Can formate dehydrogenase from: Candida boidinii catalytically reduce carbon dioxide, bicarbonate, or carbonate to formate?

Article abstract of DOI:10.1039/d0nj01183e

Formate dehydrogenase from Candida boidinii (CbFDH) reversibly catalyzes formate to carbon dioxide with the redox couple NAD+/NADH. There have been many studies on CbFDH-catalyzed oxidation of formate to carbon dioxide in the presence of NAD+. On the othe

Full text of DOI:10.1039/d0nj01183e

View Article Online
View Journal
NJC
New Journal of Chemistry
Accepted Manuscript
A journal for new directions in chemistry
This article can be cited before page numbers have been issued, to do this please use: R. Sato and Y.
Amao, New J. Chem., 2020, DOI: 10.1039/D0NJ01183E.
Volume 42
This is an Accepted Manuscript, which has been through the
Number
6
21 March 2018
Pages 3963-4776
Royal Society of Chemistry peer review process and has been
accepted for publication.
NJC
New Journal of Chemistry
rsc.li/njc
A journal for new directions in chemistry
Accepted Manuscripts are published online shortly after acceptance,
before technical editing, formatting and proof reading. Using this free
service, authors can make their results available to the community, in
citable form, before we publish the edited article. We will replace this
Accepted Manuscript with the edited and formatted Advance Article as
soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes to the
text and/or graphics, which may alter content. The journal’s standard
Terms & Conditions and the Ethical guidelines still apply. In no event
shall the Royal Society of Chemistry be held responsible for any errors
or omissions in this Accepted Manuscript or any consequences arising
from the use of any information it contains.
ISSN 1144-2546
PAPER
Chechia Hu, Tzu-Jen Lin et al.
Yellowish and blue luminescent graphene oxide quantum dots
prepared via a microwave-assisted hydrothermal route using
H2O2 and KMnO4 as oxidizing agents
rsc.li/njc

Please do not adjust margins
Page 1 of 5
New Journal of Chemistry
1
2
3
4
5
6
7
8
9
View Article Online
DOI: 10.1039/D0NJ01183E
Journal Name
COMMUNICATION
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Can formate dehydrogenase from Candida boidinii catalytically
reduce carbon dioxide, bicarbonate, or carbonate to formate?
Ryohei Satoa, Yutaka Amaoa,b *
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Formate dehydrogenase from Candida boidinii (CbFDH) dehydrogenases and natural co-enzyme NAD(P)+ or NAD(P)H is
reversibly catalyzes the formate to carbon dioxide with the considered. For example, the kinetic parameters for the
redox coupling NAD+/NADH. There have been many studies on Michaelis constants (Km) of NAD+ and NADH for CbFDH in the
CbFDH-catalyzed oxidation of formate to carbon dioxide in the formate oxidation to carbon dioxide and the reverse reaction
presence of NAD+. On the other hand, there are few studies on are determined. The Km values for NAD+ in the formate
the detailed mechanism of the carbon dioxide reduction to oxidation and NADH in the carbon dioxide reduction to CbFDH
formate catalyzed with CbFDH in the presence of NADH. In are estimated to be 50 and 2087 mM, respectively.6-8 Thus,
addition, it is not clear whether CbFDH can reduce carbon CbFDH can be activated by the lower concentration of NAD+,
dioxide, bicarbonate or carbonate to formate. In this study, compared with that of NADH (1/400), and the affinity of NAD+
formate production with CbFDH in the presence of NADH was for CbFDH is higher than that of NADH. There have been many
investigated in solutions with different ratios of carbon dioxide, studies on CbFDH-catalyzed oxidation of formate to carbon
bicarbonate and carbonate. The reaction rate of formate dioxide in the presence of NAD+.9-12 CdFDH catalyzes the stable
production with CbFDH increased in proportion to the oxidation of formate to carbon dioxide in the pH range of 5 to
concentration of carbon dioxide in the reaction solution. On the 10.11 Figure 1 shows the molar fraction of formic acid and
other hand, the formate production with CbFDH was formate under various pH range.13
suppressed in proportion to the concentration of bicarbonate
or carbonate in the reaction solution. Thus, CbFDH was found
to catalytically reduce only carbon dioxide to formate among
the three types of carbonate species.
