Xanthine Oxidase-mediated Decomposition of S-Nitrosothiols*

Madia TrujilloDagger , María Noel AlvarezDagger §, Gonzalo PeluffoDagger , Bruce A. Freemanpar , and Rafael RadiDagger

From the Dagger  Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Montevideo 11800, Uruguay, and the Departments of  Anesthesiology and par  Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35233

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

S-Nitrosothiols (RSNO) occur in vivo and have been proposed as nitric oxide (·NO) storage and transport biomolecules. Still, the biochemical mechanisms by which RSNO release ·NO in biological systems are not well defined, and in particular, the interactions between reactive oxygen species and RSNO have not been studied. In this work, we show that xanthine oxidase (XO), in the presence of purine (hypoxanthine, xanthine) or pteridine (lumazine) substrates, induces S-nitrosocysteine (CysNO) and S-nitrosoglutathione (GSNO) decomposition under aerobic conditions. The decomposition of RSNO by XO was inhibitable by copper-zinc superoxide dismutase, in agreement with the participation of superoxide anion (Obardot 2) in the process. However, while superoxide dismutase could totally inhibit aerobic decomposition of GSNO, it was only partially inhibitory for CysNO. Competition experiments indicated that Obardot 2 reacted with GSNO with a rate constant of 1 × 104 M-1·s-1 at pH 7.4 and 25 °C. The decomposition of RSNO was accompanied by peroxynitrite formation as assessed by the oxidation of dihydrorhodamine and of cytochrome c2+. The proposed mechanism involves the Obardot 2-dependent reduction of RSNO to yield ·NO, which in turn reacts fast with a second Obardot 2 molecule to yield peroxynitrite. Under anaerobic conditions, CysNO incubated with xanthine plus XO resulted in CysNO decomposition, ·NO detection, and cysteine and uric acid formation. We found that CysNO is an electron acceptor substrate for XO with a Km of 0.7 mM. In agreement with this concept, the enzymatic reduction of CysNO by XO was inhibitable by oxypurinol and diphenyliodonium, inhibitors that interfere with the catalytic cycle at the molybdenum and flavin sites, respectively. In conclusion, XO decomposes RSNO by Obardot 2-dependent and -independent pathways, and in the presence of oxygen it leads to peroxynitrite formation.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The natural occurrence and signal transduction actions of S-nitrosothiols (RSNO)1 have been demonstrated in different biological systems, including human plasma (1), airways (2), and cells (3, 4). It has been suggested that the formation and decomposition of low molecular weight RSNO, such as S-nitrosoglutathione (GSNO) and S-nitrosocysteine (CysNO), may represent a mechanism for the storage and transport of nitric oxide (·NO) (5, 6). Furthermore, S-nitrosylation can regulate protein function, as has been described for an expanding number of proteins (7-11). S-Nitrosothiols are sensitive to both photolytic (12, 13) and transition metal ion-dependent (14) decay but are stable in the presence of transition metal ion chelators in the dark. The biochemical pathways that lead to RSNO decomposition are under active investigation; nonenzymatic pathways including transnitrosation reactions in the presence of thiols (15, 16) and reductive decomposition (e.g. by ascorbate and thiols) (17, 18) are probably important physiological mechanisms. Interactions between ·NO and reactive oxygen species (i.e. superoxide, Obardot 2 (Ref. 19); lipid peroxyl and alkoxyl radicals (Ref. 20)) have been recognized as being of critical importance for modulating the signal transduction actions of ·NO as well as oxidative damage (21). However, the reactions of these species with RSNO have not been studied. This is of particular importance in the case of Obardot 2, since it is a continuously formed free radical in aerobic cells that can also act as a reductant in many cases (E'0 O2/Obardot 2 = -0.33 V, Ref. 22); thus, the question readily arises as to whether Obardot 2 can promote RSNO decomposition, i.e. ·NO release. Since Obardot 2 reacts with ·NO at almost diffusion-controlled rates (19) to form peroxynitrite anion (ONOO-), Obardot 2-mediated RSNO decomposition could in turn lead to ONOO- formation. Both ONOO- and its conjugated acid, peroxynitrous acid (ONOOH) (pKa = 6.8) (23-24) are powerful oxidants that are formed in vivo (25, 26) and contribute to tissue oxidative damage (26, 27).

In this study, we evaluated the reaction between RSNO and Obardot 2 formed from the reaction catalyzed by xanthine oxidase (XO) (xanthine:oxygen oxidoreductase, EC 1.2.3.2) in the presence of purine or pteridine substrates and molecular oxygen (28). In vessel walls, where RSNO exerts signal-transducing actions, XO is present in high concentrations (29), and in human atherosclerotic arteries it impairs EDRF (endothelial derived relaxing factor) activity (29, 30). Xanthine oxidase is composed of two identical subunits, each containing one atom of molybdenum, two iron-sulfur centers [Fe2S2], and one FAD, which function as sequential redox groups in the intramolecular transfer of electrons (31). Since XO presents a relatively broad specificity for electron acceptor substrates, we also studied whether low molecular weight RSNO such as GSNO and CysNO could serve as XO substrates. We report herein that xanthine oxidase decomposes low molecular weight S-nitrosothiols by Obardot 2-dependent and -independent mechanisms and, according to the availability of oxygen in the system, secondarily leads to peroxynitrite2 formation.

