger than those of the analogous complexes of the two subunits of hemoglobin. No free peroxynitrite is formed during these reactions, and nitrate is formed quantitatively, at both pH 7.0 and 9.0. This result indicates that, as confirmed from protein analysis after reacting the proteins with NO* for 10 times, when peroxynitrite is coordinated to the heme of myoglobin or hemoglobin it rapidly isomerizes to nitrate without nitrating the globins in physiologically significant amounts.     

Reactions of PTIO and carboxy-PTIO with *NO, *NO2, and O2-*

Nitronyl nitroxides, such as derivatives of 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIOs), react with *NO to form the corresponding imino nitroxides (PTIs) and *NO2. PTIOs are considered as monitors of *NO, stoichiometric sources of *NO2, biochemical and physiological effectors, specific tools for the elimination of *NO, and potential therapeutic agents. However, a better understanding of the chemical properties of PTIOs, especially following their reaction with *NO, is necessary to resolve many of the reported discrepancies surrounding the effects of PTIOs and to better characterize their potential therapeutic activity. We have generated electrochemically the oxidized and reduced forms of PTIO and carboxy-PTIO (C-PTIO), characterized their absorption spectra, and determined the reduction potentials for the oxoammonium/nitroxide and nitroxide/hydroxylamine couples. The rate constants for the reaction of *NO2 with PTIO and C-PTIO to form the corresponding oxoammonium cations (PTIO+s) and nitrite were determined to be (1.5 - 2) x 10(7) m-1 s-1. We have also shown that the reactions of PTIO+s with *NO form PTIOs and NO2-. The rate constants for these reactions are approximately 30-fold higher than those for the reactions of PTIOs with *NO or O2-*. The present results show that (i) the reaction of PTIOs with *NO forms solely PTIs and NO2- where [NO2-]/[PTI] varies between 1 and 2 depending on the steady-state concentrations of *NO. Consequently, quantitation of *NO is valid only at sufficiently low fluxes of *NO; (ii) the reaction of PTIOs with *NO can be used as a valid source of *NO2 only when the latter is effectively scavenged by an appropriate reductant; and (iii) the formation of peroxynitrite cannot be efficiently inhibited by PTIOs even under relatively low fluxes of *NO and O2-* and millimolar levels of PTIOs.     

Chain scission of hyaluronan by peroxynitrite

The reaction of peroxynitrite with the biopolymer hyaluronan has been studied using stopped-flow techniques combined with detection of molecular weight changes using the combination of gel permeation chromatography and multiangle laser light scattering. From the effect of peroxynitrite on the yield of hyaluronan chain breaks, it was concluded that the chain breaks were caused by hydroxyl radicals which escape a cage containing the *OH NO*(2) radical pair. The yield of free hydroxyl radicals was determined as 5+/-1% (as a proportion of the total peroxynitrite concentration). At high peroxynitrite concentrations, it was observed that the yield of chain breaks leveled out, an effect largely attributable to the scavenging of hydroxyl radicals by nitrite ions present in the peroxynitrite preparation. These experiments also provided some support for a previous proposal that the adduct formed between ONOOH and ONOO(-) might itself produce hydroxyl radicals. The rate of this reaction would have to be of the order of 0.05 s(-1) to produce hydroxyl radical yields that would account quantitatively for chain break yields at high peroxynitrite concentrations. By carrying out experiments at higher hyaluronan concentrations, it was also concluded that an additional yield of chain breaks was produced by the bimolecular reaction of the polymer with ONOOH at a rate constant of about 10 dm(3)mol(-1)s(-1). At 5.3 x 10(-3)mol dm(-3) hyaluronan, this amounted to 3.5% chain breaks (per peroxynitrite concentration). These conclusions support the proposal that the yield of hydroxyl radicals arising from the isomerization of ONOOH to nitrate ions is relatively low.     

