Transformation of a mononitrosyl iron complex to a [2Fe-2S] cluster by a cysteine analogue

Article abstract of DOI:10.1021/ja5024207

Reversible modification of iron-sulfur clusters by nitric oxide acts as a genetic switch in a group of regulatory proteins. While the conversion of [Fe-S] clusters to iron-nitrosyls has been widely studied in the past, little is known about the reverse process, the repair of [Fe-S] clusters. Reported here is a system in which a mononitrosyl iron complex (MNIC), (PPN)[Fe(S ^tBu)₃(NO)] (1), is converted to a [2Fe-2S] cluster, (PPN)₂[Fe₂S₂(SCH₂CH ₂C(O)OMe)₄] (2). This conversion requires only the addition of a cysteine analogue, 3-mercaptomethylpropionate (MMP), at room temperature without the need for any other reagents. The identity of 2 was confirmed spectroscopically, chemically, crystallographically, and analytically. Mass spectrometry and ³⁴S labeling studies support that the bridging sulfides in 2 derive from the added MMP, the cysteine analogue. The NO lost during the conversion of 1 to 2 is trapped in a dinitrosyl iron side product, (PPN)[Fe(SCH₂CH₂C(O)OMe)₂(NO)₂] (4). The present system implies that MNICs are likely intermediates in the repair of NO-damaged [2Fe-2S] clusters and that cysteine is a viable molecule responsible for the destabilization of MINCs and the formation of [2Fe-2S] clusters.

Full text of DOI:10.1021/ja5024207

Communication
pubs.acs.org/JACS
Transformation of a Mononitrosyl Iron Complex to a [2Fe-2S] Cluster
by a Cysteine Analogue
Jessica Fitzpatrick,† Harris Kalyvas,† Milos R. Filipovic,‡ Ivana Ivanovic-Burmazovic,‡
́
́
John C. MacDonald,§ Jason Shearer,¶ and Eunsuk Kim*,†
†Department of Chemistry, Brown University, Providence, Rhode Island 02912, United States
‡Department of Chemistry and Pharmacy, University of Erlangen-Nuremberg, 91058 Erlangen, Germany
§Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, Massachusetts 01609-2280, United States
¶Department of Chemistry, University of Nevada, Reno, Nevada 89557, United States
S
* Supporting Information
[2Fe-2S] protein by a mechanism that remains unknown in
vivo.5b
ABSTRACT: Reversible modification of iron-sulfur clus-
ters by nitric oxide acts as a genetic switch in a group of
regulatory proteins. While the conversion of [Fe-S]
clusters to iron-nitrosyls has been widely studied in the
past, little is known about the reverse process, the repair of
[Fe-S] clusters. Reported here is a system in which a
mononitrosyl iron complex (MNIC), (PPN)[Fe-
(StBu)3(NO)] (1), is converted to a [2Fe-2S] cluster,
(PPN)2[Fe2S2(SCH2CH2C(O)OMe)4] (2). This conver-
sion requires only the addition of a cysteine analogue, 3-
mercaptomethylpropionate (MMP), at room temperature
without the need for any other reagents. The identity of 2
was confirmed spectroscopically, chemically, crystallo-
graphically, and analytically. Mass spectrometry and 34S
labeling studies support that the bridging sulfides in 2
derive from the added MMP, the cysteine analogue. The
NO lost during the conversion of 1 to 2 is trapped in a
dinitrosyl iron side product, (PPN)[Fe(SCH2CH2C(O)-
OMe)2(NO)2] (4). The present system implies that
MNICs are likely intermediates in the repair of NO-
damaged [2Fe-2S] clusters and that cysteine is a viable
molecule responsible for the destabilization of MINCs and
the formation of [2Fe-2S] clusters.
