Dioxygen Sensitivity of [Fe]-Hydrogenase in the Presence of Reducing Substrates

Article abstract of DOI:10.1002/anie.201712293

Mono-iron hydrogenase ([Fe]-hydrogenase) reversibly catalyzes the transfer of a hydride ion from H₂ to methenyltetrahydromethanopterin (methenyl-H₄MPT⁺) to form methylene-H₄MPT. Its iron guanylylpyridinol (FeGP)

Full text of DOI:10.1002/anie.201712293

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Communications
Chemie
International Edition: DOI: 10.1002/anie.201712293
German Edition: DOI: 10.1002/ange.201712293
Iron Hydrogenases
Dioxygen Sensitivity of [Fe]-Hydrogenase in the Presence of Reducing
Substrates
Gangfeng Huang, Tristan Wagner, Ulrich Ermler, Eckhard Bill, Kenichi Ataka, and
Seigo Shima*
Abstract: Mono-iron hydrogenase ([Fe]-hydrogenase) rever-
sibly catalyzes the transfer of a hydride ion from H2 to
+
methenyltetrahydromethanopterin (methenyl-H MPT ) to
4
form methylene-H MPT. Its iron guanylylpyridinol (FeGP)
4
cofactor plays a key role in H activation. Evidence is presented
2
for O sensitivity of [Fe]-hydrogenase under turnover con-
2
ditions in the presence of reducing substrates, methylene-
+
H MPT or methenyl-H MPT /H . Only then, H O is gener-
4
4
2
2
2
ated, which decomposes the FeGP cofactor; as demonstrated
by spectroscopic analyses and the crystal structure of the
deactivated enzyme. O reduction to H O requires a reductant,
2
2
2
which can be a catalytic intermediate transiently formed during
the [Fe]-hydrogenase reaction. The most probable candidate is
an iron hydride species; its presence has already been predicted
by theoretical studies of the catalytic reaction. The findings
support predictions because the same type of reduction
reaction is described for ruthenium hydride complexes that
hydrogenate polar compounds.
Figure 1. [Fe]-hydrogenase reaction and the FeGP cofactor structure.
a) The pro-R stereospecific hydride-transfer reaction of [Fe]-hydroge-
[6]
nase. b) Chemical structure of the FeGP cofactor and the light-
[7]
decomposed product, guanylylpyridinol.
mimic the active site of [Fe]-hydrogenase have been
[
8]
M
ono-iron hydrogenase ([Fe]-hydrogenase) reversibly cat-
alyzes the activation and heterolytic cleavage of H2 and
reported.
The stability of hydrogenases and corresponding model
[
1]
[9]
transfers the hydride anion to the substrate, methenyltetra-
hydromethanopterin (methenyl-H MPT ), to form methyl-
ene-H MPT (Figure 1a). The prosthetic group of [Fe]-
hydrogenase is the iron guanylylpyridinol (FeGP) cofactor
compounds is a critical factor for their applications. The
dinuclear metal sites of [NiFe]- and [FeFe]-hydrogenases are
redox active, and most of them react with O and are quickly
+
4
[
2]
4
2
[9,10]
deactivated.
Nevertheless, several [NiFe]-hydrogenases
[
3]
(
Figure 1b; Supporting Information, Figure S1). The iron
from aerobic bacteria are O tolerant to some degree and are
able to reduce O to H O , and further to water. The iron
2
II
[11]
center of the FeGP cofactor consists of a low-spin Fe ligated
with two CO, cysteine-S, pyridinol-N, acyl-C, and a solvent
molecule (Figure 1b). The structural and catalytic features
of [Fe]-hydrogenases are particularly useful for designing new
2
2
2
center of the FeGP cofactor in [Fe]-hydrogenase is redox
[4]
[4a]
inactive and insensitive to O under the conditions tested in
2
[12]
previous studies. However, reactive oxygen species, such as
H O , and probably superoxide anion (O ), deactivate [Fe]-
hydrogenase.
[
1,5]
À
hydrogenation catalysts.
