A catalytically active [Mn]-hydrogenase incorporating a non-native metal cofactor

Article abstract of DOI:10.1038/s41557-019-0266-1

Nature carefully selects specific metal ions for incorporation into the enzymes that catalyse the chemical reactions necessary for life. Hydrogenases, enzymes that activate molecular H₂, exclusively utilize Ni and Fe in [NiFe]-, [FeFe]- and [Fe]-hydrogeanses. However, other transition metals are known to activate or catalyse the production of hydrogen in synthetic systems. Here, we report the development of a biomimetic model complex of [Fe]-hydrogenase that incorporates a Mn, as opposed to a Fe, metal centre. This Mn complex is able to heterolytically cleave H₂ as well as catalyse hydrogenation reactions. The incorporation of the model into an apoenzyme of [Fe]-hydrogenase results in a [Mn]-hydrogenase with an enhanced occupancy-normalized activity over an analogous semi-synthetic [Fe]-hydrogenase. These findings demonstrate a non-native metal hydrogenase that shows catalytic functionality and that hydrogenases based on a manganese active site are viable.

Full text of DOI:10.1038/s41557-019-0266-1

Articles
https://doi.org/10.1038/s41557-019-0266-1
A catalytically active [Mn]-hydrogenase
incorporating a non-native metal cofactor
1
2
1,3
1
4
Hui-Jie Panꢀ ꢀ , Gangfeng Huang , Matthew D. Wodrichꢀ ꢀ , Farzaneh Fadaei Tiraniꢀ ꢀ , Kenichi Ataka ,
2
1
Seigo Shimaꢀ ꢀ * and Xile Huꢀ ꢀ *
Nature carefully selects specific metal ions for incorporation into the enzymes that catalyse the chemical reactions neces-
sary for life. Hydrogenases, enzymes that activate molecular H , exclusively utilize Ni and Fe in [NiFe]-, [FeFe]- and [Fe]-
2
hydrogeanses. However, other transition metals are known to activate or catalyse the production of hydrogen in synthetic
systems. Here, we report the development of a biomimetic model complex of [Fe]-hydrogenase that incorporates a Mn,
as opposed to a Fe, metal centre. This Mn complex is able to heterolytically cleave H as well as catalyse hydrogenation
2
reactions. The incorporation of the model into an apoenzyme of [Fe]-hydrogenase results in a [Mn]-hydrogenase with an
enhanced occupancy-normalized activity over an analogous semi-synthetic [Fe]-hydrogenase. These findings demonstrate
a non-native metal hydrogenase that shows catalytic functionality and that hydrogenases based on a manganese active site
are viable.
ature judiciously chooses metal ions to catalyse biochemical which indicates Mn as a viable metal for biological H activation in
2
reactions. Understanding these choices is not only important the enzyme.
N
for a fundamental knowledge of biocatalysis, but might also
provide guidelines to design synthetic catalysts. Structure–activ- Results
ity studies in which native metal ions are replaced by non-native Synthesis, characterization, and catalytic activity of Mn mod-
analogues represent a direct approach to unravel the choices made els. The reaction of 2-hydroxy-6-methylpyridine (1) with 2equiv.
by nature. However, abiological replacement of native metals n-BuLi gave a dilithiated intermediate (2), which was not isolated but
that maintain the activity of the native enzyme activity have rarely treated directly with Mn(CO) Br followed by acidic work-up to give
5
1–3
been reported .
the targeted Mn(ꢀ) model 3 (Fig. 1b). The infrared spectrum of 3 in
−1
When it comes to the activation of dihydrogen (H ), nature THF exhibits four CO peaks at 1,948, 1,967, 1,983 and 2,068cm
2
chooses only Fe and Ni, as in [NiFe]-, [FeFe]- and [Fe]- (Supplementary Fig. 13), indicative of a Mn tetra(carbonyl) species.
4
−1
hydrogeanses . It is not yet clear whether this choice is due solely The peak at 1,651cm was attributed to the acyl group. The NMR
to the higher bioavailability of Fe and Ni relative to other metals, spectra of 3 (in deuterated THF) is characteristic of a diamagnetic
due to their unique ability to activate H in biological systems, or species (Supplementary Fig. 8), which indicates a low-spin configu-
2
1
even to both. Many other transition metals are known to activate or ration of Mn(ꢀ). The 2-OH proton gave a broad H peak at 11.26ppm.
