Nitric Oxide Is Reduced to HNO by Proton-Coupled Nucleophilic Attack by Ascorbate, Tyrosine, and Other Alcohols. A New Route to HNO in Biological Media?

Article abstract of DOI:10.1021/ja512343w

The role of NO in biology is well established. However, an increasing body of evidence suggests that azanone (HNO), could also be involved in biological processes, some of which are attributed to NO. In this context, one of the most important and yet unanswered questions is whether and how HNO is produced in vivo. A possible route concerns the chemical or enzymatic reduction of NO. In the present work, we have taken advantage of a selective HNO sensing method, to show that NO is reduced to HNO by biologically relevant alcohols with moderate reducing capacity, such as ascorbate or tyrosine. The proposed mechanism involves a nucleophilic attack to NO by the alcohol, coupled to a proton transfer (PCNA: proton-coupled nucleophilic attack) and a subsequent decomposition of the so-produced radical to yield HNO and an alkoxyl radical. (Graph Presented).

Full text of DOI:10.1021/ja512343w

Article
pubs.acs.org/JACS
Nitric Oxide Is Reduced to HNO by Proton-Coupled Nucleophilic
Attack by Ascorbate, Tyrosine, and Other Alcohols. A New Route to
HNO in Biological Media?
†
†
†
Sebastian A. Suarez,† Nicolas I. Neuman,†,‡ Martina Munoz, Lucıa Alvarez, Damian E. Bikiel,
́
́
́
́
́
̃
Carlos D. Brondino,‡ Ivana Ivanovic-Burmazovic,§ Jan Lj. Miljkovic,§ Milos R. Filipovic,§
́
́
Marcelo A. Martí,*,†,⊥ and Fabio Doctorovich*,†
†Departamento de Química Inorgan
Aires, INQUIMAE-CONICET, Ciudad Universitaria, (C1428EGA) Buenos Aires, Argentina
‡Departamento de Física, Facultad de Bioquímica y Ciencias Biolog
icas, Universidad Nacional del Litoral, Ciudad Universitaria, Paraje
El Pozo, Santa Fe 3000, Argentina
́
ica, Analítica y Química Física, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos
́
§Department of Chemistry and Pharmacy, Friedrich-Alexander University Erlangen-Nuremberg, Egerlandstrasse 1, 91058 Erlangen,
Germany
⊥Departamento de Química Biolog
́
ica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria,
Pab. II, (C1428EGA) Buenos Aires, Argentina
S
* Supporting Information
ABSTRACT: The role of NO in biology is well established.
However, an increasing body of evidence suggests that azanone
(HNO), could also be involved in biological processes, some of
which are attributed to NO. In this context, one of the most
important and yet unanswered questions is whether and how
HNO is produced in vivo. A possible route concerns the
chemical or enzymatic reduction of NO. In the present work,
we have taken advantage of a selective HNO sensing method,
to show that NO is reduced to HNO by biologically relevant
alcohols with moderate reducing capacity, such as ascorbate or tyrosine. The proposed mechanism involves a nucleophilic attack
to NO by the alcohol, coupled to a proton transfer (PCNA: proton-coupled nucleophilic attack) and a subsequent
decomposition of the so-produced radical to yield HNO and an alkoxyl radical.
oxygen species, RNOS. These methods include chemical
trapping and HPLC product characterization,17 UV−vis,19−21
and fluorescence22,24−26 detection and electrochemical detec-
tion.27−29 In particular, our group has developed both a UV−
vis trapping-based detection method that uses manganese
porphyrins (MnP) and an azanone sensing electrode that is
able to provide time-resolved quantification of HNO at the low
nanomolar level.21,28
In a broader sense, the biological relevance of nitroxyl has at
least two important aspects. The first concerns the studies of
the pharmacological effects of HNO and the elucidation of the
similarities with and the differences from NO.30−34 Unlike NO,
HNO activates HNO-TRPA1-CGRP signaling cascade for the
regulation of blood pressure and control of cardiac contrac-
tility.31 The second is related to the possibility of its
endogenous production as a biologically relevant messenger,31
an intermediate metabolite, or an undesired enzymatic side
product.35−38 In this context, several in vivo azanone sources
INTRODUCTION
■
After over two decades of intense research, the chemical
reactivity of nitric oxide and its key roles in several biological
processes, including cardiovascular regulation, immune re-
sponse, and neuronal physiology are, in principle, well
established.1−4 Azanone (HNO/NO−), also called nitroxyl, is
the one electron reduction product of NO and its reactivity and
biological relevance are currently under intense debate.5−8 It
dimerizes rapidly (kdim = 8 × 106 M−1 s−1),9 which limits its
concentration and lifetime in the solution. Moreover, HNO
reacts quickly with its sibling NO (k = 5.6 × 106 M−1 s−1)10 and
at a moderate rate (k = 3 × 103 M−1 s−1) with oxygen.9,11,12
HNO signaling is distinct to that of NO: HNO reacts mainly
with thiols8,13 and heme Fe(III) centers.14,15 The lack of
certainty concerning its endogenous production is directly
related to its elusive nature and the difficulties surrounding
unequivocal and quantitative detection, especially when NO is
present.
