Glutathionyl- and hydroxyl radical formation coupled to the redox transitions of 1,4-naphthoquinone bioreductive alkylating agents during glutathione two-electron reductive addition.

The kinetic parameters of the redox transitions subsequent to the two-electron transfer implied in the glutathione (GSH) reductive addition to 2- and 6-hydroxymethyl-1,4-naphthoquinone bioalkylating agents were examined in terms of autoxidation, GSH consumption in the arylation reaction, oxidation of the thiol to glutathione disulfide (GSSG), and free radical formation detected by the spin-trapping electron spin resonance method. The position of the hydroxymethyl substituent in either the benzenoid or the quinonoid ring differentially influenced the initial rates of hydroquinone autoxidation as well as thiol oxidation. Thus, GSSG- and hydrogen peroxide formation during the GSH reductive addition to 6-hydroxymethyl-1,4-naphthoquinone proceeded at rates substantially higher than those observed with the 2-hydroxymethyl derivative. The distribution and concentration of molecular end products, however, was the same for both quinones, regardless of the position of the hydroxymethyl substituent. The [O2]consumed/[GSSG]formed ratio was above unity in both cases, thus indicating the occurrence of autoxidation reactions other than those involved during GSSG formation. EPR studies using the spin probe 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) suggested that the oxidation of GSH coupled to the above redox transitions involved the formation of radicals of differing structure, such as hydroxyl and thiyl radicals. These were identified as the corresponding DMPO adducts. The detection of either DMPO adduct depended on the concentration of GSH in the reaction mixture: the hydroxyl radical adduct of DMPO prevailed at low GSH concentrations, whereas the thiyl radical adduct of DMPO prevailed at high GSH concentrations. The production of the former adduct was sensitive to catalase, whereas that of the latter was sensitive to superoxide dismutase as well as to catalase. The relevance of free radical formation coupled to thiol oxidation is discussed in terms of the thermodynamic and kinetic properties of the reactions involved as well as in terms of potential implications in quinone cytotoxicity.     

Mechanism of S-nitrosation of recombinant human brain calbindin D28K

Mass spectrometry and UV-vis absorption results support a mechanism for NO donation by S-nitrosoglutathione (GSNO) to recombinant human brain calbindin D(28K) (rHCaBP) that requires the presence of trace copper, added as either Cu,Zn-superoxide dismutase (CuZnSOD) or CuSO(4). The extent of copper-catalyzed rHCaBP S-nitrosation depends on the ratio of protein to GSNO and on the reaction time, and NO-transfer is prevented when copper chelators are present. CuZnSOD is an efficient catalyst of rHCaBP S-nitrosation, and the mechanism of CuZnSOD-catalyzed S-nitrosation involves reduction of the active-site Cu(II) by a number of the five free thiols in rHCaBP, giving rise to thiyl radicals. The Cu(I)ZnSOD formed catalyzes the reductive cleavage of GSNO present in solution to give GSH and release NO. rHCaBP thiyl radicals react with NO to yield the S-nitrosoprotein. Cu(II)ZnSOD is also reduced by GSH in a concentration-dependent manner up to 5 mM but not at higher GSH concentrations. However, unlike the rHCaBP thiyl radicals, GS(*) radicals dimerize to GSSG faster than their reaction with NO. The data presented here provide a biologically relevant mechanism for protein S-nitrosation by small S-nitrosothiols. S-nitrosation is rapidly gaining recognition as a major form of protein posttranslational modification, and the efficient S-nitrosation of CaBP by CuZnSOD/GSNO is speculated to be of neurochemical importance given that CaBP and CuZnSOD are abundant in neurons.     

Glutathione oxidation during peroxidase catalysed drug metabolism

The peroxidase catalyzed oxidation of certain drugs in the presence of glutathione (GSH) resulted in extensive oxidation to oxidized glutathione (GSSG). Extensive oxygen uptake ensued and thiyl radicals could be trapped. Only catalytic amounts of drugs were required indicating a redox cycling mechanism. Active drugs included phenothiazines, aminopyrine, p-phenetidine, acetaminophen and 4-N,N-(CH3)2-aminophenol. Other drugs, including dopamine and alpha-methyl dopa, did not catalyse oxygen uptake, nor were GSSG or thiyl radicals formed. Instead, GSH was depleted by GSH conjugate formation. Drugs of the former group, e.g. acetaminophen, aminopyrine or N,N-(CH3)2-aniline have also been found by other investigators to form GSSG and hydrogen peroxide when added to hepatocytes or when perfused through an isolated liver. Although cytochrome P-450 normally catalyses a two-electron oxidation of drugs, serious consideration should be given for some one-electron oxidation resulting in radical formation, oxygen activation and GSSG formation.     

