S-nitrosothiol chemistry at the single-molecule level

Article abstract of DOI:10.1002/anie.201202365

SNO patrol: S-Nitrosothiols (RSNO) are important molecules involved in cell signaling, which control physiological processes such as vasodilation and bronchodilation. By using the protein pore α-hemolysin as a nanoreactor, the biological chemistry of RSNO has been investigated at the single-molecule level (see scheme). Copyright

Full text of DOI:10.1002/anie.201202365

Angewandte
Chemie
DOI: 10.1002/anie.201202365
Single-Molecule Chemistry
S-Nitrosothiol Chemistry at the Single-Molecule Level**
Lai-Sheung Choi and Hagan Bayley
Nitric oxide (NO) and its relatives (notably RSNO and HNO)
perform important roles as regulatory molecules in health and
disease.[1] NO activates guanylate cyclase by binding to its
ferrous heme (see the Supporting Information, Figure S1).
The lifetime of NO is short, being milliseconds to seconds,[1b]
and therefore it is unlikely that NO diffuses more than
approximately 100 mm, which is in the range of a few cells,
from the site at which it is generated. Through poorly
understood mechanisms,[1b,2] NO is also converted in vivo into
small molecule and protein S-nitrosothiols (RSNOs). RSNOs
modulate cellular activity in ways that differ from the direct
action of NO.[3] Furthermore, RSNO derivatives of albumin
and hemoglobin are sufficiently long-lived to be transported
in the circulation.[4] In addition, NO is passed from one thiol
to another by transnitrosation, which can be facilitated by
specific protein–protein interactions,[2a,5] and protein function
can be modulated by S nitrosation.[6] Protein SNOs are also
converted into other derivatives, such as S-glutathionylated
guish enantiomers[14] or determine a deuterium isotope
effect,[15] and herein we show that the two atoms, -NO, in
RSNO are readily detected. Clearly, it would not be possible
to examine the single-molecule chemistry of RSNO in
a meaningful way with bulky fluorescent reagents, the main
alternative to our approach. We have investigated the lifetime
of the proposed nitroxyl disulfide intermediate, RSN(O)SRÀ,
in transnitrosation, examined the competition between trans-
nitrosation and S thiolation by S-nitrosoglutathione, and
studied the reaction between RSNO and sulfite. The latter
is especially relevant to the exacerbating effect in asthma of
sulfite[16] acting through the NO pathway.[17]
S-Nitrosoglutathione (GSNO) mediates cell signaling
through transnitrosation,[1a,18] which is the transfer of the
proteins,[7] thus generating
a complex signaling web
(Figure S1). RSNO also act as a source of HNO (nitroxyl),[8]
which is also a locally acting regulatory molecule with
biological effects that differ from and even oppose those of
NO.[1c,8a,b]
Although the actual effectors (NO, RSNO, HNO etc.) are
not always clearly distinguished, NO has been implicated in
numerous medical disorders beyond its well-known role in
erectile function. Examples include disorders involving the
cardiovascular system,[9] neuronal cell death in trauma and
disease,[6,10] the immune system,[11] and cancer.[9] RSNOs and
several agents that generate either NO or HNO are under
development as therapeutic agents.[8a,9,12]
Here, we have used the protein nanoreactor approach[13]
to examine RSNO chemistry at the single-molecule level.
Reactant molecules are tethered within the lumen of a protein
pore, staphylococcal a-hemolysin (aHL), and a non-perturb-
ing current carried through the pore by aqueous ions registers
changes in molecular structure that occur during bond-
making and bond-breaking events, thus revealing the kinetics
of the steps involved in a series or cycle of reactions
(Figure 1a). The technique is sufficiently sensitive to distin-
Figure 1. S-Nitrosothiol chemistry observed at the single-molecule
level. a) The single-channel electrical recording technique. Cross-sec-
tion of an a-hemolysin (aHL) pore, (G137C-D8)7 (P7SH), located in
a planar lipid bilayer. Cysteine residues at position 137, near the
middle of the transmembrane b barrel, are highlighted in red. The
homoheptameric P7SH has seven cysteine residues within the lumen
of the pore. The internal diameter of the transmembrane barrel is
approximately 20 ꢀ. S-Nitrosothiols (RSNOs) were added to the cis
compartment, which is the side where the cap domain of the aHL pore
resides and is connected to ground. The trans compartment was held
at +150 mV. The structures of S-nitrosoglutathione (GSNO) and
S-nitroso-N-acetyl-d-penicillamine (SNAP) are shown on the right.
b) S-Nitrosothiol chemistry observed by using the nanopore approach.
