Spectroscopic Investigations of [FeFe] Hydrogenase Maturated with [57Fe2(adt)(CN)2(CO)4]2-

Article abstract of DOI:10.1021/jacs.5b03270

The preparation and spectroscopic characterization of a CO-inhibited [FeFe] hydrogenase with a selectively ⁵⁷Fe-labeled binuclear subsite is described. The precursor [⁵⁷Fe₂(adt)(CN)₂(CO)₄]^2- was synthesized from the ⁵⁷Fe metal, S_8, CO, (NEt₄)CN, NH₄Cl, and CH₂O. (Et₄N)₂[⁵⁷Fe₂(adt)(CN)₂(CO)₄] was then used for the maturation of the [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii, to yield the enzyme selectively labeled at the [2Fe]_H subcluster. Complementary ⁵⁷Fe enrichment of the [4Fe-4S]_H cluster was realized by reconstitution with ⁵⁷FeCl₃ and Na₂S. The H_ox-CO state of [2⁵⁷Fe]_H and [4⁵⁷Fe-4S]_H HydA1 was characterized by M?ssbauer, HYSCORE, ENDOR, and nuclear resonance vibrational spectroscopy. (Chemical Equation Presented).

Full text of DOI:10.1021/jacs.5b03270

Article
pubs.acs.org/JACS
Spectroscopic Investigations of [FeFe] Hydrogenase Maturated with
[57Fe2(adt)(CN)2(CO)4]2−
Ryan Gilbert-Wilson,#,† Judith F. Siebel,#,‡ Agnieszka Adamska-Venkatesh,‡ Cindy C. Pham,§
Edward Reijerse,‡ Hongxin Wang,§,∥ Stephen P. Cramer,*,§,∥ Wolfgang Lubitz,*,‡
and Thomas B. Rauchfuss*,†
†School of Chemical Sciences, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States
‡Max-Planck-Institut fur Chemische Energiekonversion, Stiftstrasse 34-36, 45470 Mulheim an der Ruhr, Germany
̈
̈
§Department of Chemistry, University of California, Davis, California 95616, United States
∥Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States
S
* Supporting Information
ABSTRACT: The preparation and spectroscopic character-
ization of a CO-inhibited [FeFe] hydrogenase with a selectively
57Fe-labeled binuclear subsite is described. The precursor
[57Fe2(adt)(CN)2(CO)4]2− was synthesized from the 57Fe
metal, S8, CO, (NEt4)CN, NH4Cl, and CH2O.
(Et4N)2[57Fe2(adt)(CN)2(CO)4] was then used for the
maturation of the [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii, to yield the enzyme selectively labeled at the
[2Fe]H subcluster. Complementary 57Fe enrichment of the [4Fe-4S]H cluster was realized by reconstitution with 57FeCl3 and
Na S. The H -CO state of [257Fe] and [457Fe-4S] HydA1 was characterized by Mossbauer, HYSCORE, ENDOR, and nuclear
̈
2
ox
H
H
resonance vibrational spectroscopy.
INTRODUCTION
■
Since their crystallographic identification, the hydrogenases
(H2ases) represent perfect paradigms for bioinspired energy
transformations. Utilizing only base metals and operating at
near thermodynamic potentials, these enzymes promote
reactions at extraordinary rates, in the case of [FeFe] H2ases
thousands of turnover per second.1 Further motivating this
theme are the practical implications of the H2/H+ redox
reaction, which is the basis for fuel cells.
Many [FeFe] H2ases have been studied, but the algal H2ase
from Chlamydomonas reinhardtii, HydA1,2 has received
particular attention.3 The C. reinhardtii protein is ideal for
studies on biosynthesis4 and spectroscopy because it represents
a “minimal architecture” enzyme containing only the active
site.5 In contrast, most [FeFe] H2ases feature multiple
accessory [Fe−S] clusters. The active site of all [FeFe]
H2ases is the “H-cluster”, consisting of a [4Fe-4S]H cluster
appended via a bridging cysteinyl thiolate to a diiron subunit
called [2Fe]H.6 In [2Fe]H, a diiron center is bound to CO,
CN−, and azadithiolate (adt2− = [(SCH2)2NH]2−) ligands
(Figure 1).7
Figure 1. H-cluster of HydA1 in the Hox-CO state.
