 |
INTRODUCTION |
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, O
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 O
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/O
2 =
0.33
V, Ref. 22); thus, the question readily arises as to whether
O
2 can promote RSNO decomposition, i.e. ·NO
release. Since O
2 reacts with ·NO at almost
diffusion-controlled rates (19) to form peroxynitrite anion
(ONOO
), O
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
O
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 O
2-dependent and
-independent mechanisms and, according to the availability of oxygen in
the system, secondarily leads to peroxynitrite2 formation.
 |
EXPERIMENTAL PROCEDURES |
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 M
·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 (
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 (
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
(
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
(
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 (
= 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 O
2 flux. The oxidation of DHR to rhodamine was followed spectrophotometrically at 500 nm
(
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 |
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. O
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).

View larger version (11K):
[in this window]
[in a new window]
|
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.
|
|

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 2.
Dependence of S-nitrosothiol
decomposition on XO activity. GSNO ( ) or CysNO ( ) (1 mM) was incubated in air-equilibrated buffer during 20 min
in the presence of 150 µM hypoxanthine at different XO
activities.
|
|
View this table:
[in this window]
[in a new window]
|
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.
|
|

View larger version (9K):
[in this window]
[in a new window]
|
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).
|
|
View this table:
[in this window]
[in a new window]
|
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).
View this table:
[in this window]
[in a new window]
|
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.
|
|

View larger version (13K):
[in this window]
[in a new window]
|
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
( ) or uric acid formation ( ) 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).

View larger version (12K):
[in this window]
[in a new window]
|
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 O
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
O
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 O
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).

View larger version (13K):
[in this window]
[in a new window]
|
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 O
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 |
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 O
2 was a key participating species. The
reduction of GSNO by O
2 would lead to the formation of GSH and
·NO as follows.
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, O
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 O
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
O
2 reacts at very fast rates (k = 6.7 × 109 M
1·s
1; Ref.
19) with ·NO.
Thus, O
2 would be consumed by two main processes
(Reactions 1 and 2), and maximum reaction yields imply that for every
two molecules of O
2 consumed, there should be one molecule of
GSNO decomposed and one molecule of ONOO
formed (2 O
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 O
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 O
2 (Reactions 1 and 2), leading to the same
stoichiometry of O2 return to that observed for
O
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 O
2
with GSNO. In the absence of SOD, the rate of O
2 consumption due to reactions with GSNO and ·NO is expressed by the
following.
|
(Eq. 1)
|
In the presence of SOD, the rate of O
2 consumption is
expressed by the equation,
|
(Eq. 2)
|
where kGSNO, kNO,
and kSOD represent the rate constants for the
reaction of O
2 with GSNO, ·NO, and SOD,
respectively.
Under steady state conditions, ·NO concentration can be
calculated by the following.
|
(Eq. 3)
|
Rearranging Equation 2, we get Equation 4.
|
(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.
|
(Eq. 5)
|
|
(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 O
2 only partially contributes to CysNO
decomposition along with other XO-mediated, O
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).

View larger version (15K):
[in this window]
[in a new window]
|
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
O 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 O
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 O
2 generated at different
CysNO concentrations due to the fact that O
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
O
2dependent decomposition of RSNO and ONOO
formation, we considered Reaction 3.
However, in this mechanism O
2 would directly react with
GSNO to form thiyl radical and ONOO
, and then a 1 O
2:1 GSNO:1 ONOO
stoichiometry would be
expected.
Also, while in mechanism 1 the GSNO/O
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
O
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).
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 O
2-dependent and
-independent pathways. GSNO decomposition was fully dependent on a
second order reaction with O
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 O
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 O
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 O
2. However,
our data also show that under physiological or pathological conditions
where O
2 is produced and RSNO concentrations could also be
increased (2) (i.e. at sites of inflammation), O
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.
We thank Dr. Eugenio Prodanov for helpful
comments.