Generation of H2S from Thiol-Dependent NO Reactivity of Model [4Fe-4S] Cluster and Roussin's Black Anion

Article abstract of DOI:10.1021/acs.inorgchem.1c01328

Iron-sulfur clusters (Fe-S) have been well established as a target for nitric oxide (NO) in biological systems. Complementary to protein-bound studies, synthetic models have provided a platform to study what iron nitrosylated products and byproducts are produced depending on a controlled reaction environment. We have previously shown a model [2Fe-2S] system that produced a dinitrosyl iron complex (DNIC) upon nitrosylation along with hydrogen sulfide (H2S), another important gasotransmitter, in the presence of thiol, and hypothesized a similar reactivity pattern with [4Fe-4S] clusters which have largely produced inconsistent reaction products across biological and synthetic systems. Roussin's black anion (RBA), [Fe4(μ3-S)3(NO)7]-, is a previously established reaction product from synthetic [4Fe-4S] clusters with NO. Here, we present a new reactivity for the nitrosylation of a synthetic [4Fe-4S] cluster in the presence of thiol and thiolate. [Et4N]2[Fe4S4(SPh)4] (1) was nitrosylated in the presence of excess PhSH to generate H2S and an "RBA-like"intermediate that when further reacted with [NEt4][SPh] produced a {Fe(NO)2}9 DNIC, [Et4N][Fe(NO)2(SPh)2] (2). This "RBA-like"intermediate proved difficult to isolate but shares striking similarities to RBA in the presence of thiol based on IR υ(NO) stretching frequencies. Surprisingly, the same reaction products were produced when the reaction started with RBA and thiol. Similar to 1/NO, RBA in the presence of thiol and thiolate generates stoichiometric amounts of DNIC while releasing its bridging sulfides as H2S. These results suggest not only that RBA may not be the final product of [4Fe-4S] + NO but also that RBA has unprecedented reactivity with thiols and thiolates which may explain current challenges around identifying biological nitrosylated Fe-S clusters.

Full text of DOI:10.1021/acs.inorgchem.1c01328

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Generation of H2S from Thiol-Dependent NO Reactivity of Model
[4Fe-4S] Cluster and Roussin’s Black Anion
Kady M. Oakley, Ziyi Zhao, Ryan L. Lehane, Ji Ma, and Eunsuk Kim*
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ABSTRACT: Iron−sulfur clusters (Fe−S) have been well established
as a target for nitric oxide (NO) in biological systems. Complementary
to protein-bound studies, synthetic models have provided a platform to
study what iron nitrosylated products and byproducts are produced
depending on a controlled reaction environment. We have previously
shown a model [2Fe-2S] system that produced a dinitrosyl iron
complex (DNIC) upon nitrosylation along with hydrogen sulfide (H2S),
another important gasotransmitter, in the presence of thiol, and
hypothesized a similar reactivity pattern with [4Fe-4S] clusters which
have largely produced inconsistent reaction products across biological
and synthetic systems. Roussin’s black anion (RBA), [Fe4(μ3-
S)3(NO)7]−, is a previously established reaction product from synthetic
[4Fe-4S] clusters with NO. Here, we present a new reactivity for the
nitrosylation of a synthetic [4Fe-4S] cluster in the presence of thiol and
thiolate. [Et4N]2[Fe4S4(SPh)4] (1) was nitrosylated in the presence of excess PhSH to generate H2S and an “RBA-like” intermediate
that when further reacted with [NEt4][SPh] produced a {Fe(NO)2}9 DNIC, [Et4N][Fe(NO)2(SPh)2] (2). This “RBA-like”
intermediate proved difficult to isolate but shares striking similarities to RBA in the presence of thiol based on IR υ(NO) stretching
frequencies. Surprisingly, the same reaction products were produced when the reaction started with RBA and thiol. Similar to 1/NO,
RBA in the presence of thiol and thiolate generates stoichiometric amounts of DNIC while releasing its bridging sulfides as H2S.
These results suggest not only that RBA may not be the final product of [4Fe-4S] + NO but also that RBA has unprecedented
reactivity with thiols and thiolates which may explain current challenges around identifying biological nitrosylated Fe−S clusters.
Mechanisms for the NO-H2S crosstalk can be divided into
two categories. One is the crosstalk through protein regulations
as seen with the action of NO and H2S on the secondary
messenger cGMP.9 In this case, NO facilitates the production
of cGMP by activating soluble guanylyl cyclase. Once cGMP
has been produced, H2S acts to delay cGMP’s degradation by
inhibiting phosphodiesterase.10 The other type of crosstalk is a
direct chemical interaction between H2S and NO and its
metabolites. Research into the possibility of this type of
crosstalk includes the reactivity of H2S with NO,11 nitroprus-
side,12 S-nitrosothiols,13 and peroxynitrite.14 Our research
group has focused on a different possible target for NO-H2S
crosstalk: iron sulfur clusters.