subunit on a scale and at a pace that would not be readily
achieved by in vitro maturation routes.11,12 This “artificial
maturation” allows a detailed characterization of individual
active site states and the catalytic mechanism through a variety
of spectroscopic techniques.9
The artificial maturation route in principle should allow the
selective labeling of the [2Fe]H subunit with 57Fe, a nucleus
highly responsive to Mossbauer and nuclear resonance
̈
vibrational spectroscopies (NRVS). With a nuclear spin I =
1/2, 57Fe is also ideal for the suite of EPR techniques that
provide exquisite insights into Fe-based enzymes.13 57Fe-
labeling of the [2Fe]H subunit however poses a significant
synthetic challenge because salts of [Fe2(adt)(CN)2(CO)4]2−
are prepared by multistep sequences starting from reagents that
would only be awkwardly and inefficiently labeled with 57Fe. In
this report these challenges are surmounted, as established by
the preparation of HydA1 with a selectively 57Fe-labeled [2Fe]H
Recent work has shown that functional HydA1 enzyme can
be produced through incubation of unmaturated HydA1
containing only the [4Fe-4S]H subcluster, with a synthetic
active site mimic (Et4N)2[57Fe2(adt)(CN)2(CO)4].8 This
innovation exploits the fact that the active site is attached to
the protein through few covalent bonds. Via this artificial
maturation, the enzyme is now available with wide variety of
chemically and isotopically labeled versions9,10 of the [2Fe]H
Received: March 29, 2015
© XXXX American Chemical Society
A
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a
Scheme 1. Synthesis of [57Fe2(adt)(CN)2(CO)4]2−
a
+
(i) 4 KC14H10, (ii) CO, (iii) MeOH, (iv) S8, (v) H+, (vi) CH2O, NH4 , (vii) (NEt4)CN.
site. The Hox-CO state of HydA1 (Figure 1) can be obtained as
a pure state and was therefore chosen to demonstrate the
selective labeling. Using (Et4N)2[57Fe2(adt)(CN)2(CO)4] as
precursor the [2Fe]H subsite in Hox-CO was labeled using
artificial maturation. In a complementary experiment the [4Fe-
4S] subcluster in Hox-CO was labeled using FeS reconstitution.
The two 57Fe labeled versions of Hox-CO were studied using
6)]2[Fe2(CO)8] with S2Cl2 or S8 gave complex mixtures
including [Fe2S2(S5)2]2− and intractable solids but no
Fe2S2(CO)6. A successful method for the direct synthesis of
Fe2S2(CO)6 was inspired by details in the PhD thesis of W. W.
Brennessel of the Ellis group, who describes the synthesis of
K2Fe(CO)4 from FeBr2 in ∼50% yield.20 His method involves
treatment of FeBr2 in THF with four equivalents of potassium
anthracene at low temperature, followed by carbonylation at 1
atm. The Fe(-II) derivative is proposed to form via reduction of
K2Fe2(CO)8 by the fourth equivalent of K(anthracene). This
reaction was reproduced. Treatment of the resulting K2Fe-
(CO)4 with methanol efficiently afforded KHFe(CO)4,21 which
reacted with elemental sulfur to give Fe2S2(CO)6 after standard
workup.21 In this way, starting from 500 mg of 57Fe, we
prepared 180 mg of 57Fe2S2(CO)6 (11.8% from 57Fe metal)
together with 60.8 mg (4.5% yield) of 57Fe3S2(CO)9 (Scheme
1). Repeat synthesis using the same procedure resulted in the
yield of 57Fe2S2(CO)6 improving to 371 mg (24.4% from 57Fe
metal). The 13C NMR spectra of 57Fe2S2(CO)6 and
57Fe3S2(CO)9 gives 1J(57Fe,13C) = 28.3 and 26.3 Hz,
respectively. The most problematic step in the synthesis is
the conversion of KHFe(CO)4 into Fe2S2(CO)6, which we
estimate proceeded in 30% yield.
Mossbauer, electron nuclear double resonance (ENDOR),
̈
hyperfine sublevel correlation (HYSCORE) as well as nuclear
resonance vibrational (NRVS) spectroscopy.
RESULTS AND DISCUSSION
■
Synthesis and Characterization of [57Fe2(adt)-
(CN)2(CO)4]2−. The precursor to the target [57Fe2(adt)-
(CN)2(CO)4]2− is 57Fe2(adt)(CO)6, which undergoes dicya-
nation nearly quantitatively.14 Synthesis of the diiron
hexacarbonyl, however, poses challenges because it is derived
via a series of inefficient reactions from precursors that are not
readily labeled with 57Fe. Low yielding routes to unlabeled
Fe2(adt)(CO)6 are tolerated14 because the relevant reagents,
e.g., Fe(CO)5, are inexpensive and the early steps in the
preparation can be conducted on a multigram scale. The
industrial method for production of Fe(CO)5 involves the
direct carbonylation of Fe metal at high temperatures and
pressures, e.g., 175 atm at 150 °C.15 Such reactions require
specialized autoclaves,16 which are not suited for producing
small amounts of 57Fe(CO)5. A variety of laboratory syntheses
of 57Fe(CO)5 have been described, but they suffer from low
yields and difficult separations even when using specialized
equipment.17
Conversion of 57Fe2S2(CO)6 to [57Fe2(adt)(CN)2(CO)4]2−
requires two steps, which have been described previously for
the unlabeled versions of these complexes.22 The first of these
steps is the conversion of 57Fe2S2(CO)6 to 57Fe2(adt)(CO)6.