13
evolve hydrogen in synthetic systems. A [RuRu] analogue of [FeFe]- In the C NMR spectrum (in deuterated acetonitrile (CD CN))
3
hydrogenase is the only semi-synthetic hydrogenase that contains a (Supplementary Fig. 9), three types of CO carbon were observed at
5
non-native metal, but it is inactive . [Fe]-hydrogenase is an attrac- 213.6, 215.9 and 220.3ppm. The acyl carbon gave a peak at 280.8
13
13
tive platform to address questions about the choices of metals in ppm. CO/ CO exchange was observed by C NMR when 3 was
6
13
biological H activation . This enzyme catalyses the hydrogenation treated with an excess of CO in deuterated THF or CD CN, which
2
3
+
of methenyltetrahydromethanopterin (methenyl-H MPT ) to form suggests that one or more CO ligands readily dissociate from the
4
methylene-H MPT, a reaction involved in microbial methanogene- Mn centre (Supplementary Figs. 1 and 2). The solid-state molecular
4
7
sis . Unlike [NiFe]- and [FeFe]-hydrogenases, [Fe]-hydrogenase has structure of 3 was determined by X-ray crystallography (Fig. 1c). The
only one Fe centre per active site. In its resting state, the low-spin Mn ion possesses an octahedral coordination geometry. Three CO
Fe(ꢀꢀ) centre is coordinated by one H O and two cis-CO molecules, ligands are coplanar, whereas the fourth CO ligand occupies a posi-
2
a cysteine thiolate, as well as an acyl carbon and a pyridinyl nitrogen tion orthogonal to the other three. The acylmethylpyridinol ligand
8,9
from a guanylylpyridinol moiety (Fig. 1a) . During catalysis, water coordinates in a bidentate fashion via the acyl C and pyridonal N.
probably dissociates, and thereby opens a binding site for H . Given
A H /D exchange assay was employed to test the activity of 3
2
2
2
11
that Mn(ꢀ) and Fe(ꢀꢀ) are isoelectronic, and further inspired by the towards H activation . The reaction was conducted in a high-pres-
2
10
recent development of Mn-catalysed hydrogenation , we decided sure NMR tube, where D (8bar) and H (12bar; total pressure of
2
2
to prepare a Mn(ꢀ) model of the active site of [Fe]-hydrogenase. the mixed gases 20bar) were added to a solution of 3 in THF-d . The
8
Through reconstitution of the apoenzyme of [Fe]-hydrogenase with reaction was monitored by NMR spectroscopy. Complex 3 alone
this Mn(ꢀ) model, we obtained a [Mn]-hydrogenase. This artificial did not mediate the H/D exchange after two days at 25°C. However,
hydrogenase is active for the native [Fe]-hydrogenase reactions, in the presence of a base (for example, 1-methylpyrrolidine (MP)
1
Laboratory of Inorganic Synthesis and Catalysis, Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), ISIC-
2
3
LSCI, Lausanne, Switzerland. Max Planck Institute for Terrestrial Microbiology, Marburg, Germany. Laboratory for Computational Molecular Design,
Institute of Chemical Science and Engineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland. Department of Physics, Freie
4
Universität Berlin, Berlin, Germany. *e-mail: shima@mpi-marburg.mpg.de; xile.hu@epfl.ch
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a
O
N
b
N
N
NH
(
i) Mn(CO)5Br,
O
2.0 equiv. n-BuLi
THF,
N
OH
CO
O
O
NH2
P
0 °C, THF
–78 °C to 50 °C
O
OH
C
N
OLi
O
Mn
(
ii) 1.5 equiv. 37% HCl
OH OH
N
OH
CO
Li
OC
1
2
N
OH
CO
CO
3
O
Fe
S
OH2
CO
Cys176
c
d
e
N
OK
CO
1
.0 equiv. KH,
THF, 23 °C
1.1 equiv. 18-crown-6,
THF, 23 °C, 2 hours
3
C
4(18-crown-6)
O
Mn
CO
OC
CO
4
H2 (50 bar),
R1
3 (1–10 mol%), MP (20–100 mol%),
R1
THF, 100 °C, 16 hours
R2
X
R2
XH
5
6
f
g
PMP
Ph
O
N
N
H2 (50 bar),
3
(1 mol%), MP (20 mol%),
THF, 80 °C, 16 hours
O
OH
5
b
5c
5d
60%
5e
5a
6
00%
a
1
Yields of 6:
40%
69%
33%
Fig. 1 | Active site of [Fe]-hydrogenase and synthesis, structure and catalytic activity of its Mn models. a, The active site in [Fe]-hydrogenase.
b, Synthesis of complex 3, which is a non-native metal mimic of the active site of [Fe]-hydrogenase. c, X-ray structure of complex 3; the thermal eliposoids
are displayed at a 50% probability. d, Synthesis of complexes 4 and 4(18-crown-6), which are deprotonated forms of 3; the reactions prove that the
2
-OH group in 3 is prone to deprotonation. e, X-ray structure of complex 4(18-crown-6), which confirms the deprotonation of the 2-OH group; the thermal
eliposoids are displayed at a 50% probability. f, Optimized conditions for the hydrogenation of benzaldehyde. g, Other suitable substrates for catalytic
hydrogenation. Supplementary Fig. 6 gives the experimental details. PMP, para-methoxylphenyl. Complex 3 is a catalyst for hydrogenation of various
unsaturated organic compounds.
or triethylamine (Et N)), the formation of HD was observed within deprotonated species. To facilitate the isolation and crystalliza-
3
+
3
h at 25°C (Supplementary Fig. 3), which indicates H activation. tion, 4 was treated with a K -selective crown ether, 18-crown-6, to
2
+
A D /H exchange experiment was then conducted, in which give complex 4(18-crown-6) (Fig.1d). 4 and 4(18-crown-6) exhibit
2
2
0bar of D was introduced to a CD CN solution of 3 that con- similar NMR spectra (Supplementary Figs. 10 and 11). The infra-
2
3
tained 3.0equiv. MP at 25°C. H and HD were detected after 1h red spectrum of 4(18-crown-6) in THF exhibits four ν(CO) peaks
2
−1
(
Supplementary Fig. 5). This result indicates that H splitting is a at 1,901, 1,944, 1,972 and 2,052cm , and a ν(acyl C=O) peak at
2
−1
heterolytic process, which is the same as in the enzymatic reaction.