INTRODUCTION
■
Nitric oxide (NO), originally thought to simply be a toxic gas,1
has been actively investigated for its role in cellular signaling in
the past several decades.2 Along with carbon monoxide (CO)
and hydrogen sulfide (H2S), NO has been labeled as one of the
three important gasotrasmitters in biological systems. Specif-
ically, NO is known to be most active in the central nervous
system, where it effects brain development, memory, and
learning;3 in immune response, where it plays a part in the
inflammatory response;4 and in the circulatory system, where it
acts as a vasorelaxer and vasodilater to aid circulation.5
Currently known mechanisms of H2S production stem from
both enzymatic and nonenzymatic pathways. At least three
enzymes, cystathionine β-synthase (CBS), cystathione γ-lyase
(CSE), and 3-mercaptopyruvate sulfurtransferase (MST), are
involved in the biosynthesis of H2S from L-cysteine (CBS and
CSE) or 3-mercaptopyruvate (MST).6 H2S can be stored as
sulfane sulfur via oxidative post-translation modification of
cysteines to form persulfies that can release H2S under
reducing conditions.6 Interestingly, many of the biological
functions performed by H2S overlap with the functions of
NO.7 Due to this overlap, researchers have been looking for a
point of crosstalk for the two for years.8
Iron sulfur ([Fe−S]) clusters are ubiquitous in biological
systems. Common to the most ancient and modern forms of
Special Issue: Renaissance in NO Chemistry
Received: May 2, 2021
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life, they carry out many functions required for survival:
electron transport, substrate activation and catalysis, and
cellular sensing and signaling.15 Among the functions of
[Fe−S] clusters in biological systems, they have been found to
be a major target for reactivity of nitric oxide to form various
iron nitrosyl species (Chart 1). The conversion of [Fe−S]
anion (RBA) consistent with the previous report by the
Lippard group (Scheme 1a).20 The crude product generated
Scheme 1
Chart 1. Most Common Forms of Iron Nitrosyls
cluster to iron-nitrosyls results in significant changes in protein
function that can lead to NO-derived physiological signal
transduction16 or severe toxicity.17 To study the reactivity of
[Fe−S] clusters with NO, researchers have broadly taken two
different routes: protein bound clusters to study reactivity in
their native environment, and synthetic model clusters to allow
the chemical reactivity and mechanisms to be more easily
studied. Synthetic models are rarely stable or soluble in
biologically relevant aqueous or buffer systems and cannot
emulate the structural constrains imposed by the large protein
backbones. However, discrete synthetic models that are not
being masked by the bulky protein residues and buffer
molecules make it possible to study chemical reactivities
intrinsic to the [Fe−S] cofactors. These model systems are
especially valuable when the reactions involve small gaseous
molecules such as O2, NO, H2S, etc.
In the past decade, our laboratory used this synthetic
modeling approach to provide chemical insights into how
cellular redox components such as [Fe−S] clusters, NO, O2,
and H2S propagate their redox signals.18 One of the conceptual
advances we made was the identification of new reaction
conditions that lead to the formation of previously unrecog-
nized products during the reaction of [Fe−S] clusters with
NO.18,19 We found that the bridging sulfides from [2Fe-2S]
clusters can be released as H2S upon nitrosylation if the
environment can provide a formal equivalent of H• (e−/H+)
from donors such as thiols or phenols.18,19b These results led
us to hypothesize that [Fe−S] clusters can act as a point of
direct crosstalk between NO and H2S. In this Forum article, we
report our recent studies probing whether this reactivity can be
expanded to other clusters including the cubane-type [4Fe-4S]
cluster: the most common cluster form.
from the reaction of 1 with NO in the presence of thiol (10
equiv) displayed a very similar IR spectrum to that of RBA
with ν(NO) stretching frequencies of 1799, 1739, and 1708
cm−1 that are slightly shifted lower in energy from RBA
(Figure 1A). However, this “RBA-like” product, labeled Int. A
(Scheme 1b), appears to have drastically different stability
from RBA as it constantly changes in morphology and color
along with its IR spectrum every recrystallization attempt,
hampering us from isolating a purified product. The X-band
EPR spectrum of the crude product showed that the major
reaction product was EPR silent (at 77 K or RT) while a
dinitrosyl iron complex (DNIC), [Et4N][Fe(NO)2(SPh)2]
(2), was formed as a minor product (∼31%); Scheme 1b.
DNIC ν(NO) stretching frequencies of 1743 and 1681 cm−1
were also observed in the IR spectrum, with the former being
encompassed in the broad feature at 1739 cm−1 (Figures 1A
and S8). Additionally, the fingerprint region of the DNIC
thiolate ligands between 1573 and 1388 cm−1 is present.