This conversion was achieved by reduction of 57Fe2S2(CO)6
with LiBEt3H to give 57Fe2(SLi)2(CO)6, followed by
protonation to 57Fe2(SH)2(CO)6 and subsequent adt bridge
formation through reaction with a mixture of ammonia and
formaldehyde to yield 57Fe2(adt)(CO)6. Conversion of
57Fe2(adt)(CO)6 to [57Fe2(adt)(CN)2(CO)4]2− was achieved
by reaction with 2 equiv of (NEt4)CN. The isotopic purity of
the anion [57Fe2(adt)(CN)2(CO)4]2− was established by
negative ion ESI mass spectrometry (see Supporting
Information (SI), Figure S1).
Selective Labeling of HydA1. The insertion of
[57Fe2(adt)(CN)2(CO)4]2− into unmaturated HydA1 was
monitored by Fourier transform infrared spectroscopy (FT-
IR). The precursor undergoes substantial rearrangement upon
incorporation into unmaturated HydA1 (Figure S2A and S2B,
SI). One CO ligand migrates to the bridging position and the
CN− groups shift from apical to basal sites. Furthermore, in a
The above considerations led to a focus on routes that avoid
the intermediacy of Fe(CO)5. Retrosynthetic analysis reminds
one that Fe2S2(CO)6, the immediate precursor to Fe2(adt)-
(CO)6, is formed from the [HFe(CO)4]− anion, not the
pentacarbonyl. Thus, syntheses of [H57Fe(CO)4]− from 57FeX2
are of interest. Literature methods18 for generating [HFe-
(CO)4]− from iron halides proved low-yielding in our hands.
Relevant to possible routes to [H57Fe(CO)4]− is the fact that it
is easily derived from [Fe(CO)4]2− by protonation. The anion
[Fe(anthracene)2]−, prepared by Ellis and co-workers in 61%
yield from FeBr2, carbonylates at ambient pressures.19 The
product, obtained in 81% isolated yield, is [K(18-crown-
6)]2[Fe2(CO)8]. Unfortunately, attempts to convert this salt
into Fe2S2(CO)6 were unfruitful. Treatment of [K(18-crown-
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major desymmetrizing event, one of the two previously
equivalent Fe sites binds to cysteinyl sulfur, which links the
[2Fe]H and [4Fe-4S]H clusters. Under the usual reductive
maturation conditions one terminal CO ligand of the distal Fe
dissociates affording an open coordination site where substrate
and inhibitors can bind. After oxidation with thionin (Figure
S2C, SI) and flushing with CO, a pure Hox-CO state is created
(Figure S2D, SI). In the Hox-CO state, the [2Fe]H subunit is in
a delocalized mixed valence Fe(II)Fe(I) configuration.
In order to realize complementary 57Fe enrichment, in a
separate experiment the [4Fe-4S]H cluster was 57Fe-labeled as
described in the Experimental Section. In a first step, unfolding
and thereby loss of the unlabeled [4Fe-4S]H cluster was
observed by subsequent loss of the brownish color of the
solution. The [4Fe-4S]H cluster was reconstituted with 57FeCl3
and Na2S, accompanied by recovery of the brownish color.
Successful reconstitution was confirmed by activity measure-
ments, UV−vis and EPR spectroscopy (see SI for detailed
information).
EPR Characterization. The selective labeling of the two
components of the H-cluster with 57Fe greatly simplifies the
assignment of the observed pulse EPR signals (see Figure 2).
For the hybrid in which the [2Fe]H subcluster is labeled, two
spectral features in the HYSCORE spectra can be observed
(Figure 2B,C). The correlation ridges in the right-hand section
of the spectra (positive frequencies) are assigned to a weakly
coupled 57Fe nucleus while the ones in the left-hand section
(negative frequencies) originate from a strongly coupled 57Fe
nucleus. These signals can both be simulated with axial
hyperfine tensors (see Table S1 in the SI). Their Aiso values are
4.4 and 1.3 MHz, respectively, suggesting that one iron in the
[2Fe]H subsite has ∼4 times more spin density than the other.
