Mechanism of superoxide dismutase/H(2)O(2)-mediated nitric oxide release from S-nitrosoglutathione--role of glutamate.

S-Nitrosoglutathione (GSNO), a physiologically relevant nitric oxide ((*)NO) donor, exhibits antioxidant, anti-ischemic, and antiplatelet properties. The exact mechanism of (*)NO release from GSNO in biological systems has not been determined. Both copper ions and copper-containing enzymes have been shown to catalyze (*)NO release from GSNO. In this study we observed that copper-zinc superoxide dismutase (Cu,ZnSOD) in the presence of H(2)O(2) caused a rapid decomposition of GSNO, forming oxidized glutathione (GSSG) and (*)NO. The cupric ions (Cu(2+)) released from Cu,ZnSOD were bound to the glutamate moiety of GSNO, yielding a 2:1 (GSNO)(2)Cu(2+) complex. Strong chelators of cupric ions, such as histidine and diethylenetriaminepentaacetic acid, inhibited the formation of (GSNO)(2)Cu(2+) complex, GSSG, and (*)NO. GSSG alone inhibited Cu(2+)-induced decomposition of GSNO. This effect is attributed to complexation of copper by GSSG. We conclude that binding of copper to GSNO is obligatory for (*)NO release from GSNO; however, the rate of this reaction was considerably slowed due to binding of Cu(2+) by GSSG. The glutamate moiety in GSNO and GSSG controls copper-catalyzed (*)NO release from GSNO. Cu,ZnSOD and H(2)O(2) enhanced peroxidation of unsaturated lipid that was inhibited by GSNO. The antioxidant function of GSNO is related to the sequestering of copper by GSNO and its ability to slowly release (*)NO. Implications of these findings are discussed in relation to GSNO-induced cardioprotection and to neuropathological processes.

Bicarbonate enhances the peroxidase activity of Cu,Zn-superoxide dismutase. Role of carbonate anion radical.

We examined the effect of bicarbonate on the peroxidase activity of copper-zinc superoxide dismutase (SOD1), using the nitrite anion as a peroxidase probe. Oxidation of nitrite by the enzyme-bound oxidant results in the formation of the nitrogen dioxide radical, which was measured by monitoring 5-nitro-gamma-tocopherol formation. Results indicate that the presence of bicarbonate is not required for the peroxidase activity of SOD1, as monitored by the SOD1/H(2)O(2)-mediated nitration of gamma-tocopherol in the presence of nitrite. However, bicarbonate enhanced SOD1/H(2)O(2)-dependent oxidation of tocopherols in the presence and absence of nitrite and dramatically enhanced SOD1/H(2)O(2)-mediated oxidation of unsaturated lipid in the presence of nitrite. These results, coupled with the finding that bicarbonate protects against inactivation of SOD1 by H(2)O(2), suggest that SOD1/H(2)O(2) oxidizes the bicarbonate anion to the carbonate radical anion. Thus, the amplification of peroxidase activity of SOD1/H(2)O(2) by bicarbonate is attributed to the intermediary role of the diffusible oxidant, the carbonate radical anion. We conclude that, contrary to a previous report (Sankarapandi, S., and Zweier, J. L. (1999) J. Biol. Chem. 274, 1226-1232), bicarbonate is not required for peroxidase activity mediated by SOD1 and H(2)O(2). However, bicarbonate enhanced the peroxidase activity of SOD1 via formation of a putative carbonate radical anion. Biological implications of the carbonate radical anion in free radical biology are discussed.

The effect of alpha-tocopherol on the nitration of gamma-tocopherol by peroxynitrite.

It has been proposed (S. Christen et al. Proc. Natl. Acad. Sci. USA 94, 3217-3222, 1997) that although alpha-tocopherol (alpha-TH) is an efficient antioxidant, the presence of gamma-tocopherol (gamma-TH) may be required to scavenge peroxynitrite-derived reactive nitrogen species. To investigate the reactions between alpha-TH, gamma-TH, and peroxynitrite, endogenous levels of both alpha-TH and gamma-TH were monitored when low-density lipoprotein was oxidized in the presence of the peroxynitrite generator 5-amino-3-(4-morpholinyl)-1, 2,3-oxadiazolium (SIN-1). SIN-1 oxidized alpha-TH while gamma-TH levels remained constant. The sparing of gamma-TH was also demonstrated when 1,2-dilauroyl-sn-glycero-3-phosphocholine liposomes containing alpha-TH and gamma-TH were incubated with either SIN-1 or peroxynitrite. Our data show that alpha-TH inhibits peroxynitrite-mediated gamma-TH nitration, i.e., 5-NO2-gamma-tocopherol formation. The rate constants for the reactions between both alpha-TH and gamma-TH with peroxynitrite suggest that the sparing of gamma-TH by alpha-TH does not occur by competitive scavenging, but may be due to the formation of a transient gamma-TH intermediate. Nitration of gamma-TH becomes significant only after alpha-TH levels have been depleted. We conclude alpha-TH alone is sufficient to remove any peroxynitrite-derived reactive nitrogen species, as the presence of alpha-TH attenuates nitration of both gamma-TH and tyrosine. The present results also indicate that a bolus addition of peroxynitrite or SIN-1 to liposomes containing gamma-TH forms 5-NO2-gamma-tocopherol in similar yields. This is in contrast to their reaction profile with tyrosine in aqueous solution. Under these conditions, SIN-1 does not form nitrotyrosine at detectable yields.

