Artificial co-enzyme based on carbamoyl-modified viologen derivative cation radical for formate dehydrogenase in the catalytic CO2reduction to formate

Article abstract of DOI:10.1039/d0nj04375c

Formate dehydrogenase (CbFDH) from Candida boidinii is a useful biocatalyst for CO2 reduction to formate in the photoredox system, and consists of a visible-light sensitizer and an electron mediator. The electron mediator, single-electron reduced 4,4′-bipyridinium salts (4,4′-BPs) represented by methylviologen act as the co-enzyme for CbFDH in the CO2 reduction to formate. Considering that the single-electron reduced 4,4′- or 2,2′-BPs activate the CbFDH-mediated CO2 reduction to formate, the architecture of the effective co-enzyme based on the chemical modification of BP is useful for the development of the catalytic reduction of CO2 to formate with CbFDH. NAD+ has a carbamoyl group or nicotinamide moiety, which can form hydrogen bonds with some amino residues in CbFDH. Thus, we predicted that the affinity of 4,4′-BP for CbFDH could be improved by introducing a carbamoyl group or nicotinamide moiety into 4,4′-BP. In this work, the interaction between the single-electron reduced 1-carbamoylmethyl-1′-methyl-4,4′-bipyridinium salt, 1,1′-dicarbamoylmethyl-4,4′-bipyridinium salt and 1-nicotinamidethyl-1′-methyl-4,4′-bipyridinium salt and CbFDH in the CO2 reduction to formate is elucidated by enzymatic kinetic analysis, the docking simulation and density functional theory calculation.

Full text of DOI:10.1039/d0nj04375c

NJC
View Article Online
View Journal
PAPER
Artificial co-enzyme based on carbamoyl-
modified viologen derivative cation radical for
formate dehydrogenase in the catalytic CO2
reduction to formate
Cite this:DOI: 10.1039/d0nj04375c
a
bc
Akimitsu Miyaji and Yutaka Amao
*
Formate dehydrogenase (CbFDH) from Candida boidinii is a useful biocatalyst for CO
2
reduction to
formate in the photoredox system, and consists of a visible-light sensitizer and an electron mediator.
0
0
The electron mediator, single-electron reduced 4,4 -bipyridinium salts (4,4 -BPs) represented by
methylviologen act as the co-enzyme for CbFDH in the CO reduction to formate. Considering that the
single-electron reduced 4,4 - or 2,2 -BPs activate the CbFDH-mediated CO reduction to formate, the
2
0
0
2
architecture of the effective co-enzyme based on the chemical modification of BP is useful for the
+
development of the catalytic reduction of CO
2
to formate with CbFDH. NAD has a carbamoyl group or
nicotinamide moiety, which can form hydrogen bonds with some amino residues in CbFDH. Thus, we
0
Received 1st September 2020,
Accepted 9th October 2020
predicted that the affinity of 4,4 -BP for CbFDH could be improved by introducing a carbamoyl group or
0
nicotinamide moiety into 4,4 -BP. In this work, the interaction between the single-electron reduced
0
0
0
0
1
1
-carbamoylmethyl-1 -methyl-4,4 -bipyridinium salt, 1,1 -dicarbamoylmethyl-4,4 -bipyridinium salt and
DOI: 10.1039/d0nj04375c
0
0
-nicotinamidethyl-1 -methyl-4,4 -bipyridinium salt and CbFDH in the CO
2
reduction to formate is
rsc.li/njc
elucidated by enzymatic kinetic analysis, the docking simulation and density functional theory calculation.
and widely used in the bio-based CDU technologies. Among the
bio-based CDU technologies with CbFDH, some studies on the
Introduction
2 2
Recently, many CO -captured storage (CCS) and utilization visible light-driven CO reduction to formate with the photo-
(CDU) technologies have received much attention for the CO2 redox system consisting of an electron donor (ED), a photo-
curtailment. In CDU technologies, CO is used as feedstock for sensitizer (PS), an electron mediator (EM) and CbFDH as shown
2
1
–10
32–51
C1 based fuels, chemicals and polymers.
nology, the survey and development of an effective catalyst for
CO
useful organic molecules) is an important key issue.
catalysts for the CO conversion, some biocatalysts also have phyrins as a photosensitizer, the reduction potentials of the
received much attention for CDU technologies.
formate dehydrogenase (FDH) catalyzes formate oxidation to CO
In the CDU tech- in Fig. 1 have been reported.
A potential of ꢀ0.9 V (vs. Ag/AgCl) is required for the
+
2
conversion to chemical raw material (such as CO and reduction of NAD to NADH. In other words, in systems using
1
1–20
In ruthenium polypyridyl coordination complexes or metallopor-
2
2
1–25
For example, excited state of these photosensitizers are insufficient and
+
NAD cannot directly reduce NADH. Even when a sufficient
2
+
with the co-enzyme NAD (dotted line), and catalyzes the reverse reduction potential is obtained in the excited state of the
reaction of CO reduction to formate with NADH (solid line), as photosensitizer, the single-electron reduced NAD is produced
+
2
26–29
shown in Scheme 1.
and the irreversible NAD dimer is formed between the single-
+
+ 52
Thus, NAD -dependent FDH is an attractive biocatalyst for electron reduced NAD s. As the irreversible NAD dimer is an
CDU using an electron mediator redox system.
3
0,31
In particular, inactive co-enzyme for CbFDH, it is difficult to build the
FDH from Candida boidini (CbFDH) is commercially available
a
School of Materials and Chemical Technology, Tokyo Institute of Technology,
4
259 G1-14, Nagatsuda, Midori-Ku, Yokohama 226-8502, Japan
b
c
Graduate School of Science, Osaka City University, 3-3-138 Sugimoto,
Sumiyoshi-ku, Osaka 558-8585, Japan. E-mail: amao@osaka-cu.ac.jp
Research Centre of Artificial Photosynthesis (ReCAP), Osaka City University,
Scheme 1 Formate – CO
using NAD /NADH redox.
2
mutual conversion catalyzed with CbFDH
+
3
-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2020
New J. Chem.

View Article Online
Paper
NJC
2
Fig. 1 Visible light-driven CO reduction to formate with the photoredox
system, consisting of an electron donor (ED), a photosensitizer (PS), an
electron mediator (EM) and CbFDH.
+
0
0
photoredox system using a combination of the NAD direct Fig. 2 Chemical structures of 1-carbamoylmethyl-1 -methyl-4,4 -bipyridinium
0
0
salt (MCABP), 1,1 -dicarbamoylmethyl-4,4 -bipyridinium salt (DCABP) and
photoreduction and CbFDH. In contrast, some studies on the
visible light-driven CO reduction to formate with CbFDH using
the photoreduction of NAD by a photocatalyst via the rhodium
0
0
1
-nicotinamidethyl-1 -methyl-4,4 -bipyridinium salt (NEMBP).
2
+
3
2–41
complex as a hydride generator have been reported.