According to the spin-exchange model describing the electronic
structure of Hox-CO (see below), the proximal iron in [2Fe]H
has the largest spin density. The obtained hyperfine parameters
are in agreement with previously reported EPR data on the
[FeFe] H2ases from Desulfovibrio desulfuricans (DdH).13 Earlier
Mossbauer studies on the [FeFe] H ases from Desulfovibrio
̈
2
vulgaris Hildenborough (DvH) and Clostridium pasteurianum
(CpI) provided the first estimates for the hyperfine 57Fe
interactions in the [2Fe]H subsite.23,25 These measurements,
however, suffered from overlapping signals from the accessory
[4Fe-4S] clusters and are therefore less accurate (see Table S1).
The complete hyperfine tensor information on Fe1 and Fe2 as
obtained from HYSCORE (Table S1) accurately defines the
electronic structure of the binuclear subsite and can be used to
validate quantum chemical calculations on the H-cluster
properties.
As described above, it is also possible to selectively label the
[4Fe-4S]H subcluster with 57Fe. For this sample in the Hox-CO
state, no signal in the HYSCORE spectra was observed. In the
ENDOR spectra between 10−20 MHz signals from strongly
coupled Fe nuclei are observed. As it is shown in Figure 2A,
these signals are not observed when only the [2Fe]H subcluster
is labeled. This unequivocally confirms that these signals
originate from the [4Fe-4S]H subcluster. As presented in Figure
3, these signals can be simulated with four distinct anisotropic
hyperfine interactions corresponding to the four Fe centers in
the [4Fe-4S]H subcluster (hyperfine interaction parameters are
Figure 2. Q-band 57Fe ENDOR and HYSCORE spectra (20 K) of
selectively labeled HydA1 Hox-CO recorded at field positions indicated
in the EPR spectrum (inset); for experimental parameters see SI. (A)
Davies ENDOR of Hox-CO 57Fe labeled at [4Fe-4S]H (black) or
[2Fe]H (blue); simulations are indicated in red. (B) and (C)
HYSCORE spectra recorded for Hox-CO 57Fe labeled at [2Fe]H at g
= 2.008 and g = 2.053 respectively; for all simulation parameters see
Table S1.
coupling between the two subclusters.23 The four hyperfine
interactions were predicted to be divided in two groups
representing the two S = 9/2 Fe(II)Fe(III) pairs which are
mutually antiferromagnetically coupled to effective spin S = 0.
Later ENDOR studies on DdH confirmed this analysis and
showed that the hyperfine interaction of the two groups have
opposite signs.13 The current data on HydA1 are fully in line
with these studies. The two groups are defined by pair 1 with
A
iso = 33.2 MHz (brown) and 30.0 MHz (orange), pair 2 Aiso =
listed in Table S1). In earlier Mossbauer studies on CpI, these
̈
28.0 MHz (blue) and 27.6 MHz (green).
relatively large hyperfine interactions of the formally
diamagnetic [4Fe-4S]H subcluster were interpreted as originat-
ing from spin density induced by a substantial exchange
Mossbauer Characterization. The selective 57Fe-labeling
of the [2Fe]H site enables the investigation of this specific
̈
subsite by Mossbauer spectroscopy. Previous Mossbauer
̈
̈
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Figure 3. Q-band Davies ENDOR spectra and simulations of Hox-CO
HydA1 selectively labeled with 57Fe at the [4Fe-4S]H cluster. Spectra
were recorded with an RF pulse of 45 μs, shot repetition time 800 μs,
microwave frequency 33.93396 GHz, temperature 20 K at field
positions: (A) 1182.9 mT ≈ g1, (B) 1189.65 mT, (C) 1196.4 mT, (D)
1203.15 mT, (E) 1209.9 mT ≈ g2. The black line represents
experimental data and the red line the sum of the simulations. The
colored lines below each experimental spectrum are the components
of the simulation corresponding to the four hyperfine couplings:
brown Fe3, orange Fe4, blue Fe5, green Fe6 (see Table S1).
Figure 4. (A) Mossbauer spectrum and simulations of H -CO HydA1
̈
ox
selectively labeled with 57Fe at the [4Fe-4S]H subcluster. (B)
Mossbauer spectrum and simulations of H -CO HydA1 selectively
̈
ox
labeled with 57Fe at the [2Fe]H subcluster. (C) Superimposition of the
simulations of the 57Fe-labeled [2Fe]H subunit and the [4Fe-4S]H
cluster, ratio 0.5:1. All spectra were measured at 160 K in zero
magnetic field.
