Biocatalytic photosynthesis with water as an electron donor

Article abstract of DOI:10.1002/chem.201403301

Efficient harvesting of unlimited solar energy and its conversion into valuable chemicals is one of the ultimate goals of scientists. With the ever-increasing concerns about sustainable growth and environmental issues, numerous efforts have been made to develop artificial photosynthetic process for the production of fuels and fine chemicals, thus mimicking natural photosynthesis. Despite the research progress made over the decades, the technology is still in its infancy because of the difficulties in kinetic coupling of whole photocatalytic cycles. Herein, we report a new type of artificial photosynthesis system that can avoid such problems by integrally coupling biocatalytic redox reactions with photocatalytic water splitting. We found that photocatalytic water splitting can be efficiently coupled with biocatalytic redox reactions by using tetracobalt polyoxometalate and Rh-based organometallic compound as hole and electron scavengers, respectively, for photoexcited [Ru(bpy)₃]²⁺. Based on these results, we could successfully photosynthesize a model chiral compound (L-glutamate) using a model redox enzyme (glutamate dehydrogenase) upon in situ photoregeneration of cofactors.

Full text of DOI:10.1002/chem.201403301

DOI: 10.1002/chem.201403301
Communication
&
Photosynthesis
Biocatalytic Photosynthesis with Water as an Electron Donor
Jungki Ryu,[a, b] Dong Heon Nam,[a] Sahng Ha Lee,[a] and Chan Beum Park*[a]
Abstract: Efficient harvesting of unlimited solar energy
and its conversion into valuable chemicals is one of the ul-
timate goals of scientists. With the ever-increasing con-
cerns about sustainable growth and environmental issues,
numerous efforts have been made to develop artificial
photosynthetic process for the production of fuels and
fine chemicals, thus mimicking natural photosynthesis. De-
spite the research progress made over the decades, the
technology is still in its infancy because of the difficulties
in kinetic coupling of whole photocatalytic cycles. Herein,
we report a new type of artificial photosynthesis system
that can avoid such problems by integrally coupling bio-
catalytic redox reactions with photocatalytic water split-
ting. We found that photocatalytic water splitting can be
efficiently coupled with biocatalytic redox reactions by
using tetracobalt polyoxometalate and Rh-based organo-
metallic compound as hole and electron scavengers, re-
spectively, for photoexcited [Ru(bpy)3]2+. Based on these
results, we could successfully photosynthesize a model
chiral compound (l-glutamate) using a model redox
enzyme (glutamate dehydrogenase) upon in situ photore-
Figure 1. Illustrative comparison between a) natural photosynthesis and
generation of cofactors.
b) enzyme-coupled artificial photosynthesis for green synthesis of valuable
chemicals. In the light reaction, dye molecules (chlorophylls and carotenoids
in natural photosynthesis versus [Ru(bpy)3]2+ in the present study) are elec-
tronically excited upon visible-light irradiation and transfer electrons to the
reaction center (NADP+ oxidoreductases versus M) to regenerate nicotina-
mide cofactor NAD(P)H by reducing its oxidized counterpart NAD(P)+. The
photocatalytic system oxidized by donating electrons to the reaction center
is reduced (and recycled) with electrons supplied from the oxidation of
water with a tetranuclear water splitting catalyst (Mn3CaO4 cluster versus
[Co4(H2O)2(PW9O34)2]10À). The regenerated cofactors are then consumed in
Photosynthesis is a series of photophysical and photochemical
processes crafted by nature to produce essential biological
fuels by converting solar energy into the form of chemical
bonds.[1] It has many fascinating features such as efficient har-
vesting of unlimited solar energy and its use, capabilities to
the synthesis of complex organic compounds (sugar versus chiral com-
pounds) by redox enzymes in the dark reaction.