Antioxidant effects of nitric oxide and nitric oxide donor compounds on low-density lipoprotein oxidation.

Nitric oxide, when slowly released from a donor compound, has a potent inhibitory effect on the oxidative modification of LDL. This can be studied by monitoring changes in the lipid, protein, and antioxidant components of the LDL particle. In addition, the kinetics of LDL oxidation provides an insight into the mechanistic basis of the nitric oxide-dependent inhibition of LDL oxidation.

Reactions of *NO, *NO2 and peroxynitrite in membranes: physiological implications.

Nitric oxide (*NO) and nitrogen dioxide (*NO2) are hydrophobic gases. Therefore, lipid membranes and hydrophobic regions of proteins are potential sinks for these species. In these hydrophobic environments, reactive nitrogen species will exhibit different chemistry than in aqueous environments due to higher local concentrations and the lack of hydrolysis reactions. The peroxynitrite anion (ONOO-) and peroxynitrous acid (ONOOH) can freely pass through lipid membranes, making peroxynitrite-mediated reactions in a hydrophobic environment also of extreme relevance. The reactions observed by these reactive nitrogen species in a hydrophobic milieu include oxidation, nitration and even potent chain-breaking antioxidant reactions. The physiological and toxicological relevance of these reactions is discussed.

Nitration of gamma-tocopherol and oxidation of alpha-tocopherol by copper-zinc superoxide dismutase/H2O2/NO2-: role of nitrogen dioxide free radical.

Copper-zinc superoxide dismutase (Cu,ZnSOD) is the antioxidant enzyme that catalyzes the dismutation of superoxide (O2*-) to O2 and H2O2. In addition, Cu,ZnSOD also exhibits peroxidase activity in the presence of H2O2, leading to self-inactivation and formation of a potent enzyme-bound oxidant. We report in this study that lipid peroxidation of L-alpha-lecithin liposomes was enhanced greatly during the SOD/H2O2 reaction in the presence of nitrite anion (NO2-) with or without the metal ion chelator, diethylenetriaminepentacetic acid. The presence of NO2- also greatly enhanced alpha-tocopherol (alpha-TH) oxidation by SOD/H2O2 in saturated 1, 2-dilauroyl-sn-glycero-3-phosphatidylcholine liposomes. The major product identified by HPLC and UV-studies was alpha-tocopheryl quinone. When 1,2-diauroyl-sn-glycero-3-phosphatidylcholine liposomes containing gamma-tocopherol (gamma-TH) were incubated with SOD/H2O2/NO2-, the major product identified was 5-NO2-gamma-TH. Nitrone spin traps significantly inhibited the formation of alpha-tocopheryl quinone and 5-NO2-gamma-TH. NO2- inhibited H2O2-dependent inactivation of SOD. A proposed mechanism of this protection involves the oxidation of NO2- by an SOD-bound oxidant to the nitrogen dioxide radical (*NO2). In this study, we have shown a new mechanism of nitration catalyzed by the peroxidase activity of SOD. We conclude that NO2- is a suitable probe for investigating the peroxidase activity of familial Amyotrophic Lateral Sclerosis-linked SOD mutants.

The effect of nitric oxide release rates on the oxidation of human low density lipoprotein.

1-Substituted diazen-1-ium-1,2-diolates, a class of nitric oxide (. NO) donor compounds that spontaneously release .NO at different rates, were used to investigate the effect of .NO release rate upon the oxidation of low density lipoprotein (LDL). All donor compounds conferred an inhibitory effect upon the oxidation of LDL; however, the effect exhibited a biphasic dependence upon the rate of .NO release. The .NO release rate that maximally inhibited oxidation was dependent upon the rate of oxidation. When LDL was rapidly oxidized by copper(II) sulfate, a faster release rate was more effective. In contrast, when LDL was oxidized slowly by 2,2'-azobis-2-amidinopropane hydrochloride, a slower release rate was most effective. This biphasic relationship between .NO release rate and the duration of inhibition was also demonstrated when LDL oxidation was initiated with 5-amino-3-(4-morpholinyl)-1,2, 3-oxadiazolium, a peroxynitrite generator. We conclude that the antioxidant ability of .NO is dependent not only upon the rate of its release from .NO donors, but also upon the rate of oxidation. This conclusion is supported by a kinetic model of LDL oxidation in the presence of .NO.