PSH: (WT)6(G137C-D8)1, an aHL pore with a single free thiol in the
lumen; PSNO: the S-nitrosothiol derivative of PSH; PSSO3À: the
S-sulfonate derivative; PSSG: the glutathione mixed disulfide; PSOH:
the sulfenic acid; PSO2H: the sulfinic acid; DTT: dl-dithiothreitol.
[*] L.-S. Choi, H. Bayley
Department of Chemistry, University of Oxford
Oxford, OX13TA (UK)
[**] We are grateful to Dr. Mackay B. Steffensen, Dr. Hai-Chen Wu, Dr.
Dvir Rotem, and Dr. Tivadar Mach for their ideas and helpful
discussions, Dr. Barbara Odell and Dr. Timothy D. W. Claridge for
15N NMR spectroscopy, Dr. Jeffrey Harmer for EPR spectroscopy,
and Dr. Arnold J. Boersma and Dr. Sarah E. Rogers for reading the
manuscript. This work was supported by the MRC. L.-S.C. is the
recipient of a University of Oxford Croucher Scholarship (UOCS).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201202365.
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NO group to protein cysteine residues, and by S glutathiony-
lation, which involves the formation of a mixed disulfide,
again with protein cysteine residues (Figure S1).[19] We
examined the reaction of GSNO with the cysteine residues
inside the transmembrane b barrel of the homoheptameric
aHL pore (G137C-D8)7 (P7SH) (Figure 1a). The addition of
GSNO to the cis side of P7SH (Figure 1a and Figure 2a)
produced a series of small steps in the transmembrane current
(DI = À1.2 Æ 0.3 pA, 107 steps out of a total of 121 from
34 experiments). A maximum of seven steps was observed,
a number, which corresponds to the total number of cysteine
residues in each P7SH pore (Figure 2a). The heteroheptamer
(WT)6(G137C-D8)1 (PSH), which contains only one cysteine
residue, showed a single-step change (success rate 83%, n =
60 experiments, see the Supporting Information, Table S1).
Because a different RSNO, S-nitroso-N-acetyl-d-penicilla-
mine (SNAP), produced similar current steps with P7SH
(DI = À1.1 Æ 0.3 pA, 41 steps from 14 experiments), the steps
were attributed to transnitrosation reactions.
temperature (pH 6.0 and 58C). It has been suggested that
the intermediate is short-lived;[20a] and according to our
measurements, if it does exist, the lifetime must be well under
200 ms (Figure S3). It was claimed by Perissinotti et al. that
high concentrations of the N,N-dialkylsulfanyl aminoxide of
cysteine ethyl ester can be observed in methanol.[21] However,
the 1H NMR spectrum obtained by these authors is also
consistent with disulfide formation from the reaction of the
S-nitrosothiol and the thiol, which occurs slowly, at least in
aqueous solution[22] (see also the section entitled “Second-
order rate constants for S glutathionylation” in the Support-
ing Information).
In addition to the transnitrosation steps, much larger
current jumps (DI = À20 Æ 5 pA, 14 steps out of 121 steps
from 34 experiments, that is, 12%) occurred less frequently
with P7SH in the presence of GSNO at pH 7.4 (Figure 2b and
Table 1). The magnitude of these steps suggested that they
Table 1: Transnitrosation reaction rate constants, kt, measured by single-
channel current recording with P7SH.