Even if
the effective co-enzyme is crucial for the development of the
+
the direct NAD reduction to NADH with the photoredox system
could be achieved, NAD is a very expensive biological reagent,
and it is necessary to improve its usage and the turnover of the
NAD /NADH redox coupling. Now, let’s compare the kinetic
catalytic reduction of CO to formate with CbFDH. Therefore, we
2
+
+
0
focused on the chemical structure of NAD to design the 4,4 -BP
+
as a suitable co-enzyme for CbFDH. NAD has high affinity with
+
+
CbFDH in formate oxidation to CO
2
(K
m
= 90 mM). NAD has a
carbamoyl group or nicotinamide moiety, which can form
hydrogen bonds with some amino residues (threonine, serine
and histidine) in CbFDH. Hence, we predicted that the affinity
between CbFDH and 4,4 -BP could be improved by introducing
a carbamoyl group or nicotinamide moiety into 4,4 -BP. We
previously reported on the visible-light driven system for CO2
reduction to formate, involving the coupling of the photoreduction
of 1-carbamoylmethyl-1 -methyl-4,4 -bipyridinium salt (MCABP),
,1 -dicarbamoylmethyl-4,4 -bipyridinium salt (DCABP) and
parameters for the Michaelis constant (K
cat) and catalytic efficiency (kcat/K ) values of NAD and NADH
for CbFDH in the formate oxidation and CO reduction. The K
m
), catalytic constant
+
(k
m
2
m
62
+
values of NAD and NADH for CbFDH were reported to be 90
0
5
2,53
and 2087 mM, respectively.
value for the CO reduction to formate, was also reported to
be approximately 900 times lower than that of the formate
oxidation to CO , which was catalyzed with CbFDH using a
The catalytic efficiency, kcat/K
m
0
2
2
0
0
59
+
native co-enzyme. As long as the redox coupling of NAD /NADH
0
0
59
1
1
+
is used, the affinity between NAD or NADH and CbFDH does
0
0
60
-nicotinamidethyl-1 -methyl-4,4 -bipyridinium salt (NEMBP)
not change. Thus, the CbFDH catalytic activity cannot be con-
trolled in the photoredox system. Moreover, as the produced
NADH acts as a sacrificial reagent and is consumed, the redox
with water soluble zinc porphyrin and CbFDH in the presence
of an electron donor, triethanolamine (TEOA). Using an enzymatic
kinetic analysis, we also reported on the kinetic parameters of the
+
coupling of NAD /NADH is not suitable to use for the photo-
single-electron reduced MCABP for the CO reduction to formate
2
redox system.
61
with CbFDH. Furthermore, it is necessary to clarify the correla-
tion between the carbamoyl group or nicotinamide moiety bound
to 4,4 -BP and the catalytic activity of CbFDH for CO
In this paper, we carried out enzymatic kinetic studies in
order to elucidate the interaction between the single-electron
reduced MCABP, DCABP and NEMBP (chemical structures
In contrast, some studies on the visible-light driven systems
reduction to formate, which involved the coupling of
the photoreduction of various 4,4 - or 2,2 -bipyridinium salts
4,4 - or 2,2 -BPs) with ruthenium polypyridyl coordination
complexes, water soluble zinc porphyrin or chlorophyll-a and
Recently, by using an enzymatic
kinetic analysis, we were able to report on the kinetic parameters
for CO
2
0
2
reduction.
0
0
0
0
(
4
2–51
CbFDH have been reported.
shown in Fig. 2) and CbFDH in the CO reduction to formate.
2
We also aimed to clarify the direct interaction of the single-
electron reduced MCABP, DCABP and NEMBP in the substrate
binding site of CbFDH by docking simulation and density
functional theory (DFT) calculation.
0
0
of the various single-electron reduced 4,4 - or 2,2 -BPs for the CO
2
5
3–58
reduction to formate with CbFDH for the first time.
Previous studies reported that some kinds of the single-
0
0
0
electron reduced 2,2 -BP (1,1 -ethylene-2,2 -bipyridinium dibromide;
0
0
0
DB) or 4,4 -BP (1,1 -diaminoethyl-4,4 -bipyridinium dibromide;
DAV) were more effective co-enzymes for CbFDH in the CO2
reduction to formate, in comparison with that of the con-
Experimental
0
53,57,58
Materials
ventional 4,4 -BP, methylviologen (MV).
Thus, considering
0
0
62
that the single-electron reduced 4,4 - or 2,2 -BPs activate the FDH from Candida boidinii (CbFDH; M.W. 74 kDa ) was purchased
0
CbFDH-mediated CO
2
reduction to formate, the architecture of from Roche Diagnostics K.K. 4,4 -Bipyridine, methyl iodide,
New J. Chem.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2020

View Article Online
NJC
Paper
iodoacetamide, 2-bromoethylamine hydrobromide, nicotinamide molar coefficient using UV-visible absorption spectroscopy
and 2,4-dinitrochlorobenzene were supplied by Tokyo Kasei Co., (SHIMADZU, MaltiSpec-1500). The control experiment was the
+
Ltd. The other chemicals were of analytical grade or the highest formate oxidation to CO with CbFDH using NAD (100 mM).
2
grade available.
The concentration of NADH produced was determined with the
molar coefficient of NADH at 340 nm.
Preparation of MCABP, DCABP and NEMBP
0
CO
DCABP or NEMBP and CbFDH
reduction to formate with the single-electron reduced
2
reduction to formate with single-electron reduced MCABP,
1
-Methyl-4,4 -bipyridinium iodide was synthesized by following
6
3–66
0
a previously reported method.
4,4 -Bipyridine and iodo-
methane were stirred at the molar ratio of 1 : 1 in 200 ml The CO
2
acetone at room temperature for 24 h. MCABP was synthesized MCABP, DCABP or NEMBP and CbFDH was carried out as
0
by refluxing 1-methyl-4,4 -bipyridinium iodide with the molar follows. The single-electron reduced MCABP, DCABP or NEMBP
equivalent of iodoacetamide in 200 ml acetonitrile at 90 1C for was prepared with sodium dithionite as described in the
0
4
8 h. DCABP was synthesized by refluxing 4,4 -bipyridine and following method. First, sodium dithionite powder was added
iodoactamide in 200 ml acetonitrile at 90 1C for 48 h according into a quartz cell and de-aerated by vacuum pump, and then
5
9
to the previous report. NEMBP was synthesized according to flushed with CO
the previous report.