    EXPERIMENTAL PROCEDURES
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Results
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Chemicals-- GSH, GSSG, L-cysteine, hypoxanthine, xanthine, oxypurinol, lumazine, uric acid, sodium nitrite (NaNO2), sodium nitrate (NaNO3), 5,5'-dithiobis-2-nitrobenzoic acid (DTNB), diethylenetriaminepentacetic acid (dtpa), 2,4-dimethyl-1,10-phenanthroline (neocuproine), manganese dioxide, horse heart cytochrome c type VI, human hemoglobin, and porcine liver uricase type V (37 units/g of protein) were purchased from Sigma. Diphenyliodonium chloride was from Aldrich. Bovine milk xanthine oxidase was obtained from Calbiochem. Bovine copper-zinc superoxide dismutase (SOD) (3100 units/mg) was obtained from DDI Pharmaceuticals, Inc. (Mountain View, CA). Hydrogen peroxide and catalase from bovine liver were from Fluka AG (Switzerland). Argon (~99.5% pure) was purchased from AGA Gas Company (Montevideo, Uruguay). Dihydrorhodamine 123 was from Molecular Probes (Eugene, OR). All other chemicals were reagent grade. All solutions were prepared with highly pure deionized water (Barnstead D4742, resistance > 18 MOmega ·cm-1) to minimize trace metal contamination.

Synthesis of RSNO-- S-Nitrosocysteine and S-nitrosoglutathione were synthesized on the day of use by reaction of the respective thiol with acidified sodium nitrite as described previously (32) and stored on ice in the dark. Final concentrations were determined at 334 nm (epsilon 334 = 870 and 780 M-1·cm-1 for GSNO or CysNO, respectively). Reverse phase high pressure liquid chromatography analysis was performed in a Gilson Synchropack C18 column as previously reported (33) and showed that both RSNO were more than 97% pure.

Synthesis of Peroxynitrite-- Peroxynitrite was synthesized in a quenched-flow reactor from sodium nitrite and hydrogen peroxide (H2O2) under acidic conditions and quantitated as described previously (23). The H2O2 remaining from the synthesis was eliminated by treating the stock solutions of peroxynitrite with granular manganese dioxide (34).

Biochemical Analyses-- All experiments were performed in 50 mM sodium phosphate, pH 7.4, plus 0.1 mM dtpa, at 25 °C. Xanthine oxidase activity was monitored by measuring uric acid production at 292 nm (epsilon 292 = 11 mM-1·cm-1) (31) with 150 µM xanthine or 150 µM hypoxanthine as substrates. It is important to note that xanthine and hypoxanthine oxidation to uric acid involves a 2- and 4-electron oxidation, respectively. Superoxide production was determined by SOD-inhibitable reduction of cytochrome c3+ at 550 nm (Delta epsilon red-ox = 21 mM-1·cm-1)(35, 36). The univalent flux percentage was 32%, as described previously (37, 38). In some experiments, lumazine (100 µM) was used as a XO substrate. Violapterin formation from lumazine was followed at 328 nm (epsilon 328 = 10.5 mM-1·cm-1) (39), and the univalent flux percentage was 40%.

Decomposition rates of GSNO and CysNO were determined spectrophotometrically by measuring the decrease of absorbance maxima at 334 nm. For experiments requiring anaerobic conditions, solutions were deoxygenated by extensive bubbling with argon for 10 min and then injected into reaction mixtures maintained in anaerobic cuvettes using Hamilton gas-tight syringes.

The production of ·NO from GSNO or CysNO was determined electrochemically (Iso-NO, World Precision Instruments, Inc., Sarasota, FL). Nitroxyl anion (NO-) generation was assessed by the reduction of methemoglobin to nitrosyl hemoglobin as previously (40). Thiols were quantitated spectrophotometrically using the DTNB assay (41), by adding aliquots (100 µl) from the samples to 1-ml tubes containing sodium pyrophosphate 100 mM, pH 9.2, 0.5 mM DTNB, and 0.1 mM oxypurinol at different times (0-15 min), and absorbance was read at 412 nm (epsilon  = 13.6 mM-1·cm-1). At the concentrations of RSNO carried over to the DTNB assay, there was no interference with free thiol detection.

Oxygen consumption studies were performed using a water-jacketed Clark electrode (YSI model 5300).

Peroxynitrite formation was determined by two independent methods: 1) the rate of dihydrorhodamine (DHR) oxidation to rhodamine (42) and 2) the rate of cytochrome c2+ oxidation (43) as follows.

