HNO Is Produced by the Reaction of NO with Thiols

Article abstract of DOI:10.1021/jacs.7b06968

Azanone (nitroxyl, HNO) is a highly reactive compound whose biological role is still a matter of debate. One possible route for its formation is NO reduction by biological reductants. These reactions have been historically discarded due to the negative redox potential for the NO,H+/HNO couple. However, the NO to HNO conversion mediated by vitamins C, E, and aromatic alcohols has been recently shown to be feasible from a chemical standpoint. Based on these precedents, we decided to study the reaction of NO with thiols as potential sources of HNO. Using two complementary approaches, trapping by a Mn porphyrin and an HNO electrochemical sensor, we found that under anaerobic conditions aliphatic and aromatic thiols (as well as selenols) are able to convert NO to HNO, albeit at different rates. Further mechanistic analysis using ab initio methods shows that the reaction between NO and the thiol produces a free radical adduct RSNOH^?, which reacts with a second NO molecule to produce HNO and a nitrosothiol. The nitrosothiol intermediate reacts further with RSH to produce a second molecule of HNO and RSSR, as previously reported.

Full text of DOI:10.1021/jacs.7b06968

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Article
HNO is produced by the reaction of NO with thiols
Sebastian A. Suarez, Martina Muñoz, Lucia Alvarez, Mateus F. Venancio,
Willian Rocha, Damian E. Bikiel, Marcelo A. Marti, and Fabio Doctorovich
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b06968 • Publication Date (Web): 19 Sep 2017
Downloaded from http://pubs.acs.org on September 19, 2017
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HNO is produced by the reaction of NO with thiols
Sebastian A. Suarez,Ϯ,‡ Martina Muñoz,Ϯ,‡ Lucia Alvarez,Ϯ Mateus F. Venâncio,† Willian R. Rocha,†
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Damian E. Bikiel,Ϯ Marcelo A. Marti*,₶ and Fabio Doctorovich.* ,Ϯ
Ϯ Departamento de Química Inorgánica, Analítica y Química Física, Facultad de Ciencias Exactas y Naturales, Universidad
de Buenos Aires, Ciudad Universitaria, Buenos Aires, Argentina. INQUIMAEꢀCONICET, Ciudad Universitaria, Buenos
Aires, Argentina † Universidade Federal de Minas Gerais, Departamento de Química, Belo Horizonte, Brasil ₶ Departamento
de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Bueꢀ
nos Aires, Argentina. IQUIBICENꢀCONICET, Ciudad Universitaria, Buenos Aires, Argentina.
KEYWORDS: Nitric Oxide, thiols, nitroxyl, HNO, azanone
ABSTRACT: Azanone (nitroxyl, HNO) is a highly reactive compound whose biological role is still a matter of debate. One possiꢀ
ble route for its formation is NO reduction by biological reductants. These reactions have been historically discarded due to the
negative redox potential for the NO,H+/HNO couple. However, the NO to HNO conversion mediated by vitamins C, E and aroꢀ
matic alcohols has been recently shown to be feasible from a chemical standpoint. Based on these precedents we decided to study
the reaction of NO with thiols as potential sources of HNO. Using two complementary approaches, trapping by a Mn porphyrin,
and an HNO electrochemical sensor, we found that under anaerobic conditions aliphatic and aromatic thiols (as well as selenols) are
able to convert NO to HNO, albeit at different rates. Further mechanistic analysis using abꢀinitio methods show that the reaction
between NO and the thiol produces a free radical adduct RSNOH*, which reacts with a second NO molecule to produce HNO and a
nitrosothiol. The nitrosothiol intermediate reacts further with RSH to produce a second molecule of HNO and RSSR, as previously
reported.