Formate dehydrogenases (FDHs) are a set of biocatalysts that
catalyze the oxidation of formate to carbon dioxide, donating
the electrons to a second substrate (Sox) and the reverse
reaction of carbon dioxide to formate, donating the electrons to
a second substrate (Sred).1-6 Second substrates of FDH are
classified into (1) nicotinamide type (NAD+ or NADP+), (2)
cytochrome type (cytochrome b1 or c553), (3) quinone type and
(4) NAD+-ferredoxin type. Among these FDHs, FDH from
Candida boidinii (EC.1.2.1.2; CbFDH) is commercially available
and can be easily handled as a catalyst for the CO2 reduction to
formate. The relationship between the NAD(P)+-depend
Fig. 1. The molar fraction of formic acid and formate under various pH.
This means that the catalytic function of CbFDH for formate
oxidation is exhibited within the pH range in the stable presence
of formate ion. Moreover, the binding sites of NAD+ and
formate in CbFDH are clarified using crystal structure analysis
and docking simulation.11 The detailed kinetic parameters for
a. Graduate School of Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-
ku, Osaka 558-8585, Japan
b. Research Centre of Artificial Photosynthesis (ReCAP), Osaka City University, 3-3-138
Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan,
Email:amao@ocarina.osaka-cu.ac.jp
the catalytic formate oxidation with CbFDH in the presence of
NAD+ also are determined by the enzymatic reaction analysis.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 2 of 5
COMMUNICATION
Journal Name
1
2
3
4
5
6
7
8
Plummer and Busenberg.18 From these equations, theVcioewncAertinclteraOtniloinne
DOI: 10.1039/D0NJ01183E
of CO2*, bicarbonate and carbonate are represented as follows.
In contrast, there are few studies on the detailed
mechanism of the carbon dioxide reduction to formate
catalyzed with CbFDH in the presence of NADH. One of the
reasons is that the reaction rate with CbFDH in the presence of
NADH is low compared with that of NAD+ because of its low
carbon dioxide reduction activity. The catalytic efficiency, kcat
/
Km value for conversion of carbon dioxide to formate (170 × 103
M-1 min-1) was approximately 900 times lower than that of the
reverse reaction, i.e., the conversion of formate to carbon
dioxide (0.19 × 103 M-1 min-1), catalyzed with CbFDH using a
natural co-enzyme NAD+/NADH.6 We also found that the single-
electron reduced of MV (MV•) only acts as a co-enzyme for
CbFDH, and oxidized form of MV is not recognized for a co-
enzyme of CbFDH. Thus, the oxidation process with CbFDH
suppressed using oxidized form of MV as an electron carrier
molecule. Given that MV• effectively activates CbFDH, an
enzymatic kinetic analysis of carbon dioxide reduction to
formate with CbFDH in the presence of various reduced forms
of 4,4’- and 2,2’-bipyridinium salts are determined.14,15
Moreover, it is not clear whether carbon dioxide, bicarbonate
or carbonate is reduced in the formate production process
catalyzed by CbFDH. This is because, as shown in Figure 2,
carbon dioxide, bicarbonate or carbonate are mixed in the
reaction solution at a wide range of pH.16
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In this study, to clarify the carbonate species that CbFDH can
reduce, the formate production with CbFDH in the presence of
NADH was investigated in solutions with different ratios of
carbon dioxide.
In the carbon dioxide reduction to formate with CbFDH and
NADH(200 μM) and CbFDH (0 - 40 μM) obtained from Sigma-
Aldrich
in
carbon
dioxide
saturated
2-(N-
morpholino)ethanesulfonic acid (MES) buffer (pH 6.3), 50 mM
sodium bicarbonate containing MES buffer (pH 6.9) or 0.1 M
carbonate-bicarbonate buffer (pH 9.3) was reacted for 60 min
at 30.5 °C. The estimated formate concentration was calculated
using the absorbance change at 340 nm with a molar absorption
19
coefficient of NADH (ε340 = 6300 M-1 cm-1) using UV-visible
absorption spectroscopy (SHIMADZU, MultiSpec-1500) as
equivalent to the reduced concentration of NADH during
reaction. The total carbonate species Ctotal in the solution can
be measured as bicarbonate ions by adjusting the pH of the
eluent to 8.0 using an ion chromatography (Dionex ICS-1100;
electrical conductivity detector) with an ion exclusion column
(Thermo ICE AS1; column length: 9 x 150 mm; composed of a
Fig. 2. The molar fraction of carbon dioxide, bicarbonate and carbonate under
various pH.