The redox pathway of S-nitrosoglutathione, glutathione and nitric oxide in cell to neuron communications

Recent results demonstrated that S-nitrosoglutathione (GSNO) and nitric oxide (*NO) protect brain dopamine neurons from hydroxyl radical (*OH)-induced oxidative stress in vivo because they are potent antioxidants. GSNO and *NO terminate oxidant stress in the brain by (i) inhibiting iron-stimulated hydroxyl radicals formation or the Fenton reaction, (ii) terminating lipid peroxidation, (iii) augmenting the antioxidative potency of glutathione (GSH), (iv) mediating neuroprotective action of brain-derived neurotrophin (BDNF), and (v) inhibiting cysteinyl proteases. In fact, GSNO--S-nitrosylated GSH--is approximately 100 times more potent than the classical antioxidant GSH. In addition, S-nitrosylation of cysteine residues by GSNO inactivates caspase-3 and HIV-1 protease, and prevents apoptosis and neurotoxicity. GSNO-induced antiplatelet aggregation is also mediated by S-nitrosylation of clotting factor XIII. Thus the elucidation of chemical reactions involved in this GSNO pathway (GSH GS* + *NO-->[GSNO]-->GSSG + *NO-->GSH) is necessary for understanding the biology of *NO, especially its beneficial antioxidative and neuroprotective effects in the CNS. GSNO is most likely generated in the endothelial and astroglial cells during oxidative stress because these cells contain mM GSH and nitric oxide synthase. Furthermore, the transfer of GSH and *NO to neurons via this GSNO pathway may facilitate cell to neuron communications, including not only the activation of guanylyl cyclase, but also the nitrosylation of iron complexes, iron containing enzymes, and cysteinyl proteases. GSNO annihilates free radicals and promotes neuroprotection via its c-GMP-independent nitrosylation actions. This putative pathway of GSNO/GSH/*NO may provide new molecular insights for the redox cycling of GSH and GSSG in the CNS.     

S-nitrosation of glutathione by nitric oxide,peroxynitrite, and (*)NO/O(2)(*-)

To elucidate potential mechanisms of S-nitrosothiol formation in vivo, we studied nitrosation of GSH and albumin by nitric oxide ((*)NO), peroxynitrite, and (*)NO/O(2)(*)(-). In the presence of O(2), (*)NO yielded 20% of S-nitrosoglutathione (GSNO) at pH 7.5. Ascorbate and the spin trap 4-hydroxy-[2,2,4,4-tetramethyl-piperidine-1-oxyl] (TEMPOL) inhibited GSNO formation by 67%. Electron paramagnetic resonance spectroscopy with 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) demonstrated intermediate formation of glutathionyl radicals, suggesting that GSNO formation by (*)NO/O(2) is predominantly mediated by (*)NO(2). Peroxynitrite-triggered GSNO formation (0.06% yield) was stimulated 10- and 2-fold by ascorbate and TEMPOL, respectively. Co-generation of (*)NO and O(2)(*)(-) at equal fluxes yielded less GSNO than (*)NO alone, but was 100-fold more efficient (8% yield) than peroxynitrite. Moreover, in contrast to the reaction of peroxynitrite, GSNO formation by (*)NO/O(2)(*)(-) was inhibited by ascorbate. Similar results were obtained with albumin instead of GSH. We propose that sulfhydryl compounds react with O(2)(*)(-) to initiate a chain reaction that forms radical intermediates which combine with (*)NO to yield GSNO. In RAW 264.7 macrophages, S-nitrosothiol formation by (*)NO/O(2) and (*)NO/O(2)(*)(-) occurred with relative efficiencies comparable to those in solution. Our results indicate that concerted generation of (*)NO and O(2)(*)(-) may essentially contribute to nitrosative stress in inflammatory diseases.     