The proposed associative N,N-dialkylsulfanyl aminoxide
intermediate (RSN(O)SRÀ, see the Supporting Information,
Figure S3a)[20] was not observed even in experiments (n = 3)
performed with GSNO at both lower pH and reduced
pH Number of
Total number Number of kt [mÀ1 sÀ1 [c]
]
experiments[a] of reaction
S thiolation
steps
steps[b]
GSNO 7.4 34
8.4 14
SNAP 7.4 14
8.4 12
121
51
41
14
1
0
1.0Æ0.2
2.5Æ0.7
1.4Æ0.2
4.5Æ1.1
45
0
[a] One P7SH pore was used in each experiment. So, the number of
experiments represents the number of P7SH pores examined. [b] This is
the sum of the transformations (transnitrosation and S thiolation)
observed in all the experiments. [c] kt is the single-molecule bimolecular
rate constant for the transnitrosation between RSNO and the protein
thiol group. The mean value Æs.d. is given. The kt values were
statistically corrected for the number of remaining thiol groups in P7SH
(see the Supporting Information). Conditions: 2m KCl, 80 mm MOPS,
100 mm EDTA, pH 7.4 or 8.4, 22Æ18C and +150 mV.
might arise by S glutathionylation and this was confirmed by
generating the S-glutathionyl derivative of PSH by an
independent route (see the Supporting Information,
Figure S4, DI = À20 Æ 3 pA (n = 3)). S Glutathionylation by
GSNO was reduced to a single observation in 51 steps at
pH 8.4. The pH dependence of S glutathionylation by GSNO
suggests that the acidity of the microenvironment of the
reacting thiolate might dictate whether transnitrosation or
S thiolation takes place, with S thiolation favored by lower
pH. SNAP did not undergo S thiolation at pH 7.4 or pH 8.4
(Table 1), presumably owing to greater steric hindrance at the
tertiary SNO group.[23]
Figure 2. Transnitrosation, S thiolation and S sulfonation. a) Seven
successive transnitrosations by GSNO observed with P7SH. Similar
changes in transmembrane current were observed when SNAP was
used instead of GSNO. GSNO or SNAP was added to the cis
compartment. b) S Glutathionylation by GSNO under the same con-
ditions as in (a). Two transnitrosation steps (indicated) can also been
seen. c) Three successive S sulfonations by sulfite ion on the
S-nitrosothiols of P7SNO. Sodium sulfite (12 mm) was added to the
cis side of preformed P7SNO obtained from the reaction of GSNO
(2 mm, cis) with P7SH. The three S sulfonation steps are of different
amplitudes because the environment within the pore is altered after
each reaction. Note the increase in pore conductance upon S sulfona-
tion. Conditions: 2m KCl, 80 mm 3-morpholinopropane-1-sulfonic acid
(MOPS), 100 mm ethylenediaminetetraacetate (EDTA), pH 7.4, at
+150 mV and 22Æ18C.
By using spatially separated reagents,[24] multiple turn-
overs of transnitrosation of the thiol group in PSH were
observed at pH 8.4. GSNO and dl-dithiothreitol (DTT) were
added to the cis and trans compartments, respectively (Fig-
ure 3a). Titration experiments (Figure 3d) showed linear
dependencies of the reciprocals of the mean dwell times at the
unreacted pore level PSH (tPSH) and at the S-nitrosothiol level
PSNO (tPSNO) on the concentrations of GSNO and DTT,
respectively (Figure 3b and c); these data allowed the
estimation of second-order rate constants for the forward
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transnitrosation reaction between PSH and GSNO (kt = 10 Æ
1mÀ1 sÀ1) and the reverse transnitrosation between PSNO and
DTT (kt = 17 Æ 1mÀ1 sÀ1).
The reaction between RSNO and sulfite ion (SO32À) has
been reported previously,[25] with RSH as a proposed product.
However, the reaction products were not characterized
Figure 3. Reversible transnitrosation.
a) Reversible transnitrosations in the
PSH nanoreactor in the presence of
GSNO (cis) and DTT (trans). Condi-
tions: 2m KCl, 80 mm MOPS, 100 mm
EDTA, pH 8.4, at +150 mV and
22Æ18C. b) Reciprocals of the mean
dwell times for both the PSH (1/tPSH
)
and PSNO (1/tPSNO) levels versus the
concentration of GSNO. The same
concentration of DTT (8 mm) was
used throughout this set of experi-
ments. c) As in (b), but the concentra-
tion of DTT was varied and a fixed
concentration of GSNO (16 mm)
used. Each point represents the mean
Æs.d. for three experiments. d) Single-
channel recordings under the above
conditions at various concentrations
of GSNO and DTT. The levels corre-
sponding to PSH and PSNO are
marked. The two conductance levels
differ by 0.7Æ0.1 pA (n=6).
