Unique Oxidative Mechanisms for the Reactive Nitrogen Oxide Species, Nitroxyl Anion*

Katrina M. MirandaDagger §, Michael G. EspeyDagger , Kenichi YamadaDagger , Murali KrishnaDagger , Natalie Ludwick, SungMee KimDagger , David Jourd'heuil||, Matthew B. Grisham||, Martin Feelisch||, Jon M. Fukuto, and David A. WinkDagger

From the Dagger  Tumor Biology Section, Radiation Biology Branch, NCI, National Institutes of Health, Bethesda, Maryland 20892, the  Department of Molecular Pharmacology, University of California, Los Angeles, California 90269, and the || Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130

Received for publication, July 12, 2000, and in revised form, September 26, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The nitroxyl anion (NO-) is a highly reactive molecule that may be involved in pathophysiological actions associated with increased formation of reactive nitrogen oxide species. Angeli's salt (Na2N2O3; AS) is a NO- donor that has been shown to exert marked cytotoxicity. However, its decomposition intermediates have not been well characterized. In this study, the chemical reactivity of AS was examined and compared with that of peroxynitrite (ONOO-) and NO/N2O3. Under aerobic conditions, AS and ONOO- exhibited similar and considerably higher affinities for dihydrorhodamine (DHR) than NO/N2O3. Quenching of DHR oxidation by azide and nitrosation of diaminonaphthalene were exclusively observed with NO/N2O3. Additional comparison of ONOO- and AS chemistry demonstrated that ONOO- was a far more potent one-electron oxidant and nitrating agent of hydroxyphenylacetic acid than was AS. However, AS was more effective at hydroxylating benzoic acid than was ONOO-. Taken together, these data indicate that neither NO/N2O3 nor ONOO- is an intermediate of AS decomposition. Evaluation of the stoichiometry of AS decomposition and O2 consumption revealed a 1:1 molar ratio. Indeed, oxidation of DHR mediated by AS proved to be oxygen-dependent. Analysis of the end products of AS decomposition demonstrated formation of NO2- and NO3- in approximately stoichiometric ratios. Several mechanisms are proposed for O2 adduct formation followed by decomposition to NO3- or by oxidation of an HN2O3- molecule to form NO2-. Given that the cytotoxicity of AS is far greater than that of either NO/N2O3 or NO + O&cjs1138;2, this study provides important new insights into the implications of the potential endogenous formation of NO- under inflammatory conditions in vivo.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In biological systems, nitric oxide (NO) primarily reacts with heme proteins such as guanylate cyclase (summarized in Ref. 1). Although NO is a radical, it is relatively unreactive toward other biomolecules including thiols and amines. However, a variety of redox reactions can convert NO into reactive nitrogen oxide species (RNOS)1 that have a myriad of effects in biological systems. For example, N2O3 and peroxynitrite (ONOO-), which are produced by the reaction of NO with O2 or superoxide (O&cjs1138;2), respectively, can lead to oxidation, hydroxylation, nitration, and nitrosation of biomolecules (1).

Although a substantial literature exists on the putative biological effects of other RNOS, few studies have focused on nitroxyl (NO-), the one electron reduction product of NO. Several reports suggest that NO- (or its conjugate acid, HNO) can be generated from chemical reactions that occur in vivo (2, 3) including oxidation of L-arginine by tetrahydrobiopterin-free nitric oxide synthase (NOS) (4-6) and decomposition of S-nitrosothiols (7, 8). Taken together, these studies indicate that the chemistry of NO- is an essential component of the redox chemistry of NO in biological systems.

Angeli's salt (AS) is the most commonly used synthetic donor in the study of NO- effects under biological conditions (9). At physiological pH and temperature, AS spontaneously decomposes to HNO and nitrite with a half-life of 2.5 min,
<UP>N<SUB>2</SUB>O<SUB>3</SUB><SUP>2−</SUP>+H<SUP> + </SUP>→ HNO + NO<SUB>2</SUB><SUP>−</SUP></UP> (Eq. 1)
The cytotoxic effects of AS are several orders of magnitude greater than those of other RNOS and are comparable to alkylhydroperoxides (10), suggesting that NO- formation in vivo could have deleterious consequences. In a myocardial ischemia-reperfusion model, treatment with AS markedly increased infarct area (11). In contrast, NO, either from a donor or from oxidation of NO- in the presence of an electron acceptor, afforded protection in the same model. In the present report, the chemistry of AS is compared with that of ONOO- and NO/N2O3 to gain insight into the biological mechanisms in which the chemistry of NO- could be involved.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Angeli's salt (Na2N2O3) was synthesized as described previously (10). The NONOate, DEA/NO (NaEt2NN(O)NO), was a generous gift from Dr. Joseph Saavedra (National Cancer Institute, Frederick, MD). Stock solutions (~10 mM) of AS and DEA/NO were prepared in 10 mM NaOH and stored at -20 °C (12). Peroxynitrite was synthesized by mixing solutions of 0.5 M NO2- in 0.5 M HCl and 0.5 M hydrogen peroxide (H2O2) followed by rapid quenching in 1 M NaOH, as described previously (13). The resulting basic solution was exposed to MnO2 to remove excess H2O2, which was reduced to <1% per mol of ONOO-. After filtering, aliquots were stored at -20 °C for less than 2 weeks. Directly prior to use, the concentrations of these RNOS donors in 10 mM NaOH were determined from the absorbance values at 250 nm for AS and DEA/NO (epsilon  = 8000 M-1 cm-1 (12)) and 302 nm for ONOO- (epsilon  = 1670 M-1 cm-1 (14)).

