FEBS Letters 584 (2010) 638–642
journal homepage: www.FEBSLetters.org
The [FeFe]-hydrogenase maturase HydF from Clostridium acetobutylicum contains
a CO and CN ligated iron cofactor
Ilka Czech a,1, Alexey Silakov b,1, Wolfgang Lubitz b, Thomas Happe a,*
a
b
Lehrstuhl Biochemie der Pflanzen, AG Photobiotechnologie, Ruhr Universität Bochum, Universitätsstrasse 150, 44801 Bochum, Germany
Max-Planck-Institut für Bioanorganische Chemie, 45470 Mülheim an der Ruhr, Germany
a r t i c l e
i n f o
Article history:
Received 16 November 2009
Revised 9 December 2009
Accepted 10 December 2009
Available online 14 December 2009
Edited by Stuart Ferguson
Keywords:
[FeFe] Hydrogenase
H-Cluster
Maturation
Fourier-transform infrared spectroscopy
Electron paramagnetic resonance
spectroscopy
Clostridium acetobutylicum
a b s t r a c t
Biosynthesis of the [FeFe] hydrogenases active site (H-cluster) requires three maturation factors
whose respective roles are not understood yet. The clostridial maturation enzymes (CaHydE, CaHydF
and CaHydG) were homologously overexpressed in their native host Clostridium acetobutylicum.
CaHydF was able to activate Chlamydomonas reinhardtii [FeFe] hydrogenase apoprotein (CrHydA1apo) to almost 100% compared to the native specific hydrogen evolution activity. Based on electron paramagnetic resonance spectroscopy and Fourier-transform infrared spectroscopy data the
existence of a [4Fe4S] cluster and a CO and CN ligand coordinated di-iron cluster is suggested. This
study contains the first experimental evidence that the bi-nuclear part of the H-cluster is assembled
in HydF.
Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction
[FeFe] hydrogenases are [FeS] proteins that catalyze the reversible reduction of protons to molecular hydrogen. They have been
found in hydrogen evolving anaerobic bacteria and unicellular
eukaryotes [1,2]. The active site of [FeFe] hydrogenases is commonly known as H-cluster. This unique prosthetic group consists
of a ferredoxin-type [4Fe4S] cluster, connected via a single bridging cysteine to a [2Fe2S] moiety ([2Fe]H). The iron atoms of the
[2Fe]H cluster are coordinated by CO- and CN-ligands and a dithiolate bridge [3]. Based on a pulse electron paramagnetic resonance
Abbreviations: EPR, electron paramagnetic resonance spectroscopy; FTIR, Fourier-transform infrared spectroscopy; HYSCORE, Hyperfine sublevel correlation
spectroscopy; CrHydA1, [FeFe] hydrogenase protein of C. reinhardtii; CaHydE,
maturation factor HydE of C. acetobutylicum; CaHydF, maturation factor HydF of C.
acetobutylicum; CaHydG, maturation factor HydG of C. acetobutylicum; TmHydE,
maturation factor HydE of Thermotoga maritima; TmHydF, maturation factor HydF
of Thermotoga maritima; [2Fe]H, [2Fe2S] moiety of the H-cluster; GTP, Guanosintriphosphate; GDP, Guanosindiphosphate; SAM, S-adenosyl methionine; AdoH, 50 Desoxyadenosine
* Corresponding author. Address: Ruhr Universität Bochum, Lehrstuhl fur
Biochemie der Pflanzen, AG Photobiotechnologie, ND2/170, 44780 Bochum,
Germany. Fax: +49 234 32 14322.
E-mail address: [email protected] (T. Happe).
1
These authors contributed equally to this work.
spectroscopy (EPR) study, the central atom of the dithiolate bridge
has been recently identified as nitrogen [4].
The green alga Chlamydomonas reinhardtii possesses a small
[FeFe] hydrogenase (CrHydA1) of 48 kDa [5]. In contrast to bacterial-type [FeFe] hydrogenases, CrHydA1 exhibits no additional
[FeS] clusters besides the active site [6]. Recent spectroscopic studies of CrHydA1 [7–9] confirmed that the general structure of the
active center is similar to other known [FeFe] hydrogenases.