We next investigated the fate of the bridging sulfides in 1
upon nitrosylation in the presence and absence of an external
thiol. After the reactions were complete, insoluble material was
separated by filtration and subsequently combined with
triphenylphosphine, PPh3, to trap and quantify elemental
sulfur that might have been generated from the reaction. 31P
NMR spectroscopy and GC-MS were used to determine the
formation of triphenylphosphine sulfide, S=PPh3, an estab-
lished reaction product of PPh3 with elemental sulfur.19 The 1/
NO reaction in the absence of thiol was found to produce one
equivalent of S=PPh3 as expected.20 The 1/NO reaction in the
presence of thiol, however, was found to produce no S=PPh3,
RESULTS AND DISCUSSION
■
Effects of Thiol Inclusion on NO reactivity of [4Fe-4S]
Cluster. The first question we sought to answer was whether
or not adding an external thiol source to the reaction of [4Fe-
4S] cluster with NO would change the reactivity pattern as was
observed with [2Fe-2S] clusters.19b To make that determi-
nation, nitric oxide was added to a synthetic [4Fe-4S] cluster,
[Et4N]2[Fe4S4(SPh)4] (1), in the presence and the absence of
an external thiol, PhSH. Both a purified NO gas and a chemical
NO donor, S-trityl thionitrite (Ph3CSNO),20 were utilized to
examine the thiol effects. It is noted that NO(g) and Ph3CSNO
resulted in no difference in the reactivity studies reported in
this study. Exposure of excess (10 equiv) NO to an acetonitrile
solution of 1 in the absence of PhSH yielded Roussin’s black
B
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Figure 1. Reaction of (Et4N)2[Fe4S4(SPh)4] (1) with NO in the presence of thiol and/or thiolate. (A) IR spectrum (in KBr) of Int. A (red)
resulting from 1/NO in the presence of HSPh and that of authentic RBA (back). (B) EPR spectra of 1 + 10 equiv PhSH + 10 equiv Ph3CSNO
(red) followed by addition of 10 equiv PhS− (black) at 298 K. (C) Fluorescence spectra of a turn-on H2S sensor following incubation with the
headspace gas of the 1/NO reaction in the absence (green) and presence (blue) of HSPh. Inset: Response of lead acetate paper detecting H2S from
1/NO in the absence (left) and presence (right) of HSPh. (D) IR spectrum (in KBr) of Int. A + 10 equiv PhS− to generate
[Et4N][Fe(NO)2(SPh)2] (2) (red) and that of authentic 2 (black).
indicating no elemental sulfur was produced during the
reaction.
which allowed us to quantify the amount of H2S produced
during the course of a reaction using an independently
prepared calibration curve using NaSH/HCl. Interestingly, the
H2S quantification revealed that all four equivalents of bridging
sulfides in 1 are released as H2S upon nitrosylation with thiol
present, which indicates Int. A has no bridging sulfide in its
structure.
The negative Sx detection from 1/NO in the presence of
thiol led us to examine H2S evolution from the thiol containing
reaction. We first used lead acetate paper to qualitatively detect
H2S since the interaction of lead acetate with H2S even in trace
concentrations (as low as 5 ppm)21 can produce dark-brown
lead sulfide (H2S + Pb(OAc)2 → PbS + 2 AcOH). The lead
acetate paper strip was hung over the headspace of the reaction
flask for the 1/NO reactions in the absence and presence of
excess (10 equiv) PhSH. No color change on the lead acetate
paper was observed from 1/NO in the absence of an external
thiol source. However, there was a clear reaction with the lead
acetate paper from the 1/NO reaction with thiol (Figure 1C),
indicating that H2S was produced during the nitrosylation of 1
with PhSH present. Emboldened by this result, we next
quantified the amount of H2S by employing a turn-on H2S
fluorescence sensor, 7-azido-4-methylcoumarin (C7Az).22 The
conversion of the azido group of C7Az to an amino group by
H2S has been reported to enhance a fluorescent emission signal
at λ = 434 nm.22 The headspace gas of the 1/NO reaction in
the presence and absence of thiol was transferred to an
acetonitrile solution of C7Az whose fluorescence spectrum was
then subsequently analyzed. The 1/NO reaction in the absence
of thiol did not induce any fluorescence signal. However, the
same reaction in the presence of thiol led to a significant
fluorescence enhancement at 434 nm (Figure 1C). The C7Az
sensor exhibited a linear response to increasing concentration
of H2S from 0.2 mM to 1 mM in our experimental setup,
Effects of Thiolate Inclusion on NO reactivity of [4Fe-
4S] Cluster. In spite of the intriguing reactivity of 1/NO with
external thiol leading to H2S evolution, unidentified Int. A has
been a considerable challenge for us to gain further insights.