In the past decade several methods16−23 have been
developed allowing detection and quantification of azanone
with discrimination from NO and other reactive nitrogen and
Received: December 10, 2014
© XXXX American Chemical Society
A
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Article
BSHA decomposition.29 The detection limit for N2O in the present
conditions was 0.5 μmoles.
have been proposed. For example, HNO production could
result from the activity of nitric oxide synthase (NOS) in the
absence of the redox cofactor tetrahydrobiopterin.36,38−41
Another well established in vitro enzymatic azanone source
relies on the oxidation of hydroxylamine and other amino
alcohols. Several groups have shown that this reaction can be
catalyzed by heme-proteins like peroxidases, catalases, or even
myoglobin.17,42 On the other hand, chemical (nonenzymatic),
biologically compatible routes to HNO have been, to our
knowledge, much less pursued.31,43 The most direct route,
chemical reduction of NO, has been historically discarded,
possibly due to the reduction potential of −0.8 V for the
(NO/3NO¯) couple, which is outside the biological range.
Amperometry. Measurements of HNO concentration were
carried out with our previously described method based on a three-
electrode system consisting of platinum counter electrode, Ag/AgCl
reference electrode, and a gold working electrode modified with a
monolayer of cobalt porphyrin with 1-decanethiol covalently attached.
The method has been demonstrated to be specific for HNO, showing
no interference or spurious signal due to the presence of NO, O2,
NO2 , and other RNOS.27,28,53 Signal recording was performed with a
−
TEQ 03 potentiostat.
In a typical experiment, 1.2 to 24 pmoles of ROH (0.2 to 4 μM)
were added to 1.2 μmoles of NO dissolved in 6 mL (0.2 mM) of
degassed distilled water containing 0.6 μmoles of DPTA (or EDTA) at
room temperature (r.t.) under Ar atmosphere (or vice versa). For each
case, we also confirmed that the maximum used concentrations (0.2
mM) of NO, and all H• donors produced a very small signal that can
be disregarded. We have also performed the reaction of NO with
AscH− in an oxygen-free glovebox. In this case, water was
deoxygenated by distillation under nitrogen atmosphere after addition
of sodium dithionite. The results were very similar to those obtained
with degassed water (Supporting Information, Figure SI3B).
Ion Chromatography. Measurements were recorded using a
1
However, at physiological pH, HNO is expected to be the
main species (pKa = 11.4),9 displaying an estimated E° (NO,
H+/1HNO) ≈ −0.14 V.9,44 Moreover, it is important to note
that the reduction of NO to HNO (reaction 1) could be driven
forward by coupling with subsequent thermodynamically
favorable reactions, such as N2O production (reaction 2) or
reactions between radical intermediates (reaction 3).
NO + ROH → RO• + HNO
(1)
DIODEX DX-100 system, with an AS4A-SC (4 mm × 250 mm)
2−
column and an AG4A-SC guard column. The carrier was CO3
HCO3 1.8/1.7 mM, with a flow rate of 2 mL/min
/
−
2NO + HNO → N2O + NO2 + H+
−
(2)
EPR Measurements. CW-EPR measurements were performed at
X-band (9.75 GHz) on a Bruker EMX-Plus Spectrometer with a
rectangular cavity with 100 kHz field modulation. Solutions and
buffers were prepared using high purity reagents and milli-Q grade
water. All glassware was previously washed with HNO3 and abundant
milli-Q water and silicone tubing and plastic syringes were used to
transfer solutions. Diethylene triamine pentaacetic acid (DTPA) (0.5
mM) and/or ethylene diamine tetraacetic acid (EDTA) (8 mM) were
used as chelating agents to remove possible traces of catalytic metal
ions. O2 was eliminated from all solutions through vacuum-Ar cycles
and a positive Ar pressure was maintained by bubbling Ar gas on the
solutions throughout all handling.