[4Fe-4S]H cluster and the [2Fe]H site, which would require 9
parameters for its simulation (apart from the background
contributions).
studies on the active [FeFe] H2ases CpI23,24 and DvH25 are
complicated by the presence of accessory [4Fe-4S] clusters in
addition to the H-cluster. Using our approach of selective
The isomer shift (δ = 0.42 mm/s) and the quadrupole
splitting (ΔEQ = 1.04 mm/s) found for the [457Fe-4S]H cluster
are typical for oxidized [4Fe-4S] clusters.26 The spectrum of
the 57Fe-labeled [2Fe]H site can be analyzed assuming two
nonequivalent Fe sites. A small amount of an Fe(II) impurity is
apparent as well. The data can be fitted assuming a slight
nonequivalence either more pronounced in the quadrupole
splitting (simulation 1, Figure 4B) or in the isomer shift
(simulation 2, Figure S5). Simulation 1 (Figure 4B) is slightly
favored. Taking into account the different ligands coordinating
the two irons one would expect clear differences in quadrupole
splitting whereas the isomer shift is relatively insensitive to
electron density differences for low valence iron centers.27
Simulation 1 is characterized by the isomer shifts δ1(1) = 0.16
mm/s, δ1(2) = 0.08 mm/s and quadrupole splittings of
labeling, the Mossbauer spectra are simplified and Mossbauer
̈
̈
parameters can be obtained without the cosimulation of other
57Fe-components. Additionally, this method enables high
concentrations of the sample and therefore high-quality spectra.
The Mossbauer spectrum of the [457Fe-4S] cluster from
̈
H
HydA1 maturated with unlabeled [Fe2(adt)(CN)2(CO)4]2− in
the Hox-CO state is shown in Figure 4A. The counterpart
spectrum, the 57Fe-labeled [2Fe]H site in the Hox-CO state, is
presented in Figure 4B. Both spectra are quite simple and can
be simulated with relatively few components. Figure 4C
displays a superimposition of the simulated spectra 4A and
4B. It clearly shows the overlap between the signals from the
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ΔEQ1(1) = 0.89 mm/s, ΔEQ1(2) = 0.55 mm/s. The parameters
of all simulations can be found in Table S2 in the SI. The fitted
bend by comparison with assignments of previous model
complexes.29−31 This band is absent in the [257Fe]H HydA1
Hox-CO spectrum as there are no longer two CO ligands in the
basal positions of either iron center.31 The high intensity of the
Fe-CO band at 576 cm−1 in the [57Fe2(adt)(CN)2(CO)4]2−
spectrum is due to the high symmetry of this complex.
When comparing the low energy portion of the two spectra,
it is clear that the bands at 170, 198, and 326 cm−1 are largely
unmoved and there are only minor shifts for other bands in the
Fe−S region. This is an indication that there is little change in
the Fe−S geometry upon incorporation of the precursor into
the enzyme. A common feature of the NRVS of [4Fe-4S]
clusters are bands due to Fe−S bending and breathing at 150
cm−1.30 The absence of any strong band at 150 cm−1
demonstrates that there is no significant 57Fe incorporation
into the [4Fe-4S]H cluster, verifying the results from the
Mossbauer parameters are not identical to those obtained
̈
previously by Pereira et al. for DvH (δ1(1) = 0.17 mm/s, δ1(2)
= 0.13 mm/s and ΔEQ1(1) = 0.70 mm/s, ΔEQ1(2) = 0.65
mm/s, see also Table S2), but lie in the same range that is
consistent with low-spin iron in a low oxidation state. The
slight differences between the parameters obtained in this work
and those by Pereira et al. could originate from the lower
accuracy in the latter study caused by the overlapping F-cluster
signals but it is also possible that the H-cluster of [FeFe] H2ases
in different organisms (DvH vs HydA1) slightly different
electronic structures. To verify this, DvH could be maturated
using the method described here to obtain a Mossbauer
̈
spectrum from the selectively labeled [2Fe]H site. This would
also pave the way to study in detail the elusive “inactive”
oxidation states in DvH Hox-air and Htrans as well as the
“regular” active oxidation states Hox and Hred.
Mossbauer spectroscopy. Further discussion of bands and their
̈
assignments can be found in the Supporting Information.
The benefits of the selective labeling by the artificial
maturation process are illustrated also by the NRVS measure-
ments. The previously reported NRVS spectrum was recorded
on CpI, which was only partially labeled by incubation/
activation of the 56Fe-labeled inactive apo protein with 57Fe-
enriched maturation proteins (Figure 6).11 The CpI spectrum
NRVS Characterization. The [257Fe]H-HydA1 Hox-CO
sample was also analyzed by nuclear resonance vibrational
spectroscopy (NRVS). This technique is exclusively responsive
to vibrations affecting the 57Fe site. Previous attempts to record
NRVS spectra of [FeFe] H2ase (CpI)11 were hampered by
signals from other 57Fe labeled components, and not purely
originating from the [2Fe]H subcluster (see Figure 6). In this
study, NRVS spectra of (Et4N)2[57Fe2(adt)(CN)2(CO)4] and
[257Fe]H HydA1 Hox-CO were obtained (Figure 5). The NRVS
Figure 6. NRVS spectrum of HydA1 Hox-CO of this work (red) and
CpI from previous NRVS on hydrogenase (mixed states, blue).11 The
assignments are not pure vibrational modes.