Therefore, we turned our attention to convert Int. A to
another product that is more stable for isolation and
characterization. We found that an addition of excess (10
equiv) thiolate, [Et4N][SPh], to Int. A resulted in the
formation of a {Fe(NO)2}9 DNIC, [Et4N][Fe(NO)2(SPh)2]
(2) (Figure 1D). After multiple recrystallizations from MeCN/
Et2O, 2 was isolated (72% yield) as a dark red microcrystalline
solid with known spectroscopic characteristics.23 Complex 2 is
one of the most intensively studied DNICs in the
literature,20,23,24 and we capitalized on its unique g = 2.03
EPR signal to quantify the formation of 2 in each step (Scheme
1b) by employing a calibration curve prepared from
independently synthesized 2. Double integration of the g =
2.03 signal from the reaction mixture indicates that ∼31% of
iron in [Et4N]2[Fe4S4(SPh)4] (1) is converted to 2 after
nitrosylation in the presence of thiol. Upon subsequent
treatment with thiolate, there was a significant increase in
the g = 2.03 signal which corresponds to the formation of
C
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Figure 2. Reaction of RBA with thiol or thiolate. (A) IR spectra (KBr pellet) of the υNO (cm−1) region comparing RBA + 10 equiv PhSH (purple)
to [Fe4S4(SPh)4]2− (1) + 10 equiv PhSH + 10 equiv Ph3CSNO (red). (B) IR spectra comparing RBA + 10 equiv PhS− (blue) to authentic RBA
(black).
overall four equivalents of 2. The conversion of 1 to 2 can also
be achieved in one step when 1 is exposed to NO in the
presence of both thiol and thiolate. Likewise, the evolution of
four equivalents of H2S was observed from a single step
reaction of 1 with NO in the presence of both thiol and
thiolate. In order to produce the quantitative amounts of 2 and
H2S from 1, iron must receive electrons from the environment,
i.e., externally added thiol/thiolate, which led us to examine
the formation of the byproduct, PhSSPh. The GC-MS
quantification confirmed the quantitative amount (3 equiv)
of disulfide was also produced.25
modeling and semiempirical quantum chemical calculations
suggest that a reaction of RBA with thiolate would form a
pseudocubane complex, [Fe4(μ3-S)3(μ3-SPh)(NO)7]2− (Chart
2), in which the NO ligands in the thiolate adduct are more
Chart 2
Reaction of Roussin’s Black Anion with thiol/thiolate.
Roussin’s black anion (RBA), [Fe4(μ3-S)3(NO)7]−, is one of
the longest known iron nitrosyl compounds26 and it often
appears as a thermodynamic product from NO reactivity with
synthetic [Fe−S] clusters.20 Intrigued by the resemblance of
the IR spectrum of RBA to that of Int. A (Figure 1A), we
decided to investigate the reactivity of RBA with thiol and
thiolate to gain further insights into Int. A. RBA was prepared
by following a literature procedure.20 To our surprise, when
RBA was exposed to free PhSH (10 equiv), the evolution of
H2S was first noticed by the color change of the lead acetate
paper. The RBA/HSPh reaction also produces a metastable
iron nitrosyl product whose IR spectrum is remarkably similar
to that of Int. A from the reaction between
[Et4N]2[Fe4S4(SPh)4] (1) and NO in the presence of thiol
(Figure 2A). The observed similar stability and the IR features
between the two reaction products made us suspect that both
reactions might produce the same product. Therefore, we
tested whether an addition of thiolate to the product from the
reaction of RBA and PhSH would also produce a DNIC,
[Et4N][Fe(NO)2(SPh)2] (2), as was observed with the
reaction of 1/NO/thiol. Indeed, an addition of excess (20
equiv) [Et4N][SPh] to the reaction product of RBA and PhSH
produced 2 without generating any other iron nitrosyl
byproduct.
The formation of [Et4N][Fe(NO)2(SPh)2] (2) from RBA
was not sensitive to the order in which thiol and thiolate were
added. The successive addition of the reverse order, i.e.,
addition of [Et4N][SPh] followed by HSPh or the addition of
a mixture of thiol and thiolate to RBA led to the same result:
the formation of 2. When thiolate was added to RBA before
thiol addition, however, a new reaction intermediate, Int. B,
was observed by IR spectroscopy with lower ν(NO) stretching
frequencies starting at 1743 cm−1 compared to those of RBA
that begin at 1799 cm−1 (Figure 2B). Previous molecular
negative than in the RBA cluster.27 Although we were not able
to further characterize Int.B due to its limited stability, our
observed ν(NO) stretching frequencies of Int.B suggest more
reduced NO characters compared to those in RBA.
Accordingly, [Fe4(μ3-S)3(μ3-SPh)(NO)7]2− is considered as a
possible structure for Int.B. The EPR quantification revealed
that 3 equiv of iron embedded in RBA, [Fe4(μ3-S)3(NO)7]−,
are eventually converted to [Et4N][Fe(NO)2(SPh)2] (2) by
the addition of thiol and thiolate. One equivalent of NO is
presumed to be released as NO(g), while the remaining one
equiv of iron from RBA is released as [Fe(SPh)4]2−, the
presence of which was shown in the 1H NMR spectrum of the
crude product mixture (Figure S7). When extra NO is
provided to the reaction of RBA with thiol and thiolate, all
four equivalents of iron from RBA are converted to 2 as
determined by EPR quantification (Scheme 2).
