Abnormal co-enzymatic behavior of a one-electron reduced bipyridinium salt with a carbamoyl group on the catalytic activity of CO2 reduction by formate dehydrogenase

Article abstract of DOI:10.1039/c8nj03478h

One-electron reduced bipyridinium salt (BP) acts as a co-enzyme for formate dehydrogenase (FDH) from Candida boidinii in CO₂ reduction to formic acid. By using BP with a carbamoyl group (CMV), the CO₂ reduction catalytic activity of FDH was improved compared with those of other BPs. Moreover, CMV did not act as a co-enzyme for FDH in the presence of more than 20 times higher concentration of CMV against FDH. This is the first report of BP with the abnormal co-enzymatic behavior on the catalytic activity of CO₂ reduction by FDH.

Full text of DOI:10.1039/c8nj03478h

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ISSN 1144-0546
PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
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Abnormal co-enzymatic behavior of one-electron reduced
bipyridinium salt with carbamoyl-group on the CO2 reduction
catalytic activity of formate dehydrogenase
Received 00th January 20xx,
Accepted 00th January 20xx
S. Ikeyamaa, Y. Amaoa,b
DOI: 10.1039/x0xx00000x
www.rsc.org/
One-electron reduced bipyridinium salt (BP·) acts as a co-enzyme from Candida boidinii is oxygen tolerant and commercially
for formate dehydrogenase (FDH) from Candida boidinii in CO2 available. Therefore, FDH from Candida boidinii is widely used
reduction to formic acid. By using BP· with carbamoyl-group (CMV·), as a CO2-reducing catalyst in enzymatic, photochemical and
the CO2 reduction catalytic activity of FDH was improved compared electrochemical CO2 reduction. By using redox coupling of
with those of other BP·s. Moreover, CMV· did not act as a co- natural co-enzyme NAD+/NADH, FDH mainly catalyzes the
enzyme for FDH in the presence more than 20 times higher formic acid oxidation to CO2. Thus, the CO2 reduction catalytic
concentration of CMV· against FDH. This is the first report of BP· activity of FDH is low using redox coupling of NAD+/NADH.
with the abnormal co-enzymatic behavior on the CO2 reduction Thus, the effective method for utilization of the CO2 reduction
catalytic activity of FDH.
catalytic activity of FDH has been required. FDH catalyzes the
CO2 reduction to formic acid in the presence of NADH29-33 or
the one-electron reduced bipyridinium salt (BP·) as an electron
mediator.34-40
The techniques of CO2 conversion to organic compounds
have been much paid attention for CO2 reduction and
utilization in the global environmental science fields. The
catalysts for CO2 conversion systems are classified into a
photocatalyst, a molecular catalyst and a biocatalyst.1-13 In
those systems, CO2 is reduced to useful materials such as
formic acid, carbon monoxide and methanol. Among these
catalysts, electrochemical CO2 reduction with high yield by
using a simple catalyst based on copper has been reported.14,15
However, in the case of using a simple catalyst based on
copper, not only formic acid but also carbon monoxide,
methane, methanol, hydrogen, and so on due to the CO2
reduction are produced simultaneously, so control of the
selectivity for CO2 reduction is a serious problem.16
The CO2 reduction to formic acid system with FDH and
NADH that combines NADH-regenerating catalyst have been
reported.35 In this case, the efficiency of the CO2 reduction
with FDH depends on the concentration of NADH. FDH mainly
catalyzes the formic acid oxidation to CO2 in the coexistence of
NAD+ and NADH, because NAD+ has more high affinity with
FDH than that of NADH. The Michaelis constant (Km) of NAD+
and NADH in the reaction with FDH are calculated to be 90 and
2087 μM, respectively, indicating that the concentration of
NAD+ (~180 μM) to reach a maximum velocity (Vmax) for formic
acid oxidation with FDH is approximately 20 times lower than
that of NADH (~4174 μM) to reach a Vmax for CO2 reduction.