HNO (nitroxyl, or azanone), is an increasingly important biꢀ
ochemical active molecule, whose elusive nature originates in
its extremely fast dimerization (kdim = 8 x 106 Mꢀ1sꢀ1), that
limits its maximum concentration and life time.1 It also reacts
fast with NO (k = 5.6 x 106 Mꢀ1sꢀ1),2 thiols (k = 3 x 106 Mꢀ1sꢀ1),
3–8 metalloproteins (k = 0.5 – 5 x 105 Mꢀ1sꢀ1) 9–13 and at moderꢀ
ate rate with oxygen (k= 3 x 103 Mꢀ1sꢀ1).1,14,15 Given the menꢀ
tioned instability, azanone donors are used in most
(bio)chemical studies.19,20 The biological relevance of HNO
has two main aspects. The first one concerns the study of its
pharmacological effects ꢀ mainly related with the cardiovascuꢀ
lar system, including the treatment of heart failure16–18 and the
elucidation of its overlapping and differential reactivity with
NO. The second aspect, and most relevant in the present case,
concerns the possibility of its endogenous production.19,21
Enzymatic proposed physiological relevant routes to HNO,
concern oxidation of arginine,21–23 or the reaction of NO21 or
nitrosyl species24 with H2S.25 Although they make a strong
point for enzymatic HNO production, it should be stressed that
none of them has been confirmed in-vivo.19
Moreover, the reduction of NO to HNO is necessarily coupled
to other reactions that produce compounds such as N2O, which
could drive the reaction forward, overcoming an unfavorable
thermodynamic barrier.29,30 This chemical reductive route
recently received key support, showing that NO can be
converted to HNO by hydrogen sulfide (H2S)25 or aromatic
alcohols (i.e. ascorbic acid, tyrosine).29
Hꢀatom abstraction from thiols could in principle be another
reductive pathway for HNO formation. Early studies showed
that NO ‘‘slowly’’ reacts with thiols leading to formation of
disulfides, N2O and eventually N2.31,32 Pryor and coworkers
found that anaerobic aqueous solutions of thiophenol (PhSH)
and thiols exposed to NO, result in quantitative formation of
the corresponding disulfides (RSSR).32 This reaction becomes
faster to completion as the pH gets closer to the pKa of the
thiol. However, HNO for mation was ruledꢀout due to the
observation of base catalysis, despite N2O being a major reacꢀ
tion product. A few years later, Nagasawa and coworkers,7,33
studied the anaerobic reaction of albumin (a thiolꢀcontaining
protein) and other biological thiols such as glutathione with
large excess (> 10 times) of NO. These reactions, which took
place in minutes, were shown to produce N2O, and the
corresponding sulfenic acids RSꢀOH were proposed as interꢀ
mediates. In both works the initial formation of a thionitroxide
radical RSNOH● was suggested (Scheme 1), but while Pryor
proposed dimerization of the free radical leading to the
disulfide, Nagasawa suggested that it could react with excess
NO, leading to an Nꢀnitroso intermediate that would yield the
sulfenic acid and N2O. The rate constant for the anaerobic
reaction of NO with cysteine at 25 °C was determined to be
3.7 x 102 Mꢀ1sꢀ1,34 assuming the mechanism proposed
The chemical (nonꢀenzymatic), biological compatible,
routes to HNO have been much less pursued.26 The most direct
route, chemical reduction of NO, has been historically disꢀ
carded, possibly due to the negative reduction potential of –0.8
V proposed for the NO/3NOꢀ couple.27 However, at physiologꢀ
1
ical pH, HNO is expected to be the main species with an
estimated Eº (NO,H+/1HNO) ≈ −0.11 V, becoming −0.55 V at
pH 7.27 Furthermore, this value is nowadays under revision:
recently Rocha et. al. computed Eº (NO,H+/1HNO) ≈ ꢀ0.16 V
at pH 7 in aqueous solution, using quantum mechanical calcuꢀ
lations and Monte Carlo statistical mechanical simulations.28
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by Pryor.32 The fact that these authors disregarded HNO as a
reaction intermediate, possibly originates in a lack of precise
detection tools for HNO, being all indirect at the moment
(between 1982 and early 2000). In any case, the presence of
N2O as a final product and the observed NO to HNO converꢀ
sion mediated by alcohols suggest that the reaction of thiols
with NO could involve HNO as an intermediate.
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Scheme 1.
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Another important point is related to the formation of nitroꢀ
sothiols (RSNO) and their subsequent reactions, yielding
disulfides and HNO.6 On the other hand, HNO reacts with
thiols at a fast rate (ca 106 Mꢀ1sꢀ1) yielding a thiolꢀbound
hydroxylamine that decays to several thiol oxidized species
including predominantly the disulfide.35 N2O release from the
reaction of GSNO and GSH was detected (k ≈ 10ꢀ2 Mꢀ1sꢀ1),6
even in the presence of 10 equivalents of GSH. This observaꢀ
tion implies that it is possible to detect HNO in the presence
of an excess of thiol, although it is generated in a ''slow'' manꢀ
ner and reacts ’’quickly’’ with the free thiol.