has characteristic features at 353 and 382 cm−1 in the Fe−S
region (300−400 cm−1) assigned to Fe−S bands of oxidized
[4Fe-4S]2+ clusters. These bands are indicative of unselective
labeling of a [4Fe-4S] cluster. For the [257Fe]H HydA1 Hox-CO
spectrum, only low intensity features are present in this region.
Selective labeling is also evident in a notable increase in the
intensity of the Fe-CN and Fe-CO signals in the 400−600 cm−1
region.
Figure 5. NRVS spectrum of [257Fe]H HydA1 Hox-CO (red) and the
precursor [57Fe2(adt)(CN)2(CO)4]2− (blue). The peaks labeled in
black are the same energy 2 cm−1 in both spectra.
spectrum of the precursor displays intense bands in the regions
assigned to Fe-CO stretching and Fe−C−O bending (six bands
between 490 and 650 cm−1) and the Fe-CN region (two bands
between 400 and 500 cm−1).28,29 Comparing the spectrum of
the precursor to that for the HydA1 Hox-CO spectrum, one can
clearly see that the Fe−C bands are strongly affected, with Fe-
CO modes being (on average) red-shifted and Fe-CN modes
being blue-shifted. The Fe-CO/-CN shift is consistent with
major changes in the primary coordination sphere of the Fe
centers upon insertion into the enzyme and conversion to Hox-
CO.
CONCLUSIONS
■
In summary, the work demonstrates the selective 57Fe-labeling
of the HydA1 enzyme at both the [2Fe]H subunit and the
subcluster [4Fe-4S]H. The route to 57Fe-labeled iron carbonyls
is described using only conventional Schlenk techniques. The
method is clearly adaptable to labeling of other derivatives,
perhaps other hydrogenases, and their synthetic analogues.32
Focusing on the protein prepared in the Hox-CO state, all
spectroscopic measurements confirm the selectivity of the
labeling.
The notable band at 653 cm−1 is assigned as having
significant contributions from a diequatorial Fe(CO)2 stretch−
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The 57Fe hyperfine interactions from the [2Fe]H and [4Fe-
4S]H subclusters in HydA1 obtained from HYSCORE and
ENDOR spectroscopies are consistent with those obtained
earlier from the H-cluster of the H2ase DdH although slight
species dependent variations were observed. The overall
electronic structures of Hox-CO of DdH and HydA1 seem
over a period of 10 h under a constant 1.05 atm of CO. Once warmed
to room temperature the solution is a dark color with a significant
amount of pale precipitate. All solvent was removed to give a brown
solid containing the desired K2Fe(CO)4. This solid was cooled to −77
°C, and treated with MeOH (50 mL) added over the course of 20 min.
The initially brown/green solution changed to dark red as the mixture
was warmed to room temperature over the course of an hour,
indicating the formation of KHFe(CO)4. The solution was filtered
through Celite, and any remaining solid was washed with MeOH. The
volume of the solution was reduced to ∼10 mL, with the aim of
keeping as much of the red color in solution, not coprecipitating with
the colorless solids, which precipitate upon this volume reduction. The
solution was cooled to 0 °C, and solid potassium hydroxide (1.30 g,
23.2 mmol) was added against a positive pressure of argon, followed
by the addition of degassed water (15 mL). After 5 min of stirring at 0
°C, elemental sulfur (1.29 g, 40.2 mmol) was added directly against a
flow of argon. The mixture was stirred at 0 °C for 1.5 h before
degassed water (20 mL) and hexane (50 mL) were added. Solid
NH4Cl (3.22 g, 60.2 mmol) was added against a flow of argon, and a
needle with attached bubbler was attached to the flask and the solution
stirred for 16 h. Subsequent steps were performed in air. Stirring was
ceased and the red hexane layer was decanted and reserved. The
remaining aqueous solution was extract with hexane (4 × 20 mL) until
no red color remained in the hexane layer, with the extracts then
added to the previously reserved solution. The solution was passed
through a short silica plug (2 cm) and the filtrate then evaporated on a
rotary evaporator (Fe2S2(CO)6 readily sublimes under oil pump
vacuum at room temperature). An extract of the resulting red oil in
hexane (3 mL) was chromatographed on a silica gel column (2 cm ×
30 cm), eluting with hexane. The first orange band was identified as
57Fe2S2(CO)6 (180 mg, 0.520 mmol, 12.4% from 57FeBr2) and the
second dark red band as 57Fe3S2(CO)9 (60.8 mg, 4.5% from 57FeBr2).