Three highly conserved proteins HydE, HydF and HydG, which
are common in all organisms containing [FeFe] hydrogenases, are
required for the assembly of the intact H-cluster [10]. Recently,
the X-ray crystallographic structure of recombinant, reconstituted
HydE from Thermotoga maritima (TmHydE) was solved. The structure is very similar to that of biotin synthase from Escherichia coli;
in addition to the [4Fe4S] cluster of the radical-reaction center a
second [2Fe2S] cluster is present depending on the reconstitution
state [11]. HydG is proposed to be involved in the synthesis of
the dithiolate ligand. Studies of Pilet et al. demonstrated its ability
to cleave the substrate tyrosine into p-cresol in a radical-S-adenosyl methionine (SAM) reaction, further reaction steps of the second
proposed reaction product dehydroglycine could result in a dithiolate bridge with a bridging nitrogen atom [12]. HydF contains a
Guanosintriphosphate (GTP)-binding motif, shows GTPase activity
[13] and was hypothesized to act as a scaffold protein in the
0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.febslet.2009.12.016
I. Czech et al. / FEBS Letters 584 (2010) 638–642
H-cluster maturation process [14]. The steps of H-cluster biosynthesis are largely unknown. To achieve deeper insight into the maturation process, [FeFe] hydrogenase apoprotein synthesized in the
absence of maturation factors can be activated by in vitro maturation. Previous studies showed evidence that all three maturation
factors have to be expressed in concert to achieve maturation
activity [15]. The hydrogenase apoprotein needs to contain a
[4Fe4S] cluster to get activated by HydF [16]. However, there is almost nothing known about the structure of the cofactor in HydF. In
this study, a very efficient in vitro maturation system is presented,
utilizing homologously overexpressed Clostridium acetobutylicum
maturation factors (CaHydE, CaHydF and CaHydG) and CrHydA1
apoprotein (CrHydA1apo) expressed in E. coli. Hydrogen evolution
rates close to the evolution rates of native CrHydA1 were obtained.
Spectroscopic analyses by EPR and Fourier-transform infrared
spectroscopy (FTIR) techniques provide information about the
inorganic cofactors within the maturase HydF.
2. Materials and methods
2.1. Cloning of maturation factors
The genes hydE, hydF and hydG [17] were PCR-amplified from
purified C. acetobutylicum ATCC 824 genomic DNA with primers
containing adequate restriction sites. For cloning into pTCrhydA1opt-C-tag plasmid [18] the CrhydAlOpt was first excised with
BamHI and EcoRV restriction endonucleases. The maturase-encoded genes were then cloned into the vector with primers using
the same sites. The following primers, containing a BamHI restriction site in the forward primer and a EcoRV restriction site in the
reversed primer, were used: 50 -CCGGATCCGATAA TATAATAAAGTTAATTAATAAAGCAGAGGTA-30 and 50 -GCCGATATCACCAATAGAT
TCTTTGTAGCTTTTTT-30 for hydE, 50 -CCGGATCCAATGAACTTAACT
CAAC ACCCAAAGGTGAA-30 and 50 -CGGGATATCGTTCCTACTCGATT
GATTAAATATTCTA-30 for hydF, 50 -CCGGATCCTATAATGTTAAATCT
AAAGTTGCAACTGA-30 and 50 -GCGGATATCGAATCTAAAATCTCTTT
GTCCCTCTTTTAAT-30 for hydG. The resulting plasmids pThydEca,
pThydFca and pThydGca were used to transform C. acetobutylicum
ATCC824 [19].
2.2. Strains, media and growth conditions
Anaerobic overexpression of the maturation factors CaHydE, CaHydF and CaHydG in recombinant C. acetobutylicum ATCC 824
strains were performed as described earlier [18,19]. Every single
overexpressed maturase was synthesized with a background of
all three intrinsic maturases, which ensures the right assembly of
the individual overexpressed maturation factor. For overexpression of CrHydA1apo, recombinant E. coli BL21(DE3) strains were
aerobically grown in 500 ml Luria Broth (LB) medium with
100 lg ml1 ampicillin in a 2 l Erlenmeyer flask. After harvesting
by centrifugation, the pellet was washed in 100 mM Tris–HCl buffer pH 8 and frozen at 20 °C.
