A pyridinol acyl cofactor in the active site of [Fe]-hydrogenase evidenced by the reactivity of model complexes

Article abstract of DOI:10.1002/chem.201201954

Understanding the complex: The decomposition reaction of a water-soluble complex (see scheme; 1) in H₂O confirms the existence of a unique bidentate pyridinol cofactor in [Fe]-hydrogenase. This unique moiety is confirmed for the first time by the decomposition of a well-defined model complex containing a pyridinyl methyl acyl ligand. Copyright

Full text of DOI:10.1002/chem.201201954

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DOI: 10.1002/chem.201201954
A Pyridinol Acyl Cofactor in the Active Site of [Fe]-hydrogenase Evidenced
by the Reactivity of Model Complexes
Bowen Hu,[a] Dafa Chen,*[a] and Xile Hu*[b]
[Fe]-hydrogenase, containing an iron guanylylpyridinol
(FeGP) cofactor, can split H2 into H+ and HÀ in the pres-
ence of methenyltetrahydromethanopterin (methenyl-
H4MPT+).[1–3] The FeGP cofactor can be released by dena-
turation of the enzyme in the presence of 2-mercaptoetha-
nol.[4–6] The protein-free cofactor is too unstable to be iden-
tified. When irradiated by UV-A light, the extracted cofac-
tor decomposed to iron, CO, 2-mercaptoethanol, and an or-
ganic pyridinol carboxylic acid derivative (Scheme 1).[6,7]
Figure 1. Proposed structure for the active site of [Fe]-hydrogenase (A).
Proposed structure for the protein-free FeGP cofactor extracted with 2-
mercaptoethanol (B). Active site model (1).[27,28]
experimental evidence for the existence of this unique
moiety in [Fe]-hydrogenase is warranted.[12]
Based on the proposed structure of the active site of [Fe]-
Scheme 1. Decomposition of the extracted FeGP cofactor under UV-A
hydrogenase, a number of small molecular mimics have
been prepared.[13–28] Recently we reported the synthesis and
protonation reactions of a 5-coordinate model [(2-CH2CO-
6-MeOC5H3N)Fe(CO)2{S-(2,6-Me2C6H3)}] (1, Figure 1).[27,28]
The reaction with HBF4·Et2O afforded an ionic complex [(2-
light.
This observation was the basis of an earlier structural model
in which the pyridinol moiety coordinated to the Fe ion only
through its N atom.[8]
(BF4) (2).[28] We
CH2CO-6-MeOC5H3N)Fe(CO)2ACTHNUGTRENNUG(CH3CN)2]ACHTUNGTRENNUGN
This structural model was later revised based on the X-
ray structure of a mutated [Fe]-hydrogenase.[9] Currently, it
is thought that the FeII center in the active site of [Fe]-hy-
drogenase is coordinated with two cis-CO, a cysteine sulfur
atom (Cys 176), and the bidentate pyridinol acyl ligand
through its nitrogen atom and acyl donors (Figure 1A).[9–11]
The structure of the extracted FeGP cofactor was proposed
accordingly (Figure 1B).[7,12]
Recent infrared spectroscopic and mass spectrometric
studies of [Fe]-hydrogenase provide evidence for the acyl-
iron ligation.[11,12] As no other protein containing an acyl-
iron ligation has been found in the biological systems, more
found that 2 is somewhat soluble in water, which prompted
us to explore its reactivity with water. If the reaction could
occur similar to that described in Scheme 1, the acyl-iron
group in [Fe]-hydrogenase would be further proved. Fur-
thermore, to confirm the detailed geometry of the protein-
free FeGP cofactor extracted with 2-mercaptoethanol, and
which group Cu+ attacks when reacts with [Fe]-hydroge-
nase, here we also describe the reactivity of 1 with 2-mer-
captoethanol and the Cu+ ion.