Sodium azide, sodium bicarbonate, hydroxylamine (NH2OH), glutathione (GSH), methionine, uric acid, ascorbic acid, horseradish peroxidase, manganese(IV) oxide, DTPA, and dimethylformamide (DMF) were purchased from Sigma while sodium iodide, sodium nitrite, sodium nitrate, and vanadium(III) chloride were obtained from Aldrich. Stock solutions were prepared fresh daily at 100× in Milli-Q filtered H2O unless otherwise noted. The assay buffer contained the metal chelator DTPA (50 µM) in calcium and magnesium-free Dulbecco's phosphate-buffered saline (pH 7.4) ±10 mM HEPES as indicated. All experiments were performed in triplicate at 37 °C except those involving ONOO-, which were carried out at room temperature. Data reported in text and tables are mean values from n = 2-4 individual experiments. Figures are either representative data sets or mean values, if error bars are shown, from n = 2-4 individual experiments.

Instrumentation-- UV-visible spectroscopy was performed with a Hewlett-Packard 8452A diode-array spectrophotometer. Fluorescence measurements were acquired on a PerkinElmer Life Sciences LS50B fluorometer using 2.5-mm slit widths unless otherwise stated. Oxygen consumption was determined using a Medical Systems LiCOX instrument with a Revoxode oxygen catheter-microprobe electrode (Harvard Apparatus, Holliston, MA). Chemiluminescent detection of NO was accomplished with a Sievers NO analyzer (Boulder, CO).

DHR Oxidation-- Oxidation chemistry mediated by RNOS was evaluated by formation of the fluorescent dye rhodamine 123 (RH) from dihydrorhodamine 123 (DHR) (15). Stock solutions of 50 mM DHR (10 mg; Molecular Probes, Eugene, OR) were prepared in DMF (0.6 ml) immediately prior to use. Further dilutions in DMF were prepared such that the amount of DMF added to the assay buffer was minimized (<= 10 µl). Typically, addition of DHR to 1 ml of assay buffer, containing 100× quenching agent as appropriate, was followed by RNOS donor compound (50-200×). After a 30-min incubation for AS or DEA/NO at 37 °C or 5 min at room temperature for ONOO-, 1 ml of H2O was added, and the fluorescence was measured at 570 nm with excitation at 500 nm. Since DHR slowly autoxidizes to RH, background fluorescence in assay buffer alone was subtracted from the measured values with RNOS donors. Standard curves were obtained using authentic RH (Molecular Probes). The O2 dependence of DHR oxidation by AS was examined in a similar manner except that the buffer was purged with argon and transferred by syringe to a septum-capped, argon-flushed cuvette prior to AS addition.

HPA Modification-- One-electron oxidation of p-hydroxyphenylacetic acid (HPA; Aldrich) was measured as previously reported (16). Stock solutions of 100 mM HPA in H2O were diluted in assay buffer as appropriate. The procedure is similar to that described above for DHR oxidation except that fluorescent product formation was monitored at 400 nm with 326 nm excitation. Nitration of HPA was also accomplished in an analogous manner to DHR oxidation except that following decomposition of the RNOS donor, the reaction solution was diluted in 10 mM NaOH, and the yellow product was quantified from the absorption at 430 nm (epsilon  = 4400 M-1 cm-1 (17, 18)).

Hydroxylation Reactions-- Hydroxylation of benzoic acid (BA; Aldrich; 100 mM in 100 mM NaOH) was measured under similar conditions as described for DHR oxidation except that an appropriate amount of 1 M HCl was used to neutralize added NaOH. The fluorescent product was detected at 410 nm with excitation at 290 nm and slit widths of 5.0 mm (15).

Nitrosation of DAN-- Formation of N2O3 was monitored indirectly by N-nitrosation of 2,3-diaminonaphthalene (DAN; Sigma), which yields the fluorescent product 2,3-naphthotriazole (19). Stock solutions of DAN (100 mM) were prepared in DMF. After incubation with RNOS donor and dilution with 1 ml of 10 mM NaOH, fluorescence was measured at 450 nm with excitation at 375 nm.

Oxygen Measurements-- The amount of O2 consumed by 1.7 ml of assay buffer + 10 mM HEPES containing 10-60 µM AS was determined from the amplitude of the signal decay at 37 °C assuming that 1 mm Hg corresponds to 1.4 µM O2 (20). The effects of various compounds on consumption of O2 by 60 µM AS were also examined.

Nitrite and Nitrate Assay-- The concentrations of both NO2- and NO3- after complete decomposition of RNOS in assay buffer donor were determined by chemiluminscence. Sample aliquots were injected into an anaerobic (argon-purged) reaction vessel containing either NaI/glacial acetic acid (NO2- reduction) or hot VCl3, 1 M HCl (95 °C; NO2- and NO3- reduction). The resulting NO was drawn by vacuum into the detector where it reacted with O3. The chemiluminescent reaction was quantified and integrated with a photomultiplier tube/computer system. Nitrite values were validated using the Griess reaction and were found to correspond within ±5%.