perform complex catalytic reactions, and environmental com-
patibility.[1,2] These features are achieved by delicate photosyn-
thetic machinery, where various active components (e.g., light-
harvesting dyes, charge-separation components, and redox
catalysts) are assembled at nanoscale precision within mem-
brane-bound protein complexes, enabling efficient separation
of charges and transfer of redox power.[1a] Based on the under-
standing of the natural photosynthesis mechanism (also
known as the Z scheme, Figure 1a), researchers found that, in
principle, chemical fuels such as hydrogen and hydrocarbons
can be produced by photocatalytic splitting of water and the
reduction of carbon dioxide, respectively, the so-called artificial
photosynthesis.[3] With the recent energy crisis as well as ever-
increasing environmental concerns, numerous efforts have
been made to develop a highly efficient artificial photosynthe-
sis system. For example, it has been reported that efficient hy-
drogen-evolution[4] and water-splitting[5] catalysts can be devel-
oped by mimicking the metal–sulfur cluster of hydrogenase
enzymes[6] and the tetranuclear Mn3Ca(m-O)4 cluster of the
oxygen-evolving complex[2b,7] in photosystem II, respectively.
Recently, Nam and co-workers reported that the light-harvest-
ing efficiency of dyes can be significantly enhanced by assem-
[a] Dr. J. Ryu, D. H. Nam, Dr. S. H. Lee, Prof.Dr. C. B. Park
Department of Materials Science and Engineering
Korea Advanced Institute of Science and Technology (KAIST)
335 Science Road, Daejeon 305-701 (Republic of Korea)
E-mail: parkcb@kaist.ac.kr
[b] Dr. J. Ryu
Current address:
School of Energy and Chemical Engineering
Ulsan National Institute of Science and Technology (UNIST)
50 UNIST-gil, Ulsan 689-798 (Republic of Korea)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201403301.
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bling them in a desired arrange-
ment facilitating electronic cou-
pling and charge transport be-
tween them.[8]
Despite the significant prog-
ress made over the past de-
cades, current studies on artifi-
cial photosynthesis are still at
a very early stage and require
a number of scientific break-
throughs for practical applica-
tions. For example, only a few
studies have reported the devel-
opment of an integrated photo-
synthetic system,[9] while most
current studies have been limit-
ed to testing the performance of
each component in the form of
incomplete half reactions owing
to the difficulties in kinetic cou-
pling between catalytic cycles.[10]
In addition to scientific issues,
there still remain a number of
technological (e.g., CO2 capture,
separation and storage of gas-
phase solar fuels, and develop-
ment of cheap, efficient, and reli-
able fuel cells) and economic
issues (e.g., efficiency and rela-
tive production cost compared
to conventional technology) to
be addressed for practical appli-
cation of artificial photosynthe-
sis.[3b]
Figure 2. a) Diagram showing redox potentials for all the half reactions in the present study. Photoexcited
[Ru(bpy)3]2+ has a wide redox potential window, which is large enough to oxidize water and reduce M for the re-
generation of NADH cofactors. Note that redox potential of M was determined by cyclic voltammetry (see the
Supporting Information, Figure S2 and Table S1). b) Various cofactor regeneration catalysts M having different
functional groups on the bipyridine ligand. Note that M can regenerate enzymatically active 1,4-NAD(P)H and pre-
vent the formation of inactive isomers and dimers of NAD(P)H.[12a] c) Diagram showing the whole cycle for the
photocatalytic regeneration of NAD(P)H cofactors. Note that a key factor for the successful synthesis of target
chemicals through complete artificial photosynthesis—not in the form of half reactions—is how to connect the
whole catalytic cycles efficiently by facilitating the directional flow of electrons from photocatalytic oxidation of
water by Co4POM, and then the reduction of cofactors by M, to redox biocatalysis.
Herein, we report the develop-
ment of a homogeneous photo-
synthesis system that can pro-
duce fine chemicals through an integral coupling of biocatalyt-
ic reactions with photocatalytic water splitting. The new type
of artificial photosynthetic process is conceptually very similar
to natural photosynthesis, as illustrated in Figure 1. Both pro-
cesses are composed of independent photocatalytic and bio-
catalytic reactions connected by redox cycles of nicotinamide
cofactors, NAD(P)H. The artificial photosynthetic system is de-
signed to regenerate NAD(P)H from its oxidized counterpart,
NAD(P)+, through complete photocatalytic redox reaction (i.e.,
not in the form of half reactions) for the synthesis of complex
chemicals, such as chiral alcohols and natural/unnatural amino
acids, by using redox enzymes at the expense of in situ regen-
erated cofactors as a redox equivalent. By utilizing electrostatic
interactions between light-harvesting dyes and redox catalysts,
we could facilitate efficient separation of charges and direc-
tional flow of electrons throughout the overall catalytic cycles,
enabling simultaneous photocatalytic oxidation of water and
reduction of cofactors.