1) For the DHR assay, stock solutions of DHR (28.9 mM) in dimethyl sulfoxide were purged with argon and stored at -20 °C. DHR (100 µM) was exposed to GSNO or CysNO (1 mM) in the absence or presence of 150 µM hypoxanthine plus 5 milliunits/ml XO. When lumazine (100 µM), a low turnover substrate (39), was used instead of hypoxanthine as substrate for XO, the addition of 100 milliunits/ml XO was necessary in order obtain a similar Obardot 2 flux. The oxidation of DHR to rhodamine was followed spectrophotometrically at 500 nm (epsilon 500 = 78.8 mM-1·cm-1) (44).

2) Cytochrome c3+ was reduced by sodium dithionite immediately before use, and excess dithionite was removed on Sephadex G-25 using 50 mM sodium phosphate plus 0.1 mM dtpa, pH 7.4, as elution buffer. Cytochrome c2+ (50 µM) was exposed to GSNO (1 mM) in the absence or presence of 150 µM hypoxanthine plus 5 milliunits/ml XO, and initial rates of cytochrome c2+ oxidation were followed at 550 nm. The yields for methods 1 and 2 were calculated using authentic peroxynitrite as standard and were 45 and 49%, respectively, in agreement with previous reports (42, 43).

Spectrophotometric determinations were carried out either in a temperature-controlled Shimadzu UV-Vis 2401 or Milton Roy Spectronic 3000 array spectrophotometers.

Data Analysis-- All experiments reported in this manuscript were repeated and revealed reproducible results. Results are expressed as mean values with the corresponding standard deviations. Graphics and data analysis were performed using Slide-Write (Advanced Graphics Software).

    RESULTS
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Introduction
Procedures
Results
Discussion
References

Aerobic Decomposition of S-Nitrosothiols by Xanthine Oxidase

Basal decomposition of GSNO (1 mM) and CysNO (1 mM) was consistently low (0.3 and 0.7 µM/min, respectively; Fig. 1 and Table I) and depended on thermal and photolytic homolysis (12). Metal-catalyzed decomposition of RSNO, i.e. copper (14, 45), was avoided by using highly pure deionized water for preparing solutions and chelation of transition metal ions with 0.1 mM dtpa in buffers. The addition of 5 milliunits/ml XO (i.e. Obardot 2, 6.4 µM/min) plus 150 µM hypoxanthine resulted in significantly increased RSNO decomposition rates (Fig. 1, A and B). This increase in RSNO decomposition rates was larger for CysNO and proportional to XO concentration (Fig. 2). Neither hypoxanthine nor XO alone accounted for these effects (Table I). The decomposition of GSNO induced by hypoxanthine plus XO was completely inhibited by the addition of 240 nM CuZn-SOD. Fig. 3 shows the inhibitory effect of increasing SOD concentrations on the decomposition of 1 mM GSNO mediated by hypoxanthine plus XO, from which an IC50 of 10 nM SOD was obtained. Xanthine oxidase-mediated decomposition of CysNO, on the other hand, was only partially inhibited by high SOD concentrations (480 nM). The degree of inhibition by these high SOD concentrations was dependent on CysNO concentration and ranged from 50% inhibition for 0.25 mM CysNO to 0.4% inhibition for 2 mM CysNO (Table II). The addition of 0.4 µM catalase or 100 µM neocuproine had no effect on XO-dependent decomposition of both RSNO (Table I). Additionally, up to 500 µM uric acid did not influence RSNO decomposition rates (data not shown).


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Fig. 1.   Decomposition of S-nitrosothiols in the presence of hypoxanthine-xanthine oxidase. Reactions were carried out in the presence of 1 mM GSNO (A) or 1 mM CysNO (B). The additions were none (a), 5 milliunits/ml XO plus 150 µM hypoxanthine (b), and 5 milliunits/ml XO plus 150 µM hypoxanthine in the presence of 240 nM SOD (c). Both panels show typical spectrophotometric traces at 334 nm.


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Fig. 2.   Dependence of S-nitrosothiol decomposition on XO activity. GSNO (open circle ) or CysNO (bullet ) (1 mM) was incubated in air-equilibrated buffer during 20 min in the presence of 150 µM hypoxanthine at different XO activities.

                              
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Table I
CysNO and GSNO decomposition by hypoxanthine (Hx) plus xanthine oxidase under aerobic conditions
GSNO (1 mM) or CysNO (1 mM) was incubated for 20 min in 50 mM sodium phosphate, pH 7.4, plus 0.1 mM dtpa at 25 °C, either alone or in the presence of the additions indicated.


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Fig. 3.   SOD-dependent inhibition of XO-mediated GSNO decomposition. GSNO (1 mM) was incubated aerobically during 20 min with 150 µM hypoxanthine and 5 milliunits/ml xanthine oxidase in the presence of SOD. The inhibition fraction (F) of GSNO consumption by SOD was calculated as before (50).