1,616cm (Supplementary Fig. 14). These peaks are shifted to
We previously reported that deprotonation of the 2-OH group lower values compared to the corresponding peaks in 3, which indi-
was a key step in enabling H activation by a semi-synthetic [Fe]- cates a more electron-rich Mn centre on deprotonation of the 2-OH
2
12
−
hydrogenase . After deprotonation, the anionic 2-O group served group. The X-ray structure of 4(18-crown-6) confirmed the depro-
−
as an internal base to assist in the heterolytic splitting of H at the tonation of the 2-OH group (Fig. 1e). The 2-O group had a strong
2
1
2,13
Fe centre . The need for a base for H splitting by 3 suggests that interaction (2.6097(14)Å) with the K cation, which is further coor-
2
a similar mechanism was operating in the activation of H by 3. dinated by the 6 O atoms of 18-crown-6. Both 4 and 4(18-crown-6)
2
To verify this hypothesis, we sought to intentionally deprotonate were able to activate H without an external base, as observed in the
2
the 2-OH group in 3. A clean reaction was observed on treatment H /D exchange assay (Supplementary Fig. 4). This result supports
2
2
of 3 with potassium hydride (KH) in THF at room temperature that deprotonation of the 2-OH group is a key step in H activation
2
1
(
4
Fig. 1d). In the H NMR spectrum of the in situ formed complex by 3 in the presence of a base.
, all the peaks of 3 except the 2-OH peak were still observed, with
Complexes 3 and 4 are hydrogenation catalysts. To optimize
1
a significant upfield shift (Supplementary Fig. 10). H NMR spec- the reaction conditions, hydrogenation of benzaldehyde (5a) was
tra similar to that of 4 were obtained when 3 was treated with an employed as a test reaction with complex 3 as the catalyst. After
excess of MP or Et N, which suggests the formation of a similar screening various reaction parameters, the best conditions were
3
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a
b
N
OH
H+
N
O
–
CO
CO
C
C
O
+1.7
O
Mn
Mn
CO
CO
OC
OC
CO
CO
3
7a
–
+
CO
H2
+24.9
2
6.6
2
6.0
25.9
26.1
7
b
TS 7b/ 7c 7c
TS 7d/7e
N
O
CO
–
+
C7H8O
H2
C
O
Mn
–0.6
16.1
–1.7
H2
OC
7
d
CO
7e
N
O
7b
N
O
CO
CO
C
C
H
OH
O
O
Mn
CO
Mn
TS 7b/7c
7.8
H
OC
OC
7
e
6.1
CO
7
b
–0.1
1.7
–18.3
0
.0
7a
3
7c
TS 7d/ 7e
N
O
CO
N
OH
CO
H
O
C
C
O
O
Mn
Mn
H
H
7d
OC
OC
CO
CO
+10.0
–9.8
N
OH
+C7H6O
CO
C
O
O
Mn
H
OC
CO
Fig. 2 | Computational study of the mechanism of hydrogenation. a, A DFT-computed catalytic cycle for the hydrogenation of 5a catalysed by 3. The
change of free energies is marked for each step. The catalysis follows the sequence deprotonation of the 2-OH group of 3, substitution of a CO ligand by
H , heterolytic H cleavage, binding of the aldehyde, hydride and proton transfer to the aldehyde and, finally, substitution of the bound alcohol by H . The
2
2
2
deprotonation of the 2-OH group enables heterolytic H splitting. b, The reaction pathway and its associated free energy profile. The turnover limiting step
2
is either the CO dissociation or the hydride transfer.
1
8
mol% complex 3 with 20mol% MP as the base under 50bar H at Fe catalysts (Supplementary Table 2). The ligand environment of 3
2
0°C in THF (Fig. 1f). Under these conditions, the yield of hydro- is less electron donating than those of highly active Mn complexes,
genation was quantitative. Although H activation was observed at which leads to a less-hydridic Mn–H intermediate, which might be
2
2
5°C, a higher temperature (for example, 80°C) was necessary for the origin of the subdued activity.