Reductive nitrosylation and peroxynitrite-mediated oxidation of heme-hemopexin

Hemopexin (HPX), which serves as a scavenger and transporter of toxic plasma heme, has been postulated to play a key role in the homeostasis of NO. In fact, HPX-heme(II) reversibly binds NO and facilitates NO scavenging by O(2). HPX-heme is formed by two four-bladed beta-propeller domains. The heme is bound between the two beta-propeller domains, residues His213 and His266 coordinate the heme iron atom. HPX-heme displays structural features of heme-proteins endowed with (pseudo-)enzymatic activities. In this study, the kinetics of rabbit HPX-heme(III) reductive nitrosylation and peroxynitrite-mediated oxidation of HPX-heme(II)-NO are reported. In the presence of excess NO, HPX-heme(III) is converted to HPX-heme(II)-NO by reductive nitrosylation. The second-order rate constant for HPX-heme(III) reductive nitrosylation is (1.3 +/- 0.1) x 10(1) m(-1).s(-1), at pH 7.0 and 10.0 degrees C. NO binding to HPX-heme(III) is rate limiting. In the absence and presence of CO2 (1.2 x 10(-3) m), excess peroxynitrite reacts with HPX-heme(II)-NO (2.6 x 10(-6) m) leading to HPX-heme(III) and NO, via the transient HPX-heme(III)-NO species. Values of the second-order rate constant for HPX-heme(III)-NO formation are (8.6 +/- 0.8) x 10(4) and (1.2 +/- 0.2) x 10(6) m(-1).s(-1) in the absence and presence of CO2, respectively, at pH 7.0 and 10.0 degrees C. The CO2-independent value of the first-order rate constant for HPX-heme(III)-NO denitrosylation is (4.3 +/- 0.4) x 10(-1) s(-1), at pH 7.0 and 10.0 degrees C. HPX-heme(III)-NO denitrosylation is rate limiting. HPX-heme(II)-NO appears to act as an efficient scavenger of peroxynitrite and of strong oxidants and nitrating species following the reaction of peroxynitrite with CO2 (e.g. ONOOC(O)O-, CO3-, and NO2).     

Mechanisms of peroxynitrite decomposition catalyzed by FeTMPS, a bioactive sulfonated iron porphyrin

Peroxynitrite is a known cytotoxic agent that plays a role in many pathological conditions. Various peroxynitrite decomposition catalysts and pathways are being explored to develop efficient therapeutic agents that can safely remove peroxynitrite from cells and tissues. Water-soluble porphyrins, such as iron(III) meso-tetra(2,4,6-trimethyl-3,5-disulfonato)porphine chloride (FeTMPS) and iron(III) meso-tetra(N-methyl4-pyridyl)porphine chloride (FeTMPyP), have been shown to react catalytically with peroxynitrite (ONOO-). However, their mechanisms are yet to be fully understood. In this study, we have explored the reactivity of FeTMPS in the catalytic decomposition of peroxynitrite. The mechanism of this complex process has been determined. According to this mechanism, Fe(III)TMPS is oxidized by peroxynitrite to produce oxoFe(lV)TMPS and NO2 (k1 = 1.3 x 10(5) M(-1)(s(-1). The porphyrin is then reduced back to Fe(III)TMPS by nitrite, but this rate (k2 = 1.4 x 10(4) M(-1)s(-1)) is not sufficient to maintain the catalytic process at the observed rate. The overall rate of peroxynitrite decomposition catalysis, kcat, was determined to be 6 x 10(4) M(-1)s(-1), under typical conditions. We have postulated that an additional reduction pathway must exist. Kinetic simulations showed that a reaction of oxoFe(IV)TMPS with NO2 (k3 = 1.7 x 10(7) M((-1)s(-1)) could explain the behavior of this system and account for the fast reduction of oxoFe(IV)TMPS to Fe(III). Using the kinetic simulation analysis, we have also shown that two other rearrangement reactions, involving FeTMPS and peroxynitrite, are plausible pathways for peroxynitrite decay. A "cage-return" reaction between the generated oxoFe(IV)TMPS and NO2 (k8 = 5.4 x 10(4) M(-1)s(-1)), affording Fe(III)TMPS and nitrate, and a reaction between oxoFe(IV)TMPS and peroxynitrite (k7 = 2.4 x 10(4) M(-1)s(-1)) that affords oxoFe(IV)TMPS and nitrate are presented. The mechanism of FeTMPS-catalyzed peroxynitrite decay differs markedly from that of FeTMPyP, providing some insight into the reactivity of metal centers with peroxynitrite and biologically important radicals such as NO2.     