                              
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Table II
Partial inhibition of the XO-dependent decomposition of CysNO by SOD
CysNO was incubated with 150 µM hypoxanthine plus 5 milliunits/ml XO in the presence and in the absence of 480 nM SOD. Other conditions are as for Table I. The inhibition fraction (F) afforded by SOD was determined as follows: F = (va - vb)/(va - vc), where va stands for the rate of CysNO decomposition in the absence of SOD, vb is the rate of CysNO decomposition in the presence of 480 nM SOD, and vc is the rate of basal decomposition of CysNO, i.e. in the absence of XO.

The addition of 1 mM GSNO caused no effect on the rate of oxygen consumption (10 µM/min) by 150 µM hypoxanthine plus 5 milliunits/ml XO (data not shown), indicating that neither GSNO nor ·NO or secondary nitrogen oxides inhibit XO, in agreement with current observations (46).

Anaerobic Decomposition of S-Nitrosothiols by Xanthine Oxidase

When 150 µM xanthine plus 6 milliunits/ml XO were coincubated under anaerobic conditions, a very low flux of uric acid production (~6% of that under aerobic conditions) was measured, due to low residual oxygen. The addition of 1 mM CysNO led to a 10-fold increase in the initial rate of uric acid formation (from 0.4 µM/min to 4.1 µM/min), whereas up to 2 mM GSNO had no effect. This increased uric acid formation was accompanied by CysNO decomposition that varied from undetectable to 8.5 ± 2 µM/min in the absence and presence of xanthine plus XO, respectively. Neither the addition of 1 mM NaNO2 nor the addition of 1 mM NaNO3 resulted in an increase in the rate of uric acid formation by xanthine plus XO under anaerobic conditions (Table III). Since CysNO promoted the XO-dependent oxidation of xanthine to uric acid, it was evaluated whether CysNO could serve as a second substrate for XO in the presence of low oxygen concentrations. When xanthine plus XO was coincubated with different concentrations of CysNO and initial rates of uric acid formation were measured, a saturation curve was obtained (Fig. 4). The inset shows a secondary plot of vo against vo/[S] (Woolf and Hofstee plot; Ref. 47) from which a Km of 0.7 ± 0.1 mM was obtained. The initial rates of CysNO decomposition were twice the initial rates of uric acid formation (Fig. 4).

                              
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Table III
Uric acid formation by xanthine-xanthine oxidase under anaerobic conditions
Xanthine (X) (150 µM) plus 6 milliunits/ml XO were incubated anaerobically in the presence or absence of the additions shown, in 50 mM sodium phosphate, pH 7.4, plus 0.1 mM dtpa at 25 °C.


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Fig. 4.   Initial rates of S-nitrosocysteine decomposition and uric acid formation by xanthine/xanthine oxidase under anaerobic conditions. Xanthine (150 µM) plus 5 milliunits/ml XO were incubated anaerobically with CysNO (0-5 mM), and CysNO decomposition (open circle ) or uric acid formation (bullet ) was followed. The inset shows a secondary plot vo = f (vo/[S]) of the data.

To further confirm the role of CysNO as a xanthine oxidase substrate, it was observed that oxypurinol, which forms an inactive complex with XO in its reduced form at the molybdenum site (48), strongly inhibited XO-dependent CysNO decomposition (Table III). In turn, diphenyliodonium, a flavoprotein inhibitor also gave a dose-dependent inhibition of the CysNO decomposition that with 40 µM diphenyliodonium was ~80% inhibited (Table III). In addition, when 1 mM CysNO was exposed to xanthine (150 µM) plus 5 milliunits/ml XO, either under anaerobic or aerobic conditions (i.e. 255 µM O2), CysNO decomposition rates were 8.5 µM/min (Fig. 4) and 2.5 µM/min, respectively, indicating that O2 was partially inhibitory toward the enzymatic decomposition of CysNO by XO. Conversely, CysNO (2 mM) inhibited O2 consumption by 5 milliunits/ml XO plus 150 µM hypoxanthine by ~50%.

Detection of ·NO and Thiols as Products of S-Nitrosothiol Decomposition by Xanthine plus Xanthine Oxidase

Experiments were performed to characterize the decomposition products of CysNO and GSNO in the presence of xanthine plus XO.

Detection of ·NO-- Under aerobic conditions, there was no ·NO detectable when 1 mM GSNO or 1 mM CysNO were coincubated with 150 µM xanthine plus 5 milliunits/ml XO.

Under anaerobic conditions, 1 mM CysNO in buffer generated a low flux of ·NO, yielding a steady state NO concentration of <1 µM. The addition of 150 µM xanthine caused no effect on ·NO production until the addition of 5 milliunits/ml XO, which yielded an initial rate of 6 µM/min ·NO production, close to the CysNO decomposition rates reported in Fig. 4. The anaerobic production of ·NO from CysNO in the presence of xanthine plus XO was completely inhibited by 150 µM oxypurinol (Fig. 5).