A DNIC to [2Fe-2S] cluster repair process is crucial if the
interconversion between these two species constitutes a
biologically relevant signaling mechanism. Additionally, the
repair chemistry has significance in understanding how cells
might cope with global NO stress that leads to a significant
occurrence of [Fe-S] cluster degradation to DNICs beyond
NO-sensing regulatory proteins. Ding et al. have shed light on
this process using a [2Fe-2S] cluster containing ferredoxin
(Fdx). Protein-bound DNIC formed by treating Fdx with NO
can be converted back to the holo-form following incubation
with cysteine desulfurase, cysteine, and dioxygen (Scheme 1).8
Scheme 1. In Vitro Transformation of [2Fe-2S] Cluster and
DNIC in Ferredoxin
itric oxide (NO) plays a well-established role in
Nphysiological systems by acting as a regulator of
vasodilation, neurotransmission, platelet aggregation, and the
cell-mediated immune response.1 NO is capable of interacting
with a variety of chemical groups found in biological systems,
although the most prominent are thiols,2 to form nitrosothiols,
and metal centers,2b,3 to form metal nitrosyls. Iron-sulfur
clusters are protein cofactors that are known to interact with
NO leading to either elimination4 or initiation5 of biological
activity. Some [Fe-S]-containing proteins initiate gene regu-
lation upon reaction with NO.6 One of the best-studied
examples is the Escherichia coli protein SoxR, in which NO
modification of the [2Fe-2S] cluster produces SoxR-bound
dinitrosyl iron complexes (SoxR-DNICs) and upregulates the
expression of more than 60 defense genes.5b,7 When NO is
removed, the SoxR-DNICs are efficiently converted back to the
Cysteine desulfurase is an enzyme that catalyzes the conversion
of cysteine to alanine and a protein-bound persulfide (RScys-
SH).9 The resulting persulfide sulfur is then used as a source of
sulfur in a number of protein cofactors including [Fe-S]
clusters.
The details of the chemistry governing the cysteine
desulfurase-mediated DNIC repair remain unknown, although
synthetic model systems have been constructed to provide
information about the DNIC to [2Fe-2S] cluster trans-
formation.10 Model systems reported by Lippard10b and
Liaw10c describe the photolytic conversion of iron nitrosyl to
[2Fe-2S] cluster in the presence of elemental sulfur. Another
system investigated by the Liaw group exhibits the ability to
Received: March 10, 2014
Published: May 7, 2014
© 2014 American Chemical Society
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convert from DNIC to [2Fe-2S] cluster on sequential addition
of tritylthiol and an NO-sequestering iron tetrathiolate.10a
While providing valuable insight, these systems have utilized
reagents and reaction conditions that are significantly different
from the repair observed in proteins. We report here the
unprecedented transformation of MNIC to [2Fe-2S] cluster
requiring only the addition of a cysteine analogue (Scheme 2)
and bring to light the previously unexplored potential of
MNICs to act as intermediates in the repair of [2Fe-2S]
clusters from DNICs.
Figure 1. ORTEP diagram of the anion of (PPN)2[Fe2S2(SCH2CH2C-
(O)OMe)4] (2) with 50% thermal ellipsoids. Hydrogen atoms are
omitted for clarity.
Scheme 2. Transformation of MNIC to [2Fe-2S] Cluster
of 1 initially bound to the Fe or from the added MMP. In order
to distinguish between two possibilities, the starting MNIC,
(PPN)[Fe(StBu)3(NO)] (1), was prepared using 34S-labeled
tert-butylthiolate and subsequently used in the preparation of 2.
This cluster was then converted to 3 and analyzed by ESI-MS.
The data clearly indicate that the bridging sulfides in 3 are not
enriched in 34S (Figure S6). The possibility that the sulfur
atoms of the bridging sulfides exchange with those of the
phenylthiol used to derivatize the cluster was discounted by
carrying out the derivatization with Ph34SH in which a mass
shift of 8 was observed indicating the formation of
(PPN)2[Fe2(μ-S)2(34SPh)4] (Figure S7). Moreover, we ob-
served that the tert-butylthiolate ligands on 1 are released as
tert-butylthiol by 1H NMR spectroscopy (Figure S5). The only
other source of the sulfide in the Fe2S2 rhomb is the excess
MMP present in solution.
Analysis of the organic material washed from the product of
the reaction of (PPN)[Fe(StBu)3(NO)] (1) with MMP
suggests the manner by which the sulfur atom is removed
from the latter. Gas chromatography mass spectrometry (GC-
MS) measurements of the diethyl ether washings indicate that
the thioether S(CH2CH2C(O)OMe)2 is formed in the reaction.
GC-MS quantification of the amount of thioether formed over
the course of the reaction confirms that it is produced in a 2:1
stoichiometry with the product cluster. Specifically, on average
93% of the expected thioether is produced.