Reaction of superoxide and nitric oxide with peroxynitrite. Implications for peroxynitrite-mediated oxidation reactions in vivo

Peroxynitrite (ONOO(-)/ONOOH), the product of the diffusion-limited reaction of nitric oxide (*NO) with superoxide (O(-*)(2)), has been implicated as an important mediator of tissue injury during conditions associated with enhanced *NO and O(-*)(2) production. Although several groups of investigators have demonstrated substantial oxidizing and cytotoxic activities of chemically synthesized peroxynitrite, others have proposed that the relative rates of *NO and production may be critical in determining the reactivity of peroxynitrite formed in situ (Miles, A. M., Bohle, D. S., Glassbrenner, P. A., Hansert, B., Wink, D. A., and Grisham, M. B. (1996) J. Biol. Chem. 271, 40-47). In the present study, we examined the mechanisms by which excess O(-*)(2) or *NO production inhibits peroxynitrite-mediated oxidation reactions. Peroxynitrite was generated in situ by the co-addition of a chemical source of *NO, spermineNONOate, and an enzymatic source of O(-*)(2), xanthine oxidase, with either hypoxanthine or lumazine as a substrate. We found that the oxidation of the model compound dihydrorhodamine by peroxynitrite occurred via the free radical intermediates OH and NO(2), formed during the spontaneous decomposition of peroxynitrite and not via direct reaction with peroxynitrite. The inhibitory effect of excess O(-*)(2) on the oxidation of dihydrorhodamine could not be ascribed to the accumulation of the peroxynitrite scavenger urate produced from the oxidation of hypoxanthine by xanthine oxidase. A biphasic oxidation profile was also observed upon oxidation of NADH by the simultaneous generation of *NO and O(-*)(2). Conversely, the oxidation of glutathione, which occurs via direct reaction with peroxynitrite, was not affected by excess production of *NO. We conclude that the oxidative processes initiated by the free radical intermediates formed from the decomposition of peroxynitrite are inhibited by excess production of *NO or O(-*)(2), whereas oxidative pathways involving a direct reaction with peroxynitrite are not altered. The physiological implications of these findings are discussed.     

Peroxynitrite-derived carbonate and nitrogen dioxide radicals readily react with lipoic and dihydrolipoic acid

Alpha-lipoic acid (LA) and dihydrolipoic acid (DHLA) may have a role as antioxidants against nitric oxide-derived oxidants. We previously reported that peroxynitrite reacts with LA and DHLA with second-order rate constants of 1400 and 500 M(-1) s(-1), respectively, but indicated that these direct reactions are not fast enough to protect against peroxynitrite-mediated damage in vivo. Moreover, the mechanism of the reaction of peroxynitrite with LA has been recently challenged (J. Biol. Chem.279:9693-9697; 2004). Pulse radiolysis studies indicate that LA and DHLA react with peroxynitrite-derived nitrogen dioxide (*NO2) (k2 = 1.3 x 10(6) and 2.9 x 10(7) M(-1) s(-1), respectively) and carbonate radicals (CO(3-)) (k2 = 1.6 x 10(9) and 1.7 x 10(8) M(-1) s(-1), respectively). Carbonate radical-mediated oxidation of LA led to the formation of the potent one-electron oxidant LA radical cation. LA inhibited peroxynitrite-mediated nitration of tyrosine and of a hydrophobic tyrosine analog, N-t-BOC L-tyrosine tert-butyl ester (BTBE), incorporated into liposomes but enhanced tyrosine dimerization. Moreover, while LA competitively inhibited the direct oxidation of glutathione by peroxynitrite, it was poorly effective against the radical-mediated thiol oxidation. The mechanisms of reaction defined herein allow to rationalize the biochemistry of peroxynitrite based on direct and free radical-mediated processes and contribute to the understanding of the antioxidant actions of LA and DHLA.     