For ascorbate anion, time scan experiments at a fixed magnetic field
were also performed. The field B0 was chosen as the maximum of the
low-field peak corresponding to the ascorbyl radical anion doublet.
These experiments were performed with 1 G modulation amplitude,
6.33 mW microwave power, and a conversion time of 20 ms.
Computational Methods. To determine the reaction mechanism
we performed DFT calculations using the Gaussian 98 software
package. All involved species were optimized at the B3LYP level using
6-31 G(d,p) for all atoms using water (polarizable continuum model-
PCM) in order to take into account solvation effects.
Mass Spectrometry. MS experiments were performed on maXis
(Bruker Daltonics) ultrahigh resolution electron spray ionization time-
of-flight mass spectrometer equipped with cryospray ionization
module (Bruker Daltonics). Into 100 μM ascorbate solution in 80%
acetonitrile/20% 10 mM ammonium carbonate buffer pH 7.4, 500 μM
NO was added, and the reaction mixture was sprayed at −20 °C.
Spectra were recorded over 15 min time.
Cell Experiments. Bovine Aorta Endothelial cell (BAEC, CLS Cell
Lines Service GmbH, Germany) were grown in Ham’s F12 medium
supplemented with 2 mM L-glutamine and 10% fetal bovine serum at
37 °C and 5% CO2. Cells grown in ibidi dishes (Ibidi, Martinsried,
Germany) were loaded with CuBOT1 and fluorescence was recorded
as previously described.25,45 RAW 264.7 (mouse monocyte macro-
phage) from ECACC (Salisbury, UK) were grown in DMEM (Sigma-
Aldrich, USA, cat. no. D5546) cell medium supplemented with 2 mM
L-glutamine (Sigma-Aldrich, USA), 10% FBS (Sigma-Aldrich, USA),
1% penicillin-streptomycin (Sigma-Aldrich, USA), and 1% nones-
sential amino acid solution (Sigma-Aldrich, USA) in T-75 cell culture
flask at 5% CO2 and at 37 °C. Cells were stimulated with 1 μg/mL
LPS (Sigma-Aldrich, USA) overnight and next day mechanically
detached, washed once with HBSS w/o Ca2+ and Mg2, and used
immediately for analysis. We used 1 × 106 cells per sample in HBSS
RO• + NO → RONO
(3)
Interestingly, our recent results showed that HNO can be
produced in vivo by the reaction of NO31 or the nitrosyl
species30,45,46 with H2S (E°′(S•−,2H+/H2S) = E°′(S•−,H+/
HS−) = 0.92 V at pH 7).45 Also noteworthy, are several older
works which showed that NO rebinds with generated H• to
yield azanone.47−50
In this work we demonstrate that NO can actually be
reduced to azanone by several biologically relevant compounds
bearing the −OH functional group resulting in a novel potential
pathway for endogenous production of HNO.
EXPERIMENTAL SECTION
■
Reagents. Mn(III) 5,10,15,20-tetrakis(4-carboxyphenyl)-
porphyrinate was purchased from Frontier Scientific and used as
received. All reagents were purchased from Sigma-Aldrich and used as
received. Trioxodinitrate (N2O32−) was synthesized according to
published literature procedures.29,51,52 Milli-Q grade water was used in
all experiments; nitrogen and argon of high purity were used for
anaerobic experiments. NO was generated anaerobically by dropwise
addition of degassed water to a mixture of 4 g of NaNO2, 8.5 g of
FeSO4, and 8.5 g of NaBr. The so-produced NO was passed through a
NaOH solution to remove higher oxides and bubbled into degassed
water in order to get a saturated solution of NO ([NO] = 2 mM).