Hydrogen Peroxide Analysis-- Formation of H2O2 was measured using a fluorescence method described previously (21, 22). After decomposition of the RNOS donor in 1 ml of assay buffer, 100 µl of analyte solution (3 parts of 10 mg/ml HPA in H2O to 1 part of 10 mg/ml horseradish peroxidase in H2O) and 1 ml of buffer were added. The resulting fluorescence was monitored at 400 with 326 nm emission.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Oxidation of DHR-- Conversion of DHR into the fluorescent dye RH has been used as an in vitro dosimeter for two-electron oxidation reactions mediated by species such as ONOO- and Fe/H2O2 (23). When increasing concentrations of AS (5-100 µM) were exposed to buffer solutions containing 50 µM DHR, corresponding increases in RH production were observed (data not shown). Analogously, varying the DHR concentration (5-100 µM) in the presence of 10 µM AS resulted in a concentration-dependent increase in the observed fluorescence. The double-reciprocal plot of RH concentration versus DHR concentration is linear with -Xint-1 (defined as Xm) of 3.5 ± 0.5 µM and Yint-1 of 5.5 ± 1 µM (Fig. 1). These values represent the concentration of DHR that leads to 50% maximal RH production and the maximal yield of RH at infinite concentration of DHR for Xm and Yint-1, respectively. When DHR (10-100 µM) was exposed to 10 µM AS in argon-purged assay buffer + 10 mM HEPES, oxidation decreased by 85% compared with incubations carried out under aerobic conditions, indicating that O2 is required for NO--mediated DHR oxidation (Fig. 2).



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Fig. 1.   Oxidation of DHR by distinct reactive nitrogen oxide species. DEA/NO (triangles, 5 µM; m = 128, Yint = 1.34, r = 0.995), AS (circles; 10 µM; m = 6.75, Yint = 0.161, r = 0.999), or ONOO- (squares; 10 µM; m = 10.8, Yint = 0.294, r = 0.999) was added to assay buffer containing varying amounts of DHR (5-100 µM) and incubated as described under "Materials and Methods."



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Fig. 2.   Effect of O2 on AS-mediated DHR oxidation. 10 µM AS was exposed to DHR (10-100 µM) in aerated (black bars) or argon-purged (hatched bars) assay buffer + 10 mM HEPES.

As this O2 dependence could be explained by reaction between NO- and O2 to yield ONOO-, the oxidative properties of ONOO- were examined. In the presence of 10 µM ONOO-, increased DHR concentration resulted in a concentration-dependent increase in fluorescence, analogously to AS. The linear double-reciprocal plot exhibits an Xm of 3.5 ± 0.5 µM and an Yint-1 of 2.5 ± 0.5 µM (Fig. 1). The similar affinities of the oxidants produced from ONOO- and AS for DHR under similar reaction conditions further suggested that ONOO- might be an intermediate in the chemistry of AS.

Since CO2 can affect the chemistry of ONOO- (24), DHR oxidation was investigated in the presence of 25 mM HCO3-. Under these conditions, increasing DHR concentration resulted in increased fluorescence by both 10 µM AS and 10 µM ONOO- (data not shown). The Xm values with HCO3- are 4.0 ± 1 and 11 ± 3 µM, respectively, whereas the Yint-1 values were largely unchanged by HCO3-. Although the affinity for DHR was not altered in HCO3- buffer, synthetic ONOO--mediated DHR oxidation was quenched by CO2. Interestingly, DHR oxidation due to simultaneous exposure to xanthine oxidase (an O&cjs1138;2 generator) and NONOates in the presence of HEPES has previously been shown to be unaffected by CO2 (19).

The effect of the NO donor DEA/NO on DHR oxidation was examined to determine whether a RNOS derived from NO, such as N2O3, was a potential oxidative intermediate from AS decomposition. DEA/NO (5 µM; 2 mol of NO are produced per mol of DEA/NO) increased fluorescence intensity in a DHR concentration-dependent manner with Xm of 100 ± 25 µM and Yint-1 of 1 ± 0.5 µM (Fig. 1). The high Xm value for DEA/NO as compared with AS and ONOO- indicates that the species generated in the autoxidation of NO has a lower affinity for DHR. Since the reaction in question is oxidation of DHR, the high Xm value for DEA/NO further suggests that the reactive intermediate has a lower relative oxidation potential than those species formed from the other two RNOS donors.

Further elucidation of the chemical nature of the oxidizing species derived from AS was accomplished through the use of selective scavengers. Although 10 mM HEPES inhibits DHR oxidation, the effect is minimized at higher concentrations of DHR (i.e. 50 µM; data not shown). Therefore, for these assays, HEPES was included to provide further buffering capacity over phosphate-buffered saline alone. Azide (N3-), which is a good scavenger of NO+ donors such as N2O3, did not significantly affect the oxidation of 50 µM DHR mediated by either 10 µM AS or ONOO- (data not shown). In contrast, DHR oxidation by DEA/NO was completely quenched by 1 mM N3- under the same conditions. Moreover, although DAN was readily nitrosated by DEA/NO, neither AS nor ONOO- produced significant fluorescent product (data not shown). These data indicate that NO+ donating intermediates like N2O3 can be eliminated as possible candidates in AS chemistry. Additionally, both N3- and DAN were shown to provide excellent tools to distinguish N2O3-mediated modifications from those by AS or ONOO-.

Hydroxylamine reacts with HNO to form N2 and 2 H2O (25). When 10 µM AS was exposed to solutions containing varying amounts of NH2OH (0.05-2 mM), oxidation of 50 µM DHR was quenched such that the reciprocal plot of RH concentration versus NH2OH concentration is linear with an -Xint (defined as Xi) of 130 ± 10 µM NH2OH (data not shown). Conversely, oxidation by ONOO- was not substantially affected by NH2OH.