Production of Hydroxyl Free Radical in the Xanthine Oxidase System with Addition of 1-methyl-3-nitro-1-nitrosoguanidine

Recent results demonstrated that S-nitrosoglutathione (GSNO) and nitric oxide (^·NO) protect brain dopamine neurons from hydroxyl radical (^·OH)-induced oxidative stress in vivo because they are potent antioxidants. GSNO and ^·NO terminate oxidant stress in the brain by (i) inhibiting iron-stimulated hydroxyl radicals formation or the Fenton reaction, (ii) terminating lipid peroxidation, (iii) augmenting the antioxidative potency of glutathione (GSH), (iv) mediating neuroprotective action of brain-derived neurotrophin (BDNF), and (v) inhibiting cysteinyl proteases. In fact, GSNO — S-nitrosylated GSH — is approximately 100 times more potent than the classical antioxidant GSH. In addition, S-nitrosylation of cysteine residues by GSNO inactivates caspase-3 and HIV-1 protease, and prevents apoptosis and neurotoxicity. GSNO-induced antiplatelet aggregation is also mediated by S-nitrosylation of clotting factor XIII. Thus the elucidation of chemical reactions involved in this GSNO pathway (GSH → GS^· + ^·NO → [GSNO] → GSSG + ^·NO → GSH) is necessary for understanding the biology of ^·NO, especially its beneficial antioxidative and neuroprotective effects in the CNS. GSNO is most likely generated in the endothelial and astroglial cells during oxidative stress because these cells contain mM GSH and nitric oxide synthase. Furthermore, the transfer of GSH and ^·NO to neurons via this GSNO pathway may facilitate cell to neuron communications, including not only the activation of guanylyl cyclase, but also the nitrosylation of iron complexes, iron containing enzymes, and cysteinyl proteases. GSNO annihilates free radicals and promotes neuroprotection via its c-GMP-independent nitrosylation actions. This putative pathway of GSNO/GSH/^·NO may provide new molecular insights for the redox cycling of GSH and GSSG in the CNS.     

Protein S-glutathiolation triggered by decomposed S-nitrosoglutathione

Recombinant human brain calbindin D(28K) (rHCaBP), human Cu,Zn-superoxide dismutase (HCuZnSOD), rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and bovine serum albumin (BSA) were found to be S-glutathiolated in decomposed S-nitrosoglutathione (GSNO) solutions. Tryptic or Glu-C digestion and MALDI-TOF MS analyses of the digests are consistent with S-thiolation of Cys111 and Cys187 of HCuZnSOD and rHCaBP, respectively, upon exposure to decomposed GSNO. GAPDH activity analysis reveals that S-glutathiolation most likely occurs on the active site Cys149, and the single free Cys34 is assumed to be the site of S-glutathiolation in BSA. The yields of S-glutathiolation of rHCaBP, GAPDH, and BSA were much higher than those of HCuZnSOD. The latter is limited by the accessibility of Cys111 to the glutathiolating reagent in the HCuZnSOD dimer. Unlike decomposed GSNO, fresh GSNO, reduced glutathione (GSH), and oxidized glutathione (GSSG) are not efficient S-glutathiolating agents for the proteins examined here. On the basis of analysis by mass spectrometry and UV-visible absorption, GSNO decomposition in the dark at room temperature yields glutathione disulfide S-oxide [GS(O)SG], glutathione disulfide S-dioxide (GSO(2)SG), and GSSG as products. GS(O)SG is the efficient protein S-glutathiolating agent in GSNO solutions, not GSNO, which does not carry out efficient S-glutathiolation of rHCaBP, HCuZnSOD, or GAPDH in vitro. A hydrolysis pathway yielding GSOH and nitroxyl (HNO/NO(-)) as intermediates is proposed for GSNO decomposition in the dark. This is based on inhibition of GSNO breakdown by dimedone, a reagent specific for sulfenic acids, and on nitroxyl scavenging by metmyoglobin. The results presented here are contrary to numerous reports of protein S-thiolation by low-molecular weight S-nitrosothiols.     