Figure 1. A) [Mn(NO)TCPP] produced after mixing 2 mM
NO with Cys 50 µM (blue), 100 µM (green) and 200 µM
(red), duplicated runs. Inset: [Mn(NO)TCPP] (black, t= 4000
sec; grey, t=1500 sec) produced as a function of time by mixꢀ
ing NO (2 mM) with Cys (200 µM) in the presence of
Mn(III)TCPP (1 µM, orange, t=0 sec). B) [Mn(NO)TCPP]
produced after mixing 2 mM of NO with 50 µM of each thiol:
Cys (blue), R6SH (orange), PhꢀSH (green) and PhꢀSeH (red).
The reaction starts when the thiols are added (t=100 sec).
Therefore, in this complex scenario of cross reactions beꢀ
tween thiols, NO and HNO, it is of primordial relevance to be
able to detect and quantify HNO in-situ and reliably in small
concentrations to assess its presence unequivocally and underꢀ
stand the underlying reaction mechanisms. For this sake, in
the present work we investigated the rate of HNO production
from the reaction of NO with thiols.
We begin our analysis of the reactions of NO with 1ꢀ
hexanethiol (R6SH), cysteine (Cys), benzenethiol (PhꢀSH),
and benzeneselenol (PhꢀSeH), using Mn(III)TCPP as a trapꢀ
ping agent for HNO.36 Figure 1A (inset) shows the absorbance
changes observed upon addition of Cys to a solution of NO
containing Mn(III)TCPP, which are indicative of porphyrin
conversion to Mn(NO)TCPP due to HNO production (no
reaction with the porphyrin, i.e no spectral changes, are obꢀ
served either with the addition of NO or any of the thiols
alone during the experimental timescale). Figure 1A shows
that the amount of trapped HNO, as revealed by the growth in
nitrosylporphyrin (Abs424nm), increases with time and is larger
for higher Cys concentrations. Similar results are observed for
the other thiols and selenol (Figure 1B), although the rate and
amount of produced HNO varies in the following order: Phꢀ
SeH > PhꢀSH >> HexꢀSH > Cys. This order is possibly relatꢀ
ed to the decreasing stability of the corresponding RSe● or
RS● free radicals (see below).
To have an independent confirmation of HNO production
and in order to determine the corresponding reaction rates, we
measured the time courses of [HNO] for the above described
reactions using an HNO selective electrode.37,38 Figure 2A
shows the [HNO] vs. time plot obtained after addition of Cys
to an NO solution, clearly evidencing how the electrode signal
raises rapidly, revealing the presence of produced HNO (see
the other compounds in SI). Figure 2B shows that the initial
HNO production rate (vi) scales linearly with both R6SH
(orange) and NO (gray) concentrations, while the other reacꢀ
tant concentration is maintained constant and in excess. From
these plots an effective bimolecular reaction rate constant
(keff), corresponding to equation 1, can be determined. Finally,
the corresponding log vs. log plots (shown in SI) confirm that
the reaction is first order in both reactants.
v = d[HNO]/dt = keff [NO] [RSH]
(1)
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formation of nitrosothiol in the presence of O2, as previously
described,40 and subsequent reaction of RSNO with RSH to
produce HNO.6 However, the rate and amount of produced
HNO decreases as the amount of added O2 is increased by ten
times or more, as previously shown.29
Table 1. keff and N2O - nitrite ratio obtained for the reac-
tions of thiols with NO (pH = 7.4, rt, anaerobic, in the
presence of DPTA).
Figure 2. A) In blue, [HNO] determined electrochemically
keff (Mꢀ1sꢀ1)[a]
NO2‾ :N2O[b]
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Compound
Ph-SeH
Ref
after addition of Cys (20 ꢁM) to an anaerobic solution of 200
ꢁM NO in the presence of DPTA 10ꢀ4 M. kobs: is determined
from the slope of the electrode signal vs time plots. In green,
[HNO] after simultaneous addition of Cys (20 ꢁM) and O2 (20
ꢁM) to an anaerobic solution of 200 ꢁM NO. B) vi vs [R6SH]
or [NO], while the other reactant concentration is maintained
constant (10 µM).