The observed reactivity of RBA, [Fe4(μ3-S)3(NO)7]−,
displays how nitrosylation can change the chemical properties
of the bridging sulfides in [Fe−S] clusters. The [4Fe-4S]
model cluster [Et4N]2[Fe4S4(SPh)4] (1) is very stable in the
presence of large excess PhSH, and the bridging sulfides are
not prone to substitution with PhSH. However, the bridging
sulfides of RBA are easily released as H2S by PhSH at room
temperature without need for any other reagent. We suspect
that the bridging sulfides in RBA are more basic than those in
1 because they are coordinated to iron in a more reduced state
(3x {Fe(NO)2}9 and {Fe(NO)}7 in RBA) than iron (2+/3+)
of compound 1. This difference might initiate protonation of
RBA by a weak acid PhSH (pKa ∼ 6.6) to form bridging
hydrosulfide, [Fe4(μ3-SH)3(NO)7]2+, which could be sub-
sequently replaced by thiolate to form [Fe4(μ3-SPh)3(NO)7]2+,
D
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however, nitrosylated products from [4Fe-4S] clusters show no
consistency. Nuclear resonance vibrational (NRV) spectro-
scopic studies on [4Fe-4S]-ferredoxin report RBA as the main
nitrosylated product.33 Another [4Fe-4S] protein, EndoIII,
reports a 1:1 mixture of RRE and DNIC as the final
nitrosylated products analyzed by HYSCORE pulse EPR
spectroscopy and mass spectrometry.34 As for WhiD and NsrR
regulatory proteins, which are dedicated NO-sensors in
Scheme 2
̈
microorganisms, Mossbauer, NRVS, and DFT, and
14NO/15NO and 32S/34S labeling studies rule out the formation
of RBA from these proteins.35 Instead, researchers report the
nitrosylated products of WhiD and NsrR (both from S.
coelicolor) as “species related to RBA and RRE”.35 Similarly,
another ambiguous “RBA-like” description is given to the
nitrosylated product from a [4Fe-4S] HiPIP protein with
NO.36 We conjecture that the various types of unidentified
nitrosylated products observed with [4Fe-4S] proteins might
be due to the intrinsic instability of multinuclear [Fe−S]
clusters with NO ligands. The current study shows that the
bridging sulfides embedded in RBA are more reactive toward
thiol compared to the bridging sulfides in [Fe−S] clusters
without NO ligands. It also shows that the “RBA-like”
intermediates can easily convert to another type of iron
nitrosyl by thiolate. This structural vulnerability of iron
nitrosyls resulting from [4Fe-4S]/NO against environmental
factors (thiol, thiolate, etc.) might be a reason for the lack of a
general product type.
the model we currently consider a possible structure for Int. A
(Chart 2). The previous study by Lippard established that RBA
is the reaction product of 1/NO.20 Our study here suggests
that RBA may not be the final product and it could be a
reactive intermediate leading to other sulfur and iron-
containing final products in a thiol rich environment. This
unexpected chemical reactivity of RBA may explain the
challenges the biochemical and biophysical communities face
in identifying products generated from [4Fe-4S] proteins with
NO (vide infra).
Biological Implications. In spite of a considerable number
of studies that report NO reactivity of various [Fe−S] proteins,
little is known about the fate of the bridging sulfides after the
reaction. Bridging sulfides in [Fe−S] clusters are generally
acid-labile, and a strong acid can disrupt the cluster with a
release of H2S. Our study shows that the H2S evolution from
[Fe−S] clusters does not necessarily require a strong acid
when [Fe−S] clusters are exposed to NO. Consistent with our
previous studies with [2Fe-2S] clusters, we observe that the
presence of thiol alters the fate of the bridging sulfides of [4Fe-
4S] clusters upon nitrosylation and leads to the H2S evolution.
Given the high concentration of cellular thiols (1−10 mM),28
our study suggest that H2S must be a viable reaction product
when [Fe−S] cofactors are exposed to NO.
Our reactivity studies of RBA with thiol and/or thiolate
originated from a motivation to understand the 1/NO
reactivity with thiol present because RBA was the reaction
product from 1/NO in the absence of thiol.20 The H2S
evolution from RBA by PhSH came as a surprise. However,
this unexpected H2S formation from RBA has its own
intellectual merit considering that RBA has long been known
for its antimicrobial activity.29 The observed H2S liberation
from RBA by thiol suggests that the known antimicrobial effect
of RBA might be directly linked to the H2S redox
signaling.6a,9,30
The conversion of [Fe−S] clusters to iron-nitrosyl species
by NO has drawn considerable attention for the past decade.