Numerous compounds, including GSH, methionine, urate, and ascorbate react with ONOO- (26-28). Oxidation by ONOO- was quenched proportionally with increasing GSH (0.1-2 mM) such that 50% quenching occurred at 0.7 ± 0.1 mM GSH (Fig. 3A). Nearly stoichiometric quenching was observed for AS which suggests an Xi of <10 µM. In the presence of 2-20 mM methionine, AS and ONOO--mediated DHR oxidation were similarly impeded with Xi of 2.9 ± 0.6 and 4.7 ± 0.4 mM, respectively (Fig. 3B). In the presence of increasing concentrations (0.05-1 mM) of urate (dissolved in 100 mM NaOH) or ascorbate, DHR oxidation mediated by both AS and ONOO- was quenched dramatically (Xi < 100 µM; data not shown).



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Fig. 3.   Quenching of AS or ONOO--mediated DHR oxidation by GSH (A) and methionine (B). AS or ONOO- (10 µM) was incubated with DHR (50 µM) in the presence of increasing concentrations of either GSH or methionine in assay buffer + 10 mM HEPES.

Oxidation and Nitration of HPA-- The chemistry associated with reactive oxygen species and RNOS can be examined using HPA. In the presence of powerful oxidants such as those formed from peroxidases and H2O2, HPA is converted into a fluorescent dimer through an initial one-electron oxidation (16). The extent of nitration can also be evaluated through modification of HPA to the yellow product, 3-nitro-HPA (17, 18).

Exposure of ONOO- (50-500 µM) to solutions containing 1 mM HPA resulted in a concentration-dependent increase in fluorescence (Fig. 4). However, AS did not appreciably oxidize HPA under the same conditions. Chromophore bleaching by AS (0.1 and 1 mM) of HPA solutions previously exposed to ONOO- to produce the fluorescent dimer was not observed (data not shown). This suggests that AS per se did not interfere with the fluorescent product. These results show that ONOO- readily mediates one-electron oxidation while AS does not.



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Fig. 4.   Extent of one-electron oxidation as monitored by HPA dimerization. Relative fluorescence intensity obtained after incubation of ONOO- or AS (50-500 µM) with 1 mM HPA as described under "Materials and Methods."

Previous studies demonstrated that CO2 enhances nitration (29-31). High concentrations of ONOO- (>100 µM) readily nitrated 1 mM HPA, and in the presence of 25 mM HCO3- considerably more nitration product was observed (data not shown). Conversely, when HPA was exposed to AS, a peak at 430 nm was not detected in the presence or absence of HCO3-. This is consistent with a report that suggests that free tyrosine is nitrated by ONOO- but not by AS (32). Indeed, high performance liquid chromatography analysis indicated that at 5 mM RNOS donor, with or without HCO3-, significant formation of 3-nitrotyrosine occurred with ONOO- and SIN-1, which cogenerates NO and O&cjs1138;2, but not with AS (data not shown).

Hydroxylation of BA-- Modification of BA to the fluorescent compound 2-hydroxybenzoic acid (2-OHBA) was used to assess hydroxylating capacity. Exposure of 1 mM BA to increasing concentrations of ONOO- or AS (0.05-1 mM) resulted in a concentration-dependent increase in fluorescence (Fig. 5A). Double-reciprocal plots of fluorescence intensity versus BA concentration (0.5-10 mM), as a result of exposure to 500 µM RNOS donor, are linear with Xm of 1.2 ± 0.5 and 1.8 ± 0.5 mM and Yint-1 of 1055 ± 100 and 100 ± 20 for AS and ONOO-, respectively (Fig. 5B).



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Fig. 5.   Hydroxylation of BA. A, increasing concentrations of AS or ONOO- (0.05-1 mM) were exposed to 1 mM BA in assay buffer. B, increasing concentrations of BA (0.5-10 mM) were exposed to either 500 µM AS (m = 0.00114, Yint = 0.000948, r = 0.999) or 500 µM ONOO- (m = 0.0171, Yint = 0.00959, r = 0.999).

Oxygen Consumption by AS-- Under anaerobic conditions DHR oxidation by AS was considerably lower than in the presence of O2 (Fig. 2). To gain insight into the nature of the intermediate involved, the amount of O2 consumed by AS (10-60 µM) in assay buffer + 10 mM HEPES was measured electrochemically. The O2 concentration decreased with a half-life similar to that observed for decomposition of AS and with an approximate 1:1 stoichiometric ratio between AS (and thus NO-) and O2 (Fig. 6).



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Fig. 6.   Oxygen consumption by 60 µM AS in the presence of various compounds in assay buffer + 10 mM HEPES.

Further measurements were performed to indicate whether O2 is required for reaction of AS with various compounds (Fig. 6). Addition of GSH markedly inhibited O2 consumption, suggesting that NO-/HNO reacts directly with this nucleophile. Ascorbate and NH2OH also reduced O2 consumption by AS, although to a lesser degree. Conversely, DHR, methionine, urate, and BA did not substantially alter the amount of O2 consumed, indicating that these compounds either do not react directly with NO- or that the reaction rate of NO- with O2 is faster than with these compounds. These observations show that the chemistry of AS occurs through both O2-dependent and independent mechanisms.