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Fig. 5.   Nitric oxide production during xanthine oxidase-CysNO interactions. ·NO release by CysNO (1 mM) under anaerobic conditions was measured by an electrochemical probe. The arrows show the addition of 150 µM xanthine plus 5 milliunits/ml XO and of 150 µM oxypurinol, as indicated.

Detection of Nitroxyl Anion (NO-)-- No nitrosyl hemoglobin formation (i.e. an indicator of NO- formation) was detected when 1 mM GSNO was incubated, either alone or in the presence of 150 µM hypoxanthine plus 5 milliunits/ml XO, with 140 µM methemoglobin under aerobic conditions.

Detection of Thiols-- A thiol formation rate of 4 µM/min was measured when 1 mM CysNO was coincubated with xanthine plus XO (5 milliunits/ml) for 15 min under anaerobic conditions. Under aerobic conditions, due to a lower CysNO decomposition rate, it was necessary to increase XO activity and to use hypoxanthine as substrate to have enough sensitivity for DTNB assay of free thiol detection. Indeed, when 1 mM CysNO was incubated in the presence of 150 µM hypoxanthine and 10 milliunits/ml XO, a rate of 3.2 ± 0.2 µM/min thiol formation was found (data not shown).

Peroxynitrite Formation from Xanthine Oxidase and S-Nitrosothiols under Aerobic Conditions-- Under anaerobic conditions, XO-mediated decomposition of CysNO led to the production of ·NO (Fig. 5). On the other hand, there was no production of ·NO from GSNO in the presence of xanthine plus XO under anaerobic conditions. Under aerobic conditions, there was no detectable ·NO release during XO-dependent CysNO and GSNO decomposition. However, if ·NO was produced under aerobic conditions, its facile reaction with XO-derived Obardot 2 would preclude its detection, leading to the formation of ONOO-. Thus, experiments were performed to assess peroxynitrite generation in our system.

Dihydrorhodamine Oxidation-- The addition of 150 µM hypoxanthine, 5 milliunits/ml XO, and either 1 mM CysNO or GSNO to 100 µM DHR resulted in DHR oxidation rates of 0.9 µM/min (CysNO) and 1 µM/min (GSNO). Neither CysNO/GSNO nor hypoxanthine plus XO alone produced DHR oxidation. Initial rates were measured during 1.5 min, because the rate of oxidation significantly slowed down after that (Fig. 6, line a). Since Obardot 2 formation and RSNO decomposition were linear for at least 20 min, the progressive decrease in rates of DHR oxidation could be due to uric acid accumulation, since uric acid efficiently inhibits peroxynitrite-mediated DHR oxidation (42). To further investigate this point, we exposed DHR to GSNO (1 mM) in the presence of an alternative substrate for XO, lumazine (100 µM), which is oxidized to violapterin, therefore avoiding uric acid formation. Since lumazine is a low turnover substrate for XO (39) and results in a higher percentage univalent flux, the amount of XO used was adjusted to obtain the same Obardot 2 production rate as for hypoxanthine. On Fig. 6, line c, it is shown that initial rates of DHR oxidation by lumazine, XO, and GSNO were the same as with hypoxanthine but remained constant for a 12-min observation period. The addition of 0.1 milliunit/ml uricase, which catalyzes uric acid oxidation to allantoin and H2O2, increased rates of DHR oxidation by GSNO and hypoxanthine plus XO (Fig. 6, line b) and also enhanced DHR oxidation by CysNO and hypoxanthine plus XO (data not shown).


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Fig. 6.   Dihydrorhodamine oxidation during xanthine oxidase-GSNO interactions. GSNO (1 mM) was incubated with XO in the presence of 150 µM hypoxanthine (a), 150 µM hypoxanthine plus 0.1 unit/ml uricase (b), or 100 µM lumazine (c) under aerobic conditions. The activity of XO was adjusted to produce the same flux of superoxide under all conditions (4.7 µM/min) using as substrate either hypoxanthine (3.2 milliunits/ml XO) or lumazine (58 milliunits/ml XO).

Cytochrome c2+ Oxidation-- The addition of hypoxanthine (150 µM), XO (5 milliunits/ml), and of GSNO (1 mM) to cytochrome c2+ (50 µM) resulted in an initial cytochrome c2+ oxidation rate of 0.8 µM/min, with the addition of catalase (200 milliunits/ml) having no effect.