regeneration catalyst, and a water-splitting catalyst. We select-
ed [Ru(bpy)3]2+ as a light-harvesting photosensitizer because
of its near unity quantum yield, relatively long excited lifetime
(t~1 ms), chemical stability, and its excellent redox properties
(Figure 2a).[11] A Rh-based electron mediator (M)[12] and tetraco-
balt polyoxometalate [Co4(H2O)2(PW9O34)2]10À (Co4POM)[5b,c]
were used as a cofactor regeneration catalyst (Figure 2b and
Figures S1 and S2 in the Supporting Information) and a bio-
mimetic tetranuclear water-splitting catalyst (see the Support-
ing Information, Figure S3–S6), respectively. Before assembling
the integrated photosynthetic system, we studied the efficien-
cies of charge transfer from photoexcited [Ru(bpy)3]2+ dyes to
the water-splitting catalyst Co4POM and to the conventional
cofactor regeneration catalyst, [Cp*Rh(bpy)(H2O)]2+ (Mbpy), by
analyzing photophysical and chemical properties of
[Ru(bpy)3]2+ under various conditions. [Ru(bpy)3]2+ has strong
absorbance and photoluminescence (PL) in the visible light
region (at around 450 and 600 nm, respectively). By measuring
static and dynamic PL spectra (Figure 3a–b and Figure S7 in
the Supporting Information), we could calculate the PL lifetime
The photosystem for the regeneration of nicotinamide co-
factors consisted of light-harvesting dye [Ru(bpy)3]2+, a cofactor
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[Ru4(m-O)4(m-OH)2(H2O)4(g-
SiW10O36)2]10À (Ru4POM; 2.1–3.6ꢁ
109 mÀ1 À1).[5d,e] According to
s
Scandola and co-workers,[5d,e]
Ru4POM can act as an excellent
hole scavenger for photogener-
ated oxidant. Owing to the diffi-
culties in kinetically coupling
photocatalytic oxidation with re-
duction half reactions, many re-
searchers previously studied
a half reaction by using a high
concentration of sacrificial elec-
tron donor (e.g., triethylamine,
triethanolamine, and ascorbic
acid) or acceptors (e.g., H2O2,
S2O82À).[5b–e,10b,c]
We found that the kinetic cou-
pling of photocatalytic redox
cycles for the complete artificial
photosynthesis without sacrificial
agents can be achieved by en-
hancing the charge-transfer effi-
ciency of the rate-limiting step
(i.e., between photoexcited dye
and M) through chemical modifi-
cation of M (Figure 2b–c). It was
Figure 3. a) Static and b) dynamic photoluminescence spectra of [Ru(bpy)3]2+ dyes in the presence of M having
different functional groups. c) Photoregeneration of NADH cofactors achieved by water-splitting reaction with dif-
ferent M. d) Comparison between efficiencies of electrochemical and photochemical regeneration of NADH by
using different M.
Table 1. Photoluminescence lifetime of [Ru(bpy)3]2+ in the presence of
hypothesized that M compounds with negatively chargeable
functional groups, such as COOH, can facilitate the interaction
between positively charged [Ru(bpy)3]2+ and M. As a proof of
concept, we synthesized four kinds of cofactor regeneration
catalysts that have a different functional group (R) on their bi-
pyridine ligand (Mbpy, M4-dmbpy, M5-dmbpy, and M4-dcbpy; Figure 2b)
and investigated their effect on the quenching rate constant.
Note that M compounds with other common functional
groups, such as NO2, NH2, SO3H, and OH, were excluded in this
study because they exhibit poor catalytic activity and/or selec-
tivity.[12b] According to our results (Figure 3a–b and Table 1),
the nature of the functional groups on the bipyridine ligand of
M significantly influences the quenching rate constant and the
charge transfer efficiency between photoexcited [Ru(bpy)3]2+
and M. M4-dcbpy had a high quenching rate constant (1.08–
2.77ꢁ109 mÀ1 sÀ1), comparable to that of Co4POM, while other
catalysts had much lower rate constants. These results support
our hypothesis that electrostatic interaction between the dye
and M can be used to improve the charge-transfer efficiency
between them. Note that the pKa value of COOH groups in the
4-dcbpy ligand was estimated to be 3.3–3.9 by Chemicalize
(ChemAxon, Budapest, Hungary); thus, the 4-dcbpy ligand
should be negatively charged at pH 7.4, as used in the present
study. Our hypothesis is also supported by recent studies on
the quenching behaviors of [Ru(bpy)3]2+. According to litera-
ture,[5d,e,13] positively charged [Ru(bpy)3]2+ can strongly interact
different quenchers and their quenching rate coefficients.