125 ± 5 (115)
0.9 ± 0.1
This work
This work
This work
110 ± 8 (105)
1.0 ± 0.1
Ph-SH
35 ± 5 (32)
0.9 ± 0.1
R6-SH
25 ± 6 (20)
1.2 ± 0.1
This work
Cys
34
370*
0.6
8
ꢀ
Cys
BSA[c]
34
29
29
29
ꢀ
Similar results were obtained for PhꢀSH and PhꢀSeH (see
Figure SI3 and SI4). The resulting effective rate constants
reported in Table 1 show that Cys and R6ꢀSH, both aliphatic
thiols, display similar rates, about 4 times smaller than those
observed for PhꢀSH and PhꢀSeH. It should be taken into acꢀ
1.2
1.2
1.2
Ascorbic Acid
Hydroquinone
Tyrosine
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1
count that the rate constants of HNO production (kHNOꢀRSH
)
could be significantly higher, because the obtained keffs also
comprise HNO consumption (i.e. HNO reaction with NO, see
below).
The high reactivity of reactants and products results in a
complex mechanistic scheme.29,30 Therefore, to analyze possiꢀ
ble mechanisms we quantified several products, especially
those that have not been previously characterized.32 The first
important reaction to consider (Eq. 2), is that between NO and
HNO shown below (k = 6 x 106 Mꢀ1sꢀ1).2
We also tested whether O2, Fe(II), Mn(II) and Cu(II) affectꢀ
ed HNO production in the described reactions. Electrochemiꢀ
cal [HNO] measurements were performed in both the absence
and presence of various metal ions (final added concentration
of 0.02 mM). Figure 3 shows the relative peak [HNO] with
respect to the ion free reaction (performed in the presence 0.1
mM DPTA as chelating agent) for the three tested ions Cu(II)
Fe(II) and Mn(II). As shown in Figure 3, there is no catalytic
effect of iron, consistent with previous results.39 Copper has
been shown to catalyze the production of RS* and accordingꢀ
ly, there is an increase in the peak for [HNO], but the increase
is moderate (twofold). The results for Mn are in between. .
2 NO + HNO →→→ NO2¯ + N2O
(2)
For each reaction, the final concentrations of nitrite in soluꢀ
tion, and the amount of N2O in the reaction chamber headꢀ
space were determined (see SI).29,37 The results presented in
Table 1 show that in all cases N2O and NO2¯ are produced in
approximately a 1:1 ratio, as expected from eq. 2. Reaction of
HNO with thiols could lead to further reduced species such as
hydroxylamine or even ammonia.6 NH3 was quantified by
acid/base titration and its yield is ca. 20ꢀ35% based on the
total initial amount of NO (see SI). Hydroxylamine was not
detected (see SI). Accordingly, NH2OH is expected to be
further reduced to NH3 (Eqs. 3 and 4).6,41
RSH + RSNO → RSN(OH)SR
(3)
RSN(OH)SR + 4RSH →→→ RSSR + H2O + NH3
(4)
The sum of the recovered nitrogen yield based on the total
amount of reacting NO varies from 61% in the case of Cys to
96% for PhꢀSeH after 2 h of reaction (4ꢀ9% of NO was left
unreacted). We also used NMR spectroscopy to characterize
the thiolꢀderived end products. In all cases the disulfides were
observed, i.e cystine, diphenyl disulfide ꢀ as shown in previꢀ
ous works,32 diꢀNꢀhexyl disulfide, and diphenyl diselenide.
Figure 3. Relative HNO production in the described reactions
by using the electrochemical nitroxyl sensor in the presence of
Fe(II), Mn(II) and Cu(II), with respect to the ion free reaction.
When O2 was added to the reaction in 1:1 ratio with respect
to Cys (see Figure 2A – green line, Table SI2), faster producꢀ
tion of HNO was observed. This is consistent with the rapid
To gain a deeper understanding of the studied reaction we
used abꢀinitio methods to determine the energetics and strucꢀ
tures of all possible reaction steps and species, taking as startꢀ
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ing points the proposals by Pryor and Nagasawa32,33 as well as
RSNO can react with a second molecule of thiol to yield diꢀ
our previous work with aromatic alcohols.29,30 The results
presented in Scheme 2, and SI, show that the first step comꢀ
prises a protonꢀcoupled nucleophilic attack (PCNA) of the
thiol to NO,29,30 that yields a thionitrite radical intermediate
RSNO(H)●.
sulfide and HNO, as previously suggested (see SI).43 Last but
not least, as proposed by Nagasawa,33 another alternative is
rearrangement of the Oꢀprotonated diꢀNOꢀthiol (I), yielding
an intermediate sulfenic acid and N2O. Besides, a possible
HNO producing chain reaction mechanism is shown in SI.