Optical Absorbance. Measurements were recorded using an
HP8453 spectrophotometer in 1 cm path-length quartz cuvette and
using as blank the respective buffer solutions. All experiments were
performed at 25 °C in 0.1 M phosphate buffer, pH 7.4, containing
DPTA 10−4 M to avoid interferences or undesired reactions by CuII or
other divalent cations. We also checked that all reactions were
unaffected by the irradiation of the sample with the light source of the
spectrometer.
Infrared Spectrometry. Spectra from 400 to 4000 cm−1 with 1
cm−1 resolution were recorded with a research series Thermo Nicolet
FTIR spectrophotometer. All gas phase IR spectra were recorded using
an 8 cm path length gas cell with NaCl windows. The IR spectrum of
the N2O present was quantified using calibration curves for the
absorption bands showing peaks at 2212 and 2236 cm−1 for the P and
R branches, respectively.53 Under these conditions nitrous oxide
signals for each injection were compared to a calibration curve
prepared by injecting samples of N2O produced in situ by NO2−
B
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w/o Ca2+ and Mg2+ supplemented with different concentration of FBS
(fetal bovine serum) up to 5%. The temperature of the HBSS w/o
Ca2+ and Mg2 used in measurement experiments was 37 °C.
RESULTS AND DISCUSSION
■
Aromatic Alcohols and Ascorbate React with NO to
Produce HNO. Our first approach to determine the possible
production of HNO from the reaction of NO with aliphatic or
aromatic alcohols was performed by measuring the conversion
of Mn(III)TCPP to {MnNO}6 (Enemark-Feltham notation)
using UV−vis spectroscopy (see Supporting Information for
more details).54 Figure SI1A shows the absorbance changes
obtained after mixing NO solution with ascorbate (AscH−),
the predominant species under the reaction conditions. These
changes are characteristic for the reaction between Mn(III)
porphyrins and HNO, with the consequent formation of
{MnNO}6.21 Since this Mn(III) porphyrin reacts neither with
NO21 nor with ascorbate55 (see control experiments in
Supporting Information, Figure SI1 and SI2) these results
strongly suggest HNO production. Similar results were
obtained with hydroquinone (HQ), tyrosine (Y), and phenol
(PhOH), although the reaction rates varied significantly (see
Table 1). No reaction was observed with nonaromatic alcohols
Figure 1. Amperometric signal vs initial time plot after the addition of
2 μM ROH to an anaerobic aqueous solution of NO (0.2 mM): y-axis,
[HNO] after calibration. ROH = (red) AscH−; (orange) HQ; (green)
PhOH; (blue) Y.
The resulting keff are reported in Table 1, and the data show
that both diols (HQ and AscH−) react ca. 5−10 times faster
than phenols, with AscH− being the fastest.
On the other hand, Figure 2B and Figure SI3D (Supporting
Information), show that the log(vi) vs log[ROH] and log[NO]
plots are linear with a slope close to 1, confirming that the
reaction is first order in both reactants.
Table 1. Amounts of N2O and Nitrite Obtained for the
Reactions of H• Donors with NO, and the Corresponding
keff
−
keff
NO2
N2O
(μmol)
N2O
org. prod.
a
b
c
d
compound
AscH−
(M‑1 s‑1)
(μmol)
yield
yield
We also tested whether Fe(II/III), Mn(II), Cu(I/II), or
Co(II) affected HNO production in the described reactions by
using the electrochemical nitroxyl sensor. The results confirmed
that metal ions do not play any significant role in the
production of HNO (see Suppporting Information, Table SI2).
EPR Analysis. Since a formal H atom abstraction from
−OH groups by NO would produce a free radical species, the
reactions were studied by EPR. Ascorbate (0.2−2 mM),
hydroquinone (10 mM), and tyrosine (2 mM) solutions were
mixed with equal volumes of the NO saturated solutions by
simultaneous rapid injection into a quartz flat cell. The
presence of dioxygen and metal ions (DPTA or EDTA were
used as chelators) was excluded. The first two alcohols
produced clearly detectable EPR signals as shown in Figure 3
and Supporting Information, Figure SI5. Tyrosyl radicals were
not observed, presumably due to the slower reaction rate
between NO and Y and/or the lower stability of the tyrosyl
radical. Figure 3 shows the time dependence of the ascorbyl
radical concentration obtained after mixing AscH− and NO.
After mixing the reactants, an intense ascorbyl radical signal
appears which subsequently decays with a half-life of 4−8 s.