In contrast, BP· acts as a co-enzyme for FDH in CO2
reduction to formic acid instead of NADH.40 Moreover, BP
doesn’t act as a co-enzyme for FDH in formic acid oxidation to
CO2. Therefore, FDH only catalyzes the CO2 reduction to formic
acid in the coexistence of BP and BP·. In addition, the CO2
reduction catalytic efficiency with FDH is improved by using
various BP·s. Moreover, the catalytic activity of FDH can be
controlled with complete control by using BP·s based artificial
co-enzyme without changing structure of FDH. For example, by
using one-electron reduced methylviologen (MV·), widely used
as a co-enzyme for FDH, the CO2 reduction catalytic efficiency
(kcat/Km; kcat: catalytic efficiency) is improved 20 times than
that of NADH.36 Moreover, the affinity between BP· and FDH is
affected by the functional groups in BP·s. 36-40 Compared with
the Km value of the MV· for FDH in CO2 reduction to formic acid
Among the various catalysts, in contrast, the synthesis of
organic compound from CO2 with biocatalysts have been
reported.17-25 Formate dehydrogenase (FDH) has been used
for CO2 reduction to formic acid in the presence of a suitable
electron mediator as a co-enzyme.26,27 Moreover, addition of
carbonic anhydrase to FDH was shown to improve the catalytic
performance towards CO2 reduction to formic acid.28 By using
FDH, formic acid is obtained without by-products under
ambient conditions. Thus, FDH is an attractive catalyst for CO2
based material conversion. Especially, NAD+-dependent FDH
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(Km = 212 µM) as a reference, the Km value of one-electron CMV are listed in Table 1. The reduction potentials of MV and
reduced 1-aminoethly-1’-methyl-4,4’-bipyridinium salt (AMV·) NAD+ also are listed as reference. The value of first reduction
37
was 118 µM,
indicating that the affinity with FDH was potential of CMV was estimated to be -0.59 V. The value of
increased by replacing a methyl-group to an amino-group. On CMV was lower than that of MV.
the contrary, the Km value of one-electron reduced 1-
To reveal the effect of CMV as a co-enzyme on the formic
carboxymethly-1’-methyl-4,4’-bipyridinium salt (Km = 292 µM) acid oxidation catalytic activity of FDH, we checked the formic
was higher than that of MV·, indicating that carboxyl-group acid oxidation to CO2 with FDH and CMV. The reaction was
caused of decreasing the affinity of BP· with FDH. carried out as following method. The sample solution was
Furthermore, the improvement of the affinity of BP· with FDH consisted of sodium formate (16 mM) and CMV (0 - 300 μM) in
leads the enhancement of the CO2 reduction catalytic activity 1.0 mM sodium pyrophosphate buffer (pH 7.4). The solution
39-41
of FDH.
The CO2 reduction catalytic efficiency value was deaerated using freeze-pump-thaw cycles and flushed
(kcat/Km) was improved 2 times by using AMV· than using MV·. with N2 gas. FDH (a final concentration of 9.2 μM)was added
We aimed to enhance the CO2 reduction catalytic activity of to start the oxidation of formic acid to CO2. The amount of
FDH by using various BP·s. Therefore, we focused on the CMV· production was detected by measurement of the
chemical structure of NAD+ to design the BP as a suitable co- absorption of CMV· at 600 nm (molecular extinction
enzyme for FDH. NAD+ has high affinity with FDH in formic acid coefficients: ε=12,700 M·cm-1) using UV-visible absorption
oxidation to CO2 (Km = 90 µM). NAD+ has a carbamoyl-group spectroscopy (SHIMADZU, MaltiSpec-1500).
which can form hydrogen bond with some amino-residues in
Table 1. First (Ered1) and second (Ered2) reduction potentials of CMV, MV and NAD+.
FDH. Hence, we anticipated that the affinity between FDH and
BP could be improved by introducing carbamoyl-group into BP·.
Electron
mediator
CMV
Ered1 vs. Ag/AgCl
Ered2 vs Ag/AgCl
In
this
study,
1-carbamoylmethyl-1’-methyl-4,4’-
V
V
bipyridinium diiodide (CMV; chemical structure are shown in
Figure 1) was synthesized and the one-electron reduced
CMV(CMV·) was applied to the CO2 reduction to formic acid
with FDH as a co-enzyme.