Therefore, the addition of 5 milliunits/ml XO (i.e. 6 µM/min Obardot 2) in the presence of purine substrates to 1 mM GSNO produced a 2.2 µM/min GSNO decomposition rate and an oxidation rate of 1 µM/min for DHR and 0.8 µM/min for cytochrome c2+. When corrected for the yield of the techniques, this corresponded to a peroxynitrite formation rate of approximately 2 µM/min. It should be noted here that both GSNO and CysNO (1 mM) were only marginally decomposed by the addition of up to 1 mM peroxynitrite.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

S-Nitrosocysteine and S-nitrosoglutathione decomposition was promoted by XO in the presence of purine substrate (Fig. 1, A and B). GSNO was decomposed only under aerobic conditions in a process that was totally inhibited by SOD, indicating that Obardot 2 was a key participating species. The reduction of GSNO by Obardot 2 would lead to the formation of GSH and ·NO as follows.
<UP>GSNO</UP>+<UP>O&cjs1138;<SUB>2</SUB></UP> <LIM><OP><ARROW>→</ARROW></OP><UL><UP>H</UP><SUP><UP>+</UP></SUP></UL></LIM><UP> GSH</UP>+<SUP><UP>⋅</UP></SUP><UP>NO</UP>+<UP>O<SUB>2</SUB></UP>
<UP><SC>Reaction</SC> 1</UP>
It has been established that Cu1+ catalyzes GSNO decomposition and that reductants such as ascorbate, GSH, and cysteine could cause GSNO decomposition via reduction of contaminating Cu2+ to Cu1+ (49). Thus, Obardot 2 could be initially reducing copper traces in the reaction solution. To avoid this reaction, all experiments described herein were performed in the presence of dtpa to minimize transition metal redox chemistry. Moreover, the lack of effect of the Cu1+ chelator neocuproine on the XO-mediated decomposition of GSNO (Table I) rules out the possibility of Obardot 2-dependent Cu2+ reduction to Cu1+ as a contributory mechanism.

If the mechanism proposed in Reaction 1 (mechanism 1) were correct, it is predicted that ·NO and free thiol would be formed; in addition, ONOO- would be secondarily produced, since Obardot 2 reacts at very fast rates (k = 6.7 × 109 M-1·s-1; Ref. 19) with ·NO.
<UP>O&cjs1138;<SUB>2</SUB></UP>+<SUP><UP>⋅</UP></SUP><UP>NO → ONOO<SUP>−</SUP></UP>
<UP><SC>Reaction</SC> 2</UP>
Thus, Obardot 2 would be consumed by two main processes (Reactions 1 and 2), and maximum reaction yields imply that for every two molecules of Obardot 2 consumed, there should be one molecule of GSNO decomposed and one molecule of ONOO- formed (2 Obardot 2:1 GSNO:1 ONOO-). This reaction stoichiometry is supported by the data presented in Figs. 1A and 6, which shows that under aerobic conditions, the addition of 5 milliunits/ml XO (i.e. 6 µM/min Obardot 2) to 1 mM GSNO caused a 2.2 µM/min GSNO decomposition and the formation of 2 µM/min peroxynitrite measured either by DHR or by cytochrome c2+ oxidation techniques. Additionally, the lack of effect of GSNO on XO-dependent O2 consumption agrees with mechanism 1, since one O2 molecule is formed from two molecules of Obardot 2 (Reactions 1 and 2), leading to the same stoichiometry of O2 return to that observed for Obardot 2 dismutation in the absence of GSNO.

Assuming this mechanism, competition kinetics analysis (50) was performed to estimate a rate constant for the reaction of Obardot 2 with GSNO. In the absence of SOD, the rate of Obardot 2 consumption due to reactions with GSNO and ·NO is expressed by the following.
<FR><NU><UP>−</UP>d[<UP>O&cjs1138;<SUB>2</SUB></UP>]</NU><DE>dt</DE></FR>=(k<SUB><UP>GSNO</UP></SUB>[<UP>GSNO</UP>]+k<SUB><UP>NO</UP></SUB>[<SUP><UP>⋅</UP></SUP><UP>NO</UP>])[<UP>O&cjs1138;<SUB>2</SUB></UP>] (Eq. 1)
In the presence of SOD, the rate of Obardot 2 consumption is expressed by the equation,
  <FR><NU><UP>−</UP>d[<UP>O&cjs1138;<SUB>2</SUB></UP>]</NU><DE>dt</DE></FR>=(k<SUB><UP>GSNO</UP></SUB>[<UP>GSNO</UP>]+k<SUB><UP>NO</UP></SUB>[<SUP><UP>⋅</UP></SUP><UP>NO</UP>]+k<SUB><UP>SOD</UP></SUB>[<UP>SOD</UP>])[<UP>O&cjs1138;<SUB>2</SUB></UP>] (Eq. 2)
where kGSNO, kNO, and kSOD represent the rate constants for the reaction of Obardot 2 with GSNO, ·NO, and SOD, respectively.