Numerous model catalysts that
2
2
2
[12a]
Herein, we report that the FeGP cofactor of
[
Fe]-hydrogenase is decomposed under aerobic conditions in
the presence of reducing substrates. [Fe]-hydrogenase cata-
lyzes reduction of O to H O and this reactive oxygen species
[
*] G. Huang, Dr. T. Wagner, Dr. S. Shima
Max-Planck-Institut für terrestrische Mikrobiologie
Karl-von-Frisch-Straße 10, 35043 Marburg (Germany)
E-mail: shima@mpi-marburg.mpg.de
2
2
2
decomposes the iron complex of the FeGP cofactor.
According to previous reports, [Fe]-hydrogenase is not
Dr. U. Ermler
deactivated in the absence of substrate under 100% O
2
[12]
Max-Planck-Institut für Biophysik
Max-von-Laue-Straße 3, 60438 Frankfurt/Main (Germany)
(Supporting Information, Figure S2).
First information
about a deactivating effect of O on this enzyme was gained
2
Dr. E. Bill
by the enzyme assays following the oxidation of methylene-
Max-Planck-Institut für Chemische Energiekonversion
+
H MPT and reduction of methenyl-H MPT . The rate of
4
4
4
5470 Mülheim (Germany)
+
methylene-H MPToxidation to methenyl-H MPT decreases
4
4
Dr. K. Ataka
Department of Physics, Freie Universität Berlin
Berlin 14195 (Germany)
progressively under an N /O (80%/20%) gas phase com-
2
2
pared to 100% N (Figure 2a). Notably, the initial rate of the
2
reaction did not change and the exchange of the gas phase of
Supporting information, including technical details, procedures,
sequences, structures, PDB codes, and the ORCID identification
number(s) for the author(s) of this article can be found under:
https://doi.org/10.1002/anie.201712293.
the O -damaged [Fe]-hydrogenase sample to 100% N did not
2
2
reactivate the enzyme. Likewise, the enzyme activity of the
+
reverse reaction, the reduction of methenyl-H MPT with H ,
4
2
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These changes indicate a decomposition
of the FeGP cofactor because the same
UV/Vis spectral behavior was observed
when the iron site of [Fe]-hydrogenase was
[12b, 13]
degraded by light.
Initially, IR spec-
troscopic analysis confirmed the presence
À1
of the bands at 2008 and 1938 cm of the
intrinsic CO ligands of the enzyme-bound
FeGP cofactor, which were previously
reported (Supporting Information, Fig-
[
4b,d,12a]
ure S6).
In the absence of methyl-
+
ene-H MPT and methenyl-H MPT , the
4
4
bands did not change after incubating the
enzyme under O /N /H (20%/80%/0%)
Figure 2. Deactivation of [Fe]-hydrogenase by O . a) Oxidation of methylene-H MPT to
2
4
+
+
2
2
2
methenyl-H MPT . b) Reduction of methenyl-H MPT to methylene-H MPT. The gas phases
4
4
4
and O /N /H (20%/70%/10%) gas phases
were exchanged at the time indicated by arrows. c) Effect of O on [Fe]-hydrogenase activity
2
2
2
2
in the presence of methylene-H MPT under N containing 10% H and different concen-
(Supporting Information, Figure S6a). In
their presence, however, the IR peaks
decrease and almost disappear after long-
term (1 h) incubation at 408C indicating
the loss of the CO ligands (Supporting Information,
Figure S6b).
4
2
2
trations of O , as indicated.
2
also decreases with time under H /O (80%/20%) compared
2
2
to 100% H (Figure 2b). Again, no reactivation has been
2
achieved by exchanging the gas phase to 100% H . The initial
The 1.29 Š structure of O -deactivated enzyme (Support-
2
2
rate of the reaction did not appear to be changed but the O2-
deactivation rate was quicker than in the case of methylene-
H MPT oxidation. These results indicated that O does not
inhibit but slowly deactivates [Fe]-hydrogenase under the
enzyme assay conditions.
ing Information, Table S1) clearly revealed that the FeGP
cofactor is decomposed into guanylylpyridinol (Figure 3b).