catalytic hydrogenation, which suggests that hydride transfer was Density functional theory (DFT) computations at the PBE0-
more difficult than H activation. Under similar conditions using 4 dDsC/TZ2P//M06/def2-SVP theoretical level (Supplementary
2
(
5mol%) rather than 3 as the catalyst and without the base, hydroge- Computational Methods) suggest the following catalytic cycle for
nation was also achieved in a 96% yield. Similar reaction conditions the hydrogenation of 5a catalysed by 3 (Fig. 2a and Supplementary
could be applied for the hydrogenation of other substrates, which Fig. 21). Deprotonation of the 2-OH group in 3 by the base MP fol-
included a ketone (5b), primary imine (5c) and secondary imine lowed by substitution of one CO ligand (trans to the acyl ligand) by
(
5d) (Fig. 1g), and moderate yields were obtained (Supplementary H gives intermediate 7b. Heterolytic cleavage of the coordinated H
2
2
Fig. 6). The hydrogenation of an olefin substrate (5e) was less effi- in 7b leads to a hydride complex 7c, which hydrogenates 5a to give
cient (Fig. 1g). The reaction profiles of hydrogenation reactions the alcohol-bound complex 7e. Replacing the alcohol product in 7e
19
were monitored by F NMR spectroscopy using 4-CF benzalde- by H yields the product and regenerates 7b. The substitution of CO
3
2
hyde (5f) as the substrate (Supplementary Fig. 7). No induction in 7a by H as well as the transition state associated with hydride
2
period was observed, which suggests that 3 and 4 were the true transfer represent the highest points on the potential energy surface
–
1
precatalysts or catalysts. The initial rates of reactions catalysed by 3 (Fig. 2b), with similar values of about 26kcalmol . Deprotonation
and 4 were similar, but the reaction catalysed by 4 was slower, prob- and H splitting are facile processes. Given the accuracy of the DFT
2
ably due to some decomposition of 4. The catalytic activity of 3 for method, the computed reaction energy profile qualitatively agrees
hydrogenation is modest compared to some state-of-the-art Mn and with the experimental results.
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a
b
c
d
0
.0
0
0
0
.5
.4
.3
0
0
0
.4
.2
.0
1.0
–
0.1
0.2
0
0
.5
.0
0
0
.1
.0
–0.2
250
300
350
400
250
300
350
400
0
50
100 150 200
Time (s)
0
50
100 150 200
Time (s)
Wavelength (nm)
Wavelength (nm)
+
Fig. 3 | Activity of the semi-synthetic [Mn]-hydrogenase. a,b, The consumption (a) and formation (b) of methenyl-H MPT (the direction of the reactions
4
is indicated by arrows). c,d, The changes of absorbance at 336ꢀnm of the consumption (c) and formation (d) reactions are plotted. The red line shows
the activity of the reconstituted enzyme with complex 3. As negative controls, the apoenzyme alone (black), complex 3 alone (blue) and only the buffer
solution (green) were also tested. The black and blue traces in c are covered by the green trace. The increase of the absorbance of the sample with only
3
in d was due to an increase of the turbidity caused by aggregation of the Mn complex in the assay, which was confirmed by the change of the overall
ultraviolet–visible spectrum. The data show that only [Mn]-hydrogenase, but not the control samples, has substantial catalytic activity.
Table 1 | Comparison of enzyme activities of native, reconstituted and semi-synthetic [Fe]-hydrogenase and semi-synthetic
[
Mn]-hydrogenase
–1
–1
Samples
Speciꢅc activity (ꢁꢆmg ) (forward reaction: Speciꢅc activity (ꢁꢆmg ) (reverse reaction:
reduction of methenyl-H MPT with H ) dehydrogenation of methylene-H MPT)
4
2
4
+
GMPa
−GMP
b
+GMP
−GMP
Native [Fe]-hydrogenasec
Reconstituted [Fe]-hydrogenased
Semi-synthetic [Fe]-hydrogenasee
NA
520ꢀ±ꢀ30
370ꢀ±ꢀ20
1.2ꢀ±ꢀ0.3
NA
470ꢀ±ꢀ10
340ꢀ±ꢀ40
1.0ꢀ±ꢀ0.1
NA
NA
2.5ꢀ±ꢀ0.4
1.9ꢀ±ꢀ0.2
0.09ꢀ±ꢀ0.01
Semi-synthetic [Mn]-hydrogenasef
1.5ꢀ±ꢀ0.1
0.67ꢀ±ꢀ0.10
0.08ꢀ±ꢀ0.02
a
b
c
The deviations represent s.d. +GMP: reconstitution solution contains 2ꢀmM GMP. −GMP: reconstitution solution does not contain GMP. Native [Fe]-hydrogenase: The purified [Fe]-hydrogenase from
d
Methanothermobacter marburgensis. Reconstituted [Fe]-hydrogenase: [Fe]-hydrogenase was reconstituted using the apoenzyme from Methanocaldococcus jannaschii with the FeGP cofactor extracted
e
12 f
from native [Fe]-hydrogenase from M. marburgensis. Semi-synthetic [Fe]-hydrogenase: [Fe]-hydrogenase was reconstituted using from M. jannaschii with an Fe-model complex . Semi-synthetic [Mn]-
hydrogenase: [Mn]-hydrogenase was reconstituted using the apoenzyme from M. jannaschii with Mn-model complex 3. NA, not applicable.
Reconstitution and activity of [Mn]-hydrogenase. The activity of that an acetate-bound form of 3 was not the major species in the
3
in H splitting and hydrogenation catalysis contrasts the inactivity reconstitution medium, as was the case for the reconstitution of
2
1
2
1
2
of its Fe(ꢀꢀ) analogue in H activation . This reactivity difference is semi-synthetic [Fe]-hydrogenase .