2
gas. A solution containing MCABP, DCABP or
NEMBP in 5.0 ml of 50 mM sodium pyrophosphate buffer
pH 7.4) was de-aerated by freeze–pump–thaw cycles repeated
times, and then flushed with CO gas for 5 min. To prepare
the solution containing the single-electron reduced MCABP,
6
0
(
6
Determination of molar coefficient for single-electron reduced
MCABP, DCABP and NEMBP
2
Molar coefficients for the single-electron reduced MCABP, DCABP or NEMBP in sodium pyrophosphate buffer, 3.0 ml of
DCABP and NEMBP were determined by the following method. the MCABP, DCABP or NEMBP solution was added into a quartz
A solution containing MCABP, DCABP and NEMBP and sodium cell and reacted with sodium dithionite under continuous
dithionite (0.1 M) in distilled water was de-aerated with argon flowing CO
gas bubbling for 5 min. The production of the single-electron CbFDH solution (9.2 mM) to the MCABP, DCABP or NEMBP
reduced MCABP, DCABP or NEMBP form was monitored by with sodium dithionite in 3.0 ml of 1.0 mM CO -saturated
UV-vis absorption spectroscopy using a Shimadzu Multispec sodium pyrophosphate buffer (pH = 7.4). The amount of formic
500 spectrophotometer. Using the Beer–Lambert law, the acid was detected using ion chromatography system (Dionex
2
gas. The reaction was started by injection of
2
1
molar coefficients for the single-electron reduced MCABP, ICS-1100; electrical conductivity detector) with an ion exclusion
DCABP or NEMBP were obtained from the gradient in the plot column (Thermo ICE AS1; column length: 9 ꢁ 150 mm;
of the concentration of single-electron reduced MCABP, DCABP or composed of a 7.5 mm cross-linked styrene/divinylbenzene
NEMBP, and the absorbance of the maximum in the absorption resin with functionalized sulfonate groups). The 1.0 mM octane
spectrum.
sulfonic acid and 5.0 mM tetrabutylammonium hydroxide were
used as an eluent and a regenerant, respectively. The lowering
of pH by adding sodium dithionite was suppressed by using
sodium pyrophosphate buffer solution (pH 7.4), indicating that
Reduction potential measurements of MCABP, DCABP and
NEMBP
The single and double-electron reduction potentials for MCABP, the stability of CbFDH was retained in the sodium dithionite-
DCABP and NEMBP were determined by cyclic voltammetry saturated solution. For analysis of the enzyme kinetics, the CO2
(Hokuto Denko HZ-3000). All measurements were carried out reduction to formate with single-electron reduced MCABP,
under argon gas-saturated solution containing 0.2 M potassium DCABP or NEMBP and CbFDH was conducted under the
chloride and 1.0 mM sodium pyrophosphate buffer (pH 7.4) at a following conditions. The sample solution consisted of MCABP,
glassy carbon-working electrode. A platinum wire was used as DCABP or NEMBP, sodium dithionite (160 mM) and CbFDH
the counter electrode. All potentials were relative to the Ag/AgCl (9.2 mM) in CO
2
-saturated 1.0 mM sodium pyrophosphate
electrode used as the reference. The final concentration of buffer (pH 7.4). The concentration of formate production after
MCABP, DCABP or NEMBP was adjusted to 2.0 mM.
1 min was estimated as the initial rate of the CO reduction to
2
formate.
Formate oxidation to CO
CbFDH
2
with MCABP, DCABP or NEMBP and
with the oxidized form of MCABP,
Docking simulation and density functional theory (DFT)
calculation
The formate oxidation to CO
2
DCABP or NEMBP and CbFDH was carried out as follows. The For preparing the molecular model of CbFDH for docking
sample solution consisted of sodium formate (220 mM), CbFDH simulation with single-electron reduced MCABP, DCABP or
(
1
9.2 mM) and MCABP, DCABP (100 mM) or NEMBP (500 mM) in NEMBP, the crystal structure of CbFDH was obtained from
6
7
.0 mM sodium pyrophosphate buffer (pH 7.4) saturated with the Protein Data Bank (PDB) database (PDB ID: 5DN9). The
+
argon gas. The formate oxidation to CO was monitored by the protein structure of 5DN9 contains NAD and N as substrates
2
3
amount of the single-electron reduced MCABP, DCABP or and some water molecules. For constructing a CbFDH model
+
NEMBP produced. The concentration of the single-electron used for the docking simulation, the NAD , N
3
and water
reduced MCABP, DCABP or NEMBP was determined with the molecules were removed. The docking simulation using the
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2020
New J. Chem.

View Article Online
Paper
NJC
molecular models was performed by using MyPresto version 5.0
program provided by Next Generation Natural Product Chemistry
2
68
(
N PC). As the docking site of the substrate, the grid size was
set to 40 ꢁ 40 ꢁ 40 points with a grid spacing of 0.61 Å centered
on the original ligand in the crystal structure of CbFDH. The grid
box included the entire substrate-binding pocket of CbFDH, and
provided enough space for the ligand translational and rotational
walk. The standard docking protocol for the rigid and flexible
ligand docking was performed. In the binding of MCABP, DCABP
or NEMBP to CbFDH, some amino acid residues composed of the
substrate binding pocket of CbFDH possibly move flexibly and
accommodate MCABP, DCABP or NEMBP. Thus, the residues
oriented on the surface of the substrate-binding pocket of CbFDH
Fig. 3 Cyclic voltammogram of MCABP (blue), DCABP (red) and NEMBP
(
green) in argon gas-saturated sodium pyrophosphate buffer (pH 7.4)
(V93, N119, V123, R174, I175, T256, R258, H311, S313, Y358) were
containing 0.2 M potassium chloride.
chosen as the flexible amino acid residues. The molecular models
were subjected to 100 independent runs per ligand, and the other
parameters were set to the default values.
Table 1 The single and double-electron reduction potentials for MCABP,
DCABP and NEMBP (vs. Ag/AgCl)
The total energy calculations of the oxidized and single-
electron reduced MCABP, DCABP and NEMBP were performed by
density functional theory (DFT). The energy level of the highest
occupied molecular orbital (EHOMO), the lowest unoccupied mole-
Single-electron
reduction potential (V)
Double-electron
reduction potential (V)
ꢂ
ꢂ
MCABP
DCABP
NEMBP
ꢀ0.50
ꢀ0.59
ꢀ0.50
ꢀ0.91
ꢀ0.93
ꢀ0.91
cular orbital (ELUMO) of NEMBP and NEMBP , and the singly
occupied molecular orbital (ESOMO) of NEMBP were calculated
using the oB97XD functional and 6-311++g(d,p) basis set with the
conductor-like polarizable continuum model (CPCM) (diethyl
ether) in Gaussian 16.
69
DCABP and NEMBP were estimated to be ꢀ0.91, ꢀ0.93 and
0.91 V (vs. Ag/AgCl), respectively. The reduction current peak
ꢀ
at ꢀ0.10 V in the cyclic voltammogram of NEMBP was assigned
to nicotinamide, according to the previous reported literature.