Peroxidative metabolism of apigenin and naringenin versus luteolin and quercetin: glutathione oxidation and conjugation

GSH was readily depleted by a flavonoid, H(2)O(2), and peroxidase mixture but the products formed were dependent on the redox potential of the flavonoid. Catalytic amounts of apigenin and naringenin but not kaempferol (flavonoids that contain a phenol B ring) when oxidized by H(2)O(2) and peroxidase co-oxidized GSH to GSSG via a thiyl radical which could be trapped by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) to form a DMPO-glutathionyl radical adduct detected by ESR spectroscopy. On the other hand, quercetin and luteolin (flavonoids that contain a catechol B ring) or kaempferol depleted GSH stoichiometrically without forming a thiyl radical or GSSG. Quercetin, luteolin, and kaempferol formed mono-GSH and bis-GSH conjugates, whereas apigenin and naringenin did not form GSH conjugates. MS/MS electrospray spectroscopy showed that mono-GSH conjugates for quercetin and luteolin had peaks at m/z 608 [M + H](+) and m/z 592 [M + H](+) in the positive-ion mode, respectively. (1)H NMR spectroscopy showed that the GSH was bound to the quercetin A ring. Spectral studies indicated that at a physiological pH the luteolin-SG conjugate was formed from a product with a UV maximum absorbance at 260 nm that was reducible by potassium borohydride. The quercetin-SG conjugate or kaempferol-SG conjugate on the other hand was formed from a product with a UV maximum absorbance at 335 nm that was not reducible by potassium borohydride. These results suggest that GSH was oxidized by apigenin/naringenin phenoxyl radicals, whereas GSH conjugate formation involved the o-quinone metabolite of luteolin or the quinoid (quinone methide) product of quercetin/kaempferol.     

Redox-sensitivity and site-specificity of S- and N- denitrosation in proteins

Background

S-nitrosation – the formation of S-nitrosothiols (RSNOs) at cysteine residues in proteins – is a posttranslational modification involved in signal transduction and nitric oxide (NO) transport. Recent studies would also suggest the formation of N-nitrosamines (RNNOs) in proteins in vivo, although their biological significance remains obscure. In this study, we characterized a redox-based mechanism by which N-nitroso-tryptophan residues in proteins may be denitrosated.

Methodology/Principal Findings

The denitrosation of N-acetyl-nitroso Trp (NANT) by glutathione (GSH) required molecular oxygen and was inhibited by superoxide dismutase (SOD). Transnitrosation to form S-nitrosoglutathione (GSNO) was observed only in the absence of oxygen or presence of SOD. Protein denitrosation by GSH was studied using a set of mutant recombinant human serum albumin (HSA). Trp-214 and Cys-37 were the only two residues nitrosated by NO under aerobic conditions. Nitroso-Trp-214 in HSA was insensitive to denitrosation by GSH or ascorbate while denitrosation at Cys-37 was evident in the presence of GSH but not ascorbate. GSH-dependent denitrosation of Trp-214 was restored in a peptide fragment of helix II containing Trp-214. Finally, incubation of cell lysates with NANT revealed a pattern of protein nitrosation distinct from that observed with GSNO.

Conclusions

We propose that the denitrosation of nitrosated Trp by GSH occurs through homolytic cleavage of nitroso Trp to NO and a Trp aminyl radical, driven by the formation of superoxide derived from the oxidation of GSH to GSSG. Overall, the accessibility of Trp residues to redox-active biomolecules determines the stability of protein-associated nitroso species such that in the case of HSA, N-nitroso-Trp-214 is insensitive to denitrosation by low-molecular-weight antioxidants. Moreover, RNNOs can generate free NO and transfer their NO moiety in an oxygen-dependent fashion, albeit site-specificities appear to differ markedly from that of RSNOs.     

Glutathione-dependent reduction of peroxides during ferryl- and met-myoglobin interconversion: a potential protective mechanism in muscle

Met-myoglobin is oxidized both by H2O2 and other hydroperoxides to a species with a higher iron valency state and the spectral characteristics of ferryl-myoglobin. Glutathione (GSH) reduces the latter species back to met-myoglobin with parallel oxidation to its disulfide (GSSG) but cannot reduce met-myoglobin to ferrous myoglobin. Under aerobic conditions, the GSH-mediated reduction of ferry-myoglobin is associated with O2 consumption and amounts of GSSG are formed far in excess over that of the peroxide added. Under anaerobic conditions, this ratio is close to unity. These results are interpreted in terms of a one-electron redox process involving the reduction of ferryl-myoglobin to met-myoglobin and the one-electron oxidation of GSH to its thiyl radical. Further reactions of thiyl radicals are influenced by the presence of oxygen which will be the determining factor in the ratio H2O2 added/GSSG formed. It is suggested that, when oxygen is limiting, myoglobin may serve as a protector of muscle cells against peroxides and other oxidants.     