As for the [2Fe-2S] clusters, there is a clear pattern in NO
reactivity. The final nitrosylated products are either {Fe-
(NO)2}9 DNIC or its dimeric form, species known as
Roussin’s red ester (RRE), Chart 1, in most cases16c,31 with
the exception of the [2Fe-2S] clusters in mitoNEET and
miner2 that reversibly bind NO.32 Unlike [2Fe-2S] systems,
CONCLUSIONS
■
Our previous studies demonstrated that both of the bridging
sulfides in prototypical [2Fe-2S] clusters such as
[Fe2S2(SPh)4]2− can be released as H2S upon NO exposure
if the environment is capable of providing a formal equivalent
of H• (e−/H+) from donors such as thiols and phenols.18,19
The present study shows that the formation of H2S from NO/
[Fe−S] reactivity in the presence of thiol is relevant beyond
the [2Fe-2S] subclass to now include the [4Fe-4S] type, the
most common form of [Fe−S] cluster. Upon nitrosylation of
[Fe4S4(SPh)4]2− (1) in the presence of PhSH, all four
equivalents of the bridging sulfides are released as H2S with
the concomitant formation of a new EPR-silent iron nitrosyl
intermediate whose IR features resemble those of RBA. This
“RBA-like” intermediate is subsequently converted to a
{Fe(NO)2}9 DNIC, [Fe2(NO)2(SPh)2]2− (2), upon thiolate
addition. The exposure of RBA to PhSH generates H2S and the
same “RBA-like” intermediate. The latter transforms to 2 with
further treatment with thiolate. The NO binding to [Fe−S]
clusters appears to greatly alter the chemical properties of the
bridging sulfides which can be released as H2S in the presence
of thiol.
EXPERIMENTAL SECTION
■
General. All synthesized products were assumed to be air- and
moisture-sensitive. They were manipulated under argon on a standard
Schlenk line or in an atmosphere of purified nitrogen in an MBraun
Labmaster SP glovebox (O2 < 1 ppm; H2O < 1 ppm). Solvents were
purified by being passed through a series of two activated alumina
columns (MBraun solvent purification system) under an Ar
atmosphere and stored over 4 Å molecular sieves. Phenyl disulfide
(PhSSPh), thiophenol (HSPh), triphenylphosphine (TPP), triphe-
nylphosphine sulfide (TPPS), sodium hydrosulfide, Celite, 7-azido-4-
methylcoumarin (C7Az), and lead acetate paper were purchased from
Sigma and used as received. Tube sealant was purchased from Fisher
and used as received.
E
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Physical Measurements. Infrared spectra were recorded on a
Bruker Tensor 27 FT-IR spectrometer. Fluorescence spectra were
recorded on a Tecan Safire spectrometer. 1H NMR spectra were
recorded at 400 MHz on a Bruker UltraShield spectrometer and
residual solvent signals were used as an internal reference. EPR
spectra of a liquid sample were recorded on a Bruker EMX plus EPR
spectrometer at 298 K. The EPR samples were loaded into a 100 μL
glass capillary that was inserted into a 4 mm o.d. quartz EPR tube.
Spectra were recorded under the following conditions: microwave
frequency, 9.871 GHz; microwave power, 2.0 mW; modulation
amplitude and frequency, 1.000 G and 100 kHz. GC-MS data were
recorded using a Hewlett-Packard (Agilent) GCD 1800C GC-MS
spectrometer.
the side arm of the reaction flask (Figure S1). The C7Az containing
round-bottom flask was placed in liquid nitrogen to generate negative
pressure, after which the side arm of the reaction Schlenk flask was
opened to allow gas transfer for 2 min. The connection between the
C7Az containing round-bottom flask and the reaction Schlenk flask
was cut off after gas transfer. The C7Az containing round-bottom
flask was removed from liquid nitrogen, and the C7Az solution was
allowed to stir for an additional hour at room temperature. The 30-
fold diluted C7Az solution in MeCN was used for fluorescence
analysis (Figure 1A).
The calibration curve for H2S detection was made through the
same procedure using NaSH and HCl in the reaction flask to produce
H2S for analysis. By varying the amount of NaSH and HCl, H2S
between 0.2 and 1 mM was generated and used for the calibration
curve (Figure S2).
The percent yield of H2S generated during the reaction of [4Fe-4S]
cluster with Ph3CSNO in the presence of thiol and thiolate is
approximately 100% based on fluorescence intensity, signifying all 4
bridging-sulfide were released as H2S. Additionally, we found that all
H2S release was during the first step of the reaction, as in the reaction
of [4Fe-4S] cluster with Ph3CSNO and thiol, while the following
addition of [NEt4][SPh] did not result in any additional H2S
formation.
b. Iron-containing species detection. IR spectrum of the reaction
mixture of (Et4N)2[Fe4S4(SPh)4] (1) reacted with Ph3CSNO in the
presence of thiol showed the generation of a new type of iron-nitrosyl
species, Int. A (Figure 1C). The addition of [NEt4][SPh] produced
changes in the IR spectrum that suggest DNIC to be the only iron-
containing species after the addition of [NEt4][SPh] (Figure 1D).