- 0.59
- 0.67
- 0.54
- 0.93
- 0.98
NR
MV
NAD+
Figure 2 shows the relationship between CMV· production
rate and the concentration of CMV. The relationship between
NADH production rate and the concentration of NAD+ is also
shown in Fig. 2 as a reference. As a result from Fig. 2, CMV·
was not produced with formic acid oxidation to CO2 in any
conditions, showing that CMV didn’t act as a co-enzyme for
FDH. In contrast, the mechanism of formic acid oxidation to
CO2 with FDH and NAD+ has been reported.41,42 In the
mechanism, hydride transfer to NAD+ from formic acid occurs
and then formic acid is oxidized to CO2. This indicates that
hydride was not produced by formic acid oxidation with FDH
and CMV.
Fig. 1. Chemical structures of 1-carbamoylmethyl-1’-methyl-4,4’-bipyridinium
diiodide (CMV) and NAD+.
Enzyme kinetic analysis was carried out to reveal the effect
of carbamoyl-group on the function of BP· as a co-enzyme and
the CO2 reduction catalytic activity of FDH. FDH from Candida
boidinii (Mw. 74 kDa) was obtained from Roche Diagnostics
K.K. 4,4’-Bipyridyl was purchased from Tokyo Kasei Co. Ltd.
Sodium formate, 2-iodoacetoamide and sodium hydrosulfite
were procured from WAKO Chemicals Co. Ltd. The other
chemical regents were of analytical purity or the highest grade
available.1-Methyl-4,4’-bipyridinium iodide was synthesized
following method. 4,4’-Bipyridyl and iodomethane was stirred
at the molar ratio of 1:1 in 200 ml acetone at room
temperature. The residue of 1-methyl-4,4’-bipyridyl iodide was
obtained and dried under vacuum overnight. CMV was
synthesized according to the previous report.40 1-methyl-4,4’-
bipyridinium iodide and the molar equivalent of 2-
iodoacetamide were refluxed in 200 ml acetonitrile at 90 °C for
48 h. After refluxing, the precipitate of CMV was collected by
filtration and dried in a vacuum desiccator.
Fig. 2. The relationship between the reduced form production rate of CMV·
(closed circle) or NAD+ (open circle) and the concentration of NAD+ or CMV. The
sample solution was consisting of CMV or NAD+ (0 - 300 μM) and FDH (9.2 μM) in
N2 saturated 1.0 mM sodium pyrophosphate buffer (pH 7.4).
The redox potential of CMV was measured using cyclic
voltammetry (HOKUTO HZ-3000) with
electrode as a working, platinum electrode as a counter and
Ag/AgCl electrode as reference in 50 mM sodium
a glassy carbon
Next, the inhibitory effect of CMV on the formic acid
oxidation catalytic activity of FDH was checked to reveal the
interaction between CMV and FDH in more detail. The
inhibitory effect of CMV was evaluated by investigating the
a
pyrophosphate (pH 7.4). The sample solution was consisted of
CMV (2.0 mM). The first and second reduction potentials of
2 | J. Name., 2012, 00, 1-3
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relationship between NADH production rates in the presence functional group for increasing the affinity of BP· with FDH. The
of CMV. The sample solution consisting of NAD+ (20 μM), high affinity of BP· with FDH is connected the improvement of
sodium formate (16 mM) and CMV (0 - 300 μM) in 1.0 mM the CO2 reduction catalytic efficiency of FDH. The kcat/Km value
sodium pyrophosphate buffer (pH 7.4) was prepared. The of CMV· was the largest in other BP·s. The CO2 reduction
reaction was started by injecting FDH (9.2 μM). The amount of catalytic efficiency of FDH was improved approximately 3
NADH production in the presence of CMV was detected by times by using CMV· compared with that of MV·. Therefore,
measurement of absorption of NADH at 340 nm (molecular the improvement of CO2 reduction catalytic activity of FDH was
extinction coefficient: ε=6,300 M·cm-1) for 15 min using UV- achieved by using CMV·.
visible absorption spectroscopy (SHIMADZU). As a result, no
Table 2. Kinetic parameters for CO2 reduction to formic acid with BP·s and FDH
NADH production rate decrease in the presence of CMV,
showing that CMV didn’t act as an inhibitor.