2
consistent with recent observations that Mn(ꢀ) complexes are often
The activity of this [Mn]-hydrogenase was measured photo-
+
more active or stable than Fe(ꢀꢀ) complexes in hydrogenation reac- metrically for both the forward (reduction of methenyl-H MPT
4
1
0
tions . However, it was unclear whether Mn would be competent with H ) and the reverse (H production from methylene-H MPT)
2
2
4
+
for biological H activation. The study of semi-synthetic [RuRu]- reactions. The absorbance of methenyl-H MPT at 336nm was
2
4
hydrogenase showed that, even though H activation on synthetic used as a spectroscopic probe of the reaction rate. To our delight,
2
Ru complexes was facile, in a hydrogenase environment this metal the [Mn]-hydrogenase was active for the native reactions of [Fe]-
5
was inactive . To test the possible Mn-catalysed hydrogenase activ- hydrogenase (Fig. 3). The activity (Methods) largely remained
ity, complex 3 was employed to reconstitute a [Mn]-hydrogenase. identical for multiple samples; the averaged apparent specific activ-
–
1
–1
The protocol previously used for the reconstitution of semi-syn- ity was 1.5± 0.1Umg and 0.09± 0.01Umg for the forward and
12
thetic [Fe]-hydrogenases was also applicable here . 3 was first dis- reverse reaction, respectively. As observed in the reconstitution
12
solved in a solution that contained 99% methanol and 1% acetic of semi-synthetic [Fe]-hydrogenase , the absence of GMP in the
acid. The solution that contained 2equiv. 3 was mixed with a solu- reconstitution solution resulted in a twofold decrease of the activity
tion of [Fe]-hydrogenase apoenzyme from Methanocaldococcus jan- of the reconstituted enzyme (Table 1), although the infrared spec-
naschii heterologously produced in Escherichia coli in the presence trum was not changed (Supplementary Fig. 17). Interestingly, the
of 2mM guanosine monophosphate (GMP). Although 3 is soluble [Mn]-hydrogenase is biased towards the forward reaction, which
in pure methanol, the active enzyme was not reconstituted using is around 20 times faster than the reverse reaction. The native
a methanol solution of 3 in the absence of acetic acid. The role of [Fe]-hydrogenase and semi-synthetic [Fe]-hydrogenase have only
12,14
acetic acid is unclear as the infrared spectra of complex 3 in pure slightly higher rates for the forward reaction (Table 1) . The for-
methanol and in mixtures of methanol and acetic acid are essen- ward reaction was conducted at pH 7.5, whereas the reverse reac-
tially the same (Supplementary Figs. 15 and 16), which suggests tion was conducted at pH 6.0. The bias of the [Mn]-hydrogenase
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hydrogenase (Supplementary Fig. 19). Given this occupancy, the
Table 2 | Comparison of enzyme activities
–
1
actual specific activity is about 7.5Umg (forward reaction) and
–1
–1
Samples
Speciꢅc activity
Speciꢅc activity (ꢁꢆmg )
0.45Umg (reverse reaction). The corresponding turnover fre-
–1
−1
−1
(ꢁꢆmg ) (forward (reverse reaction:
quencies are 5s and 0.3s for the forward and reverse reactions,
respectively. These turnover frequencies are three orders of magni-
tude higher than those of complex 3 alone in hydrogenation reac-
tions (Supplementary Table 3), which underlines the important role
of the protein environment for catalysis. They are also significantly
higher than those of state-of-the-art synthetic Mn and Fe catalysts
reaction:
dehydrogenation of
reduction of
methylene-H MPT)
4
+
methenyl-H MPT
4
with H2)
Wild type enzymes reconstituted with
(
Supplementary Table 3).
FeGP
370ꢀ±ꢀ20
2.5ꢀ±ꢀ0.4
1.5ꢀ±ꢀ0.1
340ꢀ±ꢀ40
1.9ꢀ±ꢀ0.2
0.09ꢀ±ꢀ0.01
To probe whether the unspecifically bound Mn complex contrib-
[
[
Fe]
uted to the activity of [Mn]-hydrogenase, reconstitution was con-
ducted using a mutated apoenzyme of [Fe]-hydrogenase, in which
the binding residues for the FeGP cofactor were triply mutated
Mn]
Variant enzymes reconstituted with
(
T13V, C176A and D251A). Although unspecific binding of complex
FeGP-C176A
2.2ꢀ±ꢀ0.5
ND1
1.6ꢀ±ꢀ0.2
ND
3
to the triple mutant apoenzyme was confirmed by infrared spec-
[
[
[
Fe]-C176A
troscopy (Supplementary Fig. 20), this variant of [Mn]-hydrogenase
Mn]-C176A
0.12ꢀ±ꢀ0.19
ND
0.04ꢀ±ꢀ0.04
ND
had no enzymatic activity (Table 2). Moreover, F -dependent
420
Mn]-T13V_C176A_
methylene-H MPT dehydrogenase (Mtd), which does not bind the
4
D251A
FeGP cofactor but binds the H MPT substrates, was also used for
4
reconstitution. Unspecific binding of complex 3 by Mtd was again
observed (Supplementary Fig. 20), but this sample also showed no
enzymatic activity (Table 2). Taken together, these data indicate that
the unspecific bound Mn complex is catalytically inactive.
[
Mn]-Mtd
ND
ND
Comparison of enzyme activities of wild-type enzyme reconstituted with the FeGP cofactor, semi-
synthetic [Fe]-hydrogenases and semi-synthetic [Mn]-hydrogenases reconstituted using wild-
type and mutated apoenzymes from M. jannaschii as well as F420-dependent methylene-H
dehydrogenase (Mtd) from Archaeoglobus fulgidus. Mtd binds methenyl- and methylene-H
as substrates, but does not use the FeGP cofactor. The deviations represent the s.d. ND, not
4
MPT
4
MPT
–1
Discussion
detected (the specific activity was less than 0.015ꢀUꢀmg ).