Our results show that the reaction between NO and thiols
generates HNO. The effective bimolecular rates (reported in
Table 1) show that cysteine produces HNO about 3 times
faster than the fastest aromatic alcohol ꢀascorbic acidꢀ, and
that the aromatic PhꢀSH is even faster. To understand the
relative reactivity and overall HNO yield two important issues
stand out. First, as for the aromatic alcohols, the key and
possible rate limiting step concerns formation and stability of
the first radical intermediate, which in this case is the thioꢀ
nitrite radical RSNO(H)●. This intermediate is only stable
after proton migration to either the O or N atoms, which alꢀ
lows the radical to be localized on the XꢀS (X=Cys, Ph, R6)
and not on the nitroso group. Aromatic groups allow for better
stabilization of the unpaired spin, and thus show faster effecꢀ
tive rates. Concerning the fate of RSNO(H)●, several possibilꢀ
ities need to be considered. Spontaneous decay to HNO and
the thiyl radical is endergonic and is rendered unlikely. A
clear difference when compared to alcohols such as ascorbic
acid or hydroquinone which mainly react by this route. Diꢀ
merization of the radical ꢀas proposed by Pryor34 is expected
to be slow due to its low effective concentration. Instead,
reaction of the radical with a second molecule of NO is more
likely under the present conditions (relatively high NO conꢀ
centration), yielding HNO and RSNO. Interestingly, in the
presence of excess thiol, RSNO reacts to yield another moleꢀ
cule of HNO and the disulfide, which is the main observed S
containing organic end product. In this route, two molecules
of oneꢀelectron reductants ꢀthe thiolsꢀ behave as one molecule
of twoꢀelectron reductant ꢀlike ascorbic acidꢀ according to the
overall reaction (Eq. 5):
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Scheme 2.
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By using hydration with Zundel ions it can be shown that
the reaction varies from slightly exergonic to endergonic
depending on the protonation site (ꢀ0.68 to +5.13 kcal/mol for
R=CH3 and ꢀ2.51 to 1.88 kcal/mol for R=Ph), and that the
radical has two tautomers: one with the proton on the oxygen
atom and the other, slightly more stable, on the nitrogen atom.
For R = Ph (Figure 4), the first step barrier shows a value of
19.67 Kcal/mol, which is in reasonable agreement to the one
predicted using the kinetic constant keff (ca. 15 Kcal/mol).
Similar results were obtained using open shell DFT calculaꢀ
tions with polarizable continuum model (see SI).
2NO + 2RSH → 2HNO + RSSR
(5)
The second point is related to thermodynamics, particularly
to the reduction of NO to HNO. Recently, E°(NO,H+/HNO)
was calculated by state of the art computational methods,
rendering the value ꢀ0.16 V,10 while that for (RSSR,2H+/2
RSH) is close to ꢀ0.25 V,29 and thus an outer sphere electron
transfer is in principle thermodynamically favorable. Moreoꢀ
ver, under conditions where NO is physiologically produced
(or added pharmacologically) the relative [NO]/[HNO] ratio is
expected to be over 1000, resulting in an effective E of ca. 0
V for the (NO,H+ / HNO) couple, amenable to be reduced by
biological reductants such as cysteine, glutathione, FAD,
NAD, tyrosine and vitamin C, inter alia.
Figure 4. A) Protonꢀcoupled nucleophilic attack of the RSH
to the nitrogen atom of NO, mediated by a Zundel ion (R =
Ph). The transition state has an imaginary frequency of
510.03i cmꢀ1 and the nuclear motion associated with this mode
is the concerted motion of the hydrogen atoms from the SꢀH
group to the water and from the water to the nitrogen of the
NO molecule, forming the NꢀH bond. Results are in kcal/mol
In summary, the present work provides clear evidence for
the reaction of NO with several thiols, as an HNO source. The
reactions of NO with thiols have been studied for over thirty
years yielding a rich picture of chemical reactivity which is
still far from being completely understood. In this context,
HNO emerges as a new reaction intermediate and a key player
of this complex and highly reactive environment. The relaꢀ
tionship between HNO and thiols is dual: thiols could be
involved in HNO formation but they are also the main targets
Direct decomposition of RSNO(H)● to yield HNO and a
thiyl radical RS● is highly unfavorable (>30 kcal/mol), thereꢀ
fore the thionitrite is expected to react rapidly with a second
molecule of NO, yielding either diꢀNOꢀthiol intermediates
(species (I) in Scheme 2) or HNO and RSNO.42 The decay of
these diꢀNOꢀthiol intermediates to produce RSNO and HNO
is endergonic (7.3 kcal/mol). If an excess of thiol is present,
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E.; Doctorovich, F. Polyhedron 2007, 26, 4673–4679.