Direct measurement of nitric oxide and oxygen partitioning into liposomes and low density lipoprotein

Nitric oxide (*NO) has been proposed to play a relevant role in modulating oxidative reactions in lipophilic media like biomembranes and lipoproteins. Two factors that will regulate *NO reactivity in the lipid milieu are its diffusion and solubility, but there is no data concerning the actual diffusion (D) and partition coefficients (KP) of *NO in biologically relevant hydrophobic phases. Herein, a "equilibrium-shift" method was designed to directly determine the *NO and O2 partition coefficients in liposomes and low density lipoprotein (LDL) relative to water. It was found that *NO partitions 4.4- and 3.4-fold in liposomes and LDL, respectively, whereas O2 behaves similarly with values of 3.9 and 2.9, respectively. In addition, actual diffusion coefficients in these hydrophobic phases were determined using fluorescence quenching and found that *NO diffuses approximately 2 times slower than O2 in the core of LDL and 12 times slower than in buffer (DNOLDL=3.9 x 10(-6) cm2 s(-1),DO2LDL=7.0 x 10(-6) cm2 s(-1),DNObuffer=DO2buffer=4.5 x 10(-5) cm2 s(-1)). The influence of *NO and O2 partitioning and diffusion in membranes and lipoproteins on *NO reaction with lipid radicals and auto-oxidation is discussed. Particularly, the 3-4-fold increase in O2 and *NO concentration within biological hydrophobic phases provides quantitative support for the idea of an accelerated auto-oxidation of *NO in lipid-containing structures, turning them into sites of enhanced local production of oxidant and nitrosating species.     

Insulin-induced peroxynitrite production in human platelet-rich plasma

Recent data support the possible role of nitric oxide (NO*) in the development of insulin signalling. The aim of this study was to examine the effect of insulin on NO* production by platelets. The chemiluminescence of platelet-rich plasma prepared from the blood of healthy volunteers was measured in the presence of luminol. Indirect detection of NO* by luminol is possible in the form of peroxynitrite produced in the reaction of NO* with a superoxide free radical. Luminol oxidation induced by hydroxyl free radical and lipid peroxidation was prevented by 150 micromol/l of desferrioxamine mesylate. Insulin, in the range of 0.084-840 nmol/l, induced a concentration-dependent increase in chemiluminescence, which was inhibited both by the competitive antagonist of the NO* synthase enzyme. N(omega)-nitro-L-arginine methyl ester (at concentrations of 2.0-4.0 mmol/l, P<0.001), and by the elimination of superoxide free radicals using superoxide dismutase (72-144 IU/ml, P<0.001). In conclusion, we assume that the insulin-induced increase in chemiluminescence of platelet-rich plasma was due to increased production of NO* and superoxide free radicals forming peroxynitrite. The data are consistent with production of peroxynitrite from human platelets under insulin stimulation.     