The active sites of both the semi-synthetic [Mn]- and [Fe]-
hydrogenase lack the GMP moiety and two methyl groups in the
pyridinol group of the native cofactor (Fig. 1a), which should be
towards the forward reaction might be due to the higher pK of the principle reason for their specific activity being only a few per-
a
12
the 2-OH group compared to those of [Fe]-hydrogenase and semi- cent of that of native [Fe]-hydrogenase . Direct comparison of the
12
synthetic[Fe]-hydrogenase , which hinders its deprotonation at semi-synthetic [Mn]- and [Fe]-hydrogenases, however, reveals the
pH 6.0, and thereby decreases the base function and the rate of the relative catalytic competency of the two different metals. The occu-
reverse reaction. Control experiments showed that complex 3 or the pancy-normalized specific activity of [Mn]-hydrogenase is 25%
apoenzyme of [Fe]-hydrogenase alone did not afford the activity.
higher than that in the analogous [Fe]-hydrogenase for the forward
–
1
1
2
In [Fe]-hydrogenase, the Fe centre is bound to Cys176 thio- reaction (3 Umg at a 50% active site occupancy) , which is the
late. The reconstituted Cys176Ala variant of [Mn]-hydrogenase physiological reaction during methanogenesis. Thus, Mn appears to
–1
has a specific activity of 0.12± 0.19 Umg for the forward reac- be more active than Fe for enzymatic H activation.
2
tion, which is 8% of the native variant of the reconstituted [Mn]-
In summary, a Mn(ꢀ) model of the active site of [Fe]-hydrogenase
hydrogenase. This result contrasts the findings on semi-synthetic that is capable of splitting H and catalysing hydrogenation reac-
2
[
Fe]-hydrogenase, for which reconstituted Cys176Ala variants were tions has been prepared. Reconstitution of the apoenzyme of [Fe]-
12
inactive (Table 2) . It suggests that binding of the Mn complex hydrogenase with this Mn(ꢀ) model leads to a [Mn]-hydrogenase,
to Cys176-S is not absolutely needed for [Mn]-hydrogenase to be which is active for the [Fe]-hydrogenase reactions. These findings
active. In the native [Fe]-hydrogenase, the iron complex compo- demonstrate the catalytic functionality of a non-native metal in a
nent of the iron-guanylylpyridinol (FeGP) cofactor is connected to hydrogenase enzyme. The molar activity of this semi-synthetic
the protein via hydrogen bonds, which involves the backbone NH [Mn]-hydrogenase is higher than that of its Fe analogue, which
of Cys176, the hydroxyl group of Thr13, the carboxy group of raises an intriguing question—why does nature choose Fe over Mn?
Asp251 and water molecules (Supplementary Fig. 18). These
interactions might lead to specific binding of the Mn complex Methods
Synthesis of complex 3. 2-Hydroxy-6-methylpyridine 1 (1.3g, 12mmol) was
even in the absence of Cys176-Fe bonding, albeit with an unopti-
mized orientation.
dissolved in 60ml of dry THF in a Schlenk ꢁask. To this solution, n-BuLi (2.5M
in hexane (9.6ml, 24mmol)) was added dropwise at 0°C and the solution was
Different batches of samples of [Mn]-hydrogenase have nearly
further stirred for 30min at 0°C. In another Schlenk ꢁask, a THF (80ml) solution
identical activity, but they exhibit different infrared spectra in the
of Mn(CO) Br (3.3g, 12mmol) was cooled to −78°C. ꢂe solution of deprotonated
5
region of the ν(CO) vibrational bands. For example, the spectral 1 was then added dropwise to the Mn(CO)
Br solution at −78°C. ꢂe resulting
5
mixture was allowed to slowly warm to room temperature and further heated
to 50°C. Aꢃer stirring at 50°C overnight, the mixture was cooled to room
temperature and 1.5ml of 37% HCl aqueous solution was then slowly added.
ꢂe THF solvent was removed 30min later. ꢂe residue was further puriꢄed by
shape and positions of two samples are similar and resemble those
of complex 3 (Supplementary Fig. 19a,b), but their intensities are
different. A third sample has a spectrum similar to that of native
[
Fe]-hydrogenase (Supplementary Fig. 19). This result suggests silica gel chromatography in a glove box (1.3g, 36%) using ethyl acetate/hexane
that various amounts of Mn complexes are unspecifically bound as the eluent. A single crystal suitable for an X-ray test was obtained via the layer
diꢅusion of pentane to a THF solution of complex 3 at −22°C.