Gaseous carbon dioxide can dissolve in aqueous media.17,18 The
hydrated carbon dioxide molecule reacts with water to produce 7.5 µm cross-linked styrene/divinylbenzene resin with
functionalized sulfonate groups).
carbonic acid (H2CO3). Dissociation of H2CO3 produces bicarbonate
and carbonate depending on the pH value as shown in Fig. 2. From
the low hydration equilibrium constant of H2CO3 in aqueous media,
the dissolved carbon dioxide consists of mostly hydrated carbon
dioxide together with a small amount of H2CO3. Thus, CO2* is used to
represent the two species of hydrated carbon dioxide and H2CO3 in
the aqueous chemical equilibrium equation. The Ctotal is defined as
the total concentration of all carbonate species of CO2*, bicarbonate
and carbonate. The first (K1) and second (K2) dissociation constants
of H2CO3 can be denoted by the following equations, respectively.
The K1 and K2 depend on the solution temperature according to
The concentrations of carbon dioxide, bicarbonate and
carbonate in the solution were determined from the Ctotal and
the molar fraction of carbon dioxide, bicarbonate or carbonate
in the pH value of solution from Fig. 2. The initial rate for the
estimated formate production (v0) was determined from the
gradient of the NADH consumption up to 60 min incubation.
Figure 3 shows the time dependence of the absorbance change
at 340 nm in the NADH(200 μM) and CbFDH (40 μM) in carbon
dioxide saturated MES buffer (pH 6.3)(1), in 50 mM sodium
bicarbonate containing MES buffer (pH 6.9) (2) and in 0.1 M
carbonate-bicarbonate buffer (pH 9.3) (3), respectively.
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 3 of 5
New Journal of Chemistry
Please do not adjust margins
Journal Name
COMMUNICATION
1
2
3
4
Next, a GTA buffer consisting of consisting of 3,3-dimethyl-
glutaric acid, tris(hydroxymethyl)aminomethane and 2-amino-
View Article Online
DOI: 10.1039/D0NJ01183E
2-methyl-1,3-propanediol available at a wide range of pH (3.5 –
10) was applied to the formate production with CbFDH to
eliminate the effect of the buffer on the reaction. The fractions
of carbon dioxide and bicarbonate in the solution were adjusted
by the bubbling time of carbon dioxide gas and the pH of the
GTA buffer. The initial rate for the estimated formate
production (v0) was determined from the gradient of the NADH
consumption up to 60 min incubation in the reaction system of
NADH(200 μM) and CbFDH (40 μM). Figure 5 shows the
relationship among the initial rate for the estimated formate
production (v0), concentration of carbon dioxide (circle), of
bicarbonate (square) , and of carbonate (triangle) with CbFDH.
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Fig. 3. Time dependence of the absorbance change at 340 nm in the NADH(200
μM) and CbFDH (40 μM) in carbon dioxide saturated MES buffer (pH 6.3)(1), in 50
mM sodium bicarbonate containing MES buffer (pH 6.9) (2) and in 0.1 M
carbonate-bicarbonate buffer (pH 9.3) (3), respectively.
From the results of Figure 3, the absorbance change at 340 nm
was decreased with increasing incubation time in all cases. The
concentration of carbon dioxide in the carbon dioxide saturated
MES buffer (pH 6.3), in 50 mM sodium bicarbonate containing
MES buffer (pH 6.9) and in 0.1 M carbonate-bicarbonate buffer
(pH 9.3) were estimated to be 30.7, 11.0 and 0 mM, respectively.
Figure 4 shows the relationship between initial rate for the
estimated formate production (v0) and the concentration of
CbFDH in the carbon dioxide saturated MES buffer (pH 6.3), in
50 mM sodium bicarbonate containing MES buffer (pH 6.9) and
in 0.1 M carbonate-bicarbonate buffer (pH 9.3).