End Product Analysis-- Chemiluminescent analysis of the nitrogenous end products from the decomposition of AS under aerobic conditions provided further information as to the chemical nature of the intermediate. Significantly more NO2- than NO3- was detected. However, since AS decomposes to equimolar amounts of NO2- and NO- (Equation 1), at least 50% of the NO2- formed is not due to the chemistry of NO-. Subtracting the AS concentration from the total NO2- concentration results in approximately stoichiometric yields of NO2- and NO3- formed from NO- chemistry alone (data not shown). The NO3- values are subject to significant error particularly at the lower concentrations due to signal to noise restraints and the differential calculation involved ([NO3-] = ([NOx-] as determined from VCl3 reduction) - (total [NO2-] as determined from NaI reduction - [AS]). The product yields varied with AS concentration due to the spontaneous dimerization of HNO, which competes with the reaction of NO- and O2,


<UP>2HNO → N<SUB>2</SUB>O + H<SUB>2</SUB>O</UP> (Eq. 2)
Hydrogen peroxide and O&cjs1138;2 have been proposed as products of AS decomposition in aerated solution (2). Following AS (5-100 µM) decomposition in assay buffer, the samples were assayed for peroxide by the peroxidase/HPA assay (21, 22). Formation of H2O2 was dependent upon HEPES in a dose-dependent manner (<1% without HEPES, 10-25% with HEPES; data not shown). In comparison, decomposition of ONOO- produced 2-3-fold less H2O2 under identical conditions. As the metal chelator DTPA was always present in the assay buffer, the measured chemistry was not a result of Fenton reactions with contaminating metals.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Nitroxyl can be formed from various reactions and has been proposed to be an important intermediate in the in vivo metabolism of NO (10). However, the role of this RNOS in biological systems is not clear, partly due to the lack of understanding of its chemical properties. Several studies have suggested that NO-/HNO is a product of NOS (4-6). Although controversial, this possibility cannot as yet be discounted. Additional sources of NO- may be the decomposition of S-nitrosothiols (7, 8) or the dissociation of ONOO- to NO- and singlet oxygen (1O2) (33). Other studies examining the effects of NO- in vitro demonstrated that the synthetic NO- donor AS was orders of magnitude more cytotoxic than NO or other RNOS (10). This cytotoxicity was dependent upon molecular oxygen and was at least in part due to the ability of AS to produce double strand breaks in cellular DNA, similarly to ionizing radiation and chemotherapeutic drugs. Furthermore, AS has been shown to increase infarct size about 3-fold in an in vivo model of myocardial ischemia-reperfusion injury (11). These studies indicate that nitroxyl has proinflammatory and toxic properties. In addition, AS was reported to affect specific neuronal channels, suggesting an involvement of NO- in neurotoxicity (34).

The reactivity of AS toward several compounds was examined to gain insight into the chemistry of the intermediates formed during AS decomposition. The two-electron oxidation of DHR by AS was dependent on the presence of O2 (Fig. 2), indicating formation of a reactive intermediate from the reaction of NO- with O2. This reaction was found to be stoichiometric (Fig. 6), which suggests that ONOO- may be a participant in AS chemistry. The affinities of ONOO- and AS for DHR were similar, and significantly higher than that of the NO donor DEA/NO, although the yield of RH was ~3-fold higher with AS under all conditions examined (Fig. 1 and Table I). The possibility of intermediate formation of NO/N2O3 during AS decomposition was definitively eliminated by the lack of specific quenching of DHR oxidation by N3- and by the inability of AS to nitrosate DAN.


                              
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Table I
RNOS donor oxidation of DHR and BA

The RNOS scavengers methionine, urate, and ascorbate, in the presence of 10 mM HEPES, quenched DHR oxidation to a similar degree for both AS and ONOO- (Table II and Fig. 3B). However, DHR oxidation by AS was ~100-fold more sensitive to quenching by GSH than that by ONOO- (Fig. 3A). Our data suggest that NO-/HNO reacts directly with GSH rather than with the O2-derived product as O2 consumption by AS was also quenched by GSH (Fig. 6). In fact, GSH has a high affinity for HNO (35), and this reaction in the presence of excess GSH results in disulfide formation through the intermediate, GSNOH.
<UP>HNO + GSH → GSNHOH</UP> (Eq. 3)

<UP>GSNHOH + GSH → NH<SUB>2</SUB>OH + GSSG</UP> (Eq. 4)
This mechanism may represent the primary cellular defense against NO- toxicity in the cell, which is supported by the observation that exposure to AS results in a dramatic reduction in intracellular GSH (10). Furthermore, the selective quenching of AS-related chemistry by GSH, and that of NO/N2O3 by N3-, provides a useful tool to differentiate biological effects of NO- and NO from other RNOS.


                              
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Table II
Quenching of AS and ONOO- mediated DHR oxidation

Although similarities in the two-electron oxidation of DHR by AS and ONOO- were observed, there were notable differences in the one-electron oxidation of HPA and the hydroxylation of BA. Whereas HPA was readily oxidized by ONOO- (16), AS did not form substantial fluorescent product, suggesting that NO- does not oxidize aromatic bio-organic molecules through a radical reaction (Fig. 4). Conversely, BA hydroxylation by AS was more efficient than with ONOO- (Fig. 5 and Table I). From these data it may be deduced that NO- mediates two-electron but not one-electron oxidation, while ONOO- readily participates in either reaction.