The preparation was repeated with improved handling giving an
improved yield of 57Fe2S2(CO)6 (371 mg, 1.07 mmol, 25.4% from
57FeBr2) and a small amount of 57Fe3S2(CO)9 (15 mg, 0.031 mmol,
1.1% from 57FeBr2) 57Fe2S2(CO)6: IR (pentane): νC≡O = 2085 (m),
2045 (s), 2008 (s), 1993 (w); 13C NMR (600 MHz, d8-Toluene): δ
therefore virtually identical. The Mossbauer parameters from
̈
the [2Fe]H subsite in HydA1 revealed an intrinsic ambiguity
(i.e., two equivalent combinations of isomer shift and
quadrupole splitting) that was not detected in earlier studies
on DvH.
NRVS analysis demonstrated that through this selective 57Fe
labeling the intensity of signals from the key [2Fe]H subunit can
be improved significantly. This improvement opens up the
possibility of new modes involving the bridging CO, hydrides,
or Fe−Fe interactions being identified. This future effort would
require suitable isotopic labeling experiments of other atoms of
the [2Fe]H subunit and complementary simulations. These
labeling experiments are facilitated by the frugal use of common
reagents in this synthetic method, which allows easy
substitution for their isotopically labeled counterparts 34S8,
13CO, and 13CN−.33 Such isotope labeling of atoms bound
directly to 57Fe will facilitate assignment of the NRVS spectrum
of catalytically significant states.
Further work is underway to spectroscopically characterize
the “active” states of the enzyme, specifically Hox, Hred, and
H
sred. Through selective labeling the overlapping 57Fe ENDOR
13,34
spectra of the two subclusters in Hox
can now be
disentangled. The effects of, e.g., the bridging CO ligand and
the exchange coupling to the [4Fe-4S]H subcluster can now for
the first time be studied on selectively labeled H-clusters. In
addition, also “inactive” precursors [57Fe2(pdt)(CN)2(CO)4]2−
(pdt2− = propanedithiolate = [(SCH2)2CH2]2−) and
[57Fe2(odt)(CN)2(CO)4]2− (odt2− = oxadithiolate =
[(SCH2)2O]2−) will be studied in 57Fe labeled form as a
product of this work. Although largely inactive, these variants
show “trapped” intermediates that may be related to the
catalytic cycle.35
208.66 (d, JC−Fe = 28.3 Hz, CO); 57Fe3S2(CO)9: IR (pentane): νC≡O
=
2063 (s), 2045 (s), 2024(s), 2008 (m), 1988 (w); 13C NMR (600
MHz, d8-Toluene): δ 209.23 (d, JC−Fe = 26.3 Hz, CO). MS ESI- (m/z)
515.6 ((Et4N)[57Fe2(adt)(CN)2(CO)4]−) IR (acetonitrile): νC≡N
2075 (m) νC≡O = 1968 (s), 1924 (s), 1891 (s), 1873 (sh).
=
Preparation of 57Fe-Labeled HydA1. All samples were handled
strictly anaerobically. All buffers were carefully degassed. Unmaturated
HydA1 was prepared as described by Kuchenreuther et al.38 In order
to selectively label the [4Fe-4S]H cluster with 57Fe, unmaturated
HydA1 was treated with 6 M guanidinium chloride in 100 mM Tris/
HCl pH, 8.0, 20 mM EDTA to have a final concentration of 100−200
μM protein. After 30 min of incubation, samples were buffer
exchanged into 100 mM Tris/HCl, pH 8.0, 150 mM NaCl using
PD10 desalting columns (GE Healthcare). For the reconstitution
reaction, a protocol similar to literature procedures was used.39 57FeCl3
was prepared from 57Fe powder by dissolving in concentrated
hydrochloric acid, evaporation of the solvent and resolubilizing in
distilled water. The protein solution (50−70 μM) was incubated with
5 mM DDT for 5 min and slightly stirred. 10-fold excess of 57FeCl3
solution was added stepwise to the reaction mixture within 20 min.
Thereafter, 10-fold excess of an aqueous solution of Na2S was added
stepwise. The reaction mixture turned from reddish (after addition of
57FeCl3) to a brownish color typical for unmaturated HydA1. After
removing excess of ions using PD10 desalting columns, samples were
concentrated and kept frozen until use.
