Unique Oxidative Mechanisms for the Reactive Nitrogen Oxide
Species, Nitroxyl Anion*
Katrina M.
Miranda
§,
Michael G.
Espey
,
Kenichi
Yamada
,
Murali
Krishna
,
Natalie
Ludwick¶,
SungMee
Kim
,
David
Jourd'heuil
,
Matthew B.
Grisham
,
Martin
Feelisch
,
Jon M.
Fukuto¶, and
David A.
Wink
From the
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 |
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
2, this study
provides important new insights into the implications of the potential
endogenous formation of NO
under inflammatory conditions
in vivo.
 |
INTRODUCTION |
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
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,
|
(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 |
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
(
= 8000 M
1 cm
1 (12))
and 302 nm for ONOO
(
= 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
(
= 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 |
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
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.
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|
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
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).
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|
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.
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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,
|
(Eq. 2)
|
Hydrogen peroxide and O
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 |
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.
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.
|
(Eq. 3)
|
|
(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.
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.
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),
|
(Eq. 5)
|
|
(Eq. 6)
|
|
(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,
|
(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
2 (39))
and [O2] = 2 × 10
4 M
(40).
|
(Eq. 9)
|
|
(Eq. 10)
|
The ratio of k3 to
kISC would thus be solely dependent upon the
O2 concentration,
|
(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,
|
(Eq. 12)
|
k2[O2][HNO] would have to
exceed kISC[1NO
] as
detailed above. Substituting
Ka[HNO][H3O+]
1
for [1NO
] results in,
|
(Eq. 13)
|
which at pH 7.4 becomes
|
(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
.
|
(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),
|
(Eq. 15)
|
which simplifies to,
|
(Eq. 16)
|
or with the estimation that kISC = 103 s
1 to,
|
(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.
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.
|
(Eq. 18)
|
|
(Eq. 19)
|
|
(Eq. 20)
|
|
(Eq. 21)
|
Kinetic favorability for this pathway would require that,
|
(Eq. 22)
|
which simplifies to,
|
(Eq. 23)
|
or to,
|
(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
2, which would
dismutate to H2O2.
|
(Eq. 25)
|
|
(Eq. 26)
|
|
(Eq. 27)
|
|
(Eq. 28)
|
|
(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).
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
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
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.
 |
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