Nitration and nitrosation are also important reactions in the metabolism of NO. Nitration (NO2+ donation) by ONOO- is increased in the presence of CO2 while ONOO--mediated nitrosation reactions (NO+ donation) occur only in the presence of excess NO (15, 19, 36). Under optimal conditions, nitration of HPA was observed only with ONOO- (data not shown). However, a recent report has shown that although free tyrosine is not affected by AS, it will nitrate tyrosyl residues in proteins (32). This observation raises the possibility that NO- may be an important source of nitrated proteins in vivo, and this warrants further investigation. Both ONOO- and AS lack the ability to nitrosate DAN, which is readily accomplished by DEA/NO. In addition, the reaction of AS with RSH was reported to produce RSNHOH (Equation 3) rather than RSNO (35). Taken together, the conclusion can be made that NO- does not appreciably nitrosate or nitrate low molecular weight compounds.

In summary, our observations show that ONOO- readily mediates both one- and two-electron oxidation, while the intermediate in the reaction of NO- with O2 only efficiently mediates two-electron oxidation. Additionally, ONOO- alone can nitrate low molecular phenols while AS is a more powerful hydroxylating agent. From these comparisons it can be concluded that ONOO- is not an intermediate formed between the reaction of O2 and NO- from AS. The dissimilarities in overall chemistry also indicate that the primary pathway of ONOO- reactivity is not dissociation to NO- and 1O2 as suggested by Khan et al. (33).

Fig. 2 clearly demonstrates that two-electron oxidation occurs through an O2-dependent mechanism. Heterolytic cleavage of HN2O3- should result in 1HNO and 1NO2- formation (Equation 1) (37, 25). Numerous plausible activation mechanisms of 1HNO can be envisioned (Scheme 1), and the validity of each is discussed in detail in the following section.



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Scheme 1.  

The pathways depicted in Scheme 1 depend upon the lifetime of 1HNO relative to 1NO-. The pKa for HNO is reported to be 4.7 (38), which should result in over 99% deprotonated species at pH 7.4. Intersystem crossing of 1NO- to 3NO- followed by reaction with O2 (ground state triplet) would be predicted to produce ONOO- (37),
<SUP><UP>1</UP></SUP><UP>HNO + H<SUB>2</SUB>O ⇌<SUP> 1</SUP>NO<SUP>−</SUP></UP>(<UP>p</UP>K<SUB>a</SUB>) (Eq. 5)

<SUP><UP>1</UP></SUP><UP>NO<SUP>−</SUP> →<SUP> 3</SUP>NO<SUP>−</SUP></UP>(k<SUB><UP>ISC</UP></SUB>) (Eq. 6)

<SUP><UP>3</UP></SUP><UP>NO<SUP>−</SUP> + O<SUB>2</SUB> → ONOO<SUP>−</SUP></UP>(k<SUB><UP>4</UP></SUB>) (Eq. 7)
Since ONOO- is not formed from AS decomposition, this pathway cannot be kinetically favorable. Direct reaction of either 1HNO or its conjugate base 1NO- with 3O2 would be spin-forbidden, and thus the k2 and k3 pathways would be expected to be too slow to be significant. However, as kISC is likely to be of similar magnitude to intersystem crossing of 1O2 to 3O2, which occurs in milliseconds (k = 103 s-1), k2 and k3 may in fact exceed k4. To be a kinetically viable pathway under the experimental conditions, the rate of the appearance of the k3 product,
<SUP><UP>1</UP></SUP><UP>NO<SUP>−</SUP> + O<SUB>2</SUB> → ONOO<SUP>−</SUP></UP>(k<SUB><UP>3</UP></SUB>) (Eq. 8)
would have to exceed the rate for ONOO- production by intersystem crossing, assuming kISC is the rate-limiting step, as k4 = 109 M-1 s-1 (assuming a similar rate constant for ONOO- formation from NO and O&cjs1138;2 (39)) and [O2] = 2 × 10-4 M (40).
d[<UP>ONOO<SUP>−</SUP></UP>]<UP>/</UP>dt=k<SUB>3</SUB>[<UP>O<SUB>2</SUB></UP>][<SUP><UP>1</UP></SUP><UP>NO<SUP>−</SUP></UP>] (Eq. 9)

d[<UP>ONOO<SUP>−</SUP></UP>]<UP>/</UP>dt=k<SUB><UP>ISC</UP></SUB>[<SUP><UP>1</UP></SUP><UP>NO<SUP>−</SUP></UP>] (Eq. 10)
The ratio of k3 to kISC would thus be solely dependent upon the O2 concentration,
k<SUB>3</SUB>[<UP>O<SUB>2</SUB></UP>]>k<SUB><UP>ISC</UP></SUB> (Eq. 11)
and k3 would have to exceed kISC by 5 × 103 to be of kinetic importance.

For the reaction between 1HNO and O2 to occur by the k2 pathway,
<SUP><UP>1</UP></SUP><UP>HNO + O<SUB>2</SUB> → HN<SUP>+</SUP></UP>(<UP>O</UP>)<UP>OO<SUP>−</SUP></UP>(k<SUB>2</SUB>) (Eq. 12)
k2[O2][HNO] would have to exceed kISC[1NO-] as detailed above. Substituting Ka[HNO][H3O+]-1 for [1NO-] results in,
k<SUB>3</SUB>[<UP>O<SUB>2</SUB></UP>][<UP>HNO</UP>]>k<SUB><UP>ISC</UP></SUB>K<SUB>a</SUB>[<UP>HNO</UP>][<UP>H<SUB>3</SUB>O<SUP>+</SUP></UP>]<SUP><UP>−1</UP></SUP> (Eq. 13)
which at pH 7.4 becomes
k<SUB>3</SUB>>2×10<SUP>7</SUP> k<SUB><UP>ISC</UP></SUB> (Eq. 14)
If indeed kISC for NO- is comparable to that for O2 (103 s-1), k3 and k2 would then have to exceed 5 × 106 and 2 × 107 M-1 s-1, respectively. Both these calculated rate constants may or may not be too fast for spin forbidden reactions.