to the protein in different samples, in addition to a similar amount
of specifically bound complex, which gives rises to the hydrog-
Synthesis of complex 4 and 4(18-crown-6). To a solution of complex 3 (1.0g,
enase activity. Assuming that the infrared spectrum of Figure S19c
3
.3mmol) in THF (5ml) was added KH (132mg, 3.3mmol) slowly under stirring
originates from specifically bound Mn mimic because only two
at room temperature. When no more H gas formed, a THF solution of 18-crown-6
−
1
2
CO bands at 1,985 and 1,965cm were detected, we estimated the
occupancy of the active site of [Mn]-hydrogenase as 20% by com-
(
958mg, 3.6mmol) was added at once to the mixture. The resulting mixture was
further stirred at room temperature for 2h before it was filtered through a Teflon
2
paring its CO peak area with that of the reconstituted native [Fe]- membrane to remove all the solid impurity. A layer of Et O was then added on
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Articles
NaTure CHeMisTry
−1
top of the THF solution and the mixture was stored at −22°C. A crystal of 4(18-
(OH stretching mode) at approximately 3500cm against the amide II band of the
−1
crown-6) was obtained in 75% yield (1.5g).
protein at approximately 1550cm . Spectra were measured successively during the
concentration process. We selected a spectrum of a mildly hydrated sample that
provided enough intensity to analyze the cofactor bands. A baseline correction was
made on the selected spectrum to eliminate contributions of the broad background
Computational methods. The geometries of all the species were optimized at
the M06/def2-SVP theoretical level using the ‘ultrafine’ integration grid and the
SMD implicit solvent model for THF as implemented in Gaussian09. Refined
energy estimates were obtained using single-point computations on the optimized
M06 geometries at the PBE0-dDsC/TZ2P level as implemented in ADF. Reported
free energies are derived from the PBE0-dDsC electronic energies, M06 enthalpy
and vibrational only entropy contributions and solvation corrections using the
COSMO-RS model.
−1
from the water overtone band at approximately 2000cm . Typically, 512 spectra
were averaged to obtain a sufficient signal-to-noise ratio. Measurements were
performed in the dark by covering the spectrometer with blackout fabric to avoid
light-induced decomposition of the sample. Intensities of the observed bands from
various samples were at arbitrary concentrations. For quantitative comparison of
the obtained spectra, intensities of the CO bands were normalized by the peak
intensities of the amide II band of each spectrum.
Preparation of apoenzymes. The [Fe]-hydrogenase-encoding gene (hmd)
8
from M. jannaschii and the gene encoding F420-dependent methylene-H
4
MPT
Data availability
1
5
dehydrogenase (mtd) gene from Archaeoglobus fulgidus were used. The complete
genome sequence of the hyperthermophilic, sulfate-reducing archaeon A. fulgidus
was cloned into the expression vector pET24b. Genes of the hmd mutants (C176A
and T13V-C176A-D251A) were synthesized using the template of the wild type
hmd gene by GenScript. The E. coli strain BL21(DE3) that contained the expression
vector for hmd or mtd was cultivated and the gene expression was induced. The
The authors declare that the data supporting the findings of this study are
available from the corresponding authors upon reasonable request. CCDC-
1
856616 and CCDC-1856615 contain the supplementary crystallographic data
for 3 and 4(18-crown-6), respectively. These data can be obtained free of charge
from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
8
overproduced protein was purified as described previously .
Received: 19 July 2018; Accepted: 28 March 2019;
Published: xx xx xxxx
Reconstitution of [Mn]-hydrogenase. Complex 3 was dissolved in a solution
that contained 99% methanol and 1% acetic acid. The reconstitution of [Mn]-
2 2
hydrogenase was performed in an anaerobic tent (95% N /5% H ) at 8°C. A 2ml
aliquot the reconstituted system contains 0.5mM complex 3, 0.25mM apoenzyme,
mM GMP and 100mM sodium acetate buffer, pH 5.6. The mixture was incubated
on ice for 1h. Then the mixture was washed by 10mM MOPS buffer/KOH, pH
.0, that contained 2mM dithiothreitol through a 30kDa cutoff ultrafilter with at
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Coleman, J. E. Metal ion dependent binding of sulphonamide to carbonic
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speciꢄcity of the extracellular nuclease of Staphylococcus aureus. J. Biol. Chem.
least a total of 1,000-fold dilution to remove the unbound complex 3. Finally, the
–
1
reconstituted [Mn]-hydrogenase holoenzyme was concentrated to ~50mgml
for an enzyme activity assay. The reconstituted enzyme was quickly frozen in
2
42, 1541–1547 (1967).
3.
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Schwizer, F. et al. Artiꢄcial metalloenzymes: reaction scope and optimization
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liquid nitrogen and stored at −75°C. The reconstitution of semi-synthetic [Mn]-
hydrogenase using the mutated apoenzyme and Mtd and of semi-synthetic [Fe]-
hydrogenases was performed using the same method in the presence of 2mM
GMP, as described above. The native [Fe]-hydrogenase with the FeGP cofactor was
1
14, 4081–4148 (2014).
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8
prepared as described previously .
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of the catalytic cycle. Angew. Chem. Int. Ed. 57, 5429–5432 (2018).
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Enzyme activity assay of the [Mn]-hydrogenase holoenzyme. The reduction
6
.
.
+
of methenyl-H
4
MPT to methylene-H
4
MPT (the forward reaction) and the
+
dehydrogenation of methylene-H MPT to methenyl-H MPT (the reverse
4
4
reaction) was measured. For the forward reaction, 20µM (final concentration)
7
ꢂauer, R. K., Kaster, A. K., Seedorf, H., Buckel, W. & Hedderich, R.