Guanylylpyridinol was identified as a light-deactivated prod-
4
2
[7]
uct of the isolated FeGP cofactor (Figure 1b). The iron in
the O -deactivated enzyme has lost pyridinol-N, acyl-C, and
2
Based on these observations, the effect of O2 on the
deactivation of [Fe]-hydrogenase was tested in the presence
two CO ligands, but is still coordinated with Cys172-S, and
+
of variable O , H , methenyl-H MPT , and methylene-
2
2
4
H MPT concentrations. We incubated [Fe]-hydrogenase
4
with methylene-H MPT under a gas phase containing 10%
4
H and variable concentrations of O (0–40%) prior to testing
2
2
the activity (Figure 2c). [Fe]-hydrogenase is deactivated
progressively and the deactivation rate increases with increas-
ing O concentrations.
2
The influence of the H concentration on O -deactivation
2
2
of [Fe]-hydrogenase was tested in the presence of the
substrates methylene-H MPT (Supporting Information, Fig-
4
+
ure S3a) and methenyl-H MPT (Supporting Information,
4
Figure S3b). Increasing H2 concentrations stimulate the
deactivation of [Fe]-hydrogenase; the effect of H is more
2
+
obvious in the case of methenyl-H MPT than that of
4
methylene-H MPT. To study the impact of the substrate
4
concentration, [Fe]-hydrogenase was incubated with various
+
concentrations of methenyl-H MPT and methylene-H MPT
4
4
under an N /O /H (85%/5%/10%) gas phase. Again, higher
2
2
2
substrate concentrations and, concomitantly, a higher turn-
over rate increased the deactivation rate (Supporting Infor-
mation, Figure S4).
The irreversibility of O2 deactivation is presumably
a result of the destruction of the FeGP cofactor of [Fe]-
hydrogenase, which was investigated by UV/Vis and IR
spectroscopies, and X-ray crystallography. UV/Vis absorb-
ance peaks at 300 and 360 nm correspond to the absorbance
of the pyridinol ring and iron complex of the intact FeGP
Figure 3. Crystal structure of O
resolution. a) The active-site structure of the intact [Fe]-hydrogenase
inhibited with 2-naphthylisocyanide, which is the only available
-deactivated [Fe]-hydrogenase at 1.29 Š
2
[14]
crystal structure of [Fe]-hydrogenase derived from Methanothermo-
bacter marburgensis. b) The active-site structure of the O -deactivated
2
cofactor, respectively. Upon deactivation with O , the
2
[Fe]-hydrogenase, in which the 2F ÀF electron density contoured at
o
c
absorbance at 360 nm decreases and the absorbance at
3
.0 s is represented (Supporting Information, Table S1). GP: guanylyl-
3
00 nm increases (Supporting Information, Figure S5).
pyridinol.
4
918
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II
Fe oxidation, electron para-
magnetic resonance (EPR)
spectroscopy was used. The
II
low-spin Fe active site of
[
Fe]-hydrogenase
is
EPR
silent. Upon deactivation by
O in the presence of 0.1 mm
2
methylene-H MPT under the
4
gas phase (N /O /H (70%/
2
2
2
2
0%/10%)), only
a
minor
EPR signal at g = 4.3 from
III
high-spin Fe emerged, with
a spin concentration of less
than 7% of the total enzyme
(Supporting Information, Fig-
ure S8). An even weaker frac-
Figure 4. Kinetics of deactivation of [Fe]-hydrogenase exposed to O in the presence of methylene-H MPT
2
4
and H O formation. a) Methylene-H MPT (100 mm) and variable concentrations of [Fe]-hydrogenase
2
2
4
(
enzyme) under N /O (95%/5%). b) [Fe]-hydrogenase (100 mm) and variable concentrations of methyl-
2 2
ene-H MPT (substrate) under N /O (95%/5%) c) [Fe]-hydrogenase (100 mm) and methylene-H MPT
4
2
2
.
4
III
(
100 mm) under N with variable concentrations of O . The residual activity (*) and the respective H O
2
2
2
2
tion of low-spin Fe products
concentrations (*) were determined. The standard error of three measurements was calculated.
appeared around g = 2. Thus,
II
no significant oxidation of Fe
III
to Fe occurs and most likely
forms new bonds with the carboxymethyl-O of the guanylyl-
pyridinol, His203, and Asp189 from the partner monomer.
Thus, the number of ligands of iron is decreased from six to
four by decomposition (Figure 3).
