Reactions of synthetic [2Fe-2S] and [4Fe-4S] clusters with nitric oxide and nitrosothiols

Article abstract of DOI:10.1021/ja8054996

The interaction of nitric oxide (NO) with iron-sulfur cluster proteins results in degradation and breakdown of the cluster to generate dinitrosyl iron complexes (DNICs). In some cases the formation of DNICs from such cluster systems can lead to activation

Full text of DOI:10.1021/ja8054996

Published on Web 10/22/2008
Reactions of Synthetic [2Fe-2S] and [4Fe-4S] Clusters with
Nitric Oxide and Nitrosothiols
Todd C. Harrop, Zachary J. Tonzetich, Erwin Reisner, and Stephen J. Lippard*
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received July 19, 2008; E-mail: lippard@mit.edu
Abstract: The interaction of nitric oxide (NO) with iron-sulfur cluster proteins results in degradation and
breakdown of the cluster to generate dinitrosyl iron complexes (DNICs). In some cases the formation of
DNICs from such cluster systems can lead to activation of a regulatory pathway or the loss of enzyme
activity. In order to understand the basic chemistry underlying these processes, we have investigated the
reactions of NO with synthetic [2Fe-2S] and [4Fe-4S] clusters. Reaction of excess NO(g) with solutions of
[Fe2S2(SR)4]2- (R ) Ph, p-tolyl (4-MeC6H4), or 1/2 (CH2)2-o-C6H4) cleanly affords the respective DNIC,
[Fe(NO)2(SR)2]-, with concomitant reductive elimination of the bridging sulfide ligands as elemental sulfur.
The structure of (Et4N)[Fe(NO)2(S-p-tolyl)2] was verified by X-ray crystallography. Reactions of the
t
[4Fe-4S] clusters, [Fe4S4(SR)4]2- (R ) Ph, CH2Ph, Bu, or 1/2 (CH2)-m-C6H4) proceed in the absence of
added thiolate to yield Roussin’s black salt, [Fe4S3(NO)7]-. In contrast, (Et4N)2[Fe4S4(SPh)4] reacts with
NO(g) in the presence of 4 equiv of (Et4N)(SPh) to yield the expected DNIC. For all reactions, we could
reproduce the chemistry effected by NO(g) with the use of trityl-S-nitrosothiol (Ph3CSNO) as the nitric
oxide source. These results demonstrate possible pathways for the reaction of iron-sulfur clusters with
nitric oxide in biological systems and highlight the importance of thiolate-to-iron ratios in stabilizing DNICs.
ferric uptake regulatory protein (Fur)14,15 and the NO-responsive
transcription factor (NorR),16,17 both of which contain non-heme
Introduction
The physiological roles played by nitric oxide (NO) encom-
pass a variety of important biological functions from smooth
muscle relaxation1-3 to neurotransmission4 and the immune
response.5 Much of the biological chemistry of NO involves
transition metal centers in the active sites of proteins.6,7 Heme-
iron, in particular,8,9 has elicited a great deal of attention since
its interactions with NO were first identified as playing a central
role in the activation of guanylyl cyclase to form cGMP.10-12
Recently, non-heme targets have been identified as potential
NO reaction sites in biology.13 For instance, NO may play a
role in regulating intracellular iron levels by interaction with
iron centers. Nitric oxide has also been implicated in the
disassembly of a variety of iron-sulfur proteins leading to
the formation of dinitrosyl iron complexes (DNICs).18-20 These
DNICs are believed to take the form of tetrahedral [Fe-
(NO)2(SR)2]- species, where RS- may represent cysteinate
residues along the protein backbone21-23 or mobile ligands such
as glutathione.24 The identity and assignment of these nitrosy-
lated species are based on their observed g ) 2.03 electron
paramagnetic resonance (EPR) signals, which are characteristic
of sulfur-ligated dinitrosyl iron species. The potential for DNICs
to serve as carriers of nitric oxide in vivo has generated renewed
interest in these compounds, and several groups have demon-
(1) Ignarro, L. J.; Buga, G. M.; Wood, K. S.; Byrns, R. E.; Chaudhuri,
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9
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10.1021/ja8054996 CCC: $40.75
2008 American Chemical Society

Reactions of Synthetic [2Fe-2S] and [4Fe-4S] with NO
A R T I C L E S
Chart 1. Common Iron-Sulfur Nitrosyl Compounds
strated the ability of synthetic DNICs to transfer their NO
equivalents to other metal centers.25,26
complicating the interpretation of the reported results. Among
the species proposed and identified solely on the basis of EPR
g-values and coupling constants were DNICs containing DMF
(the reaction solvent) ligands and [Fe(NO)2(DMF)(SR)]. Forma-
tion of the nitrosylated [4Fe-3S] cluster known as Roussin’s
black salt (RBS) resulted when these clusters were allowed to
react with nitrite in aqueous alkaline medium followed by an
acidic workup (Chart 1). More recent work reveals the formation
of S-bound DNICs upon continuous purging of NO(g) through
a heterogeneous tetrahydrofuran (THF) solution of a synthetic
Although the reactivity of NO with iron-sulfur proteins
suggests that these sites represent pathological targets, recent
work has also demonstrated a possible physiological role. The
transcriptional regulator, SoxR, becomes activated when its
[2Fe-2S] cluster component is degraded in the presence of NO
to form a protein-bound DNIC.27 Subsequent work demonstrated
in vitro repair of [2Fe-2S] clusters from DNIC by treatment
with L-cysteine in the presence of cysteine desulfurase.28 Both
NO and nitrosothiols inhibit cellular respiration by degradation
of iron-sulfur clusters along the electron-transfer chain in
mitochondria.29 In certain instances this inhibition can be
reversed by light, suggesting again the possibility for repair of
nitrosylated clusters.30
cluster containing polysulfide ligands, [(S5)Fe(µ-S)2Fe(S5)]2- 25
.
The mechanism of DNIC formation and any intermediates along
the reaction pathway, however, remain to be elucidated.
Moreover, of the reported synthetic DNICs, few are produced
by direct reaction of an iron-sulfur cluster precursor with NO.
As illustrated by these examples, no systematic examination of
the reaction of stoichiometric and controlled quantities of nitric
oxide with both [2Fe-2S] and [4Fe-4S] clusters has appeared.