This behavior is consistent with disproportionation of the
ascorbyl radical into ascorbate and dehydroascorbate,56 and
also reaction of ascorbyl with NO to give O-nitrosoascor-
bate.43,57 For the reaction with HQ (shown in Supporting
Information, Figure SI5) similar results were obtained, but the
radical signal corresponding to HQ• increases 6-fold and
remains stable for several minutes, slightly decaying after 15
min. The EPR signals also allow determination of the keff for
both reactions (shown in Table 1). keff values obtained by EPR
are in the same order of magnitude as those obtained from the
electrochemical data.
8.0 0.5
(43 15)
6.1 0.4
(9)
20
11
16
9
50%
>95%
>95%
HQ
30%
PhOH
Y
3.2 0.4
0.9 0.4
8
5
6
4
20%
10%
∼ 90%
∼ 30%
a
No reaction was detected when methanol, D-mannitol or malic acid
b
were used. Determined from the slope of the electrode signal.
Between parentheses, determined by EPR, see Supporting Information
c
for details. After 24 h, based on the initial amount of NO (100 μmol).
d
Dehydroascorbate (DHA), benzoquinone (BQ), p-Ph(OH)-NO, and
o-Y-NO respectively, based on 17 μmol (initial amount).
like methanol, D-mannitol, or malic acid. The second approach
used to determine HNO production relied on the recently
developed HNO selective electrode, which allows time-resolved
nanomolar detection.27−29,53 In Figure 1 we present the
amperometric signal versus initial time plot after the addition
of each alcohol (2 μM) to an anaerobic aqueous solution of
NO (0.2 mM). The increase in the current following the
addition of the alcohol clearly proves the HNO formation. As
expected for a bimolecular reaction, the signal peak, which
reflects the HNO concentration,28 is linearly dependent on
both AscH− and NO concentrations (Supporting Information,
Figure SI3).
Figure 2A and Figure SI3C (Supporting Information), show
that vi (initial rate) versus [ROH] and [NO] plots are linear.
From the slope of these plots an effective bimolecular reaction
rate constant (keff), corresponding to reaction 1 can be
obtained.
The ubisemiquinone EPR signal has been reported during
the reaction between NO and truncated ubiquinols,58 and the
ascorbyl radical has been observed during the reaction between
v = keff[ROH][NO]
C
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Figure 2. (A) vi vs [ROH]. (B) log(vi) vs log[ROH]. [NO] = 0.2 mM. ROH = (red) AscH−; (orange) HQ; (green) PhOH; (blue) Y.
spray ionization ultrahigh-resolution mass spectrometry, the
reaction of AscH− and NO showed MS peaks (m/z 207.0368,
223.0591 and 237.0378, Supporting Information, Figure SI9),
which correspond to the first addition of NO to ascorbate, and
second addition of NO to either RO−NO−, or the ascorbyl
radical (see below for mechanistic analysis). As postulated by
Kirsch,43 once the nitrite ester [AscONO]− is formed by the
reaction of Asc•− with NO, HNO and DHA can be produced
via a radical chain mechanism as shown in eq 4,43 eq 5,60 and eq
6.43
[AscONO]− + H+ → DHA + HNO
(4)
DHA + Asc− ⇌ 2Asc•−
(5)
Asc•− + NO → [AscONO]−
(6)
Figure 3. Time dependence of ascorbyl radical concentration. Inset:
Consecutive EPR spectra of solutions of ascorbate (1 mM) alone and
with NO (1 mM). The arrow indicates beginning of the reaction.