EXPERIMENTAL SECTION
■
General Considerations. Unless otherwise indicated, reactions
were conducted using standard Schlenk techniques or in a glovebox
under an N2 atmosphere at room temperature with stirring. Synthesis
of 57FeBr2 from 57Fe,36 57Fe2(adt)(CO)6 from 57Fe2S2(CO)6,37 and
(Et4N)2[57Fe2(adt)(CN)2(CO)4] from 57Fe2(adt)(CO)6,14 were
achieved by modification of literature procedures for the equivalent
unlabeled complexes. Details can be found in the Supporting
Information.
Synthesis of 57Fe2S2(CO)6. A potassium anthracene solution was
prepared by stirring thinly sliced potassium metal (1.41 g, 36.2 mmol)
and anthracene (6.44 g, 36.1 mmol) in THF (120 mL) overnight using
a glass covered stir bar to give a deep blue solution of potassium
anthracene. In a separate flask 57FeBr2 (1.82 g, 8.40 mmol) was also
stirred overnight in THF (100 mL) giving a suspension of yellow solid
in a pale orange solution. Both solutions were chilled to −77 °C, and
the 57FeBr2 solution was cannula transferred into the potassium
anthracene solution over a period of 10 min. The solution was allowed
to warm to room temperature over a period of 5 h, resulting in a color
change from blue to very dark red/brown indicating the formation of
K[Fe(anthracene)2]. The solution was then filtered through Celite
(the solution contains very fine particles that easily block glass frits and
even block up Celite if too much pressure is applied). The volume of
the filtrate was reduced to ∼150 mL under a vacuum. The solution was
cooled to −77 °C, and the headspace was replaced by an atmosphere
of CO. The solution was then allowed to warm to room temperature
In order to selectively label unmaturated HydA1 on the [2Fe]H
subcluster, it was incubated with a 2-fold excess of (Et4N)2[57Fe2(adt)-
(CN)2(CO)4] following recent protocols.8,10 In order to obtain the
Hox-CO inhibited state, samples were first oxidized with thionine and
then flushed with CO for 20 min (Figure S2, SI). The redox states of
all samples were verified using FT-IR spectroscopy.
EPR Analysis. Field swept Q-band EPR spectra were recorded in
the pulsed mode using FID detection after a 1 μs π/2 excitation pulse.
F
DOI: 10.1021/jacs.5b03270
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
After a pseudomodulation transformation, the spectra obtained in this
way are comparable to those using CW EPR.40
Notes
The authors declare no competing financial interest.
Electron nuclear double resonance (ENDOR) was used to study the
57Fe hyperfine interactions. In this investigation the Davies ENDOR
sequence was used: [π]-td1-[RF]-td2-[π/2]-τ-[π]-τ-(ESE).41,42 The
excitation of nuclear spin transitions is detectable through an increase
in the inverted ESE intensity. The microwave preparation pulse was
set to 140 ns, whereas the length of the radiofrequency (RF) pulse was
45 μs and the shot repetition time 0.8 ms.
Q-band hyperfine sublevel correlation spectroscopy (HYSCORE)
experiments were performed using the standard HYSCORE pulse
sequence: [π/2]-τ-[π/2]-t1-[π]-t2-[π/2]-τ-(ESE).42,43 The length of
the microwave [π/2] and [π] pulses was adjusted to the maximum
available microwave power (3 W). The delay between the first two
pulses (τ) was 352 ns. The starting t1 and t2 delays in all measurements
were 100 ns and were changed by a step of 16 ns. To suppress the
effect of unwanted echoes, a four step phase cycling of the microwave
pulses was used.
ACKNOWLEDGMENTS
■
We thank Annika Gurowski and Agnes Stoer for their excellent
technical assistance, Eckhard Bill and Bernd Mienert for their
help with the Mossbauer spectroscopy, and Lars Lauterbach,
̈
Leland Gee, Yoshitaka Yoda and Kenji Tamasaku for their
expertise in NRVS. This project was supported by the National
Institutes of Health (GM061153, GM-65440), the Max-Planck
Society, the Deutsche Forschungsgemeinschaft (DIP project
LU315/17−1), and US Department of Education (CFDA-
84.200A). The NRVS experiments were performed at SPRING-
8 BL09XU (JASRI proposed no.2014B1032) and BL19LXU
(RIKEN proposal no 20140033).
Q-band experiments were performed on a Bruker ELEXYS E580
spectrometer with a SuperQ-FT microwave bridge and a home-built
resonator described earlier.44 Cryogenic temperatures (20 K) were
obtained by an Oxford CF935 flow cryostat. ENDOR experiments
were performed using the random (stochastic) acquisition technique
and making use of a 300W ENI 300 L RF amplifier. A Trilithic
H4LE35-3-AA-R high-power low pass filter (cut off frequency around
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ASSOCIATED CONTENT
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* Supporting Information
Spectra and preparative details. The Supporting Information is
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