Such information is necessary to provide a foundation for
understanding the chemistry in biological systems when protein-
bound iron-sulfur clusters encounter NO.
Despite the growing body of literature demonstrating the
reaction of NO with iron-sulfur cluster proteins, there remain
few studies exploring the reactivity of NO with synthetic cluster
analogues. Early work demonstrated that a variety of nitrosylated
iron products form upon reaction of synthetic [2Fe-2S] and
[4Fe-4S] systems with NO(g) and nitrite.31 For example, reaction
of an unspecified amount of NO(g) with a N,N-dimethylform-
In order to address this need, we recently initiated a study
of the reaction pathways of synthetic iron-sulfur compounds
with nitric oxide. We began our investigations by examining
the reactions of NO with mononuclear homoleptic iron-sulfur
amide (DMF) solution of the [4Fe-4S] analogue, [Fe4S4(SR)4]2-
,
resulted in no reaction at room temperature. Brief heating of
this reaction mixture led to the formation of a mixture of
paramagnetic mononuclear iron species that were not com-
pletely characterized. The identity of R was not specified, further
complexes of composition [Fe(SR)4]2- 32,33
The results of
.
this work provided the first structurally and spectroscopically
characterized mononitrosyl iron complexes (MNICs) of the
type [Fe(SR)3(NO)]- as well as their analogous DNICs
following stoichiometric addition of NO to [Fe(SR)4]2-
complexes. Subsequently, we have been investigating the
reactions of several synthetic [2Fe-2S] and [4Fe-4S] clusters
with NO and S-nitrosothiols. Here we report results of a
systematic study that demonstrate the formation of DNICs
as the predominant iron-containing products of nitrosylation
(25) Tsai, F.-T.; Chiou, S.-J.; Tsai, M.-C.; Tsai, M.-L.; Huang, H.-W.;
Chiang, M.-H.; Liaw, W.-F. Inorg. Chem. 2005, 44, 5872–5881.
(26) Chiang, C.-Y.; Darensbourg, M. Y. J. Biol. Inorg. Chem. 2006, 11,
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12873.
(29) Welter, R.; Yu, L.; Yu, C.-A. Arch. Biochem. Biophys. 1996, 331,
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A R T I C L E S
Harrop et al.
prepared by grinding a solid sample and mixing it with Apiezon-N
grease. Samples were placed in a 4.2 K cryostat during measure-
ment. A 57Co/Rh source was moved at a constant acceleration at
room temperature against the absorber sample. All isomer shift (δ)
and quadrupole splitting (∆EQ) values are reported with respect to
57Fe-enriched metallic iron foil that was used for velocity calibra-
tion. The displayed spectrum was folded to enhance the signal-to-
noise ratio. Fits of the data were calculated by the WMOSS version
2.5 plot and fit program.41 X-band EPR spectra were recorded on
a Bruker EMX EPR spectrometer (9.38 GHz). Temperature control
was maintained with an Oxford Instruments ESR900 liquid-He
cryostat. Spectra were recorded in 4 mm o.d. quartz EPR tubes
capped with a tight-fitting rubber septum.
in all cases and that reveal the ratio of thiolate to iron to be
critical in trapping and stabilizing these species. These
observations establish a general reactivity pattern of NO with
such cluster systems, a necessary step toward elucidating the
underlying mechanism of the cluster-to-DNIC transformation.
The availability of reaction model studies from synthetic
systems of this kind aids in the identification of reactive
species and mechanistic pathways for nitrosylation of bio-
logical iron-sulfur cluster sites. Collectively, the experiments
described here report the first detailed attempt to define such
reaction pathways. The use of trityl-S-nitrosothiol as an
organic-soluble, stoichiometric reagent for NO is also
highlighted.
Preparation of Trityl-S-nitrosothiol (Ph3CSNO). A 100 mL
round-bottom flask was charged with 2.763 g (10.0 mmol) of
Ph3CSH and 15 mL of methylene chloride and capped with a rubber
septum. No attempt was made to exclude ambient moisture or
dioxygen from the reaction. Once the thiol had dissolved, 2.0 mL
(15 mmol) of isoamyl nitrite was added via syringe, after which
the solution took on a dark green color. The flask was wrapped in
aluminum foil to exclude light and stirred at room temperature for
60 min. Methyl alcohol (30 mL) was added and the mixture allowed
to stand at -25 °C for 2 h. During this time, 2.293 g of the
compound precipitated as dark green needles. The needles were
isolated by filtration, washed with methyl alcohol, and dried in
vacuo. Yield: 75%. FTIR (KBr pellet): νNO ) 1514 cm-1. UV-vis
(CH2Cl2) (ꢀ, M-1 cm-1): 348 nm (740), 556 (sh), 602 (40). Spectra
(see Supporting Information) were identical to those previously
reported for Ph3CSNO.42
Reaction of [2Fe-2S] and [4Fe-4S] Clusters with NO(g). In a
typical reaction, a 100 mL Schlenk flask was charged with 60-80
µmol of the cluster and 15-20 mL of acetonitrile. The reaction
vessel was capped with a rubber septum, and 5-10 mL
(depending on the anticipated volume of added NO) of headspace
was removed from the flask via syringe. NO gas was then
introduced into the headspace of the flask by syringe. The
mixture was allowed to stir at ambient temperature (295-298
K) in a darkened glovebox for 2 h. All volatiles were removed
in vacuo and the resulting dark red residue was extracted
successively with diethyl ether (3 × 5 mL), THF (3 × 5 mL),
and finally MeCN (3 × 5 mL) if any insoluble material remained.
Evaporation of each extract then afforded different reaction
products that were identified by IR, UV-vis, and EPR spec-
troscopy. For reactions followed by their UV-vis spectra, excess
NO(g), typically 50-100 µL, was injected directly into the
headspace of a cuvette containing 3 mL of a 40-60 µM solution
of cluster. For reactions involving added (Et4N)(SPh), thiolate
solutions in MeCN were prepared by mixing equal molar
amounts of anhydrous Et4NCl and NaSPh followed by filtration
through glass filter paper into the reaction mixture. Reactions
employing Ph3CSNO were conducted in an identical fashion to
those using NO(g) except that nitrosothiol was introduced as a
solution in THF or MeCN.
