(Received for publication, September 10, 1996, and in revised form, November 19, 1996)
From the Institute for Environmental Medicine, the
§ Departments of Physiology and Bioengineering, and the
¶ Department of Biochemistry and Biophysics, University of
Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
The objective of this study was to investigate
the mechanism of S-nitrosothiol formation under
physiological conditions. A mechanism is proposed by which nitric oxide
(·NO) reacts directly with reduced thiol to produce a radical
intermediate, R-S-N·-O-H. This intermediate reduces an electron
acceptor to produce S-nitrosothiol. Under aerobic
conditions O2 acts as the electron acceptor and is reduced
to produce superoxide (O2). The following experimental
evidence is provided in support of this mechanism. Cysteine accelerates
the consumption of ·NO by 2.5-fold under physiological
conditions. The consumption of O2 in the presence of
·NO and cysteine is increased by 2.4-fold. The reaction orders of ·NO and cysteine are second and first order, respectively.
The second order of reaction for ·NO may result from interaction
between ·NO and O
2 to form peroxynitrite. In the
presence of Cu,Zn-superoxide dismutase, the reaction of ·NO with
cysteine generates hydrogen peroxide, indicating that the reaction
generates O
2. Finally, the formation of
S-nitrosothiol is demonstrated in an anaerobic environment
and, as predicted by the mechanism, is dependent on the presence of an
electron acceptor. These results demonstrate that under physiological
conditions ·NO reacts directly with thiols to form
S-nitrosothiol in the presence of an electron acceptor.
S-Nitrosothiols are important physiological regulators capable of producing vasodilation and inhibition of platelet aggregation (1-4). An increasing number of proteins such as albumin, glyceraldehyde-3-phosphate dehydrogenase, hemoglobin, and p21ras have been found to be S-nitrosylated in vivo (5-9). With the discovery of new S-nitrosylated proteins, it becomes evident that the formation of S-nitrosothiols may be important in such diverse processes as signal transduction, DNA repair, and blood-pressure regulation. However, at present the mechanism of the biosynthetic pathway for the formation of S-nitrosothiols is unclear (10).
It has been shown previously that the reaction of ·NO1 with sulfhydryl groups under anaerobic conditions at neutral pH does not produce S-nitrosothiol (11-13). This has led to the conclusion that S-nitrosothiols are formed by the autoxidation of ·NO, a second order reaction with respect to ·NO, to higher oxides of nitrogen (NOx), by metal catalysis (12, 14, 15), or by the action of dinitrosyl-iron complexes (16). However, the reaction of ·NO and oxygen is slow, approximately 3 to 300 pmol/s, at physiological concentrations of ·NO (0.1-1.0 µM), and the availability of redox metal is unclear (17, 18). Although dinitrosyl-iron complexes represent one possible mechanism for the formation of S-nitrosothiols, the reaction mechanism under physiological conditions also remains unclear (10).
Here we propose a novel mechanism for S-nitrosothiol formation that would operate at physiological concentrations of ·NO. In this mechanism ·NO reacts directly with a reduced thiol to produce a radical intermediate, R-S-N·-O-H (Equation 1). In the presence of an electron acceptor, such as oxygen, this intermediate can be converted to S-nitrosothiol by the reduction of the acceptor.
Therefore, the reaction of ·NO and cysteine in buffer under aerobic conditions will form superoxide via the reduction of O2 by R-S-N·-O-H (Equation 2). Superoxide reacts at a nearly diffusion-limited rate with ·NO to form peroxynitrite (19) (Equation 3). The overall reaction mechanism is shown below (Equation 4).
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(Eq. 1) |
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(Eq. 2) |
![]() |
(Eq. 3) |
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(Eq. 4) |
Cu,Zn-superoxide dismutase was obtained from Fluka (Switzerland), and DEANO was obtained from Cayman Chemical (Ann Arbor, MI). All other chemicals were obtained from Sigma. Spectrophotometric measurements were made using a UV diode array spectrophotometer (Hewlett-Packard), and ·NO in solution was measured using a ·NO-specific electrode (World Precision Instruments, Sarasota, FL).