EPR spectroscopy was used to quantify the amount of DNIC,
(Et4N)[Fe(NO)2(SPh)2] (2), generation (Figure 1B). The calibra-
tion curve for 2 (Figure S3) was prepared using independently
synthesized 2 dissolved in MeCN in the concentration range of 1−6
mM. In the glovebox, 0.2 mL of 10 mM (Et4N)2[Fe4S4(SPh)4] (1)
stock solution was mixed with 10 equiv (0.2 mL, 100 mM) of PhSH
and 10 equiv (0.2 mL, 100 mM) of Ph3CSNO in a 10 mL Schlenk
flask. After stirring in the dark for 3 h, the EPR trace showed a weak
EPR signal at g = 2.029 corresponding to ∼31% DNIC production
where the formation of 4 equiv of 2 per 1 was considered as 100%.
After the first reaction step of [4Fe-4S] with Ph3CSNO and thiol, 1
mL of [NEt4][SPh] stock solution (20 mM, 10 equiv) was added into
the reaction mixture and allowed to stir for an additional hour. The
EPR trace after the addition of [NEt4][SPh] displayed the same signal
at g = 2.029 with stronger intensity, corresponding to 93% DNIC
generation for the whole reaction of [4Fe-4S] with Ph3CSNO in the
presence of thiol and thiolate.
Synthesis. (Et4N)2[Fe4S4(SPh)4] (1),37 (Et4N)[Fe4S3(NO)7]
(RBA),20 (Et4N)[Fe(NO)2(SPh)2] (2),23 and trityl-S-nitrosothiol20
were prepared as described in the literature.
Reaction of (Et4N)2[Fe4S4(SPh)4] (1) with Ph3CSNO (a) in the
Absence of Thiol and Thiolate. This reaction has been previously
reported by Lippard and co-workers.20 In the glovebox, a solution of
50 mg (0.045 mmol) of 1 in 5 mL of MeCN was mixed with 102.2 mg
(0.243 mmol) of Ph3CSNO. The reaction was allowed to stir for 3 h
at room temperature in dark. During this time, the formation of an
insoluble solid (i.e., elemental sulfur) could be observed. This
insoluble solid was separated by filtration. The filtrate was saved, and
all volatiles were removed in vacuo. The resultant residue was washed
with Et2O and redissolved in 1 mL of THF. Crystallization from
THF/pentane gave (Et4N)[Fe4S3(NO)7] (RBA) as black crystals
(24.4 mg, 81%); its UV−vis and IR spectroscopic features were in
good agreement with those reported for RBA.20
Reaction of (Et4N)2[Fe4S4(SPh)4] (1) with Ph3CSNO (b) in the
Presence of Thiol and Thiolate. Under a N2 atmosphere, 50 mg
(0.045 mmol) of 1 was dissolved in 5 mL of MeCN, and the mixture
was transferred to a 25 mL Schlenk flask to which was added 10 equiv
of PhSH followed by addition of 10 equiv of Ph3CSNO and 10 equiv
of [NEt4][SPh]. The reaction was stirred at room temperature in the
dark for 3 h. During this time, the color of the reaction mixture turned
from brown-red to dark red and the formation of H2S could be
qualitatively observed by lead acetate paper (Figure 1A). After 3 h, all
volatiles were removed in vacuo. The residue was washed with 10 mL
of Et2O and the Et2O washing was later found to contain the PhSSPh.
Analysis of the IR spectrum of the black oily residue indicated
(Et4N)[Fe(NO)2(SPh)2] (2) to be the only NO-containing product.
The oily residue was recrystallized in 5 mL of a 1:1 MeCN:Et2O
solution in a −35 °C freezer overnight to afford 2 as dark red needles
(61.2 mg, 72%) whose UV−vis, IR, and EPR spectra were in good
agreement with those reported for 2.23
General Method for Product Detection and Quantification
for Reaction b. Under a N2 atmosphere, 0.2 mL of a 10 mM
(Et4N)2[Fe4S4(SPh)4] (1) stock solution was mixed with 10 equiv
(0.2 mL, 100 mM) of PhSH in a 10 mL Schlenk flask and sealed with
a rubber septum, and 10 equiv (0.2 mL, 100 mM) of Ph3CSNO was
injected into the flask. After 3 h of stirring, 1 mL of a [NEt4][SPh]
stock solution (20 mM, 10 equiv) was injected into the reaction
mixture. The resulting solution was allowed to further stir for 1 h at
room temperature. The reaction products were identified by IR, GS-
MS, and EPR spectroscopy.
a. Hydrogen sulfide (H2S) detection. The formation of H2S during
the reaction could be qualitatively analyzed using lead acetate paper.