Reaction of 1NO- with O2 could be expected to result in peroxynitrite formation (Equation 8). However, the difference in charge distribution between the states of nitroxyl (1HNO versus 3NOH (41-43)) may in fact lead to formation of distinct isomers of ONOO-. For example, one can envision that reaction with O2 could lead to formation of a 4-membered ring for -NO and a 3-membered ring with NO- (Scheme 2). Reaction of 1HNO with O2 may impart additional stability to either a zwitterion (Equation 12; Scheme 1) or a 4-membered ring (Scheme 2) over isomerization to ONOO-.
<SUP><UP>1</UP></SUP><UP>HNO + O<SUB>2</SUB> → HN<SUP>+</SUP></UP>(<UP>O</UP>)<UP>OO<SUP>−</SUP></UP>(K<SUB>2</SUB>) (Eq. 12)
Both DHR oxidation and O2 consumption during AS decomposition were inhibited by GSH (Figs. 3A and 6), and thus GSH (Xi < 10-5 M) directly competes with intersystem crossing. Therefore, from the rate-limiting equations (Equation 3 and 6),
k<SUB><UP>GSH</UP></SUB>[<UP>GSH</UP>][<UP>HNO</UP>]>k<SUB><UP>ISC</UP></SUB>K<SUB>a</SUB>[<UP>HNO</UP>][<UP>H<SUB>3</SUB>O<SUP>+</SUP></UP>]<SUP><UP>−1</UP></SUP> (Eq. 15)
which simplifies to,
k<SUB><UP>GSH</UP></SUB>(10<SUP>−5</SUP> <SC>m</SC>)>k<SUB><UP>ISC</UP></SUB>(2×10<SUP>−5</SUP>)(4×10<SUP>−8</SUP> <SC>m</SC>)<SUP>−1</SUP> (Eq. 16)
or with the estimation that kISC = 103 s-1 to,
k<SUB><UP>GSH</UP></SUB>>5×10<SUP>10</SUP> <SC>m</SC><SUP>−1</SUP> <UP>s<SUP>−1</SUP>.</UP> (Eq. 17)
This rate constant is unreasonably fast suggesting either that kISC < 103 s-1 or that the pKa is greater than the reported value of 4.7. In either case, reaction of HNO or 1NO- with O2 would become increasingly favorable.



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Scheme 2.  

An alternate mechanism may involve hydrolysis of HNO (Scheme 1). HNO has been demonstrated to be easily scavenged by nucleophiles such as GSH (Equation 3, Fig. 3A) (35) and NH2OH (25, 43) (Table II) and also avidly reacts with sulfhydryls (25, 44). Similarly, HNO may be hydrated to form HN(OH)2. This adduct may provide a lower energy mechanism to facilitate reaction with O2 (k1), perhaps through an outer sphere electron transfer, which would alleviate the spin forbiddeness. The direct reaction products of HNO/NO- with O2 may also react with water in this fashion. The HN(OH)2OO species, or possibly a cyclic iosmer, formed from any of these pathways, could then oxidize or hydroxylate compounds or decompose to the measured products.
<SUP><UP>1</UP></SUP><UP>HNO + H<SUB>2</SUB>O ⇌ HN</UP>(<UP>OH</UP>)<SUB><UP>2</UP></SUB> (Eq. 18)

<UP>HN</UP>(<UP>OH</UP>)<SUB><UP>2</UP></SUB><UP> + O<SUB>2</SUB> → HN</UP>(<UP>OH</UP>)<SUB><UP>2</UP></SUB><UP>OO</UP>(k<SUB>1</SUB>) (Eq. 19)

<UP>HN</UP>(<UP>OH</UP>)<SUB><UP>2</UP></SUB><UP>OO → NO<SUB>3</SUB><SUP>−</SUP> + H<SUB>2</SUB>O</UP> (Eq. 20)

<UP>HN</UP>(<UP>OH</UP>)<SUB><UP>2</UP></SUB><UP>OO + reactant → NO</UP><SUB><UP>2<SUP>−</SUP></UP></SUB><UP> + H<SUB>2</SUB>O + oxidized product</UP> (Eq. 21)
Kinetic favorability for this pathway would require that,
k<SUB>1</SUB>[<UP>O<SUB>2</SUB></UP>][<UP>HN</UP>(<UP>OH</UP>)<SUB><UP>2</UP></SUB>]>k<SUB><UP>ISC</UP></SUB>K<SUB>a</SUB>[<SUP><UP>1</UP></SUP><UP>NO<SUP>−</SUP></UP>] (Eq. 22)
which simplifies to,
k<SUB>1</SUB>[<UP>O<SUB>2</SUB></UP>]K<SUB><UP>H<SUB>2</SUB>O</UP></SUB>[<UP>HNO</UP>]>k<SUB><UP>ISC</UP></SUB>K<SUB>a</SUB>[<UP>HNO</UP>][<UP>H<SUB>3</SUB>O<SUP>+</SUP></UP>]<SUP><UP>−1</UP></SUP> (Eq. 23)
or to,
k<SUB>1</SUB>K<SUB><UP>H<SUB>2</SUB>O</UP></SUB>>1×10<SUP>11</SUP>k<SUB><UP>ISC</UP></SUB>K<SUB>a</SUB> (Eq. 24)
Unless KH2O is large or the pKa is substantially higher than 4.7, reaction with H2O would be expected to preferentially lead to formation of the conjugate base. However, either the cyclic or water adduct mechanisms can mechanistically account for the similarities and differences observed between AS and ONOO- chemistry as well as for the stoichiometric consumption of O2 and the production of the measured endproducts NO2- and NO3- in approximately equimolar ratios.