The single-electron reduction potential of methylviologen (MV),
Results and discussion
Determination of the molar coefficient for the single-electron
0
a typical example of 4,4 -BP, was estimated to be ꢀ0.67 V. For
reduced MCABP, DCABP or NEMBP
example, the single-electron potential of diphenylviologen with
0
The molar coefficients for the single-electron reduced forms of an electron-withdrawing phenyl group introduced into 4,4 -BP
MCABP, DCABP and NEMBP were determined by the absorbance was estimated to be ꢀ0.34 V, which shifts in the positive
of the maximum peak in the absorption spectra using the Beer– direction as compared with MV. Since the carbamoyl or the
Lambert law. The color in the MCABP, DCABP and NEMBP nicotine amide group is electron-withdrawing, the single-
solution was changed from colorless to blue or violet with the electron reduction potential was shifted to the positive side by
0
production of the single-electron reduced form of MCABP, DCABP introducing these substituents into 4,4 -BP. It is also expected
and NEMBP. The absorption maxima in the spectra of the single- that the single-electron potential of the asymmetric 4,4 -BP,
0
electron reduced MCABP, DCABP and NEMBP were observed at MCABP or NEMBP will shift more positively compared with that
0
5
95, 600 and 600 nm, respectively. From the absorbance results at of the symmetric 4,4 -BP and DCABP.
the maxima in the spectra, the molar coefficients for the single-
2
electron reduced MCABP, DCABP and NEMBP were determined to Formate oxidation to CO with the oxidized form of MCABP,
ꢀ1
ꢀ1
be 7800, 12 700 and 8910 M cm , respectively.
DCABP and NEMBP and CbFDH
We previously reported that the CO reduction to formate only
2
Reduction potentials for single and double-electron reduced
MCABP, DCABP and NEMBP
proceeds with CbFDH using the various single-electron reduced
28
4
2
,4-BPs, and formate oxidation to CO with CbFDH is sup-
Fig. 3 shows the cyclic voltammogram of MCABP, DCABP and pressed using the oxidized form of 4,4-BPs. Fig. 4 shows the time
NEMBP in the argon gas-saturated solution containing 0.2 M dependence of the single-electron reduced MCABP, DCABP or
potassium chloride and 1.0 mM sodium pyrophosphate buffer NEMBP and NADH production due to formate oxidation to CO
pH 7.4). with CbFDH.
Table 1 shows the reduction potentials for single and double-
2
(
+
By using NAD as a co-enzyme, NADH production in the
2
6
+
electron reduced MCABP, DCABP and NEMBP (vs. Ag/AgCl).
solution containing formate, NAD and CbFDH was observed
+
The first reduction potentials for MCABP, DCABP and NEMBP with incubation time. After 5 min incubation, full NAD
were estimated to be ꢀ0.50, ꢀ0.59 and ꢀ0.59 V (vs. Ag/AgCl), reduction to NADH due to formate oxidation proceeded with
respectively. The second reduction potentials for MCABP, CbFDH. On the other hand, in the formate oxidation to CO
2
New J. Chem.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2020

View Article Online
NJC
Paper
CO
2
reduction to formate with single-electron reduced MCABP,
DCABP and NEMBP and CbFDH
The CO reduction to formate with single-electron reduced
2
MCABP, DCABP or NEMBP and CbFDH was conducted under
the following condition. Table 2 shows the concentration of
formate produced after 1 min incubation with the single-
electron reduced MCABP, DCABP or NEMBP and CbFDH. The
typical reaction system for CO reduction to formate consisted
2
of MCABP, DCABP or NEMBP (100 mM), CbFDH (9.2 mM), and
sodium dithionite (160 mM) in CO
phate buffer (pH 7.4). After 1 min incubation, the concentration of
Fig. 4 Time dependence of single-electron reduced MCABP, DCABP or formate produced with single-electron reduced MCABP, DCABP
NEMBP and NADH production due to formate oxidation to CO
2
saturated sodium pyrophos-
2
with and NEMBP were estimated to be 9.9, 6.8 and 1.5 mM, respectively.
CbFDH in 1.0 mM sodium pyrophosphate buffer (pH 7.4). MCABP (blue),
DCABP (red), NEMBP (green) and NADH (orange).
In contrast, after 1 min incubation, the concentration of formate
produced with NADH was estimated to be 1.4 mM.
To investigate the side reactions in this system, the gas
phase was analyzed by gas chromatography (detector TCD or
FID). According to the gas chromatography analysis, no gas
using MCABP, DCABP or NEMBP, absorption of the single-
electron reduced MCABP, DCABP or NEMBP at 595, 600 or 600 nm,
respectively, was not observed in every case. Thus, no single-electron
reduced MCABP, DCABP or NEMBP in the solution containing
formate, 4,4-BPs and CbFDH was produced with incubation time.
2
other than CO was detected. In other words, no hydrogen or
CO was observed due to formate decomposition in the system
0
of 4,4 -BP, CbFDH and sodium dithionite in the CO
sodium pyrophosphate buffer.
2
-saturated
Thus, formate oxidation to CO with CbFDH is also suppressed
2
Fig. 6 shows the time dependence of formate production
with dithionite-reduced MCABP, DCABP, NEMBP or NADH and
using the oxidized form of 4,4-BPs with the carbamoyl group or
nicotinamide moiety. In particular, by using NEMBP, the nicotina-
+
CbFDH in CO -saturated sodium pyrophosphate buffer solution.
mide moiety of NEMBP was not reduced like NAD in the formate
oxidation to CO with CbFDH.
2
By using dithionite-reduced MCABP, DCABP or NEMBP, the
formate production concentration was higher than that of the
system using the natural co-enzyme NADH. After 10 min incuba-
tion, the concentrations of formate produced using dithionite-
reduced MCABP, DCABP and NEMBP were estimated to be 35,
2
Fig. 5 shows the relationship between the initial rate of
NADH or single-electron reduced MCABP, DCABP or NEMBP
+
production and NAD , MCABP, DCABP or NEMBP concentration
in the formate oxidation to CO with CbFDH.
2
24.8 and 5.0 mM, respectively. In contrast, the formate produced
Even if the concentrations of MCABP, DCABP or NEMBP
were increased, the single-electron reduced forms of MCABP,
DCABP or NEMBP were not produced with the system of formate
and CbFDH. In contrast, the NADH production rate was
using NADH was estimated to be 4.7 mM after 10 min incubation.
ꢂ
0
ꢂ
Moreover, NEMBP and 4,4 -BP showed disappointing results in
the CO reduction to formate, despite having a nicotinamide moiety
prepared for the purpose of improving the affinity for CbFDH. Next,
let us focus on the conversion yield for CO to formate with CbFDH
2
+
increased with increasing NAD concentration with the system
+
of formate and CbFDH. From Fig. 5, the K
m
value of NAD for
2
using dithionite-reduced MCABP, DCABP and NEMBP. The CO
2
CbFDH in formate oxidation was estimated to be 90 mM using
Michaelis–Menten analysis.
concentration in the reaction sample was determined as a
bicarbonate ion concentration using an ion chromatograph.