Experimental Section
General Considerations. Manipulations of air- and moisture-
sensitive materials were performed under an atmosphere of argon
gas using standard Schlenk techniques, or in an MBraun glovebox
under an atmosphere of purified nitrogen. All reagents were
purchased from commercial suppliers and used as received unless
otherwise noted. Acetonitrile, tetrahydrofuran, dichloromethane,
diethyl ether, pentane, and toluene were purified by passage through
activated alumina.34 Methyl alcohol was distilled from Mg(OMe)2
under N2 and stored over 4 Å molecular sieves prior to use.
Acetonitrile-d3 was dried over CaH2 and vacuum-distilled prior to
use. Nitric oxide (Matheson, 99%) was purified by a method adapted
from the literature.35 The NO stream was passed through an Ascarite
column (NaOH fused on silica gel) and a 6 ft coil filled with silica
gel cooled to -78 °C. NO gas was stored and transferred under an
inert atmosphere using standard gas storage bulbs and gas-tight
syringes, respectively. For reactions involving NO or nitrosothiol,
care was taken to prevent light exposure by covering reaction
glassware in aluminum foil or by performing experiments in a
darkened room.
Materials. The iron-sulfur clusters (Et4N)2[Fe2S2(SPh)4],
(nBu4N)2[Fe2S2(SPh)4], (Et4N)2[Fe2S2(S-p-tolyl)4], (nBu4N)2[Fe2S2-
(S2-o-xyl)2],(Et4N)2[Fe2S2(S-2,4,6-Me3C6H2)4],(Et4N)2[Fe4S4(SPh)4],
(Et4N)2[Fe4S4(SCH2Ph)4], (Et4N)2[Fe4S4(S-tBu)4], and (nBu4N)2[Fe4S4-
(S2-m-xyl)2] were prepared by literature procedures or slight
modifications thereof.36-38 (PPN)[Fe(NO)2I2] was prepared as
described.39 Trityl-S-nitrosothiol (Ph3CSNO) was prepared by a
modified literature procedure described below.40
Physical Measurements. IR spectra were recorded with a
ThermoNicolet Avatar 360 spectrophotometer running the OMNIC
software; solid samples were pressed into KBr disks and solution
samples were prepared in an air-tight Graseby-Specac solution cell
with CaF2 windows and 0.1 mm spacers. In situ IR spectra were
recorded on a ReactIR 1000 instrument from ASI equipped with a
1 in. diameter, 30-reflection silicon ATR (SiComp) probe. In a
typical experiment the instrument was blanked with MeCN and
the sample concentrations ranged from 15 to 30 mM. NO(g) was
added to the anaerobic sample compartment through a rubber
septum with a gas-tight syringe. UV-vis spectra were recorded
on a Cary-50 diode-array spectrophotometer in air-tight Teflon-
capped quartz cells. Samples for 57Fe Mo¨ssbauer studies were
(Et4N)[Fe(NO)2(SPh)2]. To a 15 mL MeCN solution containing
104.9 mg (120 µmol) of (Et4N)2[Fe2S2(SPh)4] was added 15.0 mL
(500 µmol) of NO(g) through a gas-tight syringe. The solution was
allowed to stir at ambient temperature for 2.5 h, during which time
the color changed from dark purple to dark red. All volatiles were
removed in vacuo, and the residue was washed with Et2O. FTIR
analysis of the residue indicated (Et4N)[Fe(NO)2(SPh)2] as the only
NO-containing species. Recrystallization of the crude product from
MeCN at -25 °C afforded 73.3 mg (66%) of dark red needles, the
(34) Pangborn, A. B.; Giardello, M. A.; Grubbs, R. H.; Rosen, R. K.;
Timmers, F. J. Organometallics 1996, 15, 1518–1520.
(35) Lorkovic´, I. M.; Ford, P. C. Inorg. Chem. 2000, 39, 632–633.
(36) Hagen, K. S.; Reynolds, J. G.; Holm, R. H. J. Am. Chem. Soc. 1981,
103, 4054–4063.
(37) Christou, G.; Garner, C. D. J. Chem. Soc., Dalton Trans. 1979, 1093–
1094.
(38) Ueyama, N.; Ueno, S.; Sugawara, T.; Tatsumi, K.; Nakamura, A.;
Yasuoka, N. J. Chem. Soc., Dalton Trans. 1991, 2723–2727.
(39) Connelly, N. G.; Gardner, C. J. Chem. Soc., Dalton Trans. 1976, 1525–
1527.
(40) Josephy, P. D.; Rehorek, D.; Janzen, E. G. Tetrahedron Lett. 1984,
25, 1685–1688.
(41) Kent, T. A. WMOSS, v. 2.5: Mo¨ssbauer Spectral Analysis Software;
WEB Research Co.: Minneapolis, MN, 1998.
(42) Arulsamy, N.; Bohle, D. S.; Butt, J. A.; Irvine, G. J.; Jordan, P. A.;
Sagan, E. J. Am. Chem. Soc. 1999, 121, 7115–7123.
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Reactions of Synthetic [2Fe-2S] and [4Fe-4S] with NO
A R T I C L E S
spectral features of which matched those of previously published
benzenethiolate DNICs.25,43
Synthesis of Dinitrosyl Iron Complex (DNIC) (Et4N)-
[Fe(NO)2(SPh)2] from (Et4N)2[Fe4S4(SPh)4]. To a 10 mL MeCN
solution containing 72.5 mg (69.1 µmol) of (Et4N)2[Fe4S4(SPh)4]
and 276 µmol of (Et4N)(SPh) was added 16.0 mL (563 µmol) of
NO(g) to the headspace of the reaction flask. The reaction was
stirred at ambient temperature in the dark for 2 h, during which
time the color turned from brown-red to dark red. All volatiles were
removed in vacuo, and the residue was treated with Et2O to remove
elemental sulfur. Analysis of the crude product by FTIR and
UV-vis spectroscopy indicated (Et4N)[Fe(NO)2(SPh)2] to be the
sole NO-containing product. The crude product was recrystallized
from a minimal amount of MeCN at -25 °C to afford 69.4 mg of
the DNIC (54%) as dark red needles.
(Et4N)[Fe(NO)2(S-p-tolyl)2]. To a 5 mL MeCN solution contain-
ing 26.0 mg (28.1 µmol) of (Et4N)2[Fe2S2(S-p-tolyl)4] was added
4.0 mL (178.6 µmol) of NO(g) through a gas-tight syringe, resulting
in a rapid color change from violet to red. After the solution was
stirred under an NO atmosphere for 4 h at room temperature, a
very small amount of a pale yellow precipitate was observed and
filtered off. The MeCN was then evaporated to afford a dark, oily
residue. This residue was washed several times with Et2O to aid in
solidifying the material, affording 23.7 mg (86% yield) of product.
FTIR (KBr, cm-1): 3056 (w), 2975 (w), 1732 (vs, νNO), 1691 (vs,
ν
NO), 1486 (m), 1434 (w), 1392 (w), 1364 (w), 1170 (w), 1085
(m), 1018 (w), 997 (w), 809 (w), 782 (w), 630 (w), 492 (w). FTIR
(THF, cm-1): 1738, (s, νNO), 1694 (s, νNO). UV-vis (THF) (ꢀ,
M-1 cm-1): 483 (3100), 795 (560). Anal. Calcd for
C22H34N3O2S2Fe: C, 53.65; H, 6.96; N, 8.53. Found: C, 54.03; H,
6.87; N, 8.62.
X-ray Data Collection and Structure Solution Refinement.
Black blocks of (Et4N)[Fe(S-p-tolyl)2(NO)2] were grown anaero-
bically at -25 °C by slow diffusion of pentane into a solution of
the compound dissolved in THF. A suitable crystal was mounted
in Paratone N oil on the tip of a glass capillary and frozen under
a 110 K nitrogen cold stream maintained by a KRYO-FLEX low-
temperature apparatus. Data were collected on a Bruker APEX CCD
X-ray diffractometer with Mo KR radiation (λ 0.71073 Å) controlled
by the SMART software package.45 Empirical absorption correc-
tions were calculated with SADABS,46 and the structures were
checked for higher symmetry by the PLATON software.47 The
structure was solved by direct methods with refinement by full-
matrix least-squares based on F2 using the SHELXTL-9748 software
incorporated in the SHELXTL software package.49,50 All non-
hydrogen atoms were located and their positions refined anisotro-
pically. Hydrogen atoms were assigned to idealized positions and
given thermal parameters equal to either 1.5 (methyl hydrogen
atoms) or 1.2 (non-methyl hydrogen atoms) times the thermal
parameters of the atoms to which they were attached.
(PPN)[Fe(NO)2({SCH2}2-o-C6H4)]. A flask was charged with
0.327 g (0.36 mmol) of (PPN)[Fe(NO)2I2] and 15 mL of MeCN.
To the brown solution was added 0.127 g (0.59 mmol) of
Na2[(SCH2)2-o-C6H4] as a solid in one portion. The resulting
mixture was allowed to stir at ambient temperature for 2 h. All
volatiles were removed in vacuo, and the remaining residue was
extracted into 20 mL of THF and filtered through a plug of
Celite. The THF solution was concentrated to 5 mL in vacuo
and layered with several volumes of diethyl ether. After the
solution was left to stand at -25 °C for 24 h, the DNIC
precipitated as 0.115 g (39%) of brown needles. FTIR (KBr,
cm-1): 2955 (w), 2933 (m), 2854 (w), 1734 (s, νNO), 1688 (s,
ν
NO), 1588 (w), 1574 (w), 1484 (w), 1438 (m), 1383 (w), 1285
(m), 1266 (m), 1184 (w), 1115 (m), 1070 (w), 1046 (w), 1027
(w), 998 (w), 768 (w), 746 (w), 724 (m), 692 (m). FTIR (MeCN,
cm-1): 1725 (νNO), 1682 (νNO). UV-vis (MeCN) (ꢀ, M-1 cm-1):
379 (3650), 425 (sh), 584 (950), 868 (450). EPR (10 K,
Results and Discussion
2-MeTHF glass): gav
C44H38N3O2P2S2Fe: C, 64.24; H, 4.66; N, 5.11. Found: C, 63.73;
H, 4.71; N, 5.29.
)
2.029. Anal. Calcd for
[2Fe-2S] Clusters. Reaction of the synthetic [2Fe-2S] cluster,
(Et4N)2[Fe2S2(SPh)4], with 4 equiv of NO(g) in the absence of
light and dioxygen afforded the previously described DNIC,
(Et4N)[Fe(NO)2(SPh)2], as the sole iron-containing product.
Verification of the DNIC was provided by IR, UV-vis, and
EPR spectroscopy.33 The only detectable byproduct was a white
solid that precipitated from the acetonitrile reaction solution.
This ether-soluble solid displayed no IR spectral features.
Subsequent treatment of the white material with triphenylphos-
Synthesis of Roussin’s Black Salt (RBS, (Et4N)[Fe4S3(NO)7])
from (Et4N)2[Fe4S4(SPh)4]. a. From NO(g). To a 5 mL MeCN
solution containing 51.2 mg (48.8 µmol) of (Et4N)2[Fe4S4(SPh)4]
was added 9.0 mL (401.8 µmol) of NO(g) to the headspace of the
reaction flask. The reaction mixture gradually changed from a deep
dark red-brown to a brown solution. After 2 h of stirring at ambient
temperature in the dark, the solution was filtered to remove a small
amount of a yellow precipitate (sulfur), after which the filtrate was
stripped to dryness. The brown residue was washed with several
portions of Et2O, which were found by GC-MS to contain PhSSPh,
and the residue was dissolved in 5 mL of THF and filtered. To the
filtrate was added an equivalent volume of pentane to precipitate
30.2 mg (93%) of black needles, the spectral features of which
were identical to those reported for RBS.44
b. From Ph3CSNO. To a solution containing 70.0 mg (66.7
µmol) of (Et4N)2[Fe4S4(SPh)4] in 10 mL of MeCN was added a
solution of 143.2 mg (360 µmol) of Ph3CSNO dissolved in 10 mL
of MeCN. The reaction mixture was allowed to stir in the dark at
ambient temperature for 3 h, after which time all volatiles were
removed in vacuo. The residue was washed with 10 mL of Et2O
and extracted into 10 mL of THF. Evaporation of the THF afforded
86.3 mg of black solid that was found by IR spectroscopy to be a
mixture of RBS, (Et4N)(SPh), and some trityl-containing species.