O is not reduced in an iron-bound state.
2
The presented study indicates that the deactivation of
[Fe]-hydrogenase upon O exposure does not occur directly in
2
response to O but via a reduction to H O , which subse-
2
2
2
Based on the presented findings and previous data
regarding decomposition of [Fe]-hydrogenase by reactive
quently decomposes the FeGP cofactor without loss of H O .
2 2
On the basis of the proposed catalytic mechanism of [Fe]-
hydrogenase (Supporting Information, Figure S9), the most
probable reductant is an iron hydride. The proposed type of
metal-hydride-driven hydrogenation reaction has precedent
[
12a]
oxygen species,
we hypothesized that O is reduced by this
2
À
enzyme to H O or O and that the reactive oxygen species
2
2
2
destroys the FeGP cofactor of [Fe]-hydrogenase. To test this
hypothesis, [Fe]-hydrogenase was decomposed in an assay
solution composed of [Fe]-hydrogenase (500 mm) and meth-
ylene-H MPT (500 mm) under 100% O . We found produc-
[15]
in inorganic ruthenium complex chemistry;
thereby
increasing the plausibility of the mechanism. Metal-hydride
formation in the [Fe]-hydrogenase reaction was already
4
2
[
8c,16]
tion of sub-stoichiometric amounts of H O (ca. 3 mm), only in
postulated on the basis of calculations
complex studies
and model-
2
2
[5,17]
the presence of both enzyme and substrate (Supporting
but experimental evidence was, thus far,
À
Information, Table S2). On the other hand, O2 was not
not provided. If our conclusions are correct, [Fe]-hydrogenase
stabilizes a hydride on its iron center in the catalytic cycle.
This finding may open a perspective to apply [Fe]-hydro-
genase for the reduction of non-polar compounds (which
might be polarized in the enzyme) and as a template for
developing new hydrogenating model catalysts (for example,
detected. The relationship between the enzymatic activity of
[
Fe]-hydrogenase and H O2 production under the O2 gas
2
phase was studied kinetically (Figure 4). Higher concentra-
tions of the enzyme, methylene-H MPTor O in the gas phase
4
2
increased the amount of H O2 produced, which directly
2
[18]
correlates with the decrease in enzyme activity. When the
a selective H O -forming catalyst ).
2 2
enzyme became fully deactivated, the increase of H O2
2
stopped.
To explore the dependency of the H O concentration on Acknowledgements
2
2
the deactivation of [Fe]-hydrogenase, 26, 2.6, and 0.26 mm of
H O were added to a solution of 26 mm [Fe]-hydrogenase
We thank the staff of the PXII beamline at the Swiss Light
Source, Villigen for help during data collection. U.E. thanks
Prof. Dr. Hartmut Michel for continuous support. This work
was supported by a grant from the Max Planck Society and
the Deutsche Forschungsgemeinschaft Priority Program
“Iron-Sulfur for Life” (SH87/ 1-1) to S.S.; G.H. was supported
by a fellowship from China Scholarship Council (CSC).
2
2
(
final concentrations) and incubated (Supporting Informa-
tion, Figure S7). In the presence of stoichiometric concen-
trations (26 mm) of H O , [Fe]-hydrogenase was fully deacti-
2
2
vated within 5 min, but less than 10% of H O was lost in the
2
2
deactivation assay. In the case of sub-stoichiometric concen-
trations of H O (2.6 mm and 0.26 mm H O ), substantial
2
2
2
2
amounts of [Fe]-hydrogenase were decomposed; the residual
Fe]-hydrogenase activity was only 30% and 60% within
[
1
0 min (Supporting Information, Figure S7). Altogether, Conflict of interest
these results demonstrated that [Fe]-hydrogenase is catalyti-
cally deactivated by sub-stoichiometric amounts of H O .
The authors declare no conflict of interest.
2
2
To determine whether the interaction of O with the iron
2
center and the reduction of O to H O are correlated with
2
2
2
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Keywords: [Fe]-hydrogenase · enzyme catalysis · H2 ·
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Manuscript received: November 30, 2017
Accepted manuscript online: February 20, 2018
Version of record online: March 22, 2018
4
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