Fig. 5. The relationship between the estimated formate production (v0),
concentration of carbonate species with CbFDH. ●: Carbon dioxide, ■: bicarbonate,
▲: carbonate.
As the concentration of carbon dioxide increased, the formate
production was tended to accelerate, while the increase in the
concentration of bicarbonate tended to suppress the formate
production. Moreover, no formate production was observed
regardless of the change in carbonate concentration. However, there
was no regular trend in these relationships. However, the
concentration of total carbonate species in each sample solution was
not uniform in various pH of GTA buffer.
Next, the relationship between the abundance ratio of carbon
dioxide, bicarbonate or carbonate to the total carbonate species in
the solution and the initial rate for the estimated formate
production (v0) was examined.
Figure 6 shows the relationship between the abundance ratio of
carbon dioxide (circle), of bicarbonate (square), and of carbonate
(triangle) to the total carbonate species and the initial rate for the
estimated formate production (v0) in the various pH of GTA
buffer containing NADH (200 μM) and CbFDH (40 μM).
As the abundance ratio of carbon dioxide to the total carbonate
species increased, the initial rate for the estimated formate
production (v0) was accelerated almost linearly. As the abundance
ratio of bicarbonate to the total carbonate species increased, in
contrast, the initial rate for the estimated formate production
(v0) was decelerated almost linearly. Moreover, no formate was
produced with increasing carbonate abundance to total carbonate
species. All these results suggest that CbFDH catalytically reduces
only carbon dioxide to formate, whereas bicarbonate or carbonate
Fig. 4. The relationship between the estimated formate production (v0) and the
concentration of CbFDH in the carbon dioxide saturated MES buffer (pH 6.3)
(circle), in 50 mM sodium bicarbonate containing MES buffer (pH 6.9) (square) and
in 0.1 M carbonate-bicarbonate buffer (pH 9.3) (triangle)
From Figs 3 and 4, the lower pH in the solution, the more
the formate production reaction with CbFDH proceeded. In
these experimental conditions, however, the formate
production enhancement by carbon dioxide and suppression by
bicarbonate and carbonate is trade-off because the
concentrations of each carbonate species are determined from
the mol fraction of carbonate species. Therefore, it is necessary
to investigate the effects of the concentrations and ratios of the
three carbonate species on formate production with CbFDH.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
New Journal of Chemistry
Page 4 of 5
COMMUNICATION
Journal Name
1
2
3
4
5
6
7
8
9
function as competitive inhibitors of carbon dioxide for the formate containing NAD+-dependent FDH.23,24 As far as we have surveyed,
View Article Online
production with CbFDH.
there are no reports of the detailed mecDhOanI:i1s0m.10o3f9/tDh0eNcJ0a1r1b8o3nE
dioxide reduction to formate catalyzed with CbFDH in the
presence of NADH. In addition, it is not clear whether CbFDH
can reduce carbon dioxide, bicarbonate or carbonate to
formate. In this study, we found that CbFDH catalytically
reduced only carbon dioxide to formate, and that other
carbonate species acted as inhibitors for CbFDH for the first
time.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Acknowledgement
This work was partially supported by Grant-in-Aid for Scientific
Research on Innovative Areas “Artificial Photosynthesis (2406)”,
“Innovations for Light-Energy Conversion (4906)” and
Fund for the Promotion of Joint International Research
(Fostering Joint International Research (B))(19KK0144).
Fig. 6. Relationship between the abundance ratio of carbon dioxide (●),
bicarbonate(■) or carbonate (▲) to the total carbonate species and the initial rate
for the estimated formate production (v0).
Figure 7 shows the binding model of formate (a),20-22 bicarbonate
(b), carbonate (c) and carbon dioxide (d) in the active site of CbFDH.
It has been reported that formate are trapped at three amino acid
residues (Asn119, Ile175 and Arg258) within the active site in CbFDH.
It is predicted that bicarbonate, carbonate or carbon dioxide is also
captured by the same three amino acid residues within the active site
in CbFDH as shown in Figure 7. Thus, bicarbonate and carbonate act
as competitive inhibitors of carbon dioxide for the formate
production with CbFDH.