Figure 4. Product characterization for the reaction between GSNO and sulfite ion in bulk solution. a) Reaction scheme. b) Stacked 400 MHz
1H NMR spectra (5.1–2.1 ppm) in D2O containing 2m KCl, 80 mm sodium phosphate, 100 mm EDTA, pD 7.4, at 22Æ18C. Glutathione derivatives
at 10 mm were used in each experiment. The reactions were complete within 10 min after mixing, and the spectra were collected within 1 h. Peaks
from the CH2S protons are framed with colors corresponding to the various glutathione derivatives highlighted in (a). c) Electrospray ionization
mass spectroscopy (ESI-MS, negative ion mode) showing the peak corresponding to GSSO3À. The sample was prepared by mixing GSNO and
sodium sulfite in a 1:2 ratio in water and the products were analyzed without purification. d) 15N NMR spectrum (50 MHz, 800–100 ppm) after
the reaction between GS15NO (83 mm) and sodium sulfite mixed in a 1:2 ratio in the same buffer as in (b). The peak at 160.4 ppm corresponds
to [15N]hydroxylamine-N-sulfonate (see also Figure S8).
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spectroscopically. Furthermore, 5,5’-dithiobis(2-nitrobenzoic
acid) (DTNB) was used to detect thiol formation, and the
disulfide bond of this reagent is also cleaved by sulfite. To
clarify the chemistry between sulfite and RSNO, a detailed
spectroscopic and kinetic study was performed with GSNO
(for details see the Supporting Information). 1H NMR
spectroscopy and mass spectrometry (Figure 4b and c)
À
showed that S-sulfoglutathione (GSSO3 ) was formed from
GSNO and sulfite at pH 7.4 and 228C. The reaction stoichi-
ometry determined by 1H NMR titration experiments
revealed that each GSNO consumes two sulfite ions
(Table 2). 15N NMR and mass spectrometry on the products
Table 2: Determination of the reaction stoichiometry for S sulfonation of
GSNO by 1H NMR.
Molar ratio of
GSNO to SO3
GSNO/
GSSO3À
ratio[b]
Expected GSNO/GSSO3À ratio if
GSNO reacted with SO32À in 1:2
stoichiometry
2À[a]
2:1
1:1
2:3
1:2
1:25
2.5Æ0.6
0.9Æ0.3
0.4Æ0.3
0
3.0
1.0
0.3
0
0
0
[a] GSNO (10 mm) was mixed with various concentrations of sulfite ion.
[b] The product ratio (mean Æs.d.) was determined from the peak areas
of the CH2S protons in 1H NMR spectra (Figure 4b, n=4). Owing to the
decomposition of GSNO to GSSG before the collection of NMR spectra,
À
the ratio of GSNO to GSSO3 À was calculated as the molar ratio of
(GSNO + 2GSSG) to GSSO3
.
Scheme 1. Proposed reaction mechanisms for S sulfonation of RSNO
by sulfite ion. The overall reaction observed at pH 7.4, 228C is shown
in reaction 1. The first proposed mechanism involves nucleophilic
attack at the sulfur atom of RSNO by sulfite ion with concerted
protonation at the RSNO nitrogen atom (reaction 2) to form the S-
sulfonate (RSSO3À) and HNO in a rate-determining step. The second
proposed mechanism involves the nucleophilic attack at the nitrogen
atom of RSNO by sulfite ion in the rate-determining step (reaction 4).
RDS=rate-determining step.
of the reaction between GS15NO and sulfite identified
hydroxylamine-N-sulfonate as the nitrogen-containing prod-
uct (Figure 4c and d and Figure S8). Nitric oxide (NO) or
nitroxyl (HNO), which might be formed as short-lived
intermediates, could not be detected by electron paramag-
netic resonance (EPR) after trapping with oxymyoglobin and
metmyoglobin.[8a] The kinetics of the reaction between GSNO
and sulfite were also followed by monitoring the disappear-
ance of GSNO at 334 nm with a stopped-flow UV/Vis
spectrometer (see the Supporting Information). The reaction
rate showed a first order dependence on each reactant
(d[GSNO]/dt = kSO 2À[GSNO][sulfite]); kSO 2À, at pH 7.4, was
Indeed, it has been reported that sulfite reacts rapidly with
HNO to form hydroxylamine-N-sulfonate,[26] which would
also explain our inability to trap and detect HNO by EPR.