Under steady state conditions, ·NO concentration can be calculated by the following.
[<SUP>·</SUP><UP>NO</UP>]=<FR><NU>k<SUB><UP>GSNO</UP></SUB>[<UP>GSNO</UP>]</NU><DE>k<SUB><UP>NO</UP></SUB></DE></FR> (Eq. 3)
Rearranging Equation 2, we get Equation 4.
<FR><NU><UP>−</UP>d[<UP>O&cjs1138;<SUB>2</SUB></UP>]</NU><DE>dt</DE></FR>=2k<SUB><UP>GSNO</UP></SUB>[<UP>GSNO</UP>][<UP>O&cjs1138;<SUB>2</SUB></UP>]+k<SUB><UP>SOD</UP></SUB>[<UP>SOD</UP>][<UP>O&cjs1138;<SUB>2</SUB></UP>] (Eq. 4)
At the SOD concentration (10 nM) that yields a 50% inhibition of the decomposition of GSNO (Fig. 3), IC50, then the following is true.
2k<SUB><UP>GSNO</UP></SUB>[<UP>GSNO</UP>]=k<SUB><UP>SOD</UP></SUB><UP> IC</UP><SUB>50</SUB> (Eq. 5)
k<SUB><UP>GSNO</UP></SUB>=<FR><NU>k<SUB><UP>SOD</UP></SUB><UP> IC</UP><SUB>50</SUB></NU><DE>2[<UP>GSNO</UP>]</DE></FR> (Eq. 6)
From these data, and taking kSOD as 2 × 109 M-1·s-1 (51, 52), a kGSNO of 1.0 ± 0.1 × 104 M-1·s-1 at 25 °C and pH 7.4 was estimated.

In the case of 1 mM CysNO, the decomposition produced by hypoxanthine plus XO was faster and only partially inhibited by even high concentrations of SOD (Fig. 1B). Thus, under aerobic conditions Obardot 2 only partially contributes to CysNO decomposition along with other XO-mediated, Obardot 2-independent pathways of ·NO release from CysNO. As for GSNO, neither H2O2 (Table I) nor uric acid that are being formed in the system account for the effect. Anaerobic experiments showed that CysNO, but not GSNO, can be used as an electron acceptor substrate for XO (Table III, Fig. 4), resulting in ·NO (Fig. 5) and thiol formation. However, the detected quantities of ·NO and thiol were approximately 75 and 50% of those predicted by mechanism 1, respectively, most likely due to secondary reactions of ·NO and thiols once formed (53, 54). As expected, oxypurinol inhibited XO-dependent CysNO decomposition; the process was also inhibited by diphenyliodonium, implying the participation of the flavin group of XO in the electron transfer step from XO to CysNO (Table III). We propose that under aerobic conditions XO can use one of two alternative electron acceptors: O2 (255 µM in air-equilibrated phosphate buffer at 25 °C) or CysNO (Scheme I).


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Scheme 1.   Enzymic decomposition of S-nitrosocysteine by xanthine oxidase. Free xanthine oxidase (E) initiates the two-electron oxidation of xanthine (XH2) to uric acid (UA), resulting in a reduced enzyme (EH2). The lower part of the scheme indicates that oxygen binds to the reduced enzyme as the second substrate to form the reduced enzyme-oxygen complex (EH2O2), which completes the catalytic cycle releasing H2O2 and Obardot 2. The lower cycle represents the classical ping-pong mechanism for xanthine oxidase reactions as initially proposed by Gutfreund and Sturtevant (60). In the presence of S-nitrosocysteine (Cys-SNO), this compound competes as an alternative electron acceptor for reduced enzyme with respect to oxygen; once CysNO binds to the enzyme to form a complex, electrons from the flavin site are transferred to CysNO followed by the release of cysteine and ·NO. This study shows that two molecules of CysNO are decomposed per molecule of uric acid formed, in agreement with the requirement for a single electron for reduction of a CysNO molecule. However, the intimate mechanism of electron transport from XO to CysNO remains to be established.

The electron acceptor preferentially used depends on the relationship between the Km for both substrates, 50 µM for oxygen (55) and 0.7 ± 0.1 mM for CysNO (this paper), and their actual concentrations. The different decomposition rates of CysNO by XO under anaerobic or aerobic conditions and the inhibition of XO-dependent O2 consumption in the presence of CysNO are in agreement with CysNO being an alternative substrate with respect to O2 (Scheme I). Still, significant CysNO decomposition can be measured at low CysNO concentrations (0.25 mM), but it is mainly due to a Obardot 2-dependent mechanism, as shown by the high degree of inhibition afforded by SOD (Table II). As the CysNO concentration increases, XO-mediated CysNO decomposition becomes resistant to extremely high doses of SOD (Table II), in agreement with CysNO being used directly as the electron acceptor substrate. We were unable to detect Obardot 2 generated at different CysNO concentrations due to the fact that Obardot 2 readily reacts with the ·NO produced during CysNO decomposition to form ONOO-. Indeed, under aerobic conditions, no ·NO was detected when GSNO or CysNO were exposed to XO due to the fast formation of ONOO- (Fig. 6 and cytochrome c2+ assay).