Synthesis and Measurement of ·NONitric oxide was synthesized by bubbling nitrogen through KNO2 that was acidified with HCl. Nitric oxide and other nitrogen oxides formed in the reaction flask were forced through a gas-washing column containing 1 M NaOH. Nitric oxide was then collected in a nitrogen-purged, sealed vessel containing double distilled H2O that was passed through a Chelex-100 column to remove metal contaminants.
Nitric oxide concentrations were continuously monitored by means of a specific electrode in a 1-ml solution of PBS, pH 7.4, 100 µM DTPA at a constant stirring velocity and at constant temperature (22 °C) in a 46-well plate. Electrode output was recorded on a chart recorder, and initial rates of decay were measured as tangents within 10 s of reaching peak ·NO concentration. Cysteine was added from a freshly prepared 10 mM stock solution upon reaching peak ·NO concentration. In order to measure release of ·NO from reaction products, ·NO was added to PBS, 100 µM DTPA containing cysteine. The reaction was allowed to proceed for 1.5 min, and then the solution was vortexed vigorously in three 10-s bursts to remove any free ·NO remaining from the reaction. ·NO release was then monitored as described above.
Measurement of O2 and H2O2 in SolutionHydrogen peroxide and oxygen measurements were made by
adding authentic ·NO to PBS, 100 µM DTPA, 2,400 units of Cu,Zn superoxide dismutase in the presence or absence of
cysteine. All measurements were made in an open system at a constant
stirring speed and temperature (22 °C). Oxygen was continuously
monitored by electrochemical reduction using a recessed microelectrode
with a gold cathode polarized at 0.65 V relative to an Ag/AgCl
reference electrode (20). The current sensitivity of the microelectrode
was 1.2 pA/µM. Hydrogen peroxide was measured by
electrochemical oxidation using a 25-µm diameter platinum wire sealed
in glass that was polarized at 0.65 V relative to an Ag/AgCl reference
electrode (21). The electrode was covered with a thin membrane by dip coating in a solution of 5% Nafion dissolved in aliphatic alcohols (Aldrich). The current sensitivity of the electrode was 0.32 nA/µM. Hydrogen peroxide was also measured by the
horseradish peroxidase-mediated oxidation of
o-phenylenediamine; 25 µg of horseradish peroxidase and 1 mM o-phenylenediamine were added to a 2-ml
reaction mixture, and the mixture was vortexed and incubated at room
temperature for 30 min. The mixture was acidified by adding 60 µl of
5 N HCl, and the absorbance at 420 nm was measured
(22).
2 ml of PBS, 100 µM DTPA was placed in a 4-ml sealed vial. The solution was degassed by bubbling through N2 for approximately 30 min. 750 µM cysteine and/or 10 mM NAD+ were added to the solution prior to degassing. After degassing, 2 µl of 37 mM DEANO, stored under N2, was injected into the vial via a gas-tight syringe with a Teflon seal. The solution was incubated at room temperature for 30 min. Under these conditions DEANO decomposes to release ·NO at a rate of approximately 6 µM/min (23). After incubation the vial was unstoppered and 1 ml of the solution was monitored for the release of ·NO by electrode as described above. Another 1 ml was placed immediately in a quartz cuvette, and absorbance at 336 nm was measured.
In order to test the prediction that thiol will
accelerate the decomposition of ·NO, we examined the loss of
·NO from an open aerobic system in the presence and absence of cysteine. Fig. 1 shows that cysteine accelerates the
decomposition of ·NO from buffer under aerobic conditions. In
the absence of cysteine the initial rate of ·NO loss was
approximately 1.6 nM/s, whereas in the presence of cysteine
the rate of ·NO loss was approximately 3.8 nM/s.
These experiments were performed at a constant stirring speed and
temperature (22 °C) and in the presence of a metal chelator. We have
previously found that these factors are critical in determining the
rate of loss of ·NO in an open
system.2
The acceleration of ·NO decomposition is dependent on the relative concentrations of ·NO and cysteine. It was necessary for the thiol concentration to exceed the ·NO concentration by approximately 100-fold. In addition, the effect of thiol on ·NO decay was only relevant at ·NO concentrations of less than 50 µM. At higher concentrations of ·NO the addition of thiol did not alter the decomposition of ·NO, as has been reported previously (12). The reaction of ·NO with O2, which is second order with respect to ·NO, becomes increasingly relevant at higher concentrations of ·NO (17). Therefore, at higher concentrations of ·NO a NOx-based mechanism for S-nitrosothiol formation and other nitrosation reactions becomes increasingly relevant (12, 14, 24).