Methanogenic archaea: ecologically relevant diꢅerences in energy
conservation. Nat. Rev. Microbiol. 6, 579–591 (2008).
+
methenyl-H
4
MPT was added to a 0.7ml solution that contained 120mM
potassium phosphate buffer, pH 7.5, which contained 1mM EDTA under 100% H
2
gas phase at 40°C. The reaction was started by injecting 10µl of the reconstituted
8
.
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Shima, S. et al. ꢂe crystal structure of [Fe]-hydrogenase reveals the geometry
of the active site. Science 321, 572–575 (2008).
+
[
Mn]-hydrogenase sample. Reduction of methenyl-H MPT was detected by
4
measuring the decrease of the absorbance at 336nm. For the reverse reaction,
0µM (final concentration) methylene-H MPT was added to a 0.7ml solution
that contained 120mM potassium phosphate buffer, pH 6.0, which contained
mM EDTA under a 100% N gas phase at 40°C. The reaction was started by
injecting 10µl of the reconstituted [Mn]-hydrogenase sample. Dehydrogenation
of methylene-H MPT was detected by measuring the increase in the absorbance
at 336nm. The activities were calculated using the extinction coefficient of
9
Hiromoto, T. et al. ꢂe crystal structure of C176A mutated [Fe]-hydrogenase
suggests an acyl-iron ligation in the active site iron complex. FEBS Lett. 583,
2
4
5
85–590 (2009).
1
2
1
0. Kallmeier, F. & Kempe, R. Manganese complexes for (de)hydrogenation
catalysis: A comparison to cobalt and iron Catalysts. Angew. Chem. Int. Ed.
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7, 46–60 (2018).
1
1
1
1
1. Xu, T. et al. A functional model of [Fe]-Hydrogenase. J. Am. Chem. Soc. 138,
+
−1
−1
14
methenyl-H MPT (ε336nm =21.6mM cm ) (ref. ). One unit (U) of activity is the
4
3
270–3273 (2016).
–
1
+
amount of enzyme that catalyseds a decrease of 1μmolmin methenyl-H
4
MPT
2. Shima, S. et al. Reconstitution of [Fe]-hydrogenase using model complexes.
Nat. Chem. 7, 995–1002 (2015).
+
(
the forward reaction) or an increase of methenyl-H MPT (the reverse reaction).
4
For the kinetic measurements shown in Fig. 3, the absorbance was measured
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sulphate-reducing archaeon Archaeoglobus fulgidus. Nature 390, 364 (1997).
+
Final concentrations of 20µM methenyl-H
4
MPT and 20µM methylene-H
4
MPT
were used as the substrates for the forward and reverse reaction, respectively, and
–1
0
.007mgml reconstituted enzyme (final concentration) was added to the 0.7ml
1
reaction mixture; the light path of the cuvette was 1cm. The spectra were recorded
every 10s. As a control, complex 3 (14uM, final concentration) or the apoenzyme
–1
(
0.014mgml ) was added to the assay instead of the reconstituted enzyme.
Acknowledgements
This work was supported by the Swiss National Science Foundation (to X.L.H.),
European Union Marie Sklodowska-Curie Individual Fellowships (794000 to H.-J.P.),
Max Planck Society (to S.S.) and Deutsche Forschungsgemeinschaft (SH 87/1-1, to S.S.).
M.D.W. acknowledges C. Corminboeuf (EPFL)for financial support and the Laboratory
for Computational Molecular Design (EPFL) for providing computing resources. G.H.
was supported by a fellowship from the China Scholarship Council (CSC).
Infrared spectroscopy of enzymes. The samples for infrared spectroscopy were
prepared in amber-coloured 1.5ml Eppendorf tubes. The sample solutions contained
–
1
1
50mgml (4mM) semi-synthetic [Mn]-hydrogenase in 10mM MOPS/NaOH
pH 7.0. The sample solutions were prepared in an anaerobic tent with the gas phase
9
5%N /5%H and then frozen in liquid nitrogen. The frozen samples in tubes were
2 2
stored in a Dewar filled with liquid nitrogen until the measurements were taken.
All infrared spectra were obtained with an FTIR spectrometer (Bruker, Vertex
7
4
0V) in an attenuated total reflection (ATR) optical configuration with a Si prism of Author contributions
5° incident angle and 2 active reflections (Smith Detection, DuraSamplIR IITM).
S.S. and X.L.H. directed the research. H.-J.P. conducted the experiments of the Mn
complexes; G.F.H. conducted experiments of the semi-synthetic enzymes; M.D.W. did
the computations; F.F.T. did the crystallographic study of Mn complexes; K.A. performed
infrared spectroscopy of the enzymes; All authors discussed the data. X.L.H. wrote the
manuscript with contributions from H.-J.P and S.S.
−1
Spectra were obtained with a resolution of 4cm . Five micro-litres of the sample
solutions were dropped onto the effective area of a Si prism (3mm diameter) and
concentrated by slowly evaporating the solvent under mild flow of argon gas. The
hydration of the sample was estimated by the relative intensities of the water band
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The authors declare no competing interests.
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Supplementary information is available for this paper at https://doi.org/10.1038/
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The Author(s), under exclusive licence to Springer Nature Limited 2019
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