HQ yields mainly benzoquinone (BQ), also possibly due to
further reaction of the HQ radical with NO. Finally, PhOH and
Y yield the corresponding products 4-nitrosophenol (p-
Ph(OH)-NO) and 3-nitrosotyrosine (o-Y-NO), whereas Y
also dimerizes to yield dityrosine (see Supporting Information
for experimental details); these products are consistent with the
presence of PhO• and Y• radicals. The lack of EPR signal in
these cases possibly arises because of their high reactivity and
the presence of the excess of NO, which yields the mentioned
products. The yields of the organic products (see Supporting
Information for details) are higher than the corresponding N2O
yields, indicating that these compounds are also produced by
other routes which do not afford HNO. The formation of
nitrosocompounds by reaction of phenols with NO has been
observed before.61
Computational Mechanistic Analysis. To get an addi-
tional insight into the reaction mechanisms we performed DFT
calculations using the Gaussian software package. As an
example, the results for AscH− are presented in Scheme 1,
while the other cases are shown in Supporting Information,
Figure SI11. The calculations show that the first step of the
reaction between NO and AscH− is endergonic (by 16 kcal/
mol) yielding a radical intermediate RO-N(H)O• (consistent
with one of the peaks observed in the mass spectrometer at m/z
207.0368, see Scheme 1 and Supporting Information, Figure
SI9). This step can be described as a nucleophilic attack of the
ascorbate anion to NO (reaction 7), coupled to proton transfer
from the vicinal −OH moiety or the solvent. Such a mechanism
can be described as a proton-coupled nucleophilic attack
(PCNA).
ascorbate and N-acetyl-N-nitrosotryptophan or NO donors
under normoxic and oxygen free conditions.43 The kinetic
analysis of these reactions is detailed in the Supporting
Information.
End Products Analysis. The initial products of the
reaction of NO with the alcohols are unstable and highly
reactive radical species. Thus, further reactions are expected to
occur. The main sink for HNO is expected to be its
dimerization and/or reaction with NO,10 yielding the stable
−
products N2O and NO2 . To detect and quantify N2O we
determined the IR spectra of the reaction chamber headspace.
As expected, NO reaction with HQ, AscH−, Y, and PhOH
results in the appearance of characteristic N2O IR bands at
2210 and 2230 cm−1 (see Supporting Information, Figure
SI7),53,59 and no signal is observed with either reactant alone.
The presence of nitrite was confirmed by ion chromatography
(see Figure SI8). Moreover, quantification of the relative N2O
and NO2¯ yields (Table 1) show that they are formed in a ca.
1:1 ratio, which is consistent with our mechanistic interpreta-
tion (eq 9; vide infra).
The R-O• radicals are also inherently unstable and thus react
further leading to more stable organic closed shell compounds.
To determine the corresponding end products for each
reaction, we used NMR spectroscopy, IR, UV, and MS
spectrometry (see Supporting Information). AscH− yields
dehydroascorbate (DHA) as the main end product, formed
by ascorbyl radical disproportionation. When studied by cryo-
D
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Last but not least, it is important to note that although the
first reaction step between NO and alcohol is endergonic, the
reaction is driven forward by the subsequent reactions of the
initial products (HNO and radicals). In fact, formation of N2O
overcompensates the endergonic HNO generation resulting in
an overall negative free energy balance for the global reaction 9
(see Scheme 1), which for AscH− is
Scheme 1. DFT Calculations. Energy Values Reported in
kcal/mol
−
AscH− + 6NO → DHA + 2N2O + 2NO2 + H+
(9)
The energy associated with the first step, either to yield directly
HNO by HAT or an “RON(H)O−like” radical intermediate by
PCNA, can be considered a minimum estimation of the global
reaction barrier. As shown in Table 2, the ΔE for the first two
Table 2. Ab Initio Calculated Reaction Energies (ΔE) in
kcal/mol for PCNA and HNO Release Steps
E°′ (V)
(pH 7)
RO•,
ΔE
ΔE
HNO
ΔE
a
a
a
ac
,
pKa
H+/ROH PCNA release step 1+2 global
AscH−
HQ
4.11
10
0.28
0.10
0.91
0.97
-
+16
- 5
+11
+ 8
−58
+ 18.5
+ 25.4
+25.3
+ 19.5
10.5
−109
−63
−70
-
Y
10
+ 7.4
+12.4
+ 33.7
+33
PhOH
MeOH
10
+37.7
+53.2
b
15.5
a
ΔE°PCM (kcal/mol), optimized at the B3LYP level using 6-31 G(d,p)
for all atoms using water (PCM: polarizable continuum model); step
1, PCNA; step 2, HNO release. HNO was not detected when
methanol was used. Final product was DHA, BQ, p-Ph(OH)NO, and
b
c
o-YNO, respectively (see SI).
steps (step 1+2) are smaller for AscH− and HQ, which are the
faster reactants (Table 1). The largest ΔE (+53.2 kcal/mol) is
observed for MeOH, which does not react under the tested
conditions. The calculated energies for step 1+2 and for the
global reaction are in reasonable agreement with those energies
obtained from tabulated redox potentials (Supporting In-
formation, Table SI3).