An additional possibility involves dimerization of NO- to form (NO)22-. In the presence of O2, (NO)22- could be converted to the oxidant NO(NO)- and O&cjs1138;2, which would dismutate to H2O2.
<UP>2NO<SUP>−</SUP> → </UP>(<UP>NO</UP>)<SUB><UP>2</UP></SUB><SUP><UP>2−</UP></SUP> (Eq. 25)

<UP>NO</UP>(<UP>NO</UP>)<SUP><UP>2−</UP></SUP><UP>+ 2H<SUP>+</SUP> → N<SUB>2</SUB>O + H<SUB>2</SUB>O</UP> (Eq. 26)

<UP>NO</UP>(<UP>NO</UP>)<SUP><UP>2−</UP></SUP><UP> + O<SUB>2</SUB> → NO</UP>(<UP>NO</UP>)<SUP><UP>−</UP></SUP><UP> + O<SUB>2</SUB><SUP>−</SUP></UP> (Eq. 27)

<UP>2O<SUB>2</SUB><SUP>−</SUP> + 2H<SUP>+</SUP> → H<SUB>2</SUB>O<SUB>2</SUB> + O<SUB>2</SUB></UP> (Eq. 28)

<UP>NO</UP>(<UP>NO</UP>)<SUP><UP>−</UP></SUP><UP> + H<SUP>+</SUP> → N<SUB>2</SUB>O + HO · </UP> (Eq. 29)
This mechanism would predict formation of H2O2 and NO2-, as the exclusive measured end product (from Equation 1), as well as a 1:2 ratio of O2 to AS consumption; however, such results were not obtained. Moreover, one-electron oxidation of HPA by hydroxyl radicals would be expected, but this was not observed with AS. These dissimilarities argue against NO(NO)- as the oxidizing intermediate from NO-.

It appears that NO- has two basic chemical mechanisms (Scheme 3). The first involves O2- independent reactions where NO- HNO interacts directly with biological compounds to lead to oxidation of thiols, amines, and NADPH, reductive nitrosylation of metals, and reduction of superoxide dismutase. Reactions of this type may primarily lead to quenching of NO- chemistry (e.g. by reaction with GSH) or to oxidation to NO (e.g. by reaction with oxidizing agents or with ferriheme proteins).



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Scheme 3.  

The second category of reactions occurs after combination with O2 to presumably form either O2N(OH)2- or an isomer of ONOO-, which has a distinct profile of oxidation, hydroxylation, nitrosation, and nitration than those of other RNOS. This suggests a unique biological signature for this species, which is responsible for the O2-dependent cytotoxicity observed with AS (10). Since AS is a more potent cytotoxin than donors that deliver NO/N2O3 (e.g. NONOates such as DEA/NO) or NO/O&cjs1138;2 (e.g. SIN-1 or xanthine oxidase/NONOates) (10) and may be produced endogenously via the NO synthase reaction (4-6), NO- may be involved in certain pathophysiological mechanisms.

The biological role of NO-, like that of other RNOS, is not clearly defined at this time. However, the potential for its derivation directly from NOS activity or from decomposition of S-nitrosothiols raises a number of interesting questions. Of particular interest is whether nitroxyl dissociates from biological compounds as HNO or NO-, since the resulting chemistry of these species can vary drastically. With two distinct types of reactivity, location will be critical to the implications of NO- in different disease states. Clearly, the reaction between O2 and NO- will primarily lead to two-electron oxidation and hydroxylation of compounds and does not yield the same product as the reaction of NO with O&cjs1138;2.


    ACKNOWLEDGEMENT

We are grateful to Prof. Peter C. Ford for insightful discussions.


    FOOTNOTES

* 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.

§ To whom correspondence should be addressed: Radiation Biology Branch, NCI, National Institutes of Health, Bldg. 10, Rm. B3-B69, Bethesda, MD 20892. Tel.: 301-496-7511; Fax: 301-480-2238; E-mail: kmiranda@box-k.nih.gov.

Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.M006174200


    ABBREVIATIONS

The abbreviations used are: RNOS, reactive nitrogen oxide species; AS, Angeli's salt; BA, benzoic acid; DAN, 2,3-diaminonaphthalene; DEA/NO, diethylamine/nitric oxide adduct; DHR, dihydrorhodamine 123; DMF, dimethylformamide; DTPA, diethylenetriaminepentaacetic acid; GSH, glutathione; HPA, p-hydroxyphenylacetic acid; NOS, nitric oxide synthase; 2-OHBA, 2-hydroxybenzoic acid; RH, rhodamine 123.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


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