ꢀ
The total concentration of carbonate (CO and HCO3 ) in the
2
CO2 saturated sodium pyrophosphate buffer (pH 7.4) was
estimated to be approximately 74 mM. We previously reported
that CbFDH was found to catalytically reduce only CO
2
to
formate among the three types of carbonate species (CO
2
,
ꢀ
2ꢀ 70
HCO
to HCO
3
and CO
3
). At pH 7.4, the abundance ratio of CO
2
ꢀ
ꢀ
3
2
([CO ]/[HCO
3
]) in the buffer solution was estimated
Table 2 The concentration of formate produced due to CO
2
reduction
with dithionite-reduced MCABP, DCABP and NEMBP and CbFDH
Dithionite-reduced
The concentration of formate
produced (mM)
0
4,4 -BPs
Fig. 5 The relationship between the initial rate of NADH or single-
0
+
electron reduced 4,4 -BP and the concentration of NAD , MCABP (blue),
DCABP (red) or NEMBP (green). The sample solution consisted of sodium
ꢂ
MCABP
DCABP
9.9
6.8
1.5
1.4
ꢂ
0
+
formate (500 mM), 4,4 -BP or NAD (0–300 mM) and FDH (9.2 mM) in N
saturated 1.0 mM sodium pyrophosphate buffer (pH 7.4).
2
ꢂ
NEMBP
NADH
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2020
New J. Chem.

View Article Online
Paper
NJC
Table 3 Kinetic parameters for CO
2
reduction to formate with the
dithionite reduced MCABP, DCABP and NEMBP and CbFDH
Dithionite reduced
,4-BPs
K
m
V
max
k
cat
kcat/K
m
3
ꢀ1
ꢀ1
ꢀ1
ꢀ1
4
(mM) (mM min
)
(min
)
(10 M min )
ꢂ
MCABP
DCABP
60.2 16.1
65.0 12.0
1.75
1.30
1.17
0.39
29.1
20.0
1.58
0.19
ꢂ
ꢂ
NEMBP
NADH
742
10.8
2087 1.6
The obtained kinetic parameters are listed in Table 3. We
previously reported the K , kcat, and kcat/Km values of NADH as a
m
24
natural co-enzyme for CbFDH in the CO reduction to formate.
values of MCABP and DCABP were lower than
those of NEMBP and NADH, indicating that MCABP and
2
Fig. 6 Time dependence of formate production with dithionite-reduced
MCABP (blue), DCABP (red), NEMBP (green) or NADH (orange) and CbFDH
ꢂ ꢂ
The K
m
ꢂ
ꢂ
2
in CO saturated 1.0 mM sodium pyrophosphate buffer (pH 7.4).
ꢂ
DCABP had high affinity with CbFDH. This result shows that
the carbamoyl group was an effective functional group for
0 ꢂ
to be 0.064. The concentration of CO
phate buffer (pH 7.4) was estimated to be 4.4 mM. Thus, the of 4,4 -BP with CbFDH is connected to the improvement of the
2
in the sodium pyrophos- increasing the affinity of 4,4 -BP with CbFDH. The high affinity
0
ꢂ
conversion yield for CO
2
to formate with CbFDH using CO reduction catalytic efficiency of CbFDH. We also reported
2
ꢂ
dithionite-reduced MCABP, DCABP or NEMBP after 10 min that the kinetic parameters of MV for CbFDH were estimated
incubation was estimated to be 0.80, 0.56 or 0.11%, respectively. to be 212 mM (K ), 1.9 min (kcat) and 9.0 ꢁ 10 M min
ꢀ1
3
ꢀ1
ꢀ1
m
5
6
(k
cat/K
m
). The CO
2
reduction catalytic efficiency of CbFDH was
Analysis of enzyme kinetics for CO reduction to formate with
single-electron reduced MCABP, DCABP or NEMBP and CbFDH
ꢂ
ꢂ
2
improved approximately 3 times by using MCABP or DCABP
compared with that of MV . Therefore, the improvement of the
ꢂ
CO
2
reduction catalytic activity of CbFDH was achieved by
Kinetic studies were performed to clarify how the dithionite
reduced MCABP, DCABP and NEMBP affect the CO reduction
2
0
ꢂ
using the carbamoyl group modified 4,4 -BP . In addition,
ꢂ
ꢂ
MCABP or DCABP showed excellent co-enzyme function for
CO reduction to formate with CbFDH, as compared with
to formate with CbFDH. Fig. 7 shows the relationship between the
initial rate of formate production and concentration of dithionite-
reduced MCABP, DCABP and NEMBP.
The initial rate of formate production increased with increasing
concentration of dithionite-reduced MCABP or DCABP until
2
0 ꢂ
4
1
,4 -BP introduced with an amino or a carboxy group (K :
m
57,58
5
4,55
0 ꢂ
m
and various 2,2 -BP s (K : 78–220 mM).
17–370 mM)
On the other hand, despite having a nicotinamide moiety
prepared for the purpose of improving the affinity for CbFDH,
200 mM. The plots of the dithionite-reduced MCABP or DCABP
ꢂ
0
ꢂ
NEMBP and 4,4 -BP showed disappointing results in all
kinetic parameters for the CO reduction to formate.
However, as the carbamoyl group was an effective functional
group for increasing the affinity of 4,4 -BP with CbFDH, the
formate production rate was drastically decreased when more
than 200 mM of MCABP or DCABP was in the sample solution.
This indicates that the CO reduction catalytic activity of
CbFDH was deactivated in the presence of 20 times higher
concentration of MCABP or DCABP against CbFDH. No
phenomenon of this deactivation of CbFDH was observed with
coincided with the Michaelis–Menten equation until 200 mM. On
the other hand, the plots followed the Michaelis–Menten equation
with NADH or dithionite-reduced NEMBP.
The kinetic parameters of dithionite-reduced MCABP, DCABP,
NEMBP or NADH were estimated by curve fitting using the
Michaelis–Menten equation in the concentration range from 0 to
2
0
ꢂ
ꢂ
ꢂ
2
200 mM for CO reduction to formate with CbFDH.
2
ꢂ
ꢂ
0
0
ꢂ
the other 4,4 - or 2,2 -BP s.
Here, let us focus on the reason for the initial rate of formate
production decreasing significantly when the concentration of
dithionite-reduced MCABP or DCABP exceeded 200 mM. We have
previously reported on the kinetic parameters of dithionite-reduced
0
MV, amino and carboxy groups introduced 4,4 -BP for the CbFDH-
54,55
catalyzed CO
2
reduction to formate.
The phenomenon of the
decrease observed for the initial formate production rate in the high
0
concentration dithionite-reduced 4,4 -BP region was observed only
0
in 4,4 -BP with a carbamoyl group, MCABP or DCABP. These results
suggest that the carbamoyl group of MCABP or DCABP is involved
in the decrease in the initial rate of formate production.
We previously reported that the methylpyridinium moiety of
Fig. 7 The relationship between the concentration of the dithionite-
reduced MCABP (blue), DCABP (red) or NEMBP (green), and the initial
ꢂ
ꢂ
ꢂ
2
MCABP was close to the CO or formate-binding site of CbFDH.
0
rate of formate production (v ). Orange: NADH.