The fate of the NO that is lost on transformation from
(PPN)[Fe(StBu)3(NO)] (1) to (PPN)2[Fe2S2(SCH2CH2C-
(O)OMe)4] (2) becomes apparent on investigation of the
THF-soluble side product described above. Addition of pentane
to the THF solution of this side product resulted in the
deposition of an oily residue. Spectroscopic analysis of the
residue is consistent with its formulation as (PPN)[Fe-
(SCH2CH2C(O)OMe)2(NO)2] (4), a DNIC bearing two
MMP thiolate ligands. The EPR spectrum of the side product
shows the characteristic g = 2.03 signal expected to arise for a
strongly coupled S = 1/2 {Fe(NO)2}9 DNIC.16 ESI-MS
revealed the compound to have m/z = 354.4 for [4-PPN]−
consistent with its formulation. The IR spectrum of the isolated
product shows bands at 1715 and 1670 cm−1, which coincide
with those of independently prepared 4.
The only final destination of NO derived from (PPN)[Fe-
(StBu)3(NO)] (1) appears to be the DNIC, (PPN)[Fe-
(SCH2CH2C(O)OMe)2(NO)2] (4). EPR monitoring of the
reaction solution shows that the g = 2.03 signal from 4 grows in
over the course of the reaction (Figure 2). No other NO-
containing byproduct has been observed by EPR or IR
spectroscopy nor the formation of N2O was detected in the
Incubation of (PPN)[Fe(StBu)3(NO)] (1),11 an MNIC
bearing tert-butylthiolate ligands where PPN = μ-nitrido-
bis(triphenylphosphorus), with excess methy 3-mercaptopropi-
onate (MMP) resulted in two types of Fe-containing reaction
products, one of which is a [2Fe-2S] cluster bearing MMP
thiolate ligands, (PPN)2[Fe2S2(SCH2CH2C(O)OMe)4] (2),
Scheme 2. The careful fractional purification of the reaction
mixture enabled us to identify every reaction product (vide
infra). Specifically, a solution of 1 was treated with excess (20
equiv) MMP at room temperature for 6 h in the dark. After
removing solvent in vacuo, the reaction mixture was washed
with diethyl ether and then THF. The solid that was insoluble
in the THF wash was recrystallized from acetonitrile/diethyl
ether to give 2 as a dark-brown microcrystalline solid (45%).
Although the microcrystalline solid appears stable for months if
stored at −38 °C, the cluster rapidly decomposes in solution,
even at low temperatures. The presence of excess MMP in
solution, however, is able to stabilize the cluster significantly.12
Solutions of 2 exhibit UV−vis absorption maxima at 330,
425, and 450 nm in acetonitrile, which are highly reminiscent of
those of ethylthiolate- and protein-bound [2Fe-2S] clusters.13
The IR spectrum of the solid material has an intense sharp
band at 1727 cm−1, which arises from the carbonyl unit in
MMP. Cyclic voltammetry experiments using a glassy carbon
electrode indicate that an electrochemically quasi-reversible
[2Fe-2S]2+/1+ event occurs at −870 mV vs Ag/AgNO3 in
acetonitrile. Finally, diffraction quality crystals of the compound
were grown from a concentrated acetonitrile solution of 2 in
the presence of excess MMP cooled to −38 °C and X-ray
diffraction analysis permitted solution of the crystal structure
(Figure 1). The bond metrics of the Fe2S2 rhomb are
comparable to those reported for analogous structures.14
Despite its instability, 2 can undergo expected substitution of
the peripheral MMP thiolates for phenylthiolate ions to
quantitatively produce (PPN)2[Fe2S2(SPh)4] (3), a previously
reported and well-characterized stable [2Fe-2S] cluster.15 This
reactivity not only serves to further confirm the formation of 2
but also provides a means of transferring the Fe2S2 rhomb into
a more stable molecular framework on which to carry out
subsequent characterization. The phenyl-substituted cluster can
be characterized by electrospray ionization mass spectrometry
(ESI-MS) and we capitalized on this feature to probe the origin
of the bridging sulfide. Given the starting reagents, the bridging
sulfide can only come from either the tert-butylthiolate groups
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ligand, as suggested for 1 by Lippard et al.11 Ligation of an
MMP carbonyl moiety, forming five-coordinate 5, yields a
species with an electronic structure that is better described as a
high-spin Fe2+ center antiferromagnetically coupled to a neutral
NO• ligand. Furthermore, the NO ligand is trans to a labilizing
thiolate.17 Both of these influences should lead to more facile
NO release from the iron-center.