To assess the potential role of molecular oxygen on these
reactions, we performed the reaction of AscH− and NO in the
presence of controlled amounts of oxygen (Supporting
Information, Figures SI2 and SI4). As shown in Figure SI4,
the amount of HNO produced decreases as the relative amount
of added O2 is increased. This is a strong indication that O2
does not catalyze HNO formation. Instead, the presence of O2
diminishes the observed signal, a fact that can be attributed to
its known reaction with either reactant, or even with azanone,
as shown in our previous work.28
NO binds preferably to C2−O, while ascorbate is preferably
deprotonated at C3−O (see Scheme 1). At this point it is
difficult to determine whether NO reacts with one or the other
tautomer, with the OH or O−, and how and when the protons
are transferred. However, attack of -O¯ to NO seems to be
more likely. The RO-N(H)O• radical intermediate decays to
HNO and the ascorbyl radical (reaction 8), which can then
react with another NO to produce a closed shell nitrite ester o-
nitrosoascorbate (also observed by MS at m/z 223.0591).
Reaction of the radical with the second NO prior to its HNO
release, possibly accounts for formation of di-ONO observed by
MS (m/z 237.0378, Supporting Information, Figure SI9g). The
O-nitrosoascorbate also decays after taking a proton to yield
HNO and DHA, as previously observed by Kirsch and co-
workers.57
In Vitro Cell Studies. In certain cell types, such as
endothelial cells, neuronal cells, and immune cells, vitamin C
accumulates to concentrations higher than 1 mM.62 To analyze
whether the described reactions occur under physiological
conditions, we used an HNO fluorescence sensor, CuBOT1, to
evaluate the intracellular azanone formation.22,24−26 Bovine
arterial endothelial cells were pretreated with either 1 mM
AscH− or 1 mM pBQH2 for 1 h to increase their intracellular
concentration. Cells were washed and then loaded with
CuBOT1 to assess the changes in intracellular levels of
HNO. The intensity of the fluorescence was compared with
basal fluorescence detected in the control (untreated cells).
Figure 4A shows a clear increase of the fluorescence with both
treatments. In addition we tested the ability of ascorbate to
reduce endogenously generated NO from another cell line,
Nu−(+H+) + NO• → (Nu − NH − O)•
(Nu − NH − O)• → HNO + (Nu)•
(7)
(8)
A similar mechanism is expected for HQ (see Supporting
Information), with two NO molecules reacting with each HQ
molecule. For Y and PhOH, the radical intermediates produced
after the addition of NO, formation of the RO-N(H)O
intermediate, and HNO release also yield the observed nitroso
derivatives. More importantly, taking into account the pKa of
the corresponding alcohols, in these three cases the reaction
undoubtedly occurs with a neutral OH group, where an
intramolecular proton rearrangement or solvent-assisted
protonation is required. Therefore, in these cases a PCNA is
proposed as well.
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Figure 4. (A) Intracellular HNO formation in bovine arterial endothelial cells as revealed by the HNO fluorescence sensor, CuBOT1. Hoeschst was
used to stain the nuclei, showing that there are cells in the control for which the signal is very low, and also that the position of the signal matches the
actual cells. (B) HNO formation after the addition of ascorbate to immunostimulated macrophages. The HNO electrode was immersed into a 106
cell/mL suspension of immunostimulated macrophages in Dulbecco’s Modified Eagle’s Medium (DMEM). Subsequently 1 mM ascorbate was
added, and the current was monitored.
RAW 264.7 macrophages. Macrophages were stimulated with
lipopolysaccharide(LPS)/interferon gamma to stimulate indu-
cible nitric oxide synthase to produce NO, and the HNO
electrode was immersed in the extracellular medium containing
106 cells/mL. After the addition of 1 mM ascorbate an
immediate rise in the signal was observed, showing clear HNO
formation (Figure 4B). No signal was observed when AscH−
was added into cell-free medium.