After the (Et4N)2[Fe2S2(SPh)4] (1) solution was loaded into the
Schlenk flask, a lead acetate paper strip was held in place by a rubber
septum in the flask headspace. Addition of Ph3CSNO, PhSH, and
[NEt4][SPh] were carefully carried out via syringe, while avoiding
touching the detector with the reaction solution. The color change of
the lead acetate paper was then observed (Figure 1A, inset).
A turn-on fluorescence H2S sensor, 7-azido-4-methylcoumarin
(C7Az),22 was used to quantify the amount of H2S produced. In the
presence of H2S, the azide group of C7Az is reduced to an amine and
generates 7-amino-4-methylcoumarin (C7Am) that emits at λem = 434
nm. After reaction completion, a 25 mL two-neck round-bottom flask
containing 1 mL of 10 mM C7Az solution in MeCN was connected to
c. Phenyldisulfide (PhSSPh) detection. PhSSPh quantification was
carried out using GC-MS where the calibration curve for PhSSPh
(Figure S4) was made in the concentration range of 50 to 500 μM,
while 50 μM of S=PPh3 was used as an internal standard to provide
reproducible data from day-to-day measurements. The calibration
curve was based on the normalized peak area of PhSSPh.
After reaction b was finished, a 150 μL aliquot of the reaction
solution was mixed with 20 μL of a solution of S=PPh3 (3.75 mM) in
MeCN. The final volume of the mixture was adjusted to 1.5 mL with
MeCN to make a GC sample. A control reaction without
(Et4N)2[Fe4S4(SPh)4] (1) was also tested and served as a baseline
to detect any PhSSPh formation that resulted from excess HSPh
oxidizing under the GC conditions. The amount of PhSSPh generated
from the [4Fe-4S] cluster that reacted with Ph3CSNO in the presence
of thiol and thiolate was determined as 91 3%, where 3 equiv of
PhSSPh formation per [4Fe-4S] cluster was considered as 100%.
Reaction of (Et4N)[Fe4S3(NO)7] (RBA) with thiol and thiolate
(c) in the presence of Ph3CSNO. In the glovebox, 0.2 mL of 10
mM RBA stock solution was mixed with 10 equiv (1 mL, 20 mM) of
Ph3CSNO and 20 equiv of [NEt4][SPh] stock solution (1 mL, 40
mM) in a 10 mL Schlenk flask and sealed with a rubber septum. Ten
equiv (0.2 mL, 100 mM) of PhSH was then injected into the flask.
The reaction solution was allowed to stir in the dark for 3 h, during
which the color of the reaction mixture changed from brown to dark
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red and the formation of H2S could be observed by lead acetate paper.
NMR, IR, and EPR spectra of the reaction mixture confirmed 2 to be
the only iron-containing product (Figure S5). EPR quantification
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ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.inorgchem.1c01328.
Calibration curves for H2S quantification, DNIC
quantification, and disulfide quantification. IR and 1H
NMR spectra monitoring the reaction of RBA/thiol/
thiolate with and without Ph3CSNO (PDF)
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AUTHOR INFORMATION
■
Corresponding Author
Eunsuk Kim − Department of Chemistry, Brown University,
Providence, Rhode Island 02912, United States;
orcid.org/0000-0001-8803-0666; Email: eunsuk_kim@
brown.edu
Authors
Kady M. Oakley − Department of Chemistry, Brown
University, Providence, Rhode Island 02912, United States
Ziyi Zhao − Department of Chemistry, Brown University,
Providence, Rhode Island 02912, United States
Ryan L. Lehane − Department of Chemistry, Brown
University, Providence, Rhode Island 02912, United States
Ji Ma − Department of Chemistry, Brown University,
Providence, Rhode Island 02912, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.inorgchem.1c01328
Funding
(13) Filipovic, M. R.; Miljkovic, J.; Nauser, T.; Royzen, M.; Klos, K.;
Shubina, T.; Koppenol, W. H.; Lippard, S. J.; Ivanovic-Burmazovic, I.
Chemical Characterization of the Smallest S-Nitrosothiol, HSNO;
Cellular Cross-talk of H2S and S-Nitrosothiols. J. Am. Chem. Soc.
2012, 134, 12016−12027.
The authors thank the NSF (CHE 1807845) for financial
support.
Notes
The authors declare no competing financial interest.
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Jia-Ling, S.; Wong, B. S.; Cheung, N. S.; Halliwell, B.; Moore, P. K.
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Annu. Rev. Biochem. 2005, 74, 247−281.
ACKNOWLEDGMENTS
■
The authors thank the NSF (CHE 1807845) for financial
support.
ABBREVIATIONS
■
cGMP, cyclic guanosine monophosphate; DNIC, dinitrosyl
iron complex; RBA, Roussin’s black anion; RRE, Roussin’s red
ester
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