Complex 2 reacts with water to form an isolable organic
1
product that was identified as 3 (Scheme 2). The H NMR
spectrum of 3 exhibits three signals at d=7.65, 6.83, and
6.76 ppm for the pyridine ring, one singlet at d=3.98 ppm
for the methoxy group, and one singlet at d=3.83 ppm for
the methylene group. In the 13C NMR spectrum of 3, one
[a] Dr. B. Hu, Prof. Dr. D. Chen
School of Chemical Engineering & Technology
Harbin Institute of Technology, Harbin 150001 (P.R. China)
E-mail: dafachen@hit.edu.cn
[b] Prof. Dr. X. Hu
Laboratory of Inorganic Synthesis and Catalysis
Institute of Chemical Sciences and Engineering
Ecole Polytechnique Fꢀdꢀrale de Lausanne (EPFL)
SB-ISIC-LSCI, BCH 3305, Lausanne 1015 (Switzerland)
E-mail: xile.hu@epfl.ch
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201201954.
Scheme 2. Reaction of 2 with water.
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Table 1. Selected infrared data.
Complex
signal of carboxyl group (d=171.3 ppm), five signals for the
pyridine ring (d=163.0, 151.1, 140.0, 116.3, and 109.8 ppm),
one signal for the methoxy group (d=53.9 ppm), and one
signal for the methylene group (d=40.5 ppm) are observed.
Based on the NMR data, compound 3 was assigned as [(2-
CH2COOH-6-MeOC5H3N)], which was further confirmed
by high-resolution mass spectrometry (HRMS).[29] The struc-
ture of 3 is quite similar to the organic product isolated
from the reaction of FeGP cofactor with water (Scheme 1),
which provides evidence for the existence of the iron-acyl li-
gation, and thus, the pyridinol acyl cofactor in the active site
of [Fe]-hydrogenase.
n(CO) [cmÀ1
]
4a[a]
2023, 1999, 1978, 1953
2021, 2003, 1961, 1946
2017, 1997, 1939
2018, 2000, 1955, 1943
2017, 1992, 1943, 1930
2019, 2001, 1958, 1944
2004, 1934
4a[b]
4b[a]
4b[b]
4c[a]
4c[b]
FeGP cofactor[c]
FeGP cofactor[d]
2031, 1972
[a] Spectrum of a solid sample. [b] Spectrum of a sample dissolved in
CH3CN. [c] Spectrum of solid sample; data taken from ref. [12].
a
[d] Spectrum of a sample dissolved in water; data taken from ref. [30].
Although the composition of the protein-free FeGP cofac-
tor is determined, its structure is not established.[7] To probe
the structure of the FeGP cofactor extracted by 2-mercap-
toethanol, we then attempted to prepare model complexes
with a 2-mercaptoethanol ligand. Reaction of [(2-CH2CO-6-
MeOC5H3N)Fe(CO)3I][27] with NaSCH2CH2OH was investi-
gated. However, only unidentified mixtures were produced.
The protein-free FeGP cofactor was isolated by the trans-
thiolation of [Fe]-hydrogenase.[4–6] Gratifyingly, the target
model complex could be prepared in a similar manner.
Thus, reaction of 1 with SHCH2CH2OH (2 equiv) at À208C
gave [(6-MeO-C5H3N-2-CH2CO)Fe(CO)2(SCH2CH2OH)]2
(4a) (Scheme 3). Compound 4a is unstable at room temper-
Reaction of 1 with HSCH2CH2CH2SH gave a similar
product
[(6-MeO-C5H3N-2-CH2CO)Fe(CO)2(SCH2CH2-
CH2SH)]2 (4b) (Scheme 3). Compound 4b is highly unsta-
ble; it decomposes even at À308C in solution.
Treatment of 1 with HSCH2CH2CH3 gave [(6-MeO-
C5H3N-2-CH2CO)Fe(CO)2(SCH2CH2CH3)]2
(4c)
1
(Scheme 3). The H NMR and IR spectra of 4c are similar
to those of 4a and 4b, which indicates that the -OH and
-SH groups in 4a and 4b do not coordinate to the Fe cen-
ters.
The ligands of the iron centers in 4a and in the protein-
free FeGP cofactor are similar. However, the extracted
FeGP cofactor is a monomeric iron complex according to
the IR data (Table 1). The reason for the structural discrep-
ancy is still unknown, but is probably caused by the differ-
ence of the second coordination sphere.[31]
The protein-free FeGP cofactor was shown to bind an ex-
ternal CO ligand.[32] Similarly, compound 4a reacted with
CO reversibly to give a tricarbonyl product [(6-MeO-
C5H3N-2-CH2CO)Fe(CO)3(SCH2CH2OH)] (6, Scheme 4).