Orders of Reaction for Cysteine and ·NOThe apparent
orders of reaction for the loss of ·NO were calculated from the
initial rate of decay measured at varying concentrations of ·NO
and cysteine. Fig. 2 shows the plots of
log(d[·NO]/dt) versus log[substrate],
where (d[·NO]/dt) is expressed as the initial rate
of decay in the presence of cysteine minus the initial rate in the
absence of cysteine. As stirring velocity, ionic strength, and
temperature are kept constant the difference between the initial rates
in the presence and absence of cysteine represents the rate of reaction
of ·NO with cysteine. The slopes of the linear regression lines
shown in Fig. 2 represent the apparent orders of reaction for
·NO (Fig. 2A) and cysteine (Fig. 2B). The
reaction orders for ·NO and cysteine approximate to second and
first order, respectively. The apparent second order for ·NO can
be explained by the production of O2 upon decomposition of the
radical intermediate. Superoxide will react with a second molecule of
·NO in an almost instantaneous fashion.
The Product of the Reaction RIs S-Nitrosocysteine
Spectrophotometric examination of the reaction
mixture containing cysteine and ·NO reveals an absorbance peak
at 336 nm, which is typical of S-nitrosothiol (5). The
absorbance at 336 nm is dependent on the quantity of ·NO added
(Fig. 3A). However, the product yield expected from these experiments is very low, on the order of 1 µM or less,
because of the low concentrations of ·NO in the reaction
mixture. In addition, the molar absorptivity of
S-nitrosocysteine is low, 3869 M1
cm
1. As a result the absorbances measured are at the
limit of spectrophotometric detection and are hence quite variable. In
order to confirm that the product of the reaction was
S-nitrosocysteine, we assayed its ability to act as a
·NO donor, a recognized property of
S-nitrosocysteine.
Cysteine-based nitrosothiols release ·NO in the presence of reduced thiol (25, 26). In our experiments, cysteine was in approximately 100-fold excess and, therefore, any S-nitrosothiol formed will readily decompose to release free ·NO. Fig. 3B shows that the reaction of ·NO and cysteine results in the production of a rapidly decaying ·NO donor. In this reaction ·NO is added to PBS either in the presence or absence of cysteine. After 1.5 min the reaction mixture is vortexed to remove any residual ·NO and then monitored for release of ·NO by electrode. In the absence of cysteine no release is measured, whereas in the presence of cysteine a peak of ·NO is observed. The presence of the characteristic S-nitrosothiol absorbance and the ability of the product to release ·NO indicate that the reaction of physiological concentrations of ·NO and cysteine forms S-nitrosocysteine.
Oxygen Consumption and Superoxide ProductionIn order to test
the second prediction of the model, the consumption of O2
by the decomposition of ·NO was measured by means of an oxygen
electrode. Fig. 4 shows that when ·NO was added
to buffer containing Cu,Zn superoxide dismutase alone, there was a
small consumption of O2. However, when cysteine was present
there was a 2.4-fold increase in O2 consumption. This observation is confirmed by previous work that shows that thiol will
increase O2 consumption in the presence of a ·NO
donor (17). When varying concentrations of ·NO were added to
buffer in the presence and absence of cysteine, there was a direct
correlation between ·NO added and O2 consumed (Fig.
4, inset). The slope of the regression line was 0.59 µM O2/µM ·NO in the
presence of cysteine. As Cu,Zn superoxide dismutase removes superoxide
and, therefore, there is no possibility of superoxide and ·NO
interaction, a 1:1 stoichiometry between O2 and ·NO
would be expected. However, Cu,Zn superoxide dismutase regenerates one
O2 molecule for every two superoxide molecules dismutated; therefore, these data are consistent with a 1:1 stoichiometry. The
observation that the predicted stoichiometry holds true would seem to
indicate that in the presence of superoxide dismutase all the
·NO present reacts with thiol, producing
S-nitrosothiol and superoxide anion.