The RBS could subsequently be purified by crystallization from
THF/pentane as described above.
phine in CD3CN at ambient temperature gave rise to a new 31
P
NMR resonance at 43.9 ppm after 24 h, consistent with
formation of SdPPh3.51 The solid therefore appears to be an
allotrope of elemental sulfur.
In all reactions employing gaseous nitric oxide, identical results
were obtained when trityl-S-nitrosothiol42 (Ph3CSNO) was used
in place of the NO(g). We find that Ph3CSNO, for which we
provide an improved synthesis, can deliver a stoichiometric quantity
of NO(g) equivalents in reactions with iron-sulfur compounds.
Such reactivity is important, considering that nitrosothiols are
(45) SMART, v. 5.6, Software for the CCD Detector System; Bruker AXS:
Madison, WI, 2000.
(46) Sheldrick, G. M. SADABS, Area-Detector Absorption Correction;
University of Go¨ttingen: Go¨ttingen, Germany, 2001.
(47) Spek, A. L. PLATON, A Multipurpose Crystallographic Tool; Utrecht
University: Utrecht, The Netherlands, 2000.
(48) Sheldrick, G. M. SHELXTL97, Program for Refinement of Crystal
Structures; University of Go¨ttingen: Go¨ttingen, Germany, 1997.
(49) Sheldrick, G. M. Acta Crystallogr., Sect. A 2008, A64, 112–122.
(50) SHELXTL, v. 6.10, Program Library for Structure Solution and
Molecular Graphics; Bruker AXS: Madison, WI, 2001.
(51) Moedritzer, K.; Maier, L.; Groenweghe, L. C. D. J. Chem. Eng. Data
1962, 7, 307–310.
(43) Strasdeit, H.; Krebs, B.; Henkel, G. Z. Naturforsch. 1986, 41B, 1357–
1362.
(44) D’Addario, S.; Demartin, F.; Grossi, L.; Iapalucci, M. C.; Laschi, F.;
Longoni, G.; Zanello, P. Inorg. Chem. 1993, 32, 1153–1160.
9
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A R T I C L E S
Harrop et al.
Scheme 1
believed to be carriers of NO in living systems.52 Solid Ph3CSNO
can be isolated in greater than 75% yield from the reaction of
Ph3CSH and isoamyl nitrite (see Experimental Section for details).
The resulting green crystals are stable for months when stored cold
(-25 °C) in the absence of light. The relatively high molecular
weight (305 g/mol) and good solubility of this nitrosothiol render
it an ideal source of NO in nonaqueous systems. As a test of the
nitrosylating ability of Ph3CSNO, the homoleptic thiolate complex
[Fe(SPh)4]2- was treated with 1 and 2 equiv of the nitrosothiol.
Examination of the reaction products by UV-vis spectroscopy (see
Supporting Information, Figure S1) demonstrated clean formation
of the previously reported53 MNIC (1 equiv) and DNIC (2 equiv)
species, confirming that the compound delivers stoichiometric NO
equivalents to the iron-thiolate complexes (Scheme 1).
Figure 1. X-band EPR spectrum obtained upon introduction of excess
NO(g) to a 300 µM solution of (Et4N)2[Fe2S2(SPh)4] in 1:1 MeCN/toluene
followed by immediate freezing in liquid nitrogen. The spectrum was
recorded at 10 K (microwave frequency, 9.385 GHz; microwave power,
2.0 mW; modulation amplitude, 4.00 G).
Reaction of (Et4N)2[Fe2S2(SPh)4] with fewer than 4 equiv of
NO(g) or Ph3CSNO resulted in formation of the DNIC and
recovery of the unreacted cluster. Examination of the reaction by
IR or EPR spectroscopy demonstrated immediate formation of
[Fe(NO)2(SPh)2]- with no observable intermediates. For example,
treatment of a 300 µM solution of (Et4N)2[Fe2S2(SPh)4] with an
excess of NO(g) followed by immediate freezing in liquid nitrogen
gave rise to the EPR spectrum shown in Figure 1, characteristic of
a DNIC. Similarly, addition of increasing equivalents of the
S-nitrosothiol, Ph3CSNO, to a 380 µM solution of (nBu4N)2-
[Fe2S2(SPh)4] in THF at 295 K yielded an increasingly intense
signal derived from (nBu4N)[Fe(NO)2(SPh)2], as judged by EPR
spectroscopy (see Supporting Information, Figure S2). Similar
results were obtained when the reaction was monitored by IR
spectroscopy.
Under more dilute concentrations (<100 µM), [2Fe-2S] clusters
were found to react with NO(g) to yield RBS instead of DNIC.
For example, when a 47 µM solution of (Et4N)2[Fe2S2(SPh)4] was
treated with NO(g) with the reaction monitored by UV-vis
spectroscopy, a featureless spectrum resulted following the disap-
pearance of the optical bands of the [2Fe-2S] cluster. Nearly
identical results were obtained with the bulkier mesityl thiolate
cluster, (Et4N)2[Fe2S2(S-2,4,6-Me3C6H2)4]. When the reaction with
(Et4N)2[Fe2S2(SPh)4] was repeated in the presence of additional
equivalents of (Et4N)(SPh), optical bands for the DNIC were
observed to form (see Supporting Information, Figure S3). In
contrast to these results for the aryl thiolate clusters, examination
by UV-vis spectroscopy of the reaction of NO(g) with the
[2Fe-2S] cluster containing the chelating [(SCH2)2-o-C6H4]2-
(S2-o-xyl) ligand in the absence of free thiolate initially revealed
Figure 2. UV-vis spectrum of the reaction of (nBu4N)2[Fe2S2(S2-o-xyl)2]
(64 µM) with excess (∼30 equiv) NO(g) at 298 K in MeCN. Spectra were
recorded over 45 min. Inset shows the spectral change associated with
transformation of the DNIC to RBS.