New J. Chem.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2020

View Article Online
NJC
Paper
As the methylpyridinium moiety of MCABP is also bound to the
CO or formate binding site of CbFDH at higher dithionite-
2
reduced MCABP concentrations, it is predicted that the
approach of CO to the substrate binding site of CbFDH was
2
hindered by the methylpyridinium moiety at higher dithionite-
6
1
reduced MCABP concentrations. However, the decrease in the
initial rate of formate production was observed in the high
concentration dithionite-reduced DCABP. From these results, it
is predicted that the carbamoyl group in MCABP or DCABP
contributes significantly to the observed decrease in the initial
rate of formate production. We applied several enzymatic
substrate binding inhibition models to the CbFDH-catalyzed
CO reduction to formate with dithionite-reduced MCABP and
2
DCABP, but no satisfactory fitting results were obtained. One of
the reasons why the substrate binding inhibition model could
not be applied is due to the rapid decrease of the initial rate of
formate production at the concentration of dithionite-reduced
MCABP or DCABP exceeding 200 mM. It has been reported that
acetamide functions as an inhibitor on the enzyme activity in
FDH from Candida methanolica, which has high homology with
7
1
CbFDH. It is suggested that an excess of the carbamoyl group,
that is, the ethylamide moiety of dithionite-reduced MCABP or
DCABP, also inhibits the CbFDH-catalyzed CO
formate. On the other hand, nicotinamide had no inhibitory
effect on the CbFDH-catalyzed CO reduction to formate, so no
decrease in the initial rate of formate production was observed
2
reduction to
2
Fig. 8 Relationship between the RMSD and docking score in each oxi-
dized and single-electron reduced 4,4 -BPs to CbFDH. (a) MCABP,
0
in the high concentration dithionite-reduced NAMBP. The (b) DCABP, and (c) NEMBP.
apparent kinetic parameters are obtained only by the enzymatic
kinetic analysis. From the results centering on the enzymatic
ꢂ
kinetics, the effectiveness of BP as a co-enzyme is insufficient it was suggested that there was no significant difference in the
in the reduction process of CO
2
to formate using CbFDH, as binding mode to CbFDH in all of the oxidized and single-electron
0
shown by discussing the substituent-bound BPs and the redox reduced 4,4 -BPs. Among these binding modes, all of the oxidized
potential of BPs.
0
and single-electron reduced 4,4 -BPs in 4 or 5 clusters (total
In order to clarify these problems, it is necessary to elucidate 100 conformations) were bound within 15 Å from the substrate
the steric interaction with BP at the substrate binding site of binding pocket in CbFDH.
CbFDH and to investigate the correlation between the energy
Fig. 9 shows the docking simulation for 4 or 5 clusters of the
oxidized and single-electron reduced 4,4 -BPs to the substrate
ꢂ
0
orbital level of BP and CO reduction.
2
binding site of CbFDH. Fig. 9(a) shows the structure of the
substrate binding pocket, consisting of 10 amino acid residues,
two valines, two arginines, asparagine, isoleucine, threonine,
Docking simulation for single-electron reduced MCABP,
DCABP or NEMBP and CbFDH
First, the docking simulation was investigated to clarify the histidine and serine in CbFDH. Fig. 9(b) shows the docking
+
validity of the binding of oxidized and single-electron reduced model for NAD in the substrate binding pocket of CbFDH. As
0
4
,4 -BP to the substrate binding site of CbFDH. The binding shown in Fig. 9, it was also suggested that there was no significant
mode was estimated for the oxidized or single-electron reduced difference in the binding mode to CbFDH in all of the oxidized
0
MCABP, DCABP or NEMBP to CbFDH by using MyPresto Portal5 and single-electron reduced 4,4 -BPs. It was presumed that the
0
program. From the docking simulation, a total of 100 conformations oxidized or single-electron reduced 4,4 -BPs were closely
0
+
in each oxidized and single-electron reduced 4,4 -BPs were obtained. located at the NAD binding site of CbFDH in all cases (red
Fig. 8 shows the relationship between the root mean square circle in Fig. 9).
deviation (RMSD) and docking score in each oxidized and
Thus, the carbamoyl group and nicotinamide moiety bound
0
0
single-electron reduced 4,4 -BPs. The RMSD value was the to 4,4 -BP were found to have an effect of promoting the binding to
measure of the average distance between the backbone atoms the substrate binding site of CbFDH. Next, in order to estimate the
of the superimposed proteins. The docking score was a calculated electron transfer ability to the single-electron reduced MCABP,
value of the binding affinity by a score function. The binding DCABP or NEMBP binding to CbFDH, the total energy calculations
0
mode of the oxidized and single-electron reduced 4,4 -BPs to of the oxidized and single-electron reduced MCABP, DCABP or
substrate binding site of CbFDH was evaluated from the relation- NEMBP forms were performed by density functional theory (DFT).
ship between the RMSD and docking score. From these results, Table 4 shows the summaries of the EHOMO, ELUMO and ESOMO
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2020
New J. Chem.

View Article Online
Paper
NJC
Table 4
EHOMO, ELUMO and ESOMO of the oxidized and single-electron
reduced MCABP, DCABP or NEMBP forms
E
HOMO (eV) LUMO (eV)
E
ESOMO (eV)
MCABP
MCABP
DCABP
DCABP
NEMBP
NEMBP
ꢀ11.4
—
ꢀ3.66
ꢀ0.47
ꢀ3.66
ꢀ0.48
ꢀ4.00
ꢀ2.50
—
ꢂ
ꢀ6.87
ꢀ11.4
—
ꢂ
—
ꢀ6.86
—
ꢀ7.49
ꢀ11.7
—
ꢂ
0
Fig. 10 SOMO of the single-electron reduced 4,4 -BPs and HOMO
of NADH structurally optimized by DFT calculation. (a) MCABP, (b) DCABP,
(
c) NEMBP and (d) NADH. The mesh indicates the surface of SOMO or
HOMO. The white, grey, blue, red, and orange balls show the atoms of
hydrogen, carbon, nitrogen, oxygen, and phosphorus, respectively.
NADH was localized on the nicotinamide moiety (Fig. 10). Although
ꢂ
NEMBP and NEMBP were likely to bind to the substrate binding
site of CbFDH, as well as DCABP and MCABP, it was presumed
ꢂ
that the SOMO of NEMBP is not sufficiently extended to the
nicotinamide moiety. Thus, the electron transfer to CO is more
2
difficult, and there is a lower efficiency of subsequent formate
Fig. 9 The structure of the substrate binding pocket in CbFDH (a) and production than observed for DCABP and MCABP.
+
docking model for NAD in the substrate-binding pocket of CbFDH (b).
We previously reported that it was predicted that the
approach of CO to the CO or formate binding site of CbFDH
Docking simulation for each oxidized and single-electron reduced
0
2
2
4
,4 -BPs to CbFDH. (c) MCABP, (d) DCABP and (e) NEMBP. The red circle
0
was hindered by the methyl-pyridinium moiety of the single-
electron reduced MCABP under the higher concentrations of
indicates that the oxidized or single-electron reduced 4,4 -BP is bound to
the substrate binding site closest (B3.0 Å) to CbFDH.
6
1
single-electron reduced MCABP. From the images of Fig. 9(c)
and (d), it was suggested that the carbamoyl group of the
oxidized and single-electron reduced MCABP or DCABP equally
bonded to the substrate binding site of CbFDH. Therefore, it
was predicted that in the higher concentration region of
values of the oxidized and single-electron reduced MCABP, DCABP
or NEMBP forms.