[e]
Quencher
PL lifetime[d]
Quenching rate coefficient kq [MÀ1 sÀ1
]
t [ns]
I0/I
t0/t
control[a]
370.97
330.14
352.65
264.58
350.70
349.30
N.A.
N.A.
Co4POM[b]
6.97ꢁ109
3.23ꢁ108
2.77ꢁ109
6.67ꢁ108
1.81ꢁ108
1.33ꢁ109
1.40ꢁ108
1.08ꢁ109
1.56ꢁ108
1.67ꢁ108
[c]
Mbpy
[c]
M4-dcbpy
[c]
M4-dmbpy
[c]
M5-dmbpy
[a] 250 mm [Ru(bpy)3]2+ alone. [b] 250 mm Co4POM. [c] 1 mmM. [d–e] See
the Experimental Section for details on the calculation of PL lifetime and
quenching rate coefficient. Note that [Ru(bpy)3]2+ has two different path-
ways for the quenching of its PL: oxidative quenching by electron accept-
or like M and reductive quenching by electron donor like Co4POM.[11b]
The last two columns are the values of quenching rate coefficients (kq)
calculated in terms of different unitless variables (i.e., I0/I and t0/t).
.
and the quenching rate constant of [Ru(bpy)3]2+ in the pres-
ence and absence of quenchers (as summarized in Table 1) and
indirectly compare the efficiency of charge separation from ex-
cited [Ru(bpy)3]2+ to each quencher (i.e., Co4POM and Mbpy).
According to our results, the quenching rate constant of
Co4POM (1.33–6.97ꢁ109 mÀ1
s
À1) is much higher than that of
Mbpy (1.40–3.23ꢁ108 mÀ1 À1) by approximately an order of
s
magnitude, thus implying that the rate-limiting step is the
electron transfer from photoexcited [Ru(bpy)3]2+ to Mbpy (i.e.,
reduction half reaction). The rate constant for Co4POM calculat-
ed in the present study was found to be comparable to that
reported previously for a similar tetranuclear polyoxometalate
2À
with negatively charged species (e.g., S2O8 and Ru4POM)
even at a high ionic strength through electrostatic interactions,
resulting in a high degree of PL quenching and a high quench-
ing rate constant.
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To demonstrate the kinetic coupling between the photoca-
talytic oxidation of water by Co4POM and the reduction of nic-
otinamide cofactors by M, we carried out experiments con-
cerning photocatalytic regeneration of cofactors by using M
compounds containing different functional groups in the ab-
sence and presence of Co4POM under the same conditions, so
as to compare their regeneration efficiencies. As shown in Fig-
ure 3c–d, it was found that the efficiency of the photoregener-
ation of nicotinamide cofactor highly depended on the type of
the functional group on the bipyridine ligand of M as well as
the presence of Co4POM catalyst. Nicotinamide cofactor was
efficiently photoregenerated only when M4-dcbpy and Co4POM
were used together, demonstrating the kinetic coupling be-
tween photocatalytic redox reactions. The order of the efficien-
cy of NADH photoregeneration (FM) was M4-dcbpy @M4-dmbpy
>
M
M
bpy >M5-dmbpy, whereas the order of catalytic activity (aM) was
bpy >M4-dcbpy >M5-dmbpy >M4-dmbpy (see the Supporting Infor-
mation, Figure S8). Note that the catalytic activity of each M
was defined as the efficiency of NADH regeneration by electro-
chemical methods[14] (see experimental details in the Support-
ing Information). The observed mismatch between the orders
of aM and FM is attributed to different efficiencies of charge
separation depending on the type of functional group on M.