Scheme 3. Transthiolation reactions of 1.
1
ature, and its half-life is about 0.5 h. The H NMR spectrum
of 4a exhibits three signals at d=7.84, 7.11, and 6.51 ppm
for the pyridine rings, two doublets at d=4.73 and 3.53 ppm
for the diastereotopic methylene hydrogens, one singlet at
d=3.39 ppm for the methoxy groups, and four multiplets be-
tween d=3.70–2.45 ppm for -CH2CH2OH groups.[29] The IR
spectra of 4a show four intense n(CO) absorption bands
both in the solid state and in solution (Table 1).[29] The
1H NMR and IR data indicates a dimeric structure for 4a.
Four instead of three signals were observed for the
Scheme 4. Reversible reaction of 4a with CO.
This compound might be considered as a mimic for the
extra-CO-bound extracted cofactor.
1
-CH2CH2OH groups in the H NMR spectrum. This is prob-
ably caused by the bulky environment around the Fe cen-
[Fe]-hydrogenase is sensitive to Cu+ ions, however, the
origin of the inhibition is unknown;[33] therefore, the reac-
À
ters, which restricts the rotation of the S CH2 bonds. A sim-
tion of 1 with [CuACTHNGUTREN(NUG CH3CN)4]CAHTUNGTERN(NUNG BF4) was studied (Scheme 5).
ilar phenomenon was observed in complex [(6-MeO-C5H3N-
2-CH2CO)Fe(CO)3{S-(2,6-Me2C6H3)}] (5), which was formed
from the reaction of 1 with CO.[27] At À308C, the two
methyl groups in the thiophenol ligand in 5 show two sin-
The reaction yielded complex 2[28] and an insoluble salt with
the composition of [Cu{S-(2,6-Me2C6H3)}]n.[34] Thus, the Cu+
ion attacked the thiolate ligand in 1. This result suggests
that the inhibition of [Fe]-hydrogenase by Cu+ ion might be
due to the attack of Cu+ on the Cys176 ligand.
1
glets in the H NMR spectrum due to the restricted rotation
around the S-C6H3 bond.[27]
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Pyridinol Acyl Cofactor
COMMUNICATION
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In summary, the reaction of 2 with water is a good chemi-
cal model for the decomposition of the protein-free FeGP
cofactor. Both reactions produce pyridinyl carboxylic acid
derivatives. This result gives strong evidence for the exis-
tence of the unique pyridinol acyl cofactor in the active site
of [Fe]-hydrogenase. It is the first time that evidence has
been provided by the reactivity of a well-defined model
complex. Furthermore, to model the protein-free FeGP co-
factor, 2-mercaptoethanol was introduced successfully to Fe
center, although this results in a dimer 4a instead of a mon-
omer, which is different from the FeGP cofactor. Addition-
ally, the reactivity of complex 1 with Cu+ suggests that Cu+
inhibits [Fe]-hydrogenase by attacking the Cys176 ligand.
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Acknowledgements
This work is supported by the Starting Research Fund from Harbin Insti-
tute of Technology (project no. AUGA9833213311), the Fundamental
Research Funds for the Central Universities (Grant No. HIT. NSRIF.
2013042), and the Swiss National Science Foundation (project no.
200020 134473/1). We thank Katherine M. Schultz (EPFL) for proof-
reading the manuscript.
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Keywords: enzyme models · cofactors · hydrogenase · iron ·
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Received: June 4, 2012
Published online: && &&, 2012
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D. Chen, X. Hu and B. Hu
Enzyme Models
B. Hu, D. Chen,* X. Hu* . . . &&&& — &&&&
A Pyridinol Acyl Cofactor in the
Active Site of [Fe]-hydrogenase
Evidenced by the Reactivity of Model
Complexes
Understanding the complex: The
decomposition reaction of a water-
soluble complex (see scheme; 1) in
H2O confirms the existence of a
unique bidentate pyridinol cofactor in
[Fe]-hydrogenase. This unique moiety
is confirmed for the first time by the
decomposition of a well-defined model
complex containing a pyridinyl methyl
acyl ligand.
&
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www.chemeurj.org
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!