The interaction of reduced glutathione with active oxygen species generated by xanthine-oxidase-catalyzed metabolism of xanthine

The interaction of reduced glutathione (GSH) with active oxygen species generated during xanthine-oxidase-catalyzed metabolism of xanthine was investigated. The only GSH-derived product detected in this system was oxidized glutathione (GSSG). Catalase inhibited the oxidation of GSH to GSSG by more than 80%, whereas superoxide dismutase exerted a smaller but significant inhibition of GSSG formation. Hydroxyl radical (OH) scavengers or desferrioxamine (1 mM) had no effect on GSSG formation. Using EPR spectroscopy and the spin trap 5,5-dimethylpyrroline-N-oxide (DMPO), the production of superoxide was observed by the detection of a DMPO-OOH radical adduct. This spectrum was altered by the inclusion of GSH (5 - 20 mM) in the reaction mixture, indicating the generation of a different radical species consistent with DMPO-glutathionyl radical adduct generation.     

Detection of glutathione thiyl free radical catalyzed by prostaglandin H synthase present in keratinocytes. Study of co-oxidation in a cellular system

We report here the application of the electron spin resonance technique to detect free radicals formed by the hydroperoxidase activity of prostaglandin H synthase in cells. Studies were done using keratinocytes obtained from hairless mice. These cells can be prepared in large number and possess significant prostaglandin H synthase activity. Initial attempts to directly detect free radical metabolites of several amines in cells were unsuccessful. A technique was developed based on the ability of some free radicals formed by prostaglandin hydroperoxidase to oxidize reduced glutathione (GSH) to a thiyl radical, which was trapped by 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Phenol and aminopyrine are excellent hydroperoxidase substrates for this purpose and thus were used for all further experiments. Using this approach we detected the DMPO/GS.thiyl radical adduct catalyzed by cellular prostaglandin hydroperoxidase. The formation of the radical was dependent on the addition of substrate, inhibited by indomethacin, and supported by either exogenous arachidonic acid or endogenous arachidonic acid released from phospholipid stores by Ca2+ ionophore A-23187. The addition of GSH significantly increased the intracellular GSH concentration and concomitantly stimulated the formation of the DMPO/GS.thiyl radical adduct. Phenol, but not aminopyrine, enhanced thiyl radical adduct formation and prostaglandin formation with keratinocytes while both cofactors were equally effective in incubations containing microsomes prepared from keratinocytes. These results suggest that prostaglandin hydroperoxidase-dependent co-oxidation of chemicals can result in the intracellular formation of free radical metabolites.     

Glutathione disulfide and S-nitrosoglutathione detoxification by Mycobacteriumtuberculosis thioredoxin system

Mycobacterium tuberculosis resides within alveolar macrophages. These phagocytes produce reactive nitrogen and oxygen intermediates to combat the invading pathogens. The macrophage glutathione (GSH) pool reduces nitric oxide (NO) to S-nitrosoglutathione (GSNO). Both glutathione disulfide (GSSG) and GSNO possess mycobactericidal activities in vitro. In this study we demonstrate that M. tuberculosis thioredoxin system, comprises of thioredoxin reductase B2 and thioredoxin C reduces the oxidized form of the intracellular mycothiol (MSSM) and is able to efficiently reduce GSSG and GSNO in vitro. Our study suggests that the thioredoxin system provide a general reduction mechanism to cope with oxidative stress associated with the microbe’s metabolism as well as to detoxify xenobiotics produced by the host.     