(12) Martí, M. A.; Bari, S. E.; Estrin, D. A.; Doctorovich, F. J. Am.
Chem. Soc. 2005, 127, 4680–4684.
(13) Boron, I.; Suárez, S. A.; Doctorovich, F.; Martí, M. A.; Bari, S. E.
J. Inorg. Biochem. 2011, 105, 1044–1049.
(14) Miranda, K. M.; Dutton, A. S.; Ridnour, L. a; Foreman, C. a; Ford,
E.; Paolocci, N.; Katori, T.; Tocchetti, C. G.; Mancardi, D.; Thomꢀ
as, D. D.; Espey, M. G.; Houk, K. N.; Fukuto, J. M.; Wink, D. a. J.
Am. Chem. Soc. 2005, 127, 722–731.
for HNO bioactivity. Due to the high reactivity of HNO, it is
expected that its steady state concentration inside cells should
be several orders of magnitude lower than that of NO. Thereꢀ
fore the “physiological” redox potential for the (NO,
H+/HNO) couple is probably even more positive than that of
(O2/O2●ꢀ), i.e. easily accessible. With the expanding arsenal of
analytical tools for HNO detection36,38,45–47 both in solution
and in cells, there is no doubt that we are getting closer to
understand the in vivo chemistry of HNO.
(15) Liochev, S. I.; Fridovich, I. Free Radic. Biol. Med. 2003, 34,
1399–1404.
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42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(16) Ma, X. L.; Gao, F.; Liu, G. L.; Lopez, B. L.; Christopher, T. A.;
Fukuto, J. M.; Wink, D. A.; Feelisch, M. Proc. Natl. Acad. Sci. U.
S. A. 1999, 96, 14617.
(17) Ma, X. L.; Weyrich, A. S.; Lefer, D. J.; Lefer, A. M. Circ. Res.
1993, 72, 403–412.
(18) Switzer, C. H.; FloresꢀSantana, W.; Mancardi, D.; Donzelli, S.;
Basudhar, D.; Ridnour, L. a; Miranda, K. M.; Fukuto, J. M.; Paoꢀ
locci, N.; Wink, D. a. Biochim. Biophys. Acta 2009, 1787, 835–
840.
(19) Doctorovich, F.; Bikiel, D.; Pellegrino, J.; Suárez, S. A.; Larsen,
A.; Martí, M. A. Coord. Chem. Rev. 2011, 255, 2764–2784.
(20) Fukuto, J. M.; Bartberger, M. D.; Dutton, A. S.; Paolocci, N.;
Wink, D. a; Houk, K. N. Chem. Res. Toxicol. 2005, 18, 790–801.
(21) Doctorovich, F.; Bikiel, D. E. D. E.; Pellegrino, J.; Suárez, S. A. S.
A.; Martí, M. A. M. A. In Progress in Inorganic Chemistry; Kenꢀ
neth D. Karlin, John Wiley & Sons, Inc, 2014.
(22) Stoll, S.; NejatyJahromy, Y.; Woodward, J. J.; Ozarowski, A.;
Marletta, M. A.; Britt, R. D. J. Am. Chem. Soc. 2010, 132, 11812–
11823.
(23) Donzelli, S.; Graham, M.; FloresꢀSantana, W.; Switzer, C. H.; Yeh,
G. C.; Huang, J.; Stuehr, D. J.; King, S. B.; Miranda, K. M.; Wink,
D. A.; Espey, M. G.; FloresꢀSantana, W.; Switzer, C. H.; Yeh, G.
C.; Huang, J.; Stuehr, D. J.; King, S. B.; Miranda, K. M.; Wink, D.
A. Free Radic. Biol. Med. 2008, 45, 578–584.
(24) Filipovic, M. R. M.; Miljkovic, J. L. J.; Nauser, T.; Royzen, M.;
Klos, K.; Shubina, T.; Koppenol, W. H.; Lippard, S. J.; Ivanovićꢀ
Burmazović, I.; Ivanovic, I. J. Am. Chem. Soc. 2012, 134, 12016–
12027.