As an alternative mechanism (mechanism 2) for the Obardot 2dependent decomposition of RSNO and ONOO- formation, we considered Reaction 3. 
<UP>GSNO</UP>+<UP>O&cjs1138;<SUB>2</SUB> → GS<SUP>⋅</SUP></UP>+<UP>ONOO<SUP>−</SUP></UP>
<UP><SC>Reaction</SC> 3</UP>
However, in this mechanism Obardot 2 would directly react with GSNO to form thiyl radical and ONOO-, and then a 1 Obardot 2:1 GSNO:1 ONOO- stoichiometry would be expected.

Also, while in mechanism 1 the GSNO/Obardot 2 reaction yields molecular oxygen (Reaction 1), no oxygen formation would occur in mechanism 2 (Reaction 3); thus, a net increase in O2 consumption should have been seen.

As a related mechanism, we also considered the possibility that Obardot 2 could reduce S-nitrosothiols to form thiyl radical and NO-, since it has been proposed that under high reductant concentrations nitroxyl anion (NO-) can be formed from RSNO (56). Nitroxyl anion could then react with molecular oxygen to form peroxynitrite (mechanism 3).
<UP>GSNO + O&cjs1138;<SUB>2</SUB> → GS<SUP>⋅</SUP></UP>+<UP>NO<SUP>−</SUP></UP>+<UP>O</UP><SUB>2</SUB>
<UP><SC>Reaction</SC> 4</UP>
<UP>NO<SUP>−</SUP></UP>+<UP>O<SUB>2</SUB> → ONOO<SUP>−</SUP></UP>
<UP><SC>Reaction</SC> 5</UP>
However, this last reaction is quite slow (1.2 × 103 M-1·s-1) (57) in comparison with NO- dimerization, which occurs at almost a diffusion-controlled rate (58), and would result in low ONOO- yields and in a lower rate of ONOO- formation than of GSNO decomposition. Furthermore, no NO- generation (i.e. nitrosyl hemoglobin formation) was detected during GSNO plus XO interactions in the presence of excess Hb3+ under aerobic conditions. As in mechanism 2, this mechanism implies an increase in oxygen consumption when GSNO is added to hypoxanthine plus XO, which was not observed. Thus, neither mechanism 2 nor mechanism 3 account for our observations.

In summary, xanthine oxidase was able to oxidize low molecular weight S-nitrosothiols by Obardot 2-dependent and -independent pathways. GSNO decomposition was fully dependent on a second order reaction with Obardot 2, while for CysNO there was also an enzymatic pathway of CysNO decomposition. GSNO did not serve as electron acceptor substrate for XO, possibly due to its larger size that may have impeded its access to the active site. It remains to be established whether other small RSNO can serve as XO substrates.

The reaction of Obardot 2 with RSNO is more than 105 times slower than with ·NO (1.0 × 104 M-1·s-1 versus 6 × 109 M-1·s-1, respectively), and a second Obardot 2 molecule would be needed for ONOO- formation. Thus, in this sense RSNO represents a mechanism that helps protect ·NO from its facile reaction with Obardot 2. However, our data also show that under physiological or pathological conditions where Obardot 2 is produced and RSNO concentrations could also be increased (2) (i.e. at sites of inflammation), Obardot 2 could promote ·NO release from RSNO and lead to ONOO- generation. In addition, we have described an enzymatic mechanism for CysNO decomposition when used as an electron acceptor for XO. Although the Km value for CysNO (0.7 mM) is high in comparison with the Km for O2, it strongly suggests that oxidoreductases, including glutathione peroxidase (59), may represent a widely used mechanism of biological ·NO release from S-nitrosothiols.

    ACKNOWLEDGEMENT

We thank Dr. Eugenio Prodanov for helpful comments.

    FOOTNOTES

* This work was supported in part by grants from the Consejo Nacional de Investigaciones Cientificas y Técnicas (138) and the Swedish Agency for Research and Cooperation (to R. R.) and National Institutes of Health FIRCA Award TW00489 (to B. A. F. and R. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported in part by a fellowship from Programma de Desarrollo de Ciencias Basicas.

Dagger Dagger To whom correspondence should be addressed: Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Avenida General Flores 2125, Montevideo 11800, Uruguay. Fax: 5982-9249563; E-mail: rradi{at}fmed.edu.uy.

1 The abbreviations and trivial name used are: RSNO, S-nitrosothiol(s); GSNO, S-nitrosoglutathione; XO, xanthine oxidase; CysNO, S-nitrosocysteine; DTNB, 5,5'-dithiobis-2-nitrobenzoic acid; dtpa, di-ethylenetriaminepentacetic acid; SOD, copper-zinc superoxide dismutase; DHR, dihydrorhodamine; neocuproine, 2,4-dimethyl-1,10- phenanthroline.

2 The term "peroxynitrite" is used to refer to either the anion (ONOO-) or its conjugate acid (ONOOH). The IUPAC recommended names are as follows: for peroxynitrite anion, oxoperoxonitrate (1-); for peroxynitrous acid, hydrogen oxoperoxonitrate; and for nitric oxide, nitrogen monoxide.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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