Protein thiyl radical mediates S-glutathionylation of complex I

Complex I is a critical site of O(2)(?-) production and the major host of reactive protein thiols in mitochondria. In response to oxidative stress, complex I protein thiols at the 51- and 75-kDa subunits are reversibly S-glutathionylated. The mechanism of complex I S-glutathionylation is mainly obtained from insight into GSSG-mediated thiol-disulfide exchange, which would require a dramatic decline in the GSH/GSSG ratio. Intrinsic complex I S-glutathionylation can be detected in the rat heart at a relatively high GSH/GSSG ratio (J. Chen et al., J. Biol. Chem. 285:3168-3180, 2010). Thus, we hypothesized that reactive thiyl radical is more likely to mediate protein S-glutathionylation of complex I. Here we employed immuno-spin trapping and tandem mass spectrometry (LC/MS/MS) to test the hypothesis in the 75-kDa subunit from S-glutathionylated complex I. Under the conditions of O(2)(?-) production in the presence of GSH, we detected complex I S-glutathionylation at Cys-226, Cys-367, and Cys-727 of the 75-kDa subunit. Addition of a radical trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), significantly decreased complex I S-glutathionylation and subsequently increased the protein radical adduct of complex I-DMPO as detected by immunoblotting using an anti-DMPO antibody. LC/MS/MS analysis indicated that Cys-226, Cys-554, and Cys-727 were involved in DMPO binding, confirming that formation of the complex I thiyl radical mediates S-glutathionylation. LC/MS/MS analysis also showed that Cys-554 and Cys-727 were S-sulfonated under conditions of O(2)(?-) generation in the absence of DMPO. In myocytes (HL-1 cell line) treated with menadione to trigger mitochondrial O(2)(?-) generation, complex I protein radical and S-glutathionylation were increased. Thus mediation of complex I S-glutathionylation by the protein thiyl radical provides a unique pathway for the redox regulation of mitochondrial function.     

Influence of glutathione and its derivatives on fibrin polymerization

A complex relationship exists between reduced, oxidized, and nitrosated glutathione (GSH, GSSG, and GSNO, respectively). Although previous studies have demonstrated S-nitrosoglutathione (GSNO) has potent antiplatelet efficacy, little work has examined the role of GSNO and related species on subsequent aspects of coagulation (e.g., fibrin polymerization). Herein, the effects of GSH, GSSG, and GSNO on the entire process of fibrin polymerization are described. Relative to normal fibrinogen, the addition of GSH, GSSG, or GSNO leads to prolonged lag times, slower rates of protofibril lateral aggregation and the formation of clots with lower final turbidities. Dose-dependent studies indicate the influence of GSH on fibrin formation is a function of both GSH and fibrinogen concentration. Studies with Aalpha251 recombinant fibrinogen (lacking alphaC regions) showed GSH had no influence on its polymerization, suggesting the glutathione species interact within the alphaC region of fibrinogen.     

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.     

Iron catalyzed conversion of NO into nitrosonium (NO+) and nitroxyl (HNO/NO-) species.

The conversion of NO into its congeners, nitrosonium (NO+) and nitroxyl (HNO/NO-) species, has important consequences in NO metabolism. Dinitrosyl iron complex (DNIC) combined with thiol ligands was shown to catalyze the conversion of NO into NO+, resulting in the synthesis of S-nitrosothiols (RSNO) both in vitro and in vivo. The formation mechanism of DNIC was proposed to involve the intermediate release of nitroxyl. Since the detection of hydroxylamine (as the product of a rapid reaction of HNO/NO- with thiols) is taken as the evidence for nitroxyl generation, we examined the formation of hydroxylamine, RSNO, and nitrite (the product of a rapid reaction of NO+ with water) in neutral solutions containing iron ions and thiols exposed to NO under anaerobic conditions. Hydroxylamine was detected in NO treated solutions of iron ions in the presence of cysteine, but not glutathione (GSH). The addition of urate, a major "free" iron-binding agent in humans, to solutions of GSH and iron ions, and the subsequent treatment of these solutions with NO increased the synthesis of GSNO and resulted in the formation of hydroxylamine. This caused a loss of urate and yielded a novel nitrosative/nitration product. GSH attenuated the urate decomposition to such a degree that it could be reflected as the function of GSH:urate. Results described here contribute to the understanding of the role of iron ions in catalyzing the conversion of NO into HNO/NO- and point to the role of uric acid not previously described.     

Proteins protect lipid membranes from oxidation by thiyl radicals

Oxidation of polyunsaturated fatty acids by thiyl radicals derived from GSH or Cys is believed to be responsible for some of the biological damage resulting from lipid oxidation under oxidative stress. However, this has not been demonstrated in complex biological systems. In this study, we measured the formation of lipid hydroperoxides in liposomes exposed to radicals generated by gamma radiation from GSH, GSSG, GSMe, Cys and Met. In the absence of proteins, the radicals oxidized the liposome lipids. In the presence of proteins, the thiyl radicals failed to react with the liposomes, even though the protein radicals efficiently oxidized the S-compounds. It appears that the thiyl and other S-radicals were effectively scavenged by the protein before initiating lipid oxidation. The results suggest that membrane lipid oxidation in vivo by thiyl radicals is unlikely to be a significant event.     

Intramolecular electron transfer between tyrosyl radical and cysteine residue inhibits tyrosine nitration and induces thiyl radical formation in model peptides treated with myeloperoxidase, H2O2, and NO2-: EPR SPIN trapping studies

We investigated the effects of a cysteine residue on tyrosine nitration in several model peptides treated with myeloperoxidase (MPO), H(2)O(2), and nitrite anion (NO(2)(-)) and with horseradish peroxidase and H(2)O(2). Sequences of model peptides were acetyl-Tyr-Cys-amide (YC), acetyl-Tyr-Ala-Cys-amide (YAC), acetyl-Tyr-Ala-Ala-Cys-amide (YAAC), and acetyl-Tyr-Ala-Ala-Ala-Ala-Cys-amide (YAAAAC). Results indicate that nitration and oxidation products of tyrosyl residue in YC and other model peptides were barely detectable. A major product detected was the corresponding disulfide (e.g. YCysCysY). Spin trapping experiments with 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) revealed thiyl adduct (e.g. DMPO-SCys-Tyr) formation from peptides (e.g. YC) treated with MPO/H(2)O(2) and MPO/H(2)O(2)/NO(2)(-). The steady-state concentrations of DMPO-thiyl adducts decreased with increasing chain length of model peptides. Blocking the sulfydryl group in YC with methylmethanethiosulfonate (that formed YCSSCH(3)) totally inhibited thiyl radical formation as did substitution of Tyr with Phe (i.e. FC) in the presence of MPO/H(2)O(2)/NO(2)(-). However, increased tyrosine nitration, tyrosine dimerization, and tyrosyl radical formation were detected in the MPO/H(2)O(2)/NO(2)(-)/YCSSCH(3) system. Increased formation of S-nitrosated YC (YCysNO) was detected in the MPO/H(2)O(2)/(*)NO system. We conclude that a rapid intramolecular electron transfer reaction between the tyrosyl radical and the Cys residue impedes tyrosine nitration and induces corresponding thiyl radical and nitrosocysteine product. Implications of this novel intramolecular electron transfer mechanism in protein nitration and nitrosation are discussed.     

Evidence for a free radical mechanism of styrene-glutathione conjugate formation catalyzed by prostaglandin H synthase and horseradish peroxidase

We have proposed, using styrene as a model, a new mechanism for the formation of glutathione conjugates that is independent of epoxide formation but dependent on the oxidation of glutathione to a thiyl radical by peroxidases such as prostaglandin H synthase or horseradish peroxidase. The thiyl radical reacts with styrene to yield a carbon-centered radical which subsequently reacts with molecular oxygen to give the styrene-glutathione conjugate. We have used electron spin resonance spin trapping techniques to detect the proposed free radical intermediates. A styrene carbon-centered radical was trapped using the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and t-nitrosobutane. The position of the carbon-centered radical was confirmed to be at carbon 7 by the use of specific 2H-labeled styrenes. The addition of the spin trap DMPO inhibited both the utilization of molecular oxygen and the formation of styrene-glutathione conjugates. Under anaerobic conditions additional styrene-glutathione conjugates were formed, one of which was identified by fast atom bombardment mass spectrometry as S-(2-phenyl)ethylglutathione. The glutathione thiyl radical intermediate was observed by spin trapping with DMPO. These results support the proposed free radical-mediated formation of styrene-glutathione conjugates by peroxidase enzymes.