Scheme 2. Proposed Mechanism for HNO Formation by the
Reaction of NO with ROH
These data strongly suggest that HNO could be produced in
the reaction of NO and AscH− under physiological conditions.
membranes63 and its physiological role in plant and animal
mitochondria, the following picture emerges:64,65 under
hypoxia, respiratory chain intermediate quinones accumulate
and NO production increases, through nitrite reductase activity
of myoglobin among others,66 creating an ideal opportunity for
the presented reaction to take place. In addition, the presented
proof of concept for physiological NO conversion to HNO,
suggests that it is not unlikely that some of the protective
effects assigned to NO, are indeed mediated by its “younger”
sibling HNO,67 as shown in our recent work.31
CONCLUSIONS
■
The present work provides clear evidence of a possible
biochemically relevant HNO source, resulting from the reaction
of NO with aromatic or “pseudoaromatic” alcohols such as
tyrosine, ascorbic acid, and hydroquinone. Mechanistically, it is
clear that the reaction does not involve a simple outer sphere
reduction coupled to proton release/uptake, which is
thermodynamically unfavorable as evidenced by the alcohol
reduction potentials shown in Table 2.
Definitive proof to these hypotheses awaits further studies
and opens the way for both potential therapeutic interventions
of azanone donors and understanding of endogenous HNO
production.
Instead, our data suggest that there is a nucleophilic addition
of ROH/RO− to NO, coupled to a proton transfer (either
intramolecular or through the solvent) that results in an RO-
N(H)O• intermediate, which decays by O−N bond cleavage,
producing HNO and the corresponding radical (see Scheme 2
and Table 2). The stability of the RO• radical (bound to HNO
or free), PCNA endergonicity, and the global energy for steps 1
+ 2 (Table 2) seem to be the key factors for the reaction to
occur, explaining why no reaction is observed for MeOH or
mannitol, and why AscH− and HQ react faster.
ASSOCIATED CONTENT
* Supporting Information
Kinetic analysis, ab initio calculations, mass spectra, EPR, and
other experimental details. This material is available free of
charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Authors
doctorovich@qi.fcen.uba.ar
Beyond the chemical novelty, biological implications are
direct. For example, given the known preference for NO
partition within the hydrophobic interior of biological
■
F
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Article
(26) Wrobel, A. T.; Johnstone, T. C.; Liang, A. D.; Lippard, S. J.;
Rivera-fuentes, P. J. Am. Chem. Soc. 2014, 136, 4697.
(27) Suarez, S. A.; Fonticelli, M. H.; Rubert, A. A.; de la Llave, E.;
́
Scherlis, D.; Salvarezza, R. C.; Martí, M. A.; Doctorovich, F. Inorg.
Chem. 2010, 49, 6955.
marcelo@qi.fcen.uba.ar
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
(28) Suar
Anal. Chem. 2013, 85, 10262−10269.
(29) Sirsalmath, K.; Suarez, S. A.; Bikiel, D. E.; Doctorovich, F. J.
́
ez, S.; Bikiel, D.; Wetzler, D.; Martí, M. A.; Doctorovich, F.
■
This work was financially supported by UBA (UBACYT W583
and 2010-12), ANPCyT (PICT 2010-416), CONICET
(PIP1207 and 112-201001-00125), and from the Bunge y
Born Foundation. S.A.S. and L.A. thank CONICET for a
fellowship grant, NIN, DEB, CDB, MAM, and FAD are
members of CONICET. J.M., M.R.F., and I.I.-B acknowledge
the support by intramural funds provided by the FAU within
the Emerging Field Initiative (Medicinal Redox Inorganic
Chemistry).
́
Inorg. Biochem. 2013, 118, 134.
(30) Filipovic, M. R.; Eberhardt, M.; Prokopovic, V.; Mijuskovic, A.;
Orescanin-Dusic, Z.; Reeh, P.; Ivanovic-Burmazovic, I. J. Med. Chem.
2013, 56, 1499.
(31) Eberhardt, M.; Dux, M.; Namer, B.; Miljkovic, J.; Cordasic, N.;
Will, C.; Kichko, T. I.; Roche, J.; de la Fischer, M.; Bikiel, D.; Suar
A.; Dorsch, K.; Leffler, A.; Babes, A.; Lampert, A.; Lennerz, J. K.;
Jacobi, J.; Martí, M. A.; Doctorovich, F.; Hogestatt, E. D.; Zygmunt, P.
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M.; Ivanovic-Burmazovic, I.; Messlinger, K.; Reeh, P.; Filipovic, M. R.
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