The energy level of SOMO and its localization on the
2
MCABP or DCABP, the reduction rate of CO to formate was
molecule are important because the electron is transferred from
slowed down by the carbamoyl group of the oxidized and
single-electron reduced MCABP or DCABP competitively orient-
ing to the substrate binding site of CbFDH.
0
the SOMO of the single-electron reduced 4,4 -BPs to CO
2
. Thus,
the electron transfer ability is greater with higher SOMO level.
According to Tables 3 and 4, the kcat value for CbFDH depends on
ꢂ
ꢂ
ꢂ
ꢂ
the energy level of SOMO of MCABP , DCABP or NEMBP . The
energy level of SOMO and the k of NEMBP were lower than
those of DCABP and MCABP (Table 4). However, the correla-
cat
Conclusions
ꢂ
ꢂ
tions between the SOMO level and kcat were not high, suggesting In conclusion, the kinetic properties were studied for the CO2
that other factors (such as the localization of SOMO) affect the reduction to formate with CbFDH using the single-electron
0
ꢂ
0
electron transfer ability of 4,4 -BPs. The SOMOs of NEMBP , reduced 4,4 -BPs bonded carbamoyl group or nicotinamide
ꢂ
ꢂ
DCABP , and MCABP were localized on the bipyridine moiety, moiety as the co-enzyme via enzymatic kinetic analysis. By using
but not on the nicotinamide moiety. Conversely, the HOMO of the single-electron reduced MCABP or DCABP, the effective CO
2
New J. Chem.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2020

View Article Online
NJC
Paper
reduction to formate with CbFDH was accomplished. On the 10 T. Ohkawara, K. Suzuki, K. Nakano, S. Mori and K. Nozaki,
other hand, the single-electron reduced NEMBP showed disap-
J. Am. Chem. Soc., 2014, 136, 10728.
pointing results in all kinetic parameters for CO reduction to 11 J. Kiwi, K. Kalyanasundaram and M. Gr ¨a tzel, Struct. Bonding,
2
formate, despite having a nicotinamide moiety prepared for the
purpose of improving the affinity for CbFDH. Moreover, the Km, 12 L. A. Kelly and M. A. J. Rodgers, J. Phys. Chem., 1994, 98, 6386.
values for CbFDH depended on the energy level 13 J. R. Darwent, P. Douglas, A. Harriman, G. Porter and M. C.
of SOMO of the single-electron reduced MCABP, DCABP or
Richoux, Coord. Chem. Rev., 1982, 44, 93.
NEMBP. However, as the carbamoyl group was an effective func- 14 P. A. Brugger, P. Cuendet and M. Gr ¨a tzel, J. Am. Chem. Soc.,
1982, 49, 37.
kcat, and kcat/K
m
tional group for increasing the affinity of the single-electron reduced
4
1981, 103, 2923.
0
,4 -BP with CbFDH, the formate production rate was drastically 15 Y. Amao, ChemCatChem, 2011, 3, 458.
decreased when more than 200 mM of the single-electron reduced 16 H. Kumagai, G. Sahara, K. Maeda, M. Higashi, R. Abe and
MCABP or DCABP was in the sample solution. This indicates that
O. Ishitani, Chem. Sci., 2017, 8, 4242.
the CO reduction catalytic activity of CbFDH was deactivated in the 17 Y. Tamaki and O. Ishitani, ACS Catal., 2017, 7, 3394.
2
presence when there was 20 times higher concentration of the 18 H. Takeda, C. Cometto, O. Ishitani and M. Robert, ACS
single-electron reduced MCABP or DCABP against CbFDH. From
Catal., 2017, 7, 70.
the docking simulation results, the oxidized and single-electron 19 G. Sahara, H. Kumagai, K. Maeda, N. Kaeffer, V. Artero,
reduced MCABP or DCABP equally bonded to the substrate binding
site of CbFDH. Therefore, it was predicted that in the higher
M. Higashi, R. Abe and O. Ishitani, J. Am. Chem. Soc., 2016,
138, 14152.
concentration region of MCABP or DCABP, the reduction rate of 20 J. Rohacova and O. Ishitani, Chem. Sci., 2016, 7, 6728.
CO to formate was slowed down by the oxidized and single- 21 J. M. Bolivar, I. Eisl and B. Nidetzky, Catal. Today, 2016, 259, 66.
2
electron reduced MCABP or DCABP competitively bonded to the 22 A. Alissandratos, H. K. Kim and C. J. Easton, Bioresour.
substrate binding site of CbFDH.
Technol., 2014, 164, 7.
2
2
3 Y. Amao and M. Ishikawa, Catal. Commun., 2007, 8, 523.
4 Y. Amao, S. Ikeyama, T. Katagiri and K. Fujita, Faraday
Discuss., 2017, 198, 73.
Conflicts of interest
25 H. Zheng, Y. Ohno, T. Nakamori and S. Suye, J. Biosci.
Bioeng., 2009, 107, 16.
There are no conflicts to declare.
26 J. R. Quayle, Methods Enzymol., 1966, 9, 360.
Acknowledgements
27 J. G. Ferry, FEMS Microbiol. Rev., 1990, 7, 377.
2
2
8 V. O. Popov and V. S. Lamzin, Biochem. J., 1994, 301, 625.
9 J. S. Blanchard and W. W. Cleland, Biochemistry, 1980,
This work was partially supported by a Grant-in-Aid for Scientific
Research on Innovative Areas ‘‘Artificial Photosynthesis (2406)’’,
19, 3543.
‘‘Innovations for Light-Energy Conversion (4906)’’, Fund for the
3
3
0 R. Obert and B. C. Dave, J. Am. Chem. Soc., 1999, 121, 12192.
1 B. El-Zahab, D. Donnelly and P. Wang, Biotechnol. Bioeng.,
Promotion of Joint International Research (Fostering Joint Inter-
national Research (B)) (19KK0144), and the joint usage/research
program of the Artificial Photosynthesis, Osaka City University.
2007, 99, 508.
3
2 R. K. Yadav, G. H. Oh, N.-J. Park, A. Kumar, K.-J. Kong and
J.-O. Baeg, J. Am. Chem. Soc., 2014, 136, 16728.
3 R. K. Yadav, J.-O. Baeg, G. H. Oh, N.-J. Park, K.-J. Kong,
J. Kim, D. W. Hwang and S. K. Biswas, J. Am. Chem. Soc.,
2012, 134, 11455.
3
Notes and references
1
2
3
4
2
Smart CO Transformation (SCOT) project in EU Seventh
Framework Program, http://scotproject.org.
M. Tamura, K. Ito, M. Honda, Y. Nakagawa, H. Sugimoto
and K. Tomishige, Sci. Rep., 2016, 6, 24038.
M. Tamura, K. Ito, Y. Nakagawa and K. Tomisahige, J. Catal.,
34 W. S. Choi, S. H. Lee, J. W. Ko and C. B. Park, ChemSusChem,
2016, 9, 1559.