We presume that 5 is unstable and disproportionates into a
more stable DNIC, 4, and a thiolate-bridged diferrous
compound, 6 (Scheme 3). Thiolate-bridged species analogous
to 6 have been previously reported in the literature and shown
to form [2Fe-2S] clusters on reaction with elemental sulfur.18
In our proposed reaction, the diferrous 6 is converted to the
diferric 2 by using one equivalent of disulfide (RSSR) with
generation of two equivalents of thioether (RSR) as a
byproduct. Support for the feasibility of the reaction between
6 and MMP disulfide came from our independent investigation
on the reaction between (NEt4)2[Fe2(μ-SEt)2(SEt)4],18b an
analogue of 6, and excess MMP disulfide. In this reaction, clean
formation of 2 and the corresponding MMP thioether was
observed (see SI). Alternatively, 2 might form via a different
intermediate, [Fe2(μ-SR)2(SR)2(NO)2]2−, the Ni analogue of
which is known.20,21 We were not, however, able to synthesize
the iron analogue in order to test the feasibility of this
hypothesis. Further mechanistic investigations are beyond the
scope of this report and will be the subject of future studies.
The present studies suggest a new potential role of MNICs
as intermediate species in the repair of [2Fe-2S] clusters
following nitrosylation. In 2006, Lippard first suggested that
MNICs could be involved in reaction pathways leading to
DNIC formation in biologically relevant sulfur-rich coordina-
tion environments.11 Here we demonstrate the first one-pot
conversion of a mononuclear iron-nitrosyl species to [2Fe-2S]
cluster requiring no reagents other than a cysteine analogue.
The in vitro transformation of protein-bound DNICs into
functional [2Fe-2S] clusters is reported to require cysteine,
cysteine desulfurase, and dioxygen.8 Although MNICs were not
initially implicated in this biological transformation, the results
presented here suggest that they may play a role in it and that
MNICs may, in general, have a greater biological importance
than is typically ascribed to them. Future work is geared toward
understanding other aspects of [2Fe-2S] cluster repair
chemistry, including the conversion of DNIC to MNIC and
the role of dioxygen.
Figure 2. Room temperature X-band EPR spectral changes associated
with the formation of (PPN)[Fe(SCH2CH2C(O)OCH3)2(NO)2] (4)
from the reaction of (PPN)[Fe(StBu)3(NO)] (1) and MMP in
acetonitrile.
headspace of the reaction mixture. Moreover, the isolated yield
of 4 indicates that it comprises ∼50% of the Fe present in 1. A
2:1 stoichiometry of the DNIC and [2Fe-2S] cluster, 4 and 2, is
consistent with a scheme in which these two complexes are the
only significant Fe- and NO-containing species formed during
the reaction (Scheme 3).
Scheme 3. Working Model of the Transformation of MNIC
into [2Fe-2S] Cluster by MMP
The experiments presented above provide insight into the
events that may occur during the chemical transformation of
MNIC to [2Fe-2S] cluster in the presence of MMP. A reaction
model (Scheme 3) has been proposed based on evidence for
the formation of [2Fe-2S] cluster, DNIC, and MMP-thioether
in a 1:2:2 molar ratio. The StBu ligands on (PPN)[Fe-
(StBu)3(NO)] (1) are first substituted by MMP to initiate the
reaction, as evidenced by release of HStBu, forming the MMP
thiolate-bound MNIC, 5. In separate experiments, nearly
identical chemistry was observed in the reaction of (PPN)[Fe-
(SEt)3(NO)] with MMP, whereas no reaction was observed
with (PPN)[Fe(SPh)3(NO)] or (PPN)[FeCl3(NO)] because
MMP could not replace the original ligands of these MNICs
(data not shown). Electronic structure calculations suggest that
four-coordinate 5, as shown in Scheme 3, is likely in
equilibrium with a five-coordinate isomer in which the carbonyl
moiety of the MMP ligand interacts with the Fe center (see SI).
This feature is unique to MMP among the thiols tested and is
likely important in the overall reactivity of this species. The
electronic structure of four-coordinate 5 can be described as a
high-spin Fe3+ center antiferromagnetically coupled to an NO−
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental and computational details, UV−vis, FTIR, ESI-
MS, and 1H NMR spectra, cyclic voltammogram of 2,
calibration curve for dimethyl 3,3′-thiopropionate, CIF for 2.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
eunsuk_kim@brown.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the NSF (CHE 1254733).
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