We found that FM is almost linearly proportional to the prod-
uct of aM and the charge separation efficiency, hCS, which is as-
sumed to be proportional to the degree of PL quenching (qM)
(see the Supporting Information, Figure S8). Note that FM can
be expressed as in the following equation: FM =
khLHhCSaMaCo4POM, where k is a proportional constant, hLH is the
light-harvesting efficiency of [Ru(bpy)3]2+, and aCo4POM is the
catalytic activity of Co4POM against water splitting.
Figure 4. Photoenzymatic synthesis of l-glutamate using glutamate dehy-
drogenase. a) Scheme showing catalytic cycles of the photoenzymatic reac-
tion. b) Photoregeneration of NADH using different electron donors (i.e.,
water and TEOA). c) Photoenzymatic synthesis of l-glutamate through in
situ regeneration of NADH by complete artificial photosynthesis: simultane-
ous photocatalytic oxidation of water and reduction of NADH cofactors.
Note that before the regeneration test, reaction media were degassed.
compounds can also regenerate other enzyme cofactors, such
as NADPH and FADH,[12] in principle, our system can be applied
to any kind of cofactor-dependent redox biocatalysis for the
synthesis of complex chiral compounds and valuable drug in-
termediates reported in the literature.[15] In addition to industri-
al potential, we believe that the present study is a scientific
milestone as the first example of artificial photosynthesis ach-
ieved by the integral coupling of water splitting with a biocata-
lytic reaction.
Based on these results, we attempted a coupling of nicotina-
mide cofactor photoregeneration with redox enzymatic syn-
thesis of l-glutamate, a model chiral compound, by using l-
glutamate dehydrogenase (GDH), a model redox enzyme (Fig-
ure 4a) that critically requires NADH for the reduction reaction.
For comparison, we carried out photoenzymatic reactions with
two types of electron donors: water with a Co4POM water-
splitting catalyst and a common electron donor triethanola-
mine (TEOA) without the catalyst. According to our results (Fig-
ure 4b), M4-dcbpy has the highest efficiency for the photoregen-
eration of NADH regardless of the type of electron donor. The
regeneration efficiency (10.7%) obtained with Co4POM was
much lower than that by using TEOA (70.7%); however, con-
sidering the low concentration of Co4POM (250 mm) and the
presence of molecular oxygen when using water as an electron
donor, it was thought that the overall efficiency of the system
using Co4POM is higher than or at least comparable to that
using TEOA even though the oxidation of water (four-electron
process) is much more difficult than that of TEOA (one-electron
process). It was previously reported that the catalytic activity
of M can be significantly degraded by molecular oxygen.[12]
Based on these results, we further tested only M4-dcbpy for the
photoenzymatic synthesis of l-glutamate. As shown in Fig-
ure 4c, we could successfully photosynthesize l-glutamate
through reductive amination of a-ketoglutarate under visible
light by using water as an electron donor. Considering that M
In summary, we have developed a new type of artificial pho-
tosynthesis system that couple photocatalytic and biocatalytic
cycles by using water as an electron donor under ambient con-
ditions. The system was designed to regenerate (i.e., reduce)
cofactors by photocatalytic cycles and to consume (i.e., oxi-
dize) the regenerated cofactors by biocatalytic cycles for enzy-
matic synthesis of target chemicals. The photocatalytic system
for cofactor regeneration consisted of light-harvesting dye
[Ru(bpy)3]2+, water splitting catalyst Co4POM as a hole scav-
enger, and a cofactor regeneration catalyst M as an electron
scavenger. According to our dynamic and static photolumines-
cence studies, charge-transfer efficiency from the photoexcited
dyes to conventional M is much lower than that to Co4POM. M
compounds with a negatively chargeable COOH group led to
a significant enhancement of the efficiency of the charge trans-
fer from the dyes to M through electrostatic interaction, allow-
ing kinetic coupling of photocatalytic redox cycles. We could
successfully regenerate nicotinamide cofactors through simul-
taneous photocatalytic oxidation of water and the reduction of
cofactors, which were then used for redox enzymatic synthesis
of l-glutamate. Considering the high potential of redox bioca-
talysis for the synthesis of complex chemicals, this study can
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Information, Figure S4) and [Ru(bpy)3]2+ were measured by using
a Jasco V-650 spectrophotometer (Tokyo, Japan).
provide a new horizon for the realization of artificial photosyn-
thesis technology.