(25) Eberhardt, M.; Dux, M.; Namer, B.; Miljkovic, J.; Cordasic, N.;
Will, C.; Kichko, T. I.; de la Roche, J.; Fischer, M.; Suárez, S. A.;
Bikiel, D.; Dorsch, K.; Leffler, A.; Babes, A.; Lampert, A.; Lenꢀ
nerz, J. K.; Jacobi, J.; Martí, M. A.; Doctorovich, F.; Högestätt, E.
D.; Zygmunt, P. M.; IvanovicꢀBurmazovic, I.; Messlinger, K.;
Reeh, P.; Filipovic, M. R.; Roche, J. de la; Fischer, M.; Bikiel, D.;
Suárez, S. A.; Dorsch, K.; Leffler, A.; Babes, A.; Lampert, A.;
Lennerz, J. K.; Jacobi, J.; Martí, M. A.; Doctorovich, F.; Högestätt,
E. D.; Zygmunt, P. M.; IvanovicꢀBurmazovic, I.; Messlinger, K.;
Reeh, P.; Filipovic, M. R. Nat. Comm. 2014, 5, 4381.
(26) Kytzia, A.; Korth, H.ꢀG.; Sustmann, R.; Groot, H. De; Kirsch, M.;
de Groot, H.; Kirsch, M. Chemistry 2006, 12, 8786–8797.
(27) FloresꢀSantana, W.; Salmon, D. J.; Donzelli, S.; Switzer, C. H.;
Basudhar, D.; Ridnour, L.; Cheng, R.; Glynn, S. A.; Paolocci, N.;
Fukuto, J. M.; Miranda, K. M.; Wink, D. A. Antioxid. Redox Sig-
nal. 2011, 14, 1659–1674.
(28) Venâncio, M.; Doctorovich, F.; Rocha, W. J Phys Chem B. 2017,
121, 6618–6625.
(29) Suarez, S. A.; Neuman, N.; Muñoz, M.; Alvarez, L.; Brondino, C.;
Bikiel, D. E.; Martí, M. A.; Doctorovich, F. J. Am. Chem. Soc
2015, 137, 4720–4727.
(30) Hamer, M.; Suarez, S. A.; Neuman, N. I.; Alvarez, L.; Muñoz, M.;
Marti, M. A.; Doctorovich, F. Inorg. Chem. 2015, 54, 9342–9350.
(31) Reihlen, H.; Friedolsheim, A. V.; Oswald, W. Justus Liebigs Ann
Chem 1928, 465, 72–96.
(32) Pryor, W.; Church, D.; Govindan, C. J. Org. 1982, V (1), 156–159.
(33) DeMaster, E. G.; Quast, B. J.; Redfern, B.; Nagasawa, H. T. Bio-
chemistry 1995, 34, 11494–11499.
ASSOCIATED CONTENT
Kinetic analysis, ab initio calculations, and other experimental
details can be found in the Supporting Information. This material
is available free of charge via the Internet at http://pubs.acs.org
AUTHOR INFORMATION
Corresponding Author
* doctorovich@qi.fcen.uba.ar
* marcelo@qi.fcen.uba.ar
Author Contributions
The manuscript was written through contributions of all authors. /
All authors have given approval to the final version of the manuꢀ
script. / ‡These authors contributed equally.
Funding Sources
This work was financially supported by UBA (UBACYT
20020130100642BA, 20020150100023BA), ANPCyT (PICT
2014ꢀ1278, PICT 2015ꢀ3854, and by the Bunge y Born Foundaꢀ
tion.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
SAS, MM and LA thank UBA & CONICET for a fellowship
grant, SAS, DEB, MAM and FD are members of CONICET.
MFV and WRR would like to thank the CNPq (Conselho
Nacional de Desenvolvimento Científico e Tecnológico, INCTꢀ
Catálise) and FAPEMIG (Fundação de Amparo à Pesquisa do
Estado de Minas Gerais) for financial support and research grant.
REFERENCES
(1) Shafirovich, V.; Lymar, S. V. Proc. Natl. Acad. Sci. U. S. A. 2002,
99, 7340–7345.
(2) Lymar, S. V; Shafirovich, V.; Poskrebyshev, G. A. Inorg. Chem.
2005, 44, 5212–5221.
(3) Doyle, M. P.; Mahapatro, S. N.; Broene, R. D.; Guy, J. K. J. Am.