639
trations were determined using Quick Start Bradford Protein Assay
(Bio-Rad Laboratories GmbH, Muenchen, Germany). GTPase activity and radical-SAM activity were measured by HPLC analyses as
described by Brazzolotto et al. [13] and Zhao et al. [20] using a
Nucleodur 100-5 C18 ec EC 250/4.6 column (Macherey Nagel,
Dueren, Germany). SAM and GTP cleavage was determined at
37 °C after different incubation times between 0 h and 6 h.
For spectroscopic measurements proteins were concentrated to
achieve 100 lM in 30 ll with Vivaspin 500 centrifugal filter units
(Satorius, Goettingen, Germany). For analyses of the reduced proteins, 30 mM dithionite were added to ensure complete reduction
of the [FeS] clusters.
2.4. Maturation assay
CrHydA1apo was incubated with CaHydE, CaHydF and CaHydG
in their as-isolated form, provided in the elution buffer. The assay
had a final volume of 400 ll and was prepared in a 1.5 ml vial to a
final volume of 400 ll with 100 mM potassium phosphate buffer
pH 6.8 with 0.25 mM GTP (Acros Organics, Geel, Beligum) and
2.5 mM MgCl2 (JT Baker, Deventer, Netherlands) and incubated at
37 °C in an anaerobic chamber for 1 h. To determine the hydrogen
evolution activity, the complete 400 ll assay was added to a
hydrogenase activity assay [5].
2.5. Spectroscopic characterization
Q-band EPR spectra of CrHydA1apo, and all clostridial maturation enzymes were measured by using the 2-pulse electron spin
echo (ESE) detected EPR technique. The ESE intensity was detected
after two microwave (MW) pulses (p/2 and p) as a function of the
external magnetic field. The delay between the MW pulses was
fixed to s = 500 ns. The length of the p/2 MW pulse was set to
40 ns and the p pulse was set to 68 ns. If not mentioned, the shot
repetition time was set to 1 ms. All measurements were performed
on a Bruker ELEXSYS E580 Q-band spectrometer with a SuperQ-FT
microwave bridge, working at 34.11 GHz. Cryogenic temperatures
(10–20 K) were obtained by an Oxford CF935 flow cryostat.
FTIR measurements were performed on a Bruker IFS 66 v/s FTIR
spectrometer equipped with a Bruker MCT (mercury–cadmium–
telluride) detector. The sample chamber was under a constant flow
of N2 purge gas to expel CO2 gas and water vapour. Low temperatures were achieved by an Oxford OptistatCF continuous helium
flow cryostat. The interferograms were accumulated in the double-sided, forward–backward mode with 2000 scans. All measurements were performed with a resolution of 3 cm1. The obtained
interferograms were automatically processed by the Opus software
utilizing a 32-points phase correction and a Blackman–Harris 3term apodization window. Baseline correction was done using a
cubic spline data interpolation procedure applied to manually selected points of the experimental spectra. Data processing was
facilitated by home-written routines in the MATLAB™ programming environment.
3. Results and discussion
2.3. Purification and characterization of StrepII-tagged proteins
3.1. Maturation capability of homologously expressed HydF
All purification steps were performed under strictly anaerobic
conditions with oxygen free buffers, containing 2 mM sodium
dithionite (dithionite). The purification was performed as described in the appropriate protocol (IBA GmbH, Goettingen, Germany). The elution step was performed with 2.5 mM
desthiobiotin in 100 mM Tris–HCl buffer pH 8, each fraction was
provided with 3% glycerol, sealed and frozen at 80 °C.
10% SDS–polyacrylamide gel electrophoresis (PAGE) was used
to analyze the presence and purity of the proteins. Protein concen-
It was shown that the facultative anaerobe bacterium C. acetobutylicum is useful for the overexpression of correctly assembled [FeS]
proteins [19]. To achieve the maturation factors (HydE, HydF and
HydG) with maturation capability, it is essential to co-synthesize
all three maturation enzymes in one organism [15]. C. acetobutylicum which naturally provides all three maturation factors can ensure the correct assembly of an individually expressed maturation
factor. Each overexpressed clostridial maturase could be purified
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I. Czech et al. / FEBS Letters 584 (2010) 638–642
with a yield of approximately 2 mg protein per liter cell culture.