In vivo, the released HNO would react rapidly with thiols,
which are present at high concentrations compared to sulfite,
3
3
124 Æ 3 mÀ1 sÀ1 at 228C and 264 Æ 5 mÀ1 sÀ1 at 378C. The latter
agrees with a previously reported value for the disappearance
of GSNO.[25b]
=
converting them to disulfides or sulfinamides (RS( O)NH2)
À
and sulfinates (RSO2 ).[8b] Nucleophilic attack with concerted
With these findings in hand, the S sulfonation of protein
SNO was examined at the single-molecule level. P7SNO was
first formed by treating P7SH with GSNO. The subsequent
addition of sulfite to the cis compartment elicited several
steps in the transmembrane current (DI =+ 1.4 to + 3.7 pA,
for each step) (Figure 2c). The second-order rate constant
protonation (reaction 2, Scheme 1) circumvents the difficulty
posed by the fact that the nitroxyl anion (NOÀ) is a triplet in
the ground state, and as such is spin forbidden to act as
leaving group.[8c,27] A second mechanism involves the direct
attack of sulfite at the nitrogen atom of P7SNO in a rate-
À
determining step to form P7S-N(OH)-SO3 (reaction 4,
kSO 2À was 82 Æ 40 mÀ1 sÀ1 (11 steps from 6 experiments) at
Scheme 1). A substitution reaction at the sulfenamide sulfur
3
À
pH 7.4 and 228C. No intermediates were observed.
atom in the P7S-N(OH)-SO3 intermediate with a second
The observations above can be rationalized by two
possible mechanisms. In the first, sulfite attacks the sulfur
atom of P7SNO in a rate-determining step, with concerted
protonation at the nitrogen atom leading to the formation of
the S-sulfonate species and nitroxyl (HNO) (reaction 2,
Scheme 1). HNO is then consumed by a second sulfite ion.
sulfite ion yields the S-sulfonate product, P7SSO3À, together
with hydroxylamine-N-sulfonate as the leaving group (reac-
tion 5). Because only one step was observed in the single-
À
À
molecule experiment, P7S-N(OH)-SO3 and P7SSO3 would
have to have similar conductance values for our observations
to be consistent with this pathway. Furthermore, in the similar
4
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S thiolation reaction, the immediate products are the disulfide
and HNO.[28] Therefore, all things considered, we favor the
first mechanism for S sulfonation, which results in the
generation of free HNO (Scheme 1).
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a range of ion channels and receptor systems, leading to
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of its asthmatic effect is the depletion of GSNO (kSO 2À
=
3
264 mÀ1 sÀ1 at pH 7.4, 378C) and protein SNOs by conversion
into their S-sulfonate analogues. Furthermore, the proposed
generation of HNO, which has different physiological effects
to free NO,[8b] might alter the normal nitrergic signaling
cascade responsible for human airway dilation, leading to
breathing difficulties.
Received: March 26, 2012
Revised: June 8, 2012
Published online: && &&, &&&&
Keywords: nitroxyl · signal transduction ·
.
single-molecule studies · S-nitrosothiol · sulfite
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D. W. Shoeman, H. T. Nagasawa, Biochemistry 1998, 37, 5362 –
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Angew. Chem. Int. Ed. 2012, 51, 1 – 6
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
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These are not the final page numbers!

.
Angewandte
Communications
Communications
Single-Molecule Chemistry
L.-S. Choi, H. Bayley
&&&& — &&&&
S-Nitrosothiol Chemistry at the Single-
Molecule Level
SNO patrol: S-Nitrosothiols (RSNO) are
important molecules involved in cell
signaling, which control physiological
processes such as vasodilation and
bronchodilation. By using the protein
pore a-hemolysin as a nanoreactor, the
biological chemistry of RSNO has been
investigated at the single-molecule level
(see scheme).
6
www.angewandte.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 1 – 6
These are not the final page numbers!