the appearance of optical bands consistent with a DNIC (vide infra)
over the course of 30 min. At longer times (1 h), optical bands for
the DNIC diminished, giving rise to a less-featured spectrum similar
to that observed in the reaction of [Fe2S2(SPh)4]2- with NO (Figure
2). The featureless spectrum is consistent with the formation of
RBS, [Fe4S3(NO)7]-, the formation of which requires oxidation
of the thiolate ligands. Elemental sulfur formed during the course
of the reaction could serve as an oxidant, as might NO or one of
its disproportionation products, such as NO2. RBS could be isolated
from reactions of (Et4N)2[Fe2S2(SPh)4] with excess NO(g) when
the reaction was allowed to stir for longer than 5 h. In this instance,
the disulfide PhSSPh was identified in the reaction mixture,
consistent with oxidation of the thiolate ligands. When (Et4N)2[Fe2S2-
(S-2,4,6-Me3C6H2)4] was allowed to react with excess NO(g),
consumption of the cluster was much slower, and no DNICs were
(52) Mutus, B. In Nitric Oxide Donors; Wang, P. G., Cai, T. B., Taniguchi,
N., Eds.; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, 2005;
pp 91-109.
(53) Lu, T.-T.; Chiou, S.-J.; Chen, C.-Y.; Liaw, W.-F. Inorg. Chem. 2006,
45, 8799–8806.
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Reactions of Synthetic [2Fe-2S] and [4Fe-4S] with NO
A R T I C L E S
Figure 3. Thermal ellipsoid (50%) drawing of the anion of (Et4N)[Fe(S-
p-tolyl)2(NO)2]·0.5THF. Hydrogen atoms and cocrystallized tetrahydrofuran
are omitted for clarity. See Supporting Information for complete list of metric
parameters (Tables S1 and S2).
Figure
4.
Zero-field Mo¨ssbauer (4.2 K) spectrum of
(Et4N)[Fe(NO)2(SPh)2]. See Supporting Information (Figure S5) for the
theoretical fit from which the isomer shift and quadrupole splitting
parameters were obtained.
identified by IR spectroscopy. Dimesityl disulfide was isolated from
the reaction mixture, indicating oxidation of the thiolate ligands.
Unfortunately, attempts to isolate the previously unreported
DNIC, [Fe(NO)2(S2-o-xyl)]-, as the Et4N+ or nBu4N+ salt were
unsuccessful. The compound could be obtained, however, as
the PPN+ (PPN ) µ-nitrido-bistriphenylphosphonium) salt by
a direct salt metathesis reaction using (PPN)[Fe(NO)2I2], as
shown in eq 1.
bond angles at iron, which deviate considerably from the 109.5°
values of a perfect tetrahedron (viz. N1-Fe1-N2, 115.68(7)°;
N2-Fe1-S2, 102.15(5)°; Supporting Information, Table S2). The
Fe-S distances (Fe1-S1, 2.3329(7) Å; Fe1-S2, 2.3343(7) Å) are
nearly identical and slightly longer than those found in other
structurally characterized DNICs containing aromatic thiolate
donors.25,43 The Fe-N bond lengths are slightly variable (Fe1-N1,
1.7210(15) Å; Fe1-N2, 1.7096(15) Å), as are the N-O distances
(N1-O1, 1.1992(19) Å; N2-O2, 1.1958(19) Å). The nitrosyl
ligands are slightly bent (Fe1-N1-O1, 166.04(14)°; Fe1-N2-O2,
170.05(14)°) and flared toward each other in the typical attracto
conformation observed in DNICs.54
The DNIC thus obtained crystallized as thin brown needles
from a mixture of THF and diethyl ether. The molecule displays
νNO values in KBr at 1734 and 1688 cm-1, compared to 1743
and 1683 cm-1 for the benzenethiolate derivative. The electronic
absorption spectrum shows two relatively intense maxima at
379 and 420 nm in MeCN that are identical to those observed
in reactions with the [2Fe-2S] cluster and NO(g) (Figure 2).
In order to verify the reaction stoichiometry observed with
(Et4N)2[Fe2S2(SPh)4] and (nBu4N)2[Fe2S2(S2-o-xyl)2], mass-balance
analysis of the analogous reaction with (Et4N)2[Fe2S2(S-p-tolyl)4]
was carried out in the presence of a slight excess of NO(g) (6 mol
equiv). Reaction of (Et4N)2[Fe2S2(S-p-tolyl)4] at 295 K with excess
NO(g) in MeCN afforded the corresponding DNIC, (Et4N)[Fe-
(S-p-tolyl)2(NO)2], in 86% yield. As expected, the IR spectrum of
the DNIC displayed two strong νNO bands at 1732 and 1691 cm-1
(KBr), similar to the spectra of other known DNICs. Workup of
the byproducts of the reaction of NO(g) with (Et4N)2[Fe2S2(S-p-
tolyl)4] afforded an off-white solid that we assign as elemental
sulfur by analogy to the observations with the benzenethiolate
cluster. There was very little to no evidence of disulfide formation
in this or any of the NO reactions with the [2Fe-2S] systems when
the reactions were stopped after 1-2 h. Thus, formation of the
two DNICs upon reaction of NO with [2Fe-2S] clusters results in
reductive elimination of 2 mol equiv of elemental sulfur.
Despite the large number of reports of DNIC species, very
few studies of their Mo¨ssbauer spectra have been undertaken.
The Mo¨ssbauer spectrum of a “dimeric DNIC” prepared by in
situ generation from aqueous FeSO4, NO(g), and cysteine
displays an isomer shift of 0.14 mm/s and a quadrupole splitting
of 1.1 mm/s.55 A 296 K Mo¨ssbauer spectrum of (Et4N)-
[Fe(NO)2(SPh)2], prepared by reaction of RBS with molten
diphenyl disulfide in the presence of KOH and Et4NCl, has δ
) 0.08 mm/s and ∆EQ ) 0.78 mm/s.43 In the present work we
recorded the Mo¨ssbauer spectrum of (Et4N)[Fe(NO)2(SPh)2]
isolated from the reaction of NO with iron-sulfur clusters. The
4.2 K zero-field spectrum is shown in Figure 4. The isomer
shift of 0.18(2) mm/s (versus iron foil at 296 K) and quadrupole
splitting of 0.69(2) mm/s may be compared with those just cited,
bearing in mind the disparity in recording temperature.56
The NO chemistry of the [2Fe-2S] clusters discovered here is
somewhat surprising in the context of recent work demonstrating
that homoleptic Fe(III) thiolates react via thiolate oxidation to afford
mononitrosyl iron complexes.53 Since {Fe2S2}2+ cores formally
contain Fe(III), we might expect similar reactivity. Our observations
indicate, however, that thiolate oxidation is only a minor side
(54) Richter-Addo, G. B.; Legzdins, P. Metal Nitrosyls; Oxford University
Press: New York, 1992.