Chem. Soc. 1988, 110, 593–599.
(4) Bartberger, M. D.; Fukuto, J. M.; Houk, K. N. Proc. Natl. Acad.
Sci. U. S. A. 2001, 98, 2194–2198.
(5) Sherman, M. P.; Grither, W. R.; McCulla, R. D. J. Org. Chem.
2010, 75, 4014–4024.
(6) Wong, P. P. S.ꢀY.; Hyun, J.; Fukuto, J. M. J.; Shirota, F. N.; Deꢀ
Master, E. G.; Shoeman, D. W.; Nagasawa, H. T. Biochemistry
1998, 37, 5362–5371.
(7) Shoeman, D. W.; Shirota, F. N.; DeMaster, E. G.; Nagasawa, H. T.
Alcohol 2000, 20, 55–59.
(8) Keceli, G.; Moore, C. D.; Toscano, J. P. Bioorg. Med. Chem. Lett.
2014, 24, 3710–3713.
(9) Ford, P. C. P. C. Inorg. Chem. 2010, 49 , 6226–6239.
(10) Hoshino, M.; Laverman, L.; Ford, P. C. Coord. Chem. Rev. 1999,
187, 75–102.
(34) Aravindakumar, C. T.; Ley, M. De; Ceulemans, J. J. Chem. Soc.
Perkin Trans. 2 2002, 3, 663–669.
(35) Jackson, M. I.; Han, T. H.; Serbulea, L.; Dutton, A.; Ford, E.;
Miranda, K. M.; Houk, K. N.; Wink, D. A.; Fukuto, J. M. Free
Radic. Biol. Med. 2009, 47, 1130–1139.
(11) Suárez, S. A.; Martí, M. A.; De Biase, P. M.; Estrin, D. a.; Bari, S.
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Page 6 of 7
1
2
3
4
5
6
7
8
(36) Dobmeier, K. P.; Riccio, D. A.; Schoenfisch, M. H. Anal. Chem.
2008, 80, 1247–1254.
(37) Heinecke, J. L.; Khin, C.; Pereira, J. C. M.; Suárez, S. A.; Iretskii,
A. V; Doctorovich, F.; Ford, P. C. J. Am. Chem. Soc. 2013, 135,
4007–4017.
(38) Suárez, S. S. A.; Bikiel, D. D. E.; Wetzler, D. E. DE; Martí, M. A.
M. A.; Doctorovich, F. Anal. Chem. 2013, 85, 10262−10269.
(39) Bagiyan, G. A.; Koroleva, I. K.; Soroka, N. V.; Ufimtsev, A. V.
Russ. Chem. Bull. 2003, 52 (5), 1135–1141.
(40) Wink, D. A.; Nims, R. W.; Darbyshire, J. F.; Christodoulou, D.;
Hanbauer, I.; Cox, G. W.; Laval, F.; Laval, J.; Cook, J. A.; Krishꢀ
na, M. C.; DeGraff, W. G.; Mitchell, J. B. Chem. Res. Toxicol.
1994, 7 (4), 519–525.
(41) Singh, S. P.; Wishnok, J. S.; Keshive, M.; Deen, W. M.; Tannenꢀ
baum, S. R. Proc. Natl. Acad. Sci. U. S. A. 1996, 93 (25), 14428–
14433.
(42) Filipovic, M, In The Chemistry and Biology of Nitroxyl (HNO),
Fabio Doctorovich, Patrick Farmer and Marcelo A. Marti; Elsevier
Inc., 2017.
(43) Fukuto, J. M.; Cisneros, C. J.; Kinkade, R. L. J. Inorg. Biochem.
2013, 118, 201–208.
(44) Jocelyn, P. C. Eur. J. Biochem. 1967, 2 (3), 327–331.
(45) Wrobel, A. T.; Johnstone, T. C.; Liang, A. D.; Lippard, S. J.;
Riveraꢀfuentes, P. J. Am. Chem. Soc. 2014, 136, 4697–4705.
(46) Reisz, J. a; Zink, C. N.; King, S. B. J. Am. Chem. Soc. 2011, 133
(30), 11675–11685.
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10
11
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13
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15
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17
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20
21
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23
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32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(47) Cline, M. R.; Tu, C.; Silverman, D. N.; Toscano, J. P. Free Radic.
Biol. Med. 2011, 50 (10), 1274–1279.
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