BP·s
Km
μM
60.2
212
118
kcat
min-1
1.8
kcat/Km
Next let us focus on the function of CMV· as a co-enzyme
for FDH by enzymatic kinetic analysis, CO2 reduction to formic
acid with CMV· and FDH was carried out. The solution
M-1·min-1
2.9 ×104
9.0 ×103
1.9 ×104
CMV·
MV·
AMV·
1.9
consisting of CMV (0
- 500 μM) in 1.0 mM sodium
2.3
pyrophosphate buffer (pH 7.4) was deaerated using freeze-
pump-thaw cycles and flushed with CO2 gas. CMV was reduced
with sodium hydrosulfite (4.0 mM) as a reductant. The CO2
reduction to formic acid was started by injecting FDH (9.2 μM).
The amount of formic acid production for 1 min after starting
the reaction was detected using an ion chromatography
system (ICS-1100, Thermo).
On the other hand, formic acid production rate was
drastically decreased more than 200 μM of CMV· in the sample
solution. This indicates that CO2 reduction catalytic activity of
FDH was deactivated in the presence 20 times higher
concentration of CMV· against FDH. No phenomenon of this
deactivation of FDH was observed with the other BP·s.37
Next let us focus on the positional relationship between
the NAD+-recognition and CO2 or formic acid binding sites in
the FDH and CMV·. The structure of FDH from Candida boidinii
was reported by Labrou et al.43-46 Figure 4 show the amino-
residues of NAD+-recognition and CO2 or formic acid binding
sites and proposed position of CMV· in FDH.
Figure 3 shows the relationship between the formic acid
production rate and the concentration of CMV·. The formic
acid production rate was increasing with increasing the
concentration of CMV· until 200 μM. The plots until 200 μM of
CMV· coincide with Michaelis-Menten equation.
Fig. 4. The proposed interaction between CMV· and the amino-residues of NAD+-
recognition and CO2 or formic acid binding sites in FDH.
Fig. 3. The relationship between the formic acid production rate and the
concentration of CMV·. (Broken line: Theoretical value without inhibitory, Line:
Measured value). The sample solution was consisting of CMV (0 - 500 μM), FDH
(9.2 μM) and sodium hydrosulfite (4.0 mM) in CO2 saturated 1.0 mM sodium
pyrophosphate buffer (pH 7.4).
In FDH, NAD+-recognition field is consisted of amino-
residues like Asp 96, Asp 195 and His 232. In this field,
carbamoyl-group of NAD+ can form the hydrogen bond with
amino-residues of Asp 96, Asp 195 and His 232 to recognize
NAD+. Thus, hydrogen bond between carbamoyl-group of
CMV· and amino-residues in NAD+-recognition field in FDH also
was formed and high affinity between CMV· and FDH was
accomplished.
Table 2 shows kinetic parameters for CO2 reduction to
formic acid calculated by Lineweaver-Burk equation. The
kinetic parameters of CMV· were estimated to be 60.2 μM
(Km), 1.8 min-1 (kcat
)
and 2.9 ×104 M-1·min-1 (kcat/Km),
respectively. Those parameters of MV· and AMV· in same
condition also were shown as a reference. 36,37
The Km value of CMV· was the lowest in other BP·s,
indicating that CMV· had high affinity with FDH. The requiring
amount of CMV· (~120 μM) to reach Vmax for formic acid
production with FDH was a half of that of AMV· (~236 μM).
This result shows that carbamoyl-group was an effective
On the other hand, formic acid production rate drastically
was decreased in the presence of excess CMV· as shown in Fig.
3. There is a possibility as a cause of the deactivation of the
CO2 reduction catalytic activity of FDH with excess CMV·. In the
CO2 or formic acid binding site of FDH, Asn 119, Arg 258 and
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