The reaction between nitric oxide and alpha-tocopherol: a reappraisal.

Recently Gorbunov et al. reported that nitric oxide (.NO) can directly oxidize alpha-tocopherol to alpha-tocopheroxyl radical (Gorbunov et al., Biochem. Biophys. Res. Commun., 219, 835-841, 1996). We have reinvestigated this reaction and report that a direct reaction between .NO and alpha-tocopherol does not occur. However, the reaction between .NO and oxygen generates an oxidant which oxidizes alpha-tocopherol to alpha-tocopheryl quinone. Exposure of alpha-tocopherol to a low flux of .NO generated from spermine NONOate (100 microM) results in no consumption of alpha-tocopherol under either aerobic or anaerobic conditions. A higher flux of .NO, generated from 1 mM spermine NONOate, oxidizes alpha-tocopherol only under aerobic conditions. Artifactual oxidation of alpha-tocopherol can be observed when using commercial .NO that is contaminated with higher oxides of nitrogen, such as dinitrogen trioxide and dinitrogen tetraoxide.

Inhibition of macrophage-dependent low density lipoprotein oxidation by nitric-oxide donors.

We have previously shown that nitric oxide donors inhibit the oxidation of low density lipoprotein (LDL) initiated by copper ions or by azo-bis-amidinopropane (Hogg et al., 1993. FEBS Lett. 334: 170-174). In this study, the nitric oxide donors S-nitroso-N-acetylpenicillamine (SNAP), spermine NONOate, and sodium nitroprusside were tested for their ability to inhibit macrophage-dependent oxidation of LDL. SNAP and spermine NONOate inhibited macrophage-dependent oxidation of LDL in a time- and concentration-dependent manner. We propose that nitric oxide is acting as a chain-breaking antioxidant that can inhibit the progression of lipid peroxidation in cell dependent-oxidation of LDL. By this mechanism nitric oxide could be an endogenous defense against atherogenesis. In contrast, sodium nitroprusside enhanced cell-mediated oxidation of LDL by a mechanism dependent on superoxide production and transition metal ions. Sodium nitroprusside also enhanced LDL oxidation by cell culture medium alone by a similar mechanism. The use of sodium nitroprusside as a nitric oxide donor in cellular systems appears to be complicated by the release of iron leading to an enhanced oxidative stress. Thus the effects of sodium nitroprusside in such systems may be unrelated to nitric oxide release.

The antioxidant effect of spermine NONOate in human low-density lipoprotein.

The nitric oxide (*NO) donor N-[4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl]-1,3- propanediamine, also referred to as Spermine NONOate (SNN), inhibited the copper(II) sulfate-initiated oxidative modification of human low-density lipoprotein (LDL) as measured by the formation of thiobarbituric acid reactive substances, conjugated diene formation, and changes in electrophoretic mobility. The presence of the nitronyl nitroxide 1-oxy-2-[p-(trimethylammoniumyl)phenyl]-4,4,5,5- tetramethylimadazoline 3-oxide, a scavenger of *NO, antagonized the inhibitory activity of SNN. The inhibition of copper-dependent LDL oxidation had a nonlinear dependence on SNN concentration. Low concentrations of SNN ( < 4 microM) were only poorly effective at inhibiting LDL oxidation; however, a dramatic enhancement of inhibition occurred above 4 microM SNN. This behavior was qualitatively different from that of butylated hydroxytoluene, a phenolic chain-breaking antioxidant, which exhibited an approximately linear concentration dependence in this system. Addition of 13[S-(E,Z)]-hydroperoxy-9,11-octadecadienoic acid, a lipid hydroperoxide, to LDL diminished the antioxidant effect of 4 and 8 microM SNN, but not that of 12 microM SNN. SNN inhibited the depletion of alpha-tocopherol during both copper-dependent and 2,2'-azobis(2-amidinopropane)-dependent oxidation of LDL. We propose that a direct reaction is occurring between NO and the lipid peroxyl radical, forming a lipid-nitroso adduct. Formation of this product would not only remove the lipid peroxyl radical, thus preventing chain propagation, but would also prohibit the regeneration of lipid hydroperoxide, thereby stopping further transition metal ion-dependent initiation. The difference in the kinetic behavior between SNN and conventional antioxidants can be explained by this effect.