Further characterizations of the as-isolated proteins by HPLC analyses showed radical-SAM activity of CaHydE (0.008 AdoH min1)
and GTPase activity of CaHydF (0.017 GDP min1), which were in
the same range as the TmHydE and TmHydF activities as reported
previously [13,21]. The main elution fractions are light brown colored, which indicates the presence of iron in all proteins.
For analyzing the influence of several maturation factors in an
in vitro maturation assay, different combinations of 0.7 lg anaerobically purified CrHydA1apo, which was heterologously expressed
in E. coli BL21(DE3) with a yield of 0.8 mg per liter, with 8.4 lg
of each maturation enzyme were tested. CrHydA1apo and the three
maturases exhibited no background activity of hydrogen evolution
when tested individually. The combination of CaHydE and/or CaHydG with CrHydA1apo also did not result in H2-production.
However, by combining CrHydA1apo with CaHydF a hydrogen
evolution rate of 486 lmol H2 min1 mg1 was achieved. To evaluate the dependence of the molar CaHydF: CrHydA1apo ratio and
the hydrogen evolution, several protein ratios were tested. Maximum CrHydA1apo activity of 98% (726 lmol H2 min1 mg protein1) compared to the native enzyme CrHydA1 was achieved
by incubating 0.017 lM CrHydA1apo with 0.466 lM CaHydF (see
Fig. 1). The experiments show that the hydrogen production rate
of CrHydA1apo increases nearly linearly up to almost full native
hydrogen evolution activity at rising CaHydF:CrHydA1apo ratios.
Interestingly, an about 80-fold excess of the HydF protein is
needed to yield an H2 evolution rate of only 50 lmol H2 min1
mg1 (about 15% of the native activity) if HydF is heterologously
expressed with a background of the other heterologously introduced maturation factors in E. coli [14,15,22]. Apparently, the overexpression of the maturation factors in their native host, with
background expression of the intrinsic maturases, results in a
much more capable CaHydF enzyme. To give evidence for the assumed cluster transfer from HydF to HydA apoprotein [14,16] in
the activation process, we have examined proteins of the maturation process on presence of inorganic cofactors.
trum are very typical for a ferredoxin like (all-Cys ligated)
[4Fe4S]1+ cluster [23]. The relaxation properties and the temperature dependence are also typical for [4Fe4S]1+ clusters [23]. Based
on our EPR measurements we can confirm that the CrHydA1apo already contains the cubane part of the H-cluster as it was suggested
recently by Mulder et al. based on EPR and Mössbauer measurements on CrHydA1apo [16].
To understand the structure and functional properties of the
maturation proteins, it was important to perform spectroscopical
analyses with homologously expressed maturation enzymes with
full maturation capability. So far, only EPR studies were initiated
with the HydE, HydF and HydG enzymes from T. maritima
(TmHydE, TmHydF and TmHydG). The T. maritima maturases were
heterologously expressed in E. coli with no background expression
of the two other maturation factors, which means that they have
no maturation capability. The metal cofactors were reconstituted
with the cysteine desulfurase IscS protein from E. coli [13,21]. To
evaluate whether [FeS] clusters of active clostridial maturation factors differ from those with no maturation capability, we performed
EPR and FTIR measurements of C. acetobutylicum maturation factors expressed in concert with all maturases and with no need of
subsequent reconstitution.
The EPR spectra of CaHydE and CaHydG reduced with dithionite
are quite similar to typical spectra of [4Fe4S]1+ clusters (Fig. 2IBC).
The spectra are almost axial with principal g-values characteristic
for this class of FeS clusters (gCaHydE = 2.036, 1.932, 1.908; gCaHydG = 2.035, 1.940, 1.904). The CaHydE sample without dithionite
shows a dramatic decrease of the [4Fe4S]1+ EPR signal. This
3.2. Spectroscopic analyses of inactive hydrogenase and active
maturases
The EPR spectrum of the dithionite treated CrHydA1apo reveals a
strong rhombic signal (Fig. 2IA). The principal g-values of the spec-
Fig. 1. Hydrogen evolution rate as a function of CaHydF:CrHydA1apo molar ratio.
The specific hydrogen evolution rate is plotted against the molar ratio of
CaHydF:CrHydA1apo. The amount of both components in the maturation assay
was varied, for CaHydF from 0.011 to 0.186 nmol and for CrHydA1apo from 0.007 to
0.016 nmol.
Fig. 2. Q-band pulse EPR spectra of CrHydA1apo (panel I, A), CaHydE (panel I, B),
CaHydG (panel I, C) measured at 10 K and of CaHydF (panel II). Panel I: (A) EPR
spectrum of CrHydA1apo measured at 10 K. (B) EPR spectrum of CaHydE with
dithionite (blue) and without dithionite (red) measured at 10 K; C) EPR spectrum of
CaHydG with dithionite measured at 10 K. Panel II: EPR spectra of CaHydF (A) with
dithionite (blue) and without dithionite (red) measured at 10 K, the latter spectrum
was measured with 10 ms repetition time; (B) without dithionite measured at 20 K.
The principal components of the g-matrix are indicated for each spectrum (arrows).
Asterisks indicate artefacts in the spectra due to background signals of the EPR
resonator.
I. Czech et al. / FEBS Letters 584 (2010) 638–642
indicates that the [4Fe4S] cluster in CaHydE is mainly in its oxidized EPR silent form when no reductant is present. We did not observe appearance of any additional signals in the spectra for this
sample. Therefore, based on the obtained data we suggest that
both CaHydE and CaHydG contain a single [4Fe4S] cluster in their
structures. From the X-ray structure of TmHydE a [4Fe4S] and a
[2Fe2S] cluster have been suggested [11]. However, the presence
of the additional [2Fe2S] cluster is largely dependent on the reconstitution and soaking conditions. We did not observe any EPR signals of CaHydE that could be related to a reduced [2Fe2S] cluster. It
has to be noted that elimination of the [2Fe2S] cluster by mutagenesis of TmHydE did not affect its maturation capacity [11]. Therefore, it is possible that this cluster does not play a role in the
maturation process and might not be conserved in different HydE
proteins.
The EPR spectra of CaHydF showed some diversity for the sample with and without dithionite (Fig. 2II). The EPR spectrum of the
sample with dithionite is rather simple and resembles that of a
[4Fe4S]1+ cluster (Fig. 2IIA), similar to the signal observed in
ðredÞ
TmHydF [13] ðg CaHydF ¼ 2:049; 1:902; 1:872Þ. In the CaHydF primary
structure, three cysteine residues (Cys304, Cys353 and Cys356) together with a histidine provide the only possible motif for the ligation of an [FeS] cluster. We performed pulse EPR measurements of
14
N signals to probe His ligation on the [4Fe4S] signal using hyperfine sublevel correlation spectroscopy (HYSCORE). The measured
X-band HYSCORE spectrum (not shown) is identical to the one
measured on TmHydF [13], indicating a similar histidine ligation
of the [4Fe4S] cluster.
ðoxÞ
An additional and rather different EPR signal ðg CaHydF ¼
2:045; 2:007; 1:906Þ was detected in the CaHydF samples that had
been purified without dithionite (‘‘oxidized form”), while the
[4Fe4S] signal was found to be considerably smaller (Fig. 2II). For
convenience, the spectra in Fig. 2IIA are normalized to the
[4Fe4S] signals. Notably, the high-field component of the spectrum
has a complex structure (i.e., an extra ‘‘step” at g = 1.960). This
might indicate the presence of several overlapping signals.
We found that the longitudinal relaxation for the additional
(‘‘oxidized”) signal is about 10 times slower than for the [4Fe4S]
signal. At 20 K the spectrum shows predominantly the ‘‘oxidized”
signal while the [4Fe4S] signal is strongly suppressed due to fast
longitudinal relaxation (Fig. 2IIB). As it has been observed for various FeS clusters by EPR the longitudinal relaxation rates becomes
slower with decreasing number of Fe atoms in the FeS cluster [24].
Although the relaxation rates can depend strongly on the actual
structure and surrounding of a FeS cluster, we suggest that this
additional signal might be attributed to a cluster which contains
three or less irons.
Based on EPR data we can conclude that the Fe-cluster is EPR silent in the reduced state and EPR active in the oxidized state,
which is rather similar to the redox behaviour of the EPR signals
of the H-cluster [7]. Thus it is tempting to suggest low-valence
states of the irons in the Fe cluster (e.g., [Fe(I)Fe(I)]red and
[Fe(II)Fe(I)]ox configurations). Moreover, the extracted g-values
for the ‘‘oxidized” EPR spectrum of CaHydF are rather different
from those of the oxidized H-cluster. The electronic structure of
the EPR active states of the H-cluster is governed by a strong exchange interaction between the [2Fe]H and the [4Fe4S]H subcluster
resulting in principal g-values above ge = 2.0023. The fact that the
principal g-values for the ‘‘oxidized” CaHydF differs dramatically
from what has been observed for the oxidized H-cluster most probably indicates that in CaHydF the [2Fe2S] cluster and the [4Fe4S]
cluster are not bound like the H-cluster.
Since a part of the assembly of the bi-nuclear subcluster of the
H-cluster should involve incorporation of CN and CO ligands, we
decided to further characterize the CaHydF protein by means of IR
spectroscopy. We concentrated on the frequency range between
641
2100 cm1 and 1800 cm1 in the FTIR spectra which is predominantly governed by signals of C–O/C–N stretching vibrations [25].
It has been shown for the H-cluster and for various related biomimetic (bi-nuclear) model complexes that CN bands normally lie in
the range between 2110 cm1 and 2025 cm1, while CO bands occur at the lower frequencies between 2015 cm1 and 1850 cm1
[9,25–27]. Bridging CO ligands between two irons are usually
showing signals in a separate region of the IR spectrum between
1850 cm1 and 1770 cm1 [9,25–27].
The dithionite reduced sample showed a FTIR spectrum, consisting of four bands in the region of the terminal CO and two at
the CN region (see Fig. 3A). It is noteworthy that the position of
the IR bands is similar to the one observed in Fe(I)–Fe(I) model
complexes containing CN ligands and no bridging CO [27]. When
dithionite was removed from the sample the FTIR spectrum changed (Fig. 3B). Seven additional bands could be identified in the CO
region, while the CN bands did not change significantly. Most of
the additional bands shifted upwards in frequency, which is typical
for oxidation of the central iron atom [9,26]. Since the number of
CO bands in this sample is larger than what has been observed
for the dithionite reduced sample (which is anticipated to contain
a single reduced state of the additional Fe-cluster), we propose the
existence of more than one additional species. Future electrochemical studies could clarify the assignment of these bands. Nevertheless, as can be seen from Fig. 3B, a clear band at 1811 cm1 has
been observed which is typical for a bridging CO ligation, indicating the presence of a Fe–CO–Fe moiety. Therefore, these results
suggest a bi-nuclear nature of the additional Fe-cofactor in CaHydF.
3.3. Summary
Several hypotheses have been formulated for the role of HydF in
the maturation process, although no direct experimental evidence
has been obtained so far [14,16,28].
The fact, that CaHydF is the only responsible maturation factor
for in vitro maturation of CrHydA1apo supports the results of
McGlynn et al. and encourages the hypothesis that HydF is acting
as the key player for the assembly of the bi-nuclear site in HydA
[14]. Furthermore, we showed that homologously expressed
Fig. 3. FTIR spectra of CaHydF reduced with dithionite (A) and without dithionite
(B) recorded at 200 K. Spectrum B is scaled up 10-times (due to the lower
concentration) to facilitate the direct comparison with spectrum A (see also the
shaded area under the spectrum B). Numbers above the spectra show wavenumber
of the corresponding bands in cm1.
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I. Czech et al. / FEBS Letters 584 (2010) 638–642
CaHydF is much more active than heterologously expressed CaHydF and that it is possible to activate CrHydA1apo up to full native
hydrogen evolution activity.
Two different Fe-containing cofactors have been detected in
CaHydF. One of them can be identified as a [4Fe4S] cluster. Considering the spectroscopic observations presented here we propose
that (1) the observed additional Fe-cluster is a bi-nuclear cluster;
(2) in the oxidized form the cluster is EPR active, while in the reduced form it is EPR silent; (3) irons in the additional Fe-cluster
are coordinated by CN and CO ligands (most likely one CN and
two CO ligands per iron); (4) in at least one of the oxidized forms
the cluster contains also a bridging CO ligand. Since the properties
of this additional cluster are very similar to those of the bi-nuclear
subcluster of the H-cluster, it is anticipated that it can be used for
the activation of the hydrogenase apoprotein.