35 S. K. Kuk, R. K. Singh, D. H. Nam, R. Singh, J. K. Lee and
C. B. Park, Angew. Chem., Int. Ed., 2017, 129, 3885.
36 R. Cazelles, J. Drone, F. Fajula, O. Ersen, S. Moldovan and
A. Galarneau, New J. Chem., 2013, 37, 3721.
37 X. Ji, Z. Su, P. Wang, G. Ma and S. Zhang, ACS Nano, 2015,
9, 4600.
2016, 343, 75.
M. Honda, M. Tamura, Y. Nakagawa and K. Tomishige,
Catal. Sci. Technol., 2014, 4, 2830.
5
6
J. Takaya and N. Iwasawa, J. Am. Chem. Soc., 2017, 139, 6074.
T. Suga, T. Saitou, J. Takaya and N. Iwasawa, Chem. Sci., 38 H. Choe, J. C. Joo, D. H. Cho, M. H. Kim, S. H. Lee,
017, 8, 1454.
K. D. Jung and Y. H. Kim, PLoS One, 2014, 9, e103111.
T. Suga, H. Mizuno, J. Takaya and N. Iwasawa, Chem. Commun., 39 J. H. Kim, D. H. Nam and C. B. Park, Curr. Opin. Biotechnol.,
014, 50, 14360.
2014, 28, 1.
M. Hatazawa, K. Nakabayashi, S. Ohkoshi and K. Nozaki, 40 S. K. Kuk, R. K. Singh, D. H. Nam, R. Singh, J. K. Lee and
Chem. – Eur. J., 2016, 22, 13677.
C. B. Park, Angew. Chem., Int. Ed., 2017, 56, 3827.
M. L. Lephoto, K. Nakano, D. Appavoo, B. O. Owaga, 41 H. Wu, C. Tian, X. Song, C. Liu, D. Yang and Z. Jiang, Green
K. Nozaki and J. Darkwa, Catalysts, 2016, 6, 17.
Chem., 2013, 15, 1773.
2
7
8
9
2
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2020
New J. Chem.

View Article Online
Paper
NJC
4
2 D. Mandler and I. Willner, J. Chem. Soc., Perkin Trans. 2, 1988, 63 Y. Amao, T. Kamachi and I. Okura, J. Photochem. Photobiol., A,
97. 1996, 98, 59.
3 I. Willner and D. Mandler, J. Am. Chem. Soc., 1989, 111, 1330. 64 Y. Amao, T. Kamachi and I. Okura, J. Porphyrins Phthalocyanines,
9
4
4
4 I. Willner, N. Lapidot, A. Riklin, R. Kasher, E. Zahavy and
E. Katz, J. Am. Chem. Soc., 1994, 116, 1428.
1998, 2, 201.
65 Y. Amao, T. Hiraishi and I. Okura, J. Mol. Catal. A: Chem.,
1997, 126, 13.
45 I. Willner, I. Willner and N. Lapidot, J. Am. Chem. Soc., 1990,
1
12, 6438.
66 Y. Amao, T. Hiraishi and I. Okura, J. Mol. Catal. A: Chem.,
1997, 126, 21.
4
4
6 R. Miyatani and Y. Amao, Biotechnol. Lett., 2002, 24, 1931.
7 R. Miyatani and Y. Amao, J. Mol. Catal. B: Enzym., 2004, 67 Q. Guo, L. Gakhar, K. Wickersham, K. Francis, A. Vardi-
27, 121.
Kilshtain, D. T. Major, C. M. Cheatum and A. Kohen,
4
4
8 R. Miyatani and Y. Amao, J. Jpn. Pet. Inst., 2004, 47, 27.
Biochemistry, 2016, 55, 2760.
9 Y. Amao, R. Abe and S. Shiotani, J. Photochem. Photobiol., A, 68 MyPresto version 5.0 program provided by Next Generation
015, 313, 149. Natural Product Chemistry (N PC).
0 I. Tsujisho, M. Toyoda and Y. Amao, Catal. Commun., 2006, 69 M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria,
2
2
5
5
5
7
, 173.
1 M. Kodaka and Y. Kubota, J. Chem. Soc., Perkin Trans. 2,
999, 891.
2 R. Wienkamp and E. Steckhan, Angew. Chem., Int. Ed. Engl.,
983, 22, 497.
M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, G. A.
Petersson, H. Nakatsuji, X. Li, M. Caricato, A. V. Marenich,
J. Bloino, B. G. Janesko, R. Gomperts, B. Mennucci, H. P.
Hratchian, J. V. Ortiz, A. F. Izmaylov, J. L. Sonnenberg,
D. Williams-Young, F. Ding, F. Lipparini, F. Egidi, J. Goings,
B. Peng, A. Petrone, T. Henderson, D. Ranasinghe, V. G.
Zakrzewski, J. Gao, N. Rega, G. Zheng, W. Liang, M. Hada,
M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida,
T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven,
K. Throssell, Montgomery JA Jr, J. E. Peralta, F. Ogliaro,
M. J. Bearpark, J. J. Heyd, E. N. Brothers, K. N. Kudin,
V. N. Staroverov, T. A. Keith, R. Kobayashi, J. Normand,
K. Raghavachari, A. P. Rendell, J. C. Burant, S. S. Iyengar,
J. Tomasi, M. Cossi, J. M. Millam, M. Klene, C. Adamo,
R. Cammi, J. W. Ochterski, R. L. Martin, K. Morokuma,
O. Farkas, J. B. Foresman and D. J. Fox, Gaussian 16, Revision
A.03, Gaussian, Inc., Wallingford, CT, 2016.
1
1
5
5
5
5
5
3 Y. Amao, Chem. Lett., 2017, 46, 780.
4 S. Ikeyama and Y. Amao, Chem. Lett., 2016, 45, 1259.
5 S. Ikeyama and Y. Amao, ChemCatChem, 2017, 9, 833.
6 Y. Amao and S. Ikeyama, Chem. Lett., 2015, 44, 1182.
7 S. Ikeyama, R. Abe, S. Shiotani and Y. Amao, Chem. Lett.,
2016, 45, 979.
5
5
6
8 S. Ikeyama, R. Abe, S. Shiotani and Y. Amao, Bull. Chem. Soc.
Jpn., 2018, 91, 1369.
9 S. Ikeyama, T. Katagiri and Y. Amao, J. Photochem. Photo-
biol., A, 2018, 358, 362.
0 A. Miyaji and Y. Amao, J. Photochem. Photobiol., A, 2000,
under submission.
6
6
1 S. Ikeyama and Y. Amao, New J. Chem., 2018, 42, 15556.
2 H. Slusarczyk, S. Felber, M.-R. Kula and M. Pohl, Eur. 71 Y. Izumi, H. Kanzaki, S. Morita, H. Futazuka and
70 R. Sato and Y. Amao, New J. Chem., 2020, 44, 11922.
J. Biochem., 2000, 267, 1280.
H. Yamada, Eur. J. Biochem., 1989, 182, 333.
New J. Chem.
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2020