Notes and references
1
V. I. Tishkov and V. O. Popov, Biochemistry (Moscow), 2004,
69, 1252.
R. Wichmann, C. Wandrey, A.F. Buckmann and M.R. Kula,
Biotechnol. Bioeng., 1981, 23, 2789.
2
3
4
W. Hummel and M. R. Kula, Eur. J. Biochem., 1989, 184, 1.
H. Zhao and W. A. van der Donk, Curr. Opin. Biotechnol., 2003,
14, 583.
5
6
7
8
9
J.G. Ferry, FEMS Microbiol. Rev. 1990, 7, 377.
Y. Amao, J. CO2 Utilization 2018, 26, 623．
Y. Amao, Chem. Lett. 2017, 46, 780.
Y. Amao, Sustainable Energy & Fuels, 2018, 2, 1928.
R. Castillo, M. Oliva, S. Martı´ and V. Moliner, J. Phys. Chem. B,
2008, 112, 10012.
10 J. Shi, Y. Jiang, Z. Jiang, X. Wang, X. Wang, S. Zhang, P. Hanac
and C. Yang, Chem. Soc. Rev., 2015, 44, 5981.
11 N. E. Labrou and D. J. Rigden, Biochem. J. 2001, 354, 455.
12 N. E. Labrou, D.J. Rigden and Y. D. Clonis, Eur. J. Biochem.
2000, 267, 6657.
13 W. M. Haynes, CRC Handbook of Chemistry and Physics, 92nd
ed.; CRC Press: Cleveland, 2011–2012
14 S. Ikeyama and Y. Amao, ChemCatChem 2017, 9, 833.
15 S. Ikeyama, R. Abe, S. Shiotani and Y. Amao, Bull. Chem. Soc.
Jpn. 2018, 91, 1369.
16 K. Teramura, K. Hori, Y. Terao, Z. Huang, S. Iguchi, Z. Wang, H.
Asakura, S. Hosokawa and T. Tanaka, J. Phys. Chem. C. 2017,
121, 8711.
17 E. Wilhelm, R. Battino and R.J. Wilcock, Chem. Rev., 1977, 77,
219.
18 L.N. Plummer and E. Busenberg, Geochim. Cosmochim. Acta,
1982, 46, 1011.
19 R. B. McComb, L. W. Bond, R. W. Burnett, R. C. Keech and G.
N. Bowers Jr. Clin. Chem. 1976, 22,141.
20 Q. Guo, L. Gakhar, K. Wickersham, K. Francis, A. Vardi-
Kilshtain, D. T. Major, C. M. Cheatum and A. Kohen,
Biochemistry, 2016, 55, 2760.
Fig. 7. The binding model of formate (a), bicarbonate (b), carbonate (c) and carbon
dioxide (d) in the active site of CbFDH. The 3D structure of CbFDH used Protein
Data Bank entry-5DN9(DOI: 10.2210/pdb5dn9/pdb.
From these results, CbFDH catalytically reduced only carbon
dioxide to formate. In molybdenum containing NAD+-dependent
FDH from Ralstonia eutropha with catalytic activity of carbon
dioxide reduction like CbFDH, the optimum pH was reported around
6.5. Also, carbon dioxide reduction is progressing in a pH region
between 7.5 and 8.5, thus, it is possible that bicarbonate or
carbonate can also be reduced to formate with molybdenum
21 K. Schirwitz, A. Schmidt and V. S. Lamzin, Protein Sci. 2007, 16,
1146.
22 J. N. Bandaria, S. Dutta, S. E. Hill, A. Kohen and C. M. Cheatum,
J. Am. Chem. Soc., 2007,130, 22.
23 D. Niks, J. Duvvuru, M. Escalona and R. Hille, J. Biol. Chem.,
2016, 291, 1162.
24 X. Yu, D. Niks, A. Mulchandani and R. Hille, J. Biol. Chem., 2017,
292,16872.
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 5 of 5
New Journal of Chemistry
View Article Online
DOI: 10.1039/D0NJ01183E
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
245x240mm (72 x 72 DPI)