Acknowledgements
This work was supported by the European Union/Energy
Network (SOLAR-H2 contract 212508), the BMBF (H2-design cells)
and the Max-Planck-Gesellschaft. We are also very grateful to L.
Currell (MPI Mülheim/Ruhr) for his technical assistance.
References
[1] Vignais, P.M., Billoud, B. and Meyer, J. (2001) Classification and phylogeny of
hydrogenases. FEMS Microbiol. Rev. 25, 455–501.
[2] Happe, T. and Kaminski, A. (2002) Differential regulation of the Fehydrogenase during anaerobic adaptation in the green alga Chlamydomonas
reinhardtii. Eur. J. Biochem. 269, 1022–1032.
[3] Peters, J.W., Lanzilotta, W.N., Lemon, B.J. and Seefeldt, L.C. (1998) X-ray crystal
structure of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum to
1.8 angstrom resolution. Science 282, 1853–1858.
[4] Silakov, A., Wenk, B., Reijerse, E. and Lubitz, W. (2009) (14)N HYSCORE
investigation of the H-cluster of [FeFe] hydrogenase: evidence for a nitrogen in
the dithiol bridge. Phys. Chem. Chem. Phys. 11, 6592–6599.
[5] Happe, T. and Naber, J.D. (1993) Isolation, characterization and N-terminal
amino acid sequence of hydrogenase from the green alga Chlamydomonas
reinhardtii. Eur. J. Biochem. 214, 475–481.
[6] Melis, A. and Happe, T. (2004) Trails of green alga hydrogen research – from
Hans Gaffron to new frontiers. Photosynth. Res. 80, 401–409.
[7] Kamp, C., Silakov, A., Winkler, M., Reijerse, E.J., Lubitz, W. and Happe, T. (2008)
Isolation and first EPR characterization of the [FeFe]-hydrogenases from green
algae. Biochim. Biophys. Acta 1777, 410–416.
[8] Stripp, S., Sanganas, O., Happe, T. and Haumann, M. (2009) The structure of the
active site H-cluster of [FeFe] hydrogenase from the green alga
Chlamydomonas reinhardtii studied by X-ray absorption spectroscopy.
Biochemistry 48, 5042–5049.
[9] Silakov, A., Kamp, C., Reijerse, E., Happe, T. and Lubitz, W. (2009)
Spectroelectrochemical characterization of the active site of the [FeFe]
hydrogenase HydA1 from Chlamydomonas reinhardtii. Biochemistry 48,
7780–7786.
[10] Posewitz, M.C., King, P.W., Smolinski, S.L., Smith, R.D., Ginley, A.R., Ghirardi,
M.L. and Seibert, M. (2005) Identification of genes required for hydrogenase
activity in Chlamydomonas reinhardtii. Biochem. Soc. Trans. 33, 102–104.
[11] Nicolet, Y., Rubach, J.K., Posewitz, M.C., Amara, P., Mathevon, C., Atta, M.,
Fontecave, M. and Fontecilla-Camps, J.C. (2008) X-ray structure of the [FeFe]hydrogenase maturase HydE from Thermotoga maritima. J. Biol. Chem. 283,
18861–18872.
[12] Pilet, E., Nicolet, Y., Mathevon, C., Douki, T., Fontecilla-Camps, J.C. and
Fontecave, M. (2009) The role of the maturase HydG in [FeFe]-hydrogenase
active site synthesis and assembly. FEBS Lett. 583, 506–511.
[13] Brazzolotto, X., Rubach, J.K., Gaillard, J., Gambarelli, S., Atta, M. and Fontecave,
M. (2006) The [FeFe]-hydrogenase maturation protein HydF from Thermotoga
maritima is a GTPase with an iron–sulfur cluster. J. Biol. Chem. 281, 769–774.
[14] McGlynn, S.E., Shepard, E.M., Winslow, M.A., Naumov, A.V., Duschene, K.S.,
Posewitz, M.C., Broderick, W.E., Broderick, J.B. and Peters, J.W. (2008) HydF as
a scaffold protein in [FeFe] hydrogenase H-cluster biosynthesis. FEBS Lett. 582,
2183–2187.
[15] McGlynn, S.E., Ruebush, S.S., Naumov, A., Nagy, L.E., Dubini, A., King, P.W.,
Broderick, J.B., Posewitz, M.C. and Peters, J.W. (2007) In vitro activation of
[FeFe] hydrogenase: new insights into hydrogenase maturation. J. Biol. Inorg.
Chem. 12, 443–447.
[16] Mulder, D.W., Ortillo, D.O., Gardenghi, D.J., Naumov, A.V., Ruebush, S.S.,
Szilagyi, R.K., Huynh, B., Broderick, J.B. and Peters, J.W. (2009) Activation of
HydA(DeltaEFG) requires a preformed [4Fe-4S] cluster. Biochemistry 48,
6240–6248.
[17] King, P.W., Posewitz, M.C., Ghirardi, M.L. and Seibert, M. (2006) Functional
studies of [FeFe] hydrogenase maturation in an Escherichia coli biosynthetic
system. J. Bacteriol. 188, 2163–2172.
[18] von Abendroth, G., Stripp, S., Silakov, A., Croux, C., Soucaille, P., Girbal, L. and
Happe, T. (2008) Optimized over-expression of [FeFe] hydrogenases with high
specific activity in Clostridium acetobutylicum. Int. J. Hydrogen Energy 33,
6076–6081.
[19] Girbal, L., von Abendroth, G., Winkler, M., Benton, P.M.C., Meynial-Salles, I.,
Croux, C., Peters, J.W., Happe, T. and Soucaille, P. (2005) Homologous and
heterologous
overexpression
in
Clostridium
acetobutylicum
and
characterization of purified clostridial and algal Fe-only hydrogenases with
high specific activities. Appl. Environ. Microbiol. 71, 2777–2781.
[20] Zhao, X., Miller, J.R., Jiang, Y., Marletta, M.A. and Cronan, J.E. (2003) Assembly
of the covalent linkage between lipoic acid and its cognate enzymes. Chem.
Biol. 10, 1293–1302.
[21] Rubach, J.K., Brazzolotto, X., Gaillard, J. and Fontecave, M. (2005) Biochemical
characterization of the HydE and HydG iron-only hydrogenase maturation
enzymes from Thermatoga maritima. FEBS Lett. 579, 5055–5060.
[22] Boyer, M.E., Stapleton, J.A., Kuchenreuther, J.M., Wang, C.W. and Swartz, J.R.
(2008) Cell-free synthesis and maturation of [FeFe] hydrogenases. Biotechnol.
Bioeng. 99, 59–67.
[23] Guigliarelli, B. and Bertrand, P. (1999) Application of EPR Spectroscopy to the
Structural and Functional Study of Iron–Sulfur Proteins in: Advances in
Inorganic Chemistry (Sykes, A.G., Ed.), pp. 421–497, Academic Press.
[24] Rupp, H., Rao, K.K., Hall, D.O. and Cammack, R. (1978) Electron spin relaxation
of iron–sulphur proteins studied by microwave power saturation. Biochim.
Biophys. Acta 537, 255–260.
[25] Nakamoto, K. (1997) Infrared and Raman Spectra of Inorganic Coordination
CompoundsApplications in Coordination, Organometallic and Bioinorganic
Chemistry, Vol. 5th Edition, John Wiley and Sons, Inc., New York.
[26] Albracht, S.P.J., Roseboom, W. and Hatchikian, E.C. (2006) The active site of the
[FeFe]-hydrogenase from Desulfovibrio desulfuricans. 1. Light sensitivity and
magnetic hyperfine interactions as observed by electron paramagnetic
resonance. J. Biol. Inorg. Chem. 11, 88–101.
[27] Razavet, M., Davies, S.C., Hughes, D.L. and Pickett, C.J. (2001) {2Fe3S} clusters
related to the di-iron sub-site of the H-centre of all-iron hydrogenases. Chem.
Commun., 847–848.
[28] Peters, J.W., Szilagyi, R.K., Naumov, A. and Douglas, T. (2006) A radical solution
for the biosynthesis of the H-cluster of hydrogenase. FEBS Lett. 580, 363–367.