X-ray crystallographic analysis further confirmed the proposed
DNIC structure (Figure 3). The structure of the anion contains a
four-coordinate iron center in a distorted tetrahedral geometry. The
distortions at the metal center are most readily apparent from the
(55) Lobysheva, I. I.; Serezhenkov, V. A.; Stucan, R. A.; Bowman, M. K.;
Vanin, A. F. Biokhimiya 1997, 62, 801–808.
(56) Ellison, M. K.; Schulz, C. E.; Scheidt, W. R. Inorg. Chem. 2000, 39,
5102–5110.
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A R T I C L E S
Harrop et al.
Scheme 2. Possible Reaction Pathway for [2Fe-2S] Clusters with NO
reaction of [2Fe-2S] clusters with NO, leading to formation of
nitrosylated species other than DNICs. Instead, inorganic sulfide
provides all of the necessary reducing equivalents to transform the
cluster to 2 equiv of the DNIC. A possible pathway for this
transformation is depicted in Scheme 2. If addition of NO occurs
concomitantly with elimination of sulfur, then reaction of the first
NO equivalent will lead to elimination of 1 mol equiv of sulfur
and formation of a {Fe-NO}7 mononitrosyl iron complex (Scheme
2). Addition of the second equivalent of NO results in elimination
of a second mol-equiv of elemental sulfur and formation of a
putative dimeric {Fe-NO}8 species. Break-up of the dimer by
addition of another 2 equiv of NO affords the corresponding
DNICs. In support of this proposal, the nickel analogue of
(Et4N)2[Fe2(µ-SPh)2(NO)2(SPh)2] has recently been isolated and
crystallographically characterized by our laboratory.57
iron-nitrosyl species formed after addition of NO. IR spectra
were acquired every 30 s after introduction of NO to the
headspace of a reaction flask containing an MeCN solution
of (Et4N)2[Fe4S4(SPh)4] for a total time of 2 h. Introduction
of 1 mol-equiv of NO(g) resulted in an immediate color
change from deep red-brown to a slightly lighter brown and
an IR spectrum with two broad νNO bands at 1745 and 1708
cm-1 and a small peak at 1800 cm-1. These bands grow in
intensity until 20 min after NO addition. These values match
identically the IR spectrum of the tetraphenylarsonium salt
of the iron-sulfide-nitrosyl cluster (Ph4As)[Fe4S3(NO)7]
(RBS).58 To determine whether any other iron-nitrosyl
complexes might form during this process, additional equiva-
lents of NO(g) were added to the MeCN solution of
[Fe4S4(SPh)4]2-. As revealed by ReactIR, no other complexes
could be detected up to the addition of 8 mol-equiv of NO,
and each stoichiometric addition resulted only in growth of
IR peaks associated with RBS and no other nitrosyl species
(Figure 5). The formation of RBS from [Fe4S4(SPh)4]2- and
excess NO(g) in MeCN was also monitored by UV-vis
[4Fe-4S] Clusters. The reaction of the [4Fe-4S] cluster,
(Et4N)2[Fe4S4(SPh)4], with NO(g) was performed in a similar
fashion to that described above for the [2Fe-2S] systems.
We initially chose to monitor the reaction by in situ ReactIR
spectroscopy, which facilitates characterization of all
Figure 5. In situ ReactIR spectra following the addition of 1 (0 s), 2, 4, and 8 (indicated) mol-equiv of NO(g) into the headspace of a flask containing
(Et4N)2[Fe4S4(SPh)4] (21.0 mM) in MeCN at 298 K. The bands at 1800, 1745, and 1708 cm-1 increase in intensity as more NO equivalents are added. The
reaction was monitored over the course of 2 h. Each acquisition represents eight scans collected at 30 s intervals.
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Reactions of Synthetic [2Fe-2S] and [4Fe-4S] with NO
A R T I C L E S
spectroscopy. The characteristic intense 450 nm band of this
cluster immediately diminished and a notably featureless
spectrum resulted, having several small absorptions in the
320-380 nm range. A shoulder occurs at 425 nm with an
additional broad peak at 580 nm that matched the published
spectrum of RBS. Similar results were obtained for other
[4Fe-4S] clusters containing PhCH2S-, tBu-S-, and the
chelating (-SCH2)2-m-C6H4 (S2-m-xyl) thiolates, as well as
reactions employing trityl-S-nitrosothiol. Although we have
not performed detailed kinetic studies on these reactions, the
rate of appearance of RBS qualitatively appears to correlate
with the steric bulk of the thiolate ligand. The least sterically
demanding cluster containing the chelating S2-m-xyl thiolate
reacts in less than 5 min to yield RBS according to UV-vis
spectroscopy. In contrast, the bulkiest cluster, [Fe4S4-
(S-tBu)4]2-, required nearly 2 h to react completely with
NO(g).
Additional evidence that RBS is the sole product of this
chemistry was obtained by isolation of the cluster from a
reaction of (Et4N)2[Fe4S4(SPh)4] with NO(g) in MeCN. After
removal of the volatile components, PhSSPh was obtained as a
white solid by extraction of the reaction mixture with diethyl
ether. The black salt could be isolated by subsequent extraction
with THF and crystallization. Examination of the crude THF
extract by IR spectroscopy also revealed vibrations due to PhS-,
suggesting the presence of (Et4N)(SPh). Taken together, these
observations are consistent with the chemistry given in eq 2,
react rapidly in the presence of the elemental sulfur generated
to form RBS. To test this hypothesis, we investigated the
reaction of (Et4N)2[Fe4S4(SPh)4] with 8 mol-equiv of NO(g)
in the presence of 4 equiv of (Et4N)(SPh). After workup of
the reaction, the only nitrosyl-containing species detected by
IR,
UV-vis,
and
EPR
spectroscopy
was
(Et4N)[Fe(NO)2(SPh)2]. Importantly, there was only a very
small amount of disulfide in the reaction mixture, less than
1 equiv per cluster, indicating that very little thiolate
oxidation had taken place. The formation of DNICs in these
reactions could occur through reaction of RBS with
thiolate.60,61 This scenario is unlikely, however, since
significant quantities of disulfide would still be formed. These
results support the conclusion that the reactivity of [4Fe-4S]
clusters is similar to that of the [2Fe-2S] clusters, with the
sulfide ligands providing the electrons necessary for reduction
of the iron centers to form DNICs. A balanced equation for
[Fe4S4(SPh)4]2- reacting with NO and excess thiolate to
generate 4 equiv of DNIC is presented in eq 3. We have not
been able to identify the sulfur byproducts in any of the
reactions of this type thus far investigated.
(Et4N)2[Fe4S4(SPh)4] + 8NO + 4(Et4N)(SPh) f
4(Et4N)[Fe(NO)2(SPh)2] + 3S + (Et4N)2S (3)
Conclusions
The reactions of gaseous NO and trityl-S-nitrosothiol with
synthetic [2Fe-2S] and [4Fe-4S] clusters afford {Fe(NO)2}9
DNIC species or Roussin’s black salt as the nitrosylated iron
products. The nature of the thiolate ligand and the ratio of
thiolate to iron appear to be critical in determining which
products are formed. With synthetic [2Fe-2S] systems, a 2:1
ratio of thiolate to iron is sufficient to allow formation and
isolation of the corresponding DNICs, with (Et4N)[Fe(S-p-
tolyl)2(NO)2] being structurally characterized by X-ray
crystallography. The mechanism of [2Fe-2S] cluster nitros-
ylation appears to involve inorganic sulfide ligands as the
reducing agents, which provide all the necessary reducing
equivalents for the reaction to form elemental sulfur and the
DNIC. In contrast, reaction of stoichiometric and excess
amounts of NO(g) with [4Fe-4S] clusters in the absence of
added thiolate affords RBS. When additional equivalents of
thiolate are present, the corresponding DNIC is observed as
the sole nitrosylated product. Taken together, these results
demonstrate that the end products in iron-sulfur degradation
by NO gas or nitrosothiols are {Fe(NO)2}9 dinitrosyl iron
complexes. Disassembly of the iron-sulfur core appears to
take place readily with both two and four iron systems.
Furthermore, sufficient equivalents of thiolate are necessary
to trap and stabilize the DNIC formed during nitrosylation.
When the thiolate-to-iron ratio is too low (<2), subsequent
reactivity with sulfur occurs to afford RBS. These results
suggest that, biologically, the breakdown of iron-sulfur
clusters must occur in tandem with the sequestration of
inorganic sulfur (or sulfide) to prevent formation of thermo-
dynamically stable iron-sulfur-nitrosyl clusters. Protection
of the clusters in a folded protein may divert this chemistry,
(Et4N)2[Fe4S4(SPh)4] + 7NO f (Et4N)[Fe4S3(NO)7] +
3
2
PhSSPh + (Et4N)(SPh) + S (2)
where the tetranuclear 2Fe(II)-2Fe(III) cluster is transformed
into RBS, 1Fe(II)-3Fe(I), assuming the nitrosyl ligands to be
formally neutral, with three electrons coming from the coordi-
nated thiolates and two electrons from one of the bridging
sulfides. FTIR analysis of the crystalline product of this reaction
revealed νNO peaks at 1799, 1733, 1711, and 1693 cm-1, in
agreement with RBS formation. An X-ray crystallographic
analysis of the product confirmed it to be the tetraethylammo-
nium salt of RBS, (Et4N)[Fe4S3(NO)7]. The geometric param-
eters and unit cell dimensions match those of a previously
published structure of the Et4N+ salt of RBS.44
The formation of RBS from reactions of the [4Fe-4S]
clusters with NO may be contrasted with related results with
bacterial HiPIPs, in which treatment of the cluster with
DEANO, diethylamino NONOate, produced an EPR spectrum
characteristic of a DNIC.22 From this comparison we form
the working hypothesis that formation of [Fe4S3(NO)7]- from
[Fe4S4(SPh)4]2- outside of a protein environment most likely
proceeds by disassembly of the cluster and formation of
mononuclear iron-nitrosyl species that reassemble to gener-
ate RBS. Such reactivity has previously been proposed for
transformations involving RBS, Roussin’s red salt, and the
tetranitrosyl cluster [Fe4S4(NO)4].59 Reaction of synthetic
[4Fe-4S] iron-sulfur clusters with NO to form RBS via
mononuclear DNIC species that are too unstable to observe
might proceed in the following manner. Atf low thiolate-to-
iron (1:1) ratios, DNICs containing solvent ligands31 would
(57) Tennyson, A. G.; Dhar, S.; Lippard, S. J. J. Am. Chem. Soc. 2008,
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A R T I C L E S
Harrop et al.
tively), and E.R. is the recipient of a Schro¨dinger fellowship (Grant
J2485-B10) from the Austrian Science Foundation. We thank Ms.
S. Friedle for assistance with Mo¨ssbauer measurements.
a possibility currently under investigation with the use of
site-differentiated constructs.62-64
Acknowledgment. This work was supported by NSF grant
CHE-0611944. Z.J.T. and T.C.H. acknowledge postdoctoral fel-
lowship support from the National Institute of General Medical
Sciences (1 F32 GM082031-02 and 1 F32 GM075684-01, respec-
Supporting Information Available: Additional spectra, crys-
tallographic data, refinement details, and a fully labeled ORTEP
diagram for (Et4N)[Fe(NO)2(S-p-tolyl)2], as well as the corre-
sponding CIF file. This material is available free of charge via
the Internet at http://pubs.acs.org.
(62) Holm, R. H. Pure Appl. Chem. 1998, 70, 931–938.
(63) Walsdorff, C.; Saak, W.; Pohl, S. J. Chem. Soc., Dalton Trans. 1997,
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