To further confirm that superoxide was generated by the reaction of thiol and ·NO, the production of H2O2 in the presence of Cu,Zn superoxide dismutase was monitored by means of a specific electrode (21). The addition of approximately 6 µM ·NO had no effect on the current within the electrode in the absence of cysteine. However, in the presence of 750 µM cysteine there was an increase in the current that corresponded to an increase in H2O2 concentration of 4.9 ± 1.9 µM (n = 5).
The formation of H2O2 was confirmed independently by adding horseradish peroxidase and o-phenylenediamine 10 min after the addition of ·NO. In the presence of cysteine, ·NO, and superoxide dismutase this led to the generation of an absorbance characteristic of the oxidation of o-phenylenediamine, A420 = 0.155 ± 0.029 (n = 4). In the absence of either cysteine (A420 = 0.018) or superoxide dismutase (A420 = 0.020), oxidation of o-phenylenediamine was not observed. These results indicate that the reaction of ·NO and cysteine in the presence of O2 produces superoxide.
Formation of S-Nitrosocysteine under Anaerobic ConditionsIn
order to confirm the final prediction of the model, ·NO was
reacted with cysteine in an anaerobic environment in the presence of an
electron acceptor, NAD+. Fig. 5 shows that
in the presence of 10 mM NAD+ the reaction of
·NO and cysteine produced a compound that releases ·NO at
a steady rate. The decay of DEANO over the course of the anaerobic
period will release approximately 18 µM ·NO.
However, the vial contains both a gaseous and an aqueous phase, so a
considerable portion (approximately 80%) of the ·NO released by
DEANO may be in the gaseous phase. The ·NO measured in solution
after the anaerobic period is most likely the residual dissolved
material left from the decomposition of DEANO. It is possible that some
of the DEANO remains intact and is, therefore, releasing ·NO
after the anaerobic period. However, the half-life of DEANO under these
conditions is 2 min and thus it is unlikely that a significant quantity
of intact DEANO remains after 30 min (23).
In the absence of either cysteine or NAD+ the rate of loss of ·NO from the buffer immediately after the anaerobic incubation period was 6.7 and 6.0 nM/s, respectively. This rate probably represents the loss of dissolved ·NO from the solution. In contrast, the rate of loss of ·NO was only 3.1 nM/s in the presence of cysteine and NAD+, indicating the presence of a ·NO donor. The addition of excess reduced thiols confirmed the presence of a ·NO donor, as it elicited an increase in the concentration of ·NO within the reaction mixture only when both cysteine and NAD+ were present. Spectrophotometric analysis immediately after anaerobic incubation reveals that the product of the reaction possesses an absorbance peak of 0.05 unit at 336 nm, equivalent to 11 µM S-nitrosothiol. Previous work has shown that the anaerobic interaction of glutathione and ·NO in the absence of an electron acceptor proceeds through the same radical intermediate but that the products are disulfide and N2O (13). These results indicate that S-nitrosothiol can be formed from the anaerobic reaction of cysteine and ·NO only in the presence of an electron acceptor.
Previous work has shown that it is possible to form S-nitrosothiol via mechanisms involving either ·NO reaction with molecular O2 or metal catalysis (12, 14, 15). However, at the concentrations of ·NO used in this study, there is little or no production of higher oxides of nitrogen, and interaction with metals was avoided by using a metal chelator. Therefore, the formation of S-nitrosothiols under the conditions used in this study shows that S-nitrosothiols can be formed by a novel mechanism. An alternative to the mechanism proposed within this paper is that ·NO reacts with oxygen to form a nitrosyldioxyl radical (Equation 5). The nitrosyldioxyl radical then reacts with a reduced thiol to produce S-nitrosothiol and superoxide (Equation 6).
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(Eq. 5) |
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(Eq. 6) |
The reactions described here are significant because they operate at low concentrations of ·NO and thus allow for the formation of S-nitrosothiols under physiological conditions. Our results and the already proven mechanisms of S-nitrosothiol formation indicate that S-nitrosothiols can be formed in vivo under a wide variety of pathophysiological conditions.