Cyclic voltammetry
Experimental Section
Electrochemical activities of Co4POM (see the Supporting Informa-
tion, Figure S5) and M compounds (see the Supporting Informa-
tion, Figure S2) were examined by cyclic voltammetry by using
a 273 A potentiostat/galvanostat (Princeton Applied Research, Oak
Ridge, TN) in a 3-electrode configuration: GC disk (0.03 cm2) as
a working electrode, Pt wire as a counter electrode, and Ag/AgCl
as a reference electrode.
Materials
All chemicals, including RhCl3·3H2O, hexamethyldewarbenzene
(HMDB), 2,2’-bipyridine (bpy), 4,4’-dimethyl-2,2’-bipyridine (4-
dmbpy), 5,5’-dimethyl-2,2’-bipyridine (5-dmbpy), 4,4’-dicarboxy-
2,2’-bipyridine (4-dcbpy), methanol, N,N-dimethylformamide, dieth-
yl ether, chloroform, benzene, NAD+, triethanolamine (TEOA), a-ke-
toglutarate, glutamate dehydrogenase (GDH), Na2WO4·3H2O,
Na2HPO4·7H2O, Co(NO3)2·6H2O, and tris(2–2’-bipyridyl)dichlororu-
thenium(II) hexahydrate (Ru(bpy)3Cl2·6H2O) were purchased from
Sigma–Aldrich (St. Louis, MO).
Photocatalytic splitting of water by using Co4POM
Photocatalytic oxygen evolution from water by Co4POM was tested
under the following conditions: 1 mm [Ru(bpy)3]2+ as a light-har-
vesting dye, 10 mm Co4POM as a water-splitting catalyst, and 5 mm
Na2S2O8 as an electron scavenger in 80 mm sodium borate buffer
(pH 8.0). Before visible-light irradiation, the above solution was
purged with Ar gas for 30 min in a gas-tight vial. For the analysis
of O2 evolution, 250 mL of samples were taken from the headspace
of the vial every 10 min after light irradiation by using an air-tight
syringe and measured with an Agilent 7890 A gas chromatograph
equipped with a thermal conductivity detector (see the Supporting
Information, Figure S6).
Synthesis of [Cp*Rh(bpy)(H2O)]2+ and derivatives thereof
Details of the method for the synthesis of [Cp*Rh(bpy)(H2O)]2+ and
its derivatives can be found elsewhere.[12] Briefly, RhCl3·3H2O
(1 mmol) and hexamethyldewarbenzene (HMDB) (2.5 mmol) were
dissolved in anhydrous methanol and then refluxed at 658C for
15 h under vigorous stirring in Ar atmosphere. A brown solid prod-
uct was recovered by rotary evaporation and then redissolved in
chloroform. Byproducts were removed by washing with an excess
amount of deionized water. The intermediate product [Cp*RhCl2]
(Cp*=pentamethylcyclopentadienyl) was further purified by crys-
tallization in chloroform/benzene (1:10). [Cp*Rh(bpy)(H2O)]2+ and
its derivatives (M) were prepared by mixing [Cp*RhCl2] and two
equimolar amounts of bipyridine derivatives (e.g., bpy, 4-dmbpy, 5-
dmbpy, or 4-dcbpy) in methanol or N,N-dimethylformamide for
1 h. Final products were separated by crystallization in methanol/
diethyl ether (1:5), dried under vacuum overnight, and character-
ized by FT-IR spectroscopy (see the Supporting Information, Fig-
ure S1) and cyclic voltammetry (see the Supporting Information,
Figure S2).
Photoluminescence characterization of [Ru(bpy)3]2+
Photoluminescence (PL) quenching of [Ru(bpy)3]2+ (250 mm in
0.1m phosphate buffer, pH 7.4) was studied by measuring its static
and dynamic (time-resolved) PL spectra in the presence and ab-
sence of quenchers: M (1 mm; Figure 3) and Co4POM (250 mm; see
the Supporting Information, Figure S7). Static PL spectra were mea-
sured with a Shimadzu RF5301-PC spectrofluorometer (Tokyo,
Japan). Dynamic PL spectra were measured at 600 nm under exci-
tation at 450 nm with a pulsed laser diode (pulsed width <1.3 ns)
by using a Horiba NanoLog spectrofluorometer (Kyoto, Japan). The
PL lifetime (t) of [Ru(bpy)3]2+ was determined by fitting time-re-
solved PL decay curves by using the following equation: I=
Aexp(Àt/t) where t is a given time, A is an exponential prefactor
that was normalized to unity, and t is the lifetime (Table 1).
