Bioelectrocatalytic reduction of O2 catalyzed by CueO from escherichia coli adsorbed on a highly oriented pyrolytic graphite electrode

Article abstract of DOI:10.1246/cl.2007.132

CueO from Escherichia coli, a member of the multi-copper oxidase (MCO) family, was examined as a direct electron transfer-type bioelectrocatalyst for the four-electron reduction of O₂. Although CueO requires the fifth copper located near the type I Cu site to exhibit its oxidase activity (Roberts et al., 2003), it has been found that CueO receives electrons directly from electrodes even in the absence of the fifth copper. The fact indicates that electrons are transferred directly from electrodes to the type I Cu site. Furthermore, the catalytic current density of as large as about -4 mA cm ^-2 was successfully observed with a rotating pyrolytic graphite electrode at pH 5. CueO is found to be superior to other MCOs in view of the catalytic activity and is an important candidate as a catalyst of the cathode in biofuel cells. Copyright

Full text of DOI:10.1246/cl.2007.132

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Chemistry Letters Vol.36, No.1 (2007)
Bioelectrocatalytic Reduction of O Catalyzed by CueO from Escherichia coli
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Adsorbed on a Highly Oriented Pyrolytic Graphite Electrode
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1;2
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Yuko Miura, Seiya Tsujimura, Yuji Kamitaka, Shinji Kurose, Kunishige Kataoka,
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Takeshi Sakurai, and Kenji Kano
Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502
Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192
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(Received September 29, 2006; CL-061141; E-mail: seiya@kais.kyoto-u.ac.jp)
CueO from Escherichia coli, a member of the multi-copper
that the catalytic reaction does not generate any reactive oxygen
species.
oxidase (MCO) family, was examined as a direct electron trans-
fer-type bioelectrocatalyst for the four-electron reduction of O2.
Although CueO requires the fifth copper located near the type I
Cu site to exhibit its oxidase activity (Roberts et al., 2003), it has
been found that CueO receives electrons directly from electrodes
even in the absence of the fifth copper. The fact indicates that
electrons are transferred directly from electrodes to the type I
Cu site. Furthermore, the catalytic current density of as large
as about ꢁ4 mA cmꢁ was successfully observed with a rotating
pyrolytic graphite electrode at pH 5. CueO is found to be supe-
rior to other MCOs in view of the catalytic activity and is an
important candidate as a catalyst of the cathode in biofuel cells.
In this study, we examined the electrochemical behavior of
CueO from the viewpoint of the bioelectrocatalytic activity in
the O2 reduction. To our knowledge, this is the first paper on
the electrochemical study on CueO. The catalytic behavior of
CueO was compared with other MCOs, such as BOD and fungal
laccase.
The gene coding for precursor CueO gene was designed to
0
2
have sequence coding EcoRI restriction site at 5 -site and the
0
6ꢂHis-tag and BamHI restriction site at 3 -end. The gene was
inserted into pUC18, with which E. coli BL21 (DE3) was trans-
formed by heat shock method. Expression and purification of
1
2
CueO were carried out as described previously. The enzyme
concentrations were determined using a molar absorption coeffi-
ꢁ1
ꢁ1
ꢁ3
Copper is essential but toxic in mammals and bacteria.
CueO (Cu efflux oxidase) is proposed to work for copper homeo-
stasis in Escherichia coli, although the details of the mechanism
cient at 612 nm: "612 ¼ 5800 M cm (M = mol dm ). All
chemicals used in this study were of analytical reagent grade,
and all solutions were prepared with distilled water.
1
remain unknown. CueO is a monomeric protein with a molecu-
2
Voltammetry was performed using a BAS CV-50W electro-
chemical analyzer. Highly oriented pyrolytic graphite electrodes
(edge plane) (HOPGE) were kindly donated by Prof. Abe in
Kyoto University, were cut, and set into the PEEK tube (BAS)
with insulator. The handmade electrode was polished with an
emery paper (#600), rinsed with distilled water, sonicated in dis-
tilled water, and was attached to the shaft of an electrode rotator
(RDE-2, BAS) for rotating disk electrode (RDE) measurements.
lar mass of ca. 53.4 kDa. The enzyme belongs to the family of
3
multi-copper oxidases (MCO) including laccase, bilirubin
oxidase (BOD), ascorbate oxidase, ceruloplasmin, CotA, etc.
Copper atoms in MCOs are classified into three types based on
spectroscopic and magnetic properties: the type I Cu is EPR-
detectable and gives a blue color in the oxidized (cupric) state,
the type II Cu is also EPR-detectable but colorless, and the
type III Cu center composed of two copper ions bridged through
hydroxide is EPR-silent and shows an absorption band at 330 nm
2
The projected surface area of the HOPGE was 0.0337 cm . The
water-jacketed electrolysis cell had a platinum wire counter
3
in the optical spectrum. In MCOs, the type I Cu site is the inlet
electrode and an AgjAgCljsat. KCl reference electrode. Meas-
ꢃ
of electrons from electron-donating substrates and transfers the
electrons to the trinuclear center composed of one type II Cu
and two type III Cu atoms. The trinuclear center (type II–III
Cu cluster) serves as a catalytic site to reduce O2 into water.
CueO is markedly different from other MCOs in view of the
urements were carried at 25 C under O2-saturated or deaerated
conditions. The electrolyte solution volume was 5 mL, and the
enzyme concentration was fixed to be 0.4 mM.
Cyclic voltammogram of CueO under O2-saturated condi-
tions in the presence of CuSO4 is shown as curve (a) in
Figure 1. Clear and large cathodic waves were observed. When
CueO was added into the solution, the cathodic current starts to
increase gradually with time and reached the plateau. The behav-
ior can be interpreted by model that CueO adsorbs on the elec-
trode as a monolayer and that it catalyses the electrochemical re-
duction of O2. The appearance of the peak-shaped wave strongly
suggests the depletion of O2 in the vicinity of the electrode sur-
face and that the mass transfer (diffusion) of dissolved O2 is the
rate determining step at potential more negative than the peak
potential. Although voltammograms obtained in the absence of
O2 as controls did not exhibit the faradic current due to the redox
of the type I Cu(1+/2+) itself (curve (c) in Figure 1), the wave
is ascribed to DET-type catalytic reduction of O2, in which the
electrons are transferred directly from electrode to CueO and
CueO reduces O2 into water.
catalytic activities toward electron-donating substrates including
0
2,2 -azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS),
p-phenylenediamine, and 2,6-dimethoxyphenol. In order to
2
exhibit the oxidase activity, CueO requires excess amount of
1
copper ions in reaction solution. Recent X-ray crystallographic
study has revealed that CueO has a fifth copper binding site
4
located near the type I Cu site. The fifth copper would be
necessary to exhibit the oxidase activity for electron-donating
substrates described above, and the electrons would be transfer-
4
red from the substrate to type I Cu via the fifth copper.
Some MCOs are promising enzymes as catalysts of direct
electron-transfer (DET)-type bioelectrocatalytic four-electron
5
–10
reduction of O2 to water.
utilized in biofuel cells.
The catalytic reactions may be
One of the attractive properties of
the enzymes as catalysts for the cathode in biofuel cells is
5
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Copyright ꢀ 2007 The Chemical Society of Japan

Chemistry Letters Vol.36, No.1 (2007)
133
0
1
2
3
4
0
.0
c
0
rpm
-
-
-
-
-
-
-
0.2
0.4
0.6
0
2
4
Levich Plot
2
5
50 rpm
00 rpm
-
-
b
1
000 rpm
a
0
5
101/2 15
1/2
1500 rpm
ω
/ [rad/s]
0.0
0.2
0.4
0.6
E / V vs. Ag|AgCl
-0.5
0.0
0.5
E / V vs. Ag|AgCl
Figure 1. Cyclic voltammograms of CueO (0.4 mM) in a
McIlvaine buffer (pH 5.0, 25 C) under O2-saturated conditions
ꢃ
Figure 2. Rotating disk voltammograms of CueO catalyzing
ꢃ
reduction of O2 in a McIlvaine buffer (pH 5.0, 25 C) under
O2 saturated conditions. Rotating rates (rpm) were listed in the
figure. Inset represents the Levich plot of the plateau current
(
(
(
O2 concentration: ca. 1.2 mM) in the presence (a) and absence
b) of 1 mM of CuSO4. The electrode was not rotated. Curve
c) was obtained under deaerated conditions, and the other
ꢁ1
2
in rotating voltammetry for reduction of O on the CueO-modi-
conditions are same as curve (a). Scan rates are 20 mV s
.
ꢁ1
fied electrode. Scan rates are 20 mV s
.
Surprisingly, similar catalytic wave was observed even in
the absence of CuSO4 (curve (b) in Figure 1). The wave is almost
identical with that in the presence of CuSO4 (curve (a)) in the
shape and location. These results indicate that CueO works as
a good catalyst in DET-type catalytic reduction of O2, in which
the electrons are transferred directly from electrode to the type I
Cu site. This is in marked contrast with the fact that excess cop-
per ions are necessary for CueO to exhibit or enhance its oxidase
activity in solution (e.g., ca. 7.6% of the relative activity toward
in place of CueO, the limiting current showed characteristics of
enzyme-kinetic control at large ! under O2-saturated conditions.
The magnitude of the limiting catalytic current at 250 rpm was
about 8 and 9 times as large as that observed using BOD and
laccase, respectively. These results suggest that the catalytic
rate constant of CueO is sufficiently larger than that of other
MCOs in DET-type bioelectrocatalysis and that CueO is a great
candidate as an electrocatalyst for the cathode in biofuel cells.
6
,8
2
ABTS in the absence of copper ions ). The type I Cu is covered
with an ꢀ-helix portion, as evidenced by the X-ray crystallo-
This work was supported in part by grants from NEDO and
COE for Microbial-Process in Kyoto University.
4
graphic study. The ꢀ-helix would make difficult for substrates
to approach the type I Cu site. The fifth copper site would
function as a mediator of the electron transfer from substrates
to the type I Cu site. In DET-type bioelectrocatalysis, the
electron seems to be transferred from electrodes to the type I
Cu site in a way that is completely different from that in substrate
oxidation.
References
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2
2
ꢃ0
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ꢃ0
began to increase around E of the type I Cu site. Therefore,
ꢃ0
the E value of the type I Cu site in CueO may be located
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rotation rate (!). The limiting O2 reduction current showed
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Figure 2, where the straight broken line represents a theoretical
Levich plot for a four-electron transfer process with following
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1
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ꢁ
5
2
ꢁ1
of O2, 1:7 ꢂ 10 cm s ; kinematic viscosity of water, 0.01
2
ꢁ1
.
cm s
9
The limiting currents were controlled by the mass transfer of
O2 at ! at least up to 1500 rpm. When BOD or laccase was used
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Published on the web (Advance View) December 9, 2006; doi:10.1246/cl.2007.132