Reactivity of 2',7'-dichlorodihydrofluorescein and dihydrorhodamine 123 and their oxidized forms toward carbonate, nitrogen dioxide, and hydroxyl radicals

The aim of this study was to investigate the oxidation of two common fluorescent probes, dichlorodihydrofluorescein (DCFH2) and dihydrorhodamine (DHR), and their oxidized forms, dichlorofluorescein and rhodamine, by the radical products of peroxynitrite chemistry, *OH, NO2*, and CO3*-. At pH 8.0-8.2, rate constants for the interaction of carbonate radical with probes were estimated to be 2.6 x 10(8) x M(-1) s(-1) for DCFH2 and 6.7 x 10(8) M(-1) s(-1) for DHR. Nitrogen dioxide interacted more slowly than carbonate radical with these probes: the rate constant for the interaction between NO2* and DCFH2 was estimated as 1.3 x 10(7) M(-1) s(-1). Oxidation of DHR by nitrogen dioxide led to the production of rhodamine, but the kinetics of these reactions were complex. Hydroxyl radical interacted with both probes with rate constants close to the diffusion-controlled limit. We also found that oxidized forms of these fluorescent probes reacted rapidly with carbonate, nitrogen dioxide, and hydroxyl radicals. These data suggest that probe oxidation may often be in competition with reaction of the radicals with cellular antioxidants.     

Melatonin nitrosation promoted by NO*2; comparison with the peroxynitrite reaction

N-nitroso species have recently been detected in animal tissues. Protein N-nitrosotryptophan is the best candidate for this N-nitroso pool. N-nitrosation of N-blocked trytophan derivatives like melatonin (MelH) by N2O3 or peroxynitrite (ONOOH/ONOO- ) has been observed under conditions of pH and reagent concentrations similar to in vivo conditions. We studied the reaction of NO*2 with MelH. When NO*2 was synthesized by gamma-irradiation of aqueous neutral solutions of nitrate under anaerobic conditions, detected oxidation and nitration of MelH were negligible. In the presence of additional nitrite, when NO* was also generated, formation of 1-nitrosomelatonin increased with nitrite concentration. Nitrosation is not due to N2O3 but could proceed via successive additions of NO*2 and NO*. For comparison, peroxynitrite was infused into a solution of MelH under air leading to the same products as those detected in irradiated solutions but in different proportions. In the presence of additional nitrite, the formation of nitroderivatives increased significantly while N-formylkynuramine and 1-nitrosomelatonin were maintained at similar levels. Mechanistic implications are discussed.     

Inactivation and nitration of human superoxide dismutase (SOD) by fluxes of nitric oxide and superoxide

Human recombinant MnSOD and CuZnSOD were both inactivated when exposed to simultaneous fluxes of superoxide (JO(2)(*-)) and nitric oxide (J*NO). The inactivation was also observed with varying J*NO/JO(2)(*-) ratios. Protein-derived radicals were detected in both CuZn and MnSOD by immuno-spin trapping. The formation of protein radicals was followed by tyrosine nitration in the case of MnSOD. When MnSOD was exposed to J*NO and JO(2)(*-) in the presence of uric acid, a scavenger of peroxynitrite-derived free radicals, nitration was decreased but inactivation was not prevented. On the other hand, glutathione, known to react with both peroxynitrite and nitrogen dioxide, totally protected MnSOD from inactivation and nitration on addition of authentic peroxynitrite but, notably, it was only partially inhibitory in the presence of the more biologically relevant J*NO and JO(2)(*-). The data are consistent with the direct reaction of peroxynitrite with the Mn center and a metal-catalyzed nitration of Tyr-34 in MnSOD. In this context, we propose that inactivation is also occurring through a *NO-dependent nitration mechanism. Our results help to rationalize MnSOD tyrosine nitration observed in inflammatory conditions in vivo in the presence of low molecular weight scavengers such as glutathione that otherwise would completely consume nitrogen dioxide and prevent nitration reactions.     

Mitochondrial aconitase reaction with nitric oxide, S-nitrosoglutathione, and peroxynitrite: mechanisms and relative contributions to aconitase inactivation

Using highly purified recombinant mitochondrial aconitase, we determined the kinetics and mechanisms of inactivation mediated by nitric oxide (*NO), nitrosoglutathione (GSNO), and peroxynitrite (ONOO(-)). High *NO concentrations are required to inhibit resting aconitase. Brief *NO exposures led to a reversible inhibition competitive with isocitrate (K(I)=35 microM). Subsequently, an irreversible inactivation (0.65 M(-1) s(-1)) was observed. Irreversible inactivation was mediated by GSNO also, both in the absence and in the presence of substrates (0.23 M(-1) s(-1)). Peroxynitrite reacted with the [4Fe-4S] cluster, yielding the inactive [3Fe-4S] enzyme (1.1 x 10(5) M(-1) s(-1)). Carbon dioxide enhanced ONOO(-)-dependent inactivation via reaction of CO(3)*(-) with the [4Fe-4S] cluster (3 x 10(8) M(-1) s(-1)). Peroxynitrite also induced m-aconitase tyrosine nitration but this reaction did not contribute to enzyme inactivation. Computational modeling of aconitase inactivation by O(2)*(-) and *NO revealed that, when NO is produced and readily consumed, measuring the amount of active aconitase remains a sensitive method to detect variations in O(2)*(-) production in cells but, when cells are exposed to high concentrations of NO, aconitase inactivation does not exclusively reflect changes in rates of O(2)*(-) production. In the latter case, extents of aconitase inactivation reflect the formation of secondary reactive species, specifically ONOO(-) and CO(3)*(-), which also mediate m-aconitase tyrosine nitration, a footprint of reactive *NO-derived species.     

Peroxynitrite formation from macrophage-derived nitric oxide.

Peroxynitrite formation by rat alveolar macrophages activated with phorbol 12-myristate 13-acetate was assayed by the Cu,Zn superoxide dismutase-catalyzed nitration of 4-hydroxyphenylacetate. The inhibitor of nitric oxide synthesis N-methyl-L-arginine prevented the Cu,Zn superoxide dismutase-catalyzed nitration of 4-hydroxyphenylacetate by stimulated macrophages, while Cu-depleted Zn superoxide dismutase did not catalyze the formation of 3-nitro-4-hydroxyphenylacetate either in vitro or in the presence of activated macrophages. The rate of phenolic nitration by activated macrophages was 9 +/- 2 pmol x 10(6) cells-1 x min-1 (mean +/- STD). Only 8% of synthetic peroxynitrite was trapped by superoxide dismutase, which suggested that the rate of peroxynitrite formation may have been as high as 0.11 nmol x 10(6) cells-1 x min-1. This upper estimate was consistent with N-methyl-L-arginine increasing the amount of superoxide detected with cytochrome c by 0.12 nmol x 10(6) cells-1 x min-1. The rate of nitrite and nitrate accumulation was 0.10 +/- 0.001 nmol x 10(6) cells-1 x min-1, suggesting that the majority of nitric oxide produced by activated macrophages may have been converted to peroxynitrite. The formation of a relatively long lived, strong oxidant from the reaction of nitric oxide and superoxide in activated macrophages may contribute to inflammatory cell-mediated tissue injury.     

Mechanism of nitrite-stimulated catalysis by lactoperoxidase.

The reactions of lactoperoxidase (LPO) intermediates compound I, compound II and compound III, with nitrite (NO2(-)) were investigated. Reduction of compound I by NO2(-) was rapid (k2 = 2.3 x 10(7) M(-1) x s(-1); pH = 7.2) and compound II was not an intermediate, indicating that NO2* radicals are not produced when NO2(-) reacts with compound I. The second-order rate constant for the reaction of compound II with NO2(-) at pH = 7.2 was 3.5 x 10(5) M(-1) x s(-1). The reaction of compound III with NO2(-) exhibited saturation behaviour when the observed pseudo first-order rate constants were plotted against NO2(-) concentrations and could be quantitatively explained by the formation of a 1 : 1 ratio compound III/NO2(-) complex. The Km of compound III for NO2(-) was 1.7 x 10(-4) M and the first-order decay constant of the compound III/ NO2(-) complex was 12.5 +/- 0.6 s(-1). The second-order rate constant for the reaction of the complex with NO2(-) was 3.3 x 10(3) M(-1) x s(-1). Rate enhancement by NO2(-) does not require NO2* as a redox intermediate. NO2(-) accelerates the overall rate of catalysis by reducing compound II to the ferric state. With increasing levels of H2O2, there is an increased tendency for the catalytically dead-end intermediate compound III to form. Under these conditions, the 'rescue' reaction of NO2(-) with compound III to form compound II will maintain the peroxidatic cycle of the enzyme.     

Interactions of peroxynitrite,tetrahydrobiopterin, ascorbic acid,and thiols: implications for uncoupling endothelial nitric-oxide synthase

Tetrahydrobiopterin (BH4) serves as a critical co-factor for the endothelial nitric-oxide synthase (eNOS). A deficiency of BH4 results in eNOS uncoupling, which is associated with increased superoxide and decreased NO* production. BH4 has been suggested to be a target for oxidation by peroxynitrite (ONOO-), and ascorbate has been shown to preserve BH4 levels and enhance endothelial NO* production; however, the mechanisms underlying these processes remain poorly defined. To gain further insight into these interactions, the reaction of ONOO- with BH4 was studied using electron spin resonance and the spin probe 1-hydroxy-3-carboxy-2,2,5-tetramethyl-pyrrolidine. ONOO- reacted with BH4 6-10 times faster than with ascorbate or thiols. The immediate product of the reaction between ONOO- and BH4 was the trihydrobiopterin radical (BH3.), which was reduced back to BH4 by ascorbate, whereas thiols were not efficient in recycling of BH4. Uncoupling of eNOS caused by peroxynitrite was investigated in cultured bovine aortic endothelial cells (BAECs) by measuring superoxide and NO* using spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine and the NO*-spin trap iron-diethyldithiocarbamate. Bolus ONOO-, the ONOO- donor 3-morpholinosydnonimine, and an inhibitor of BH4 synthesis (2,4-diamino-6-hydroxypyrimidine) uncoupled eNOS, increasing superoxide and decreasing NO* production. Exogenous BH4 supplementation restored endothelial NO* production. Treatment of BAECs with both BH4 and ascorbate prior to ONOO- prevented uncoupling of eNOS by ONOO-. This study demonstrates that endothelial BH4 is a crucial target for oxidation by ONOO- and that the BH4 reaction rate constant exceeds those of thiols or ascorbate. We confirmed that ONOO- uncouples eNOS by oxidation of tetrahydrobiopterin and that ascorbate does not fully protect BH4 from oxidation but recycles BH3. radical back to BH4.     

Reaction of peroxynitrite with carbon dioxide: intermediates and determination of the yield of CO3 •– and NO2 •

CO2 catalyses the isomerization of the biological toxin ONOO- to NO3- via an intermediate, presumably ONOOCO2-, which has an absorption maximum near 650 nm. The reflection spectrum of solid NMe4+ ONOO- exposed to CO2 shows a similar band near 650 nm; this absorption decays over minutes. Stopped-flow experiments in which CO2 solutions were mixed with alkaline ONOO- solutions indicate the formation of at least one intermediate. The initial absorption at 302 nm is less than that of ONOO-, which indicates that reactions take place within the mixing time, and this absorption is dependent (but not linearly) on the ONOO- and CO2 concentrations. We found that reaction of peroxynitrite with carbon dioxide forms some trioxocarbonate(*1-) (CO3*-) and nitrogen dioxide (NO2*) radicals via homolysis of the O-O bond in ONOOCO2-. We determined the extent of radical formation by mixing peroxynitrite, carbon dioxide and nitrogen monoxide. The later reacts with CO3*- and NO2* radicals to form, effectively, three NO2- per homolysis; ONOOCO2- that does not undergo homolysis yields NO3- and CO2. Based on the NO3- and NO2- analyses, the extent of conversion to NO3- is 96 +/- 1% and that of homolysis is 3 +/- 1%, respectively, significantly less than that reported in the literature.