Quenching rate coefficients were calculated by the Stern–Volmer
relationship: I0/I=t0/t=1+kqt0[Q] where I0 and I are the PL inten-
sity of [Ru(bpy)3]2+ without and with a quencher, respectively, t0
and t are the PL lifetime of [Ru(bpy)3]2+ without and with
a quencher, respectively, kq is the quenching rate coefficient, and
[Q] is the concentration of the quencher.
Synthesis of water-splitting catalyst [Co4(H2O)2(PW9O34)2]10À
(Co4POM)
Tetracobalt polyoxometalates, [Co4(H2O)2(PW9O34)2]10À
, was pre-
pared according to the literature.[5b] In brief, the sodium salt of
Co4POM (Na10-Co4POM) was synthesized by refluxing an aqueous
solution (100 mL, pH 7) of Na2WO4·3H2O (0.108 mol), Na2WO4·3H2O
(0.012 mol), Na2HPO4·7H2O, and Co(NO3)2·6H2O (0.024 mol) at
1008C for 2 h. The resulting Co4POM was separated by precipita-
tion by addition of excessive amounts of NaCl, further purified by
recrystallization from hot water, and characterized by various ana-
lytical tools, such as FT-IR/Raman spectroscopy, UV/Vis absorption
spectroscopy, and cyclic voltammetry (see the Supporting Informa-
tion, Figures S3–S6).
Photochemical reactions
Photoregeneration of NADH was carried out in the presence and
absence of the water-splitting catalyst Co4POM. The reaction
medium (pH 7.4) consisted of NAD+ (1 mm),
M
(1 mm),
Spectroscopic analysis
[Ru(bpy)3]2+ (250 mm), and phosphate buffer (100 mm). Co4POM
(250 mm) can be additionally dissolved in the reaction medium for
the photoregeneration of NADH coupled with water splitting. For
comparison, triethanolamine (TEOA, 1m) was used as a sacrificial
electron donor in the absence of Co4POM. The regeneration of
NADH from NAD+ was monitored by measuring the change of op-
tical density of the reaction medium at 340 nm. Note that the
molar absorption coefficient of NADH at 340 nm is 6.22ꢁ
103 mÀ1 cmÀ1. For the synthesis of model chiral compound l-gluta-
mate, NADH-dependent glutamate dehydrogenase (GDH) was
Cofactor regeneration catalyst M (see the Supporting Information,
Figure S1) and water-splitting catalyst Co4POM (see the Supporting
Information, Figure S3) were analyzed by using a Hyperion 3000
FT-IR spectrometer (Bruker Optics, Ettlingen, Germany) by the KBr
pellet method under vacuum and a LabRAM HR UV/Vis/NIR disper-
sive Raman microscope (Horiba Jobin Yvon, Kyoto, Japan) under
the following conditions: number of scan, 128; scan range, 400–
4,000 cmÀ1; resolution, 4 cmÀ1; Ar ion laser (514.5 nm) for Raman
spectroscopy. Absorbance spectra of Co4POM (see the Supporting
Chem. Eur. J. 2014, 20, 12020 – 12025
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Communication
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A constant potential of À0.8 V versus Ag/AgCl was applied to elec-
trochemically regenerate NADH cofactors.[14] The reaction medium
for the electrochemical regeneration of NADH cofactors was com-
posed of 1 mm NAD+ and 1 mm M in 0.1m phosphate buffer
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Acknowledgements
This study was supported by grants from the National Re-
search Foundation (NRF) through the National Leading Re-
search Laboratory (NRF-2013R1A2A1A05005468) and the Intel-
ligent Synthetic Biology Center of Global Frontier R&D Project
(2011-0031957), Republic of Korea
Keywords: biocatalysis
enzymes · water splitting
· NADH · photosynthesis · redox
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Received: April 29, 2014
Published online on August 1, 2014
Chem. Eur. J. 2014, 20, 12020 – 12025
www.chemeurj.org
12025
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim