Efficiency of Site-Specific Clicked Laccase–Carbon Nanotubes Biocathodes towards O2 Reduction

Article abstract of DOI:10.1002/chem.201905234

A maximization of a direct electron transfer (DET) between redox enzymes and electrodes can be obtained through the oriented immobilization of enzymes onto an electroactive surface. Here, a strategy for obtaining carbon nanotube (CNTs) based electrodes covalently modified with perfectly control-oriented fungal laccases is presented. Modelizations of the laccase-CNT interaction and of electron conduction pathways serve as a guide in choosing grafting positions. Homogeneous populations of alkyne-modified laccases are obtained through the reductive amination of a unique surface-accessible lysine residue selectively engineered near either one or the other of the two copper centers in enzyme variants. Immobilization of the site-specific alkynated enzymes is achieved by copper-catalyzed click reaction on azido-modified CNTs. A highly efficient reduction of O₂ at low overpotential and catalytic current densities over ?3 mA cm^?2 are obtained by minimizing the distance from the electrode surface to the trinuclear cluster.

Full text of DOI:10.1002/chem.201905234

A Journal of
Accepted Article
Title: Efficiency of site-specific clicked laccase-carbon nanotubes
biocathodes towards O2 reduction
Authors: Solène Gentil, Pierre Rousselot-Pailley, Ferran Sancho,
Viviane Robert, Yasmina Mekmouche, Victor Guallar, Thierry
Tron, and Alan Le Goff
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201905234
Link to VoR: http://dx.doi.org/10.1002/chem.201905234
Supported by

Chemistry - A European Journal
10.1002/chem.201905234
FULL PAPER
Efficiency of site-specific clicked laccase-carbon nanotubes
biocathodes towards O reduction
2
[
a,b]
[c]
[d]
[c]
Solène Gentil,
Pierre-Rousselot-Pailley, Ferran Sancho, Viviane Robert, Yasmina
[
c]
[d, e]
[c]
[a]
Mekmouche, Victor Guallar,
Thierry Tron* and Alan Le Goff*
Abstract:
A
maximization of
a
direct electron transfer (DET)
envisioned as potential renewable non-precious-metal catalyst
for ORR. However, the bulky protein matrix surrounding the
copper centers is de facto limiting both the catalyst loadings and
the electron transfer rates. Transferring electrons between the
surface of the electrode up to the TNC is ruled by subtle
parameters. In particular, a direct electron transfer (DET), which
allows the enzyme to operate at its high potential without any
redox partners or mediators, is influenced by the distance
between the electron entry point in the enzyme and the surface
of the electrode. This distance as well as the presence of
insulating medium between the enzyme active site and the
electrode has to be minimized. This challenge requires thin-layer
modification of the electrode, the use of nanostructured material
between redox enzymes and electrodes can be obtained through the
oriented immobilization of enzymes onto an electroactive surface.
Here, we present a strategy for obtaining carbon nanotube (CNTs)
based electrodes covalently modified with perfectly control-oriented
fungal laccases. Modelisations of the laccase-CNT interaction and of
electron conduction pathways serve as guide in choosing grafting
positions. Homogeneous populations of alkyne-modified laccases
are obtained via the reductive amination of a unique surface
accessible lysine residue selectively engineered near either one or
the other of the two copper centers in enzyme variants.
Immobilization of the site-specific alkynated enzymes is achieved via
copper click on azido-modified CNTs. A highly efficient reduction of
3–8
O
2
at low overpotential and catalytic current densities over -3 mA
and a favorable orientation of the enzyme. Since the seminal
−2
9–11
cm are obtained by minimizing the distance from the electrode
surface to the trinuclear cluster.
work of Shleev and Gorton,
numerous articles describe DET
reactions of MCOs on electrodes. All together they cover
important variations of experimental conditions including enzyme
type (laccase, BOD, CueO), electrode type (stationary, or
rotating-disk electrode), electrode material (gold, graphite),
Introduction
immobilization
method
(physi-sorption,
supramolecular,
covalent), operating conditions (enzyme purity, pH, T°C).
Interpretations of results range from conclusions that electrons
enter the enzyme via the high-redox-potential T1 copper center
just as much that, at least under certain conditions, the TNC
might be directly accessible for DET without compromising the
Multi Copper Oxidases (MCO) are redox enzymes reducing
dioxygen via a 4H /4e mechanism at the expense of various
substrates oxidation. While oxidation occurs at a surface located
+
-
mononuclear type
1 (T1) copper center, electrons are
transferred from the T1 to an embedded trinuclear Type2/Type3
copper center (TNC) where dioxygen is reduced into water.
Immobilized at an electrode surface, high redox potential
enzymes (e.g. laccases, bilirubin oxidases) are able to achieve
the electrocatalytic oxygen reduction reaction (ORR) with a
small overpotential (eg. 100-300 mV), close to the overpotential
of the platinum catalyst traditionally used at the cathode of fuel
12–15
ORR.
The control of the enzyme orientation on the electrode surface
appears as a major challenge in order to avoid random
orientation generally resulting in poor electrode performances.
To gain control on the immobilization and wiring of MCOs,
strategies involving a surface modification have been developed.
Most reported attempts rely on a chemical modification of the
1–3
cells.
This is the reason why such copper enzymes are
8,16
surface of the electrode.
For example, Armstrong and
colleagues have taken advantage of the hydrophobicity of
several amino acids nearby the T1 copper center of a laccase
from Trametes versicolor to obtain its self-immobilization on a
pyrolytic graphite electrode surface modified with polycyclic
[
[
[
a]
b]
c]
Dr S. Gentil, Y. and Dr A. Le Goff
Univ. Grenoble Alpes, CNRS, DCM, 38000 Grenoble
E-mail: alan.le-goff@univ-grenoble-alpes.fr
Dr S. Gentil
Univ. Grenoble Alpes, CEA, CNRS, BIG-LCBM, 38000 Grenoble,
France
17,18
aromatics as substrate mimics.
Similar strategies have been
successfully exploited to immobilize enzymes at the surface of
Drs. P. Rousselot Pailley, V. Robert, Y. Mekmouche and T. Tron
Aix Marseille Université, Centrale Marseille, CNRS, iSm2 UMR
gold nanoparticles, carbon nanotubes (CNTs) and graphene
19–26
sheets.
Nowadays a variety of chemical reactions are
7
313,13397 Marseille, France
available to introduce different functionalities at the surface of
these conductive materials. In particular, the chemical or
electrochemical reduction of aryldiazonium salts have proven to
be a successful mean to modify the surface of the -extended
E-mail: thierry.tron@univ-amu.fr
F. Sancho, Prof. V. Guallar
Joint BSC-CRG-IRB Research Program in Computational Biology
Barcelona Supercomputing Centre, Jordi Girona 29, 08034
Barcelona, Spain
[
d]
13,27–29
network of CNT sidewalls.
[
e]
Prof. V. Guallar ICREA
Passeig Lluís Companys 23, 08010 Barcelona, Spain
Comparatively, reports targeting specific modifications of the
Supporting information for this article is given via a link at the end
of the document.
surface of MCOs – i.e. terminal tags, single surface exposed
30–32
reactive variant or unnatural amino acid – are still few.
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201905234
FULL PAPER
Probably seen as complex because a modification of the DNA to
encode a site-selective anchor point is a pre-requisite, this
strategy has yet no equivalent to generate uniform
enzyme/electrode interfaces where almost each enzyme
molecule is bound through the same single point. Moreover, in
principle, for any given enzyme/electrode couple the interface
can be as finely adjusted as moving the anchoring point (i.e. the
reactive function) from one position to an adjacent surface
exposed position. In a recent work, we have precisely introduced
a pyrene moiety at the surface of a fungal laccase. This pyrene
group has allowed us to immobilize the enzyme at the surface of
CNTs (supra-molecular interaction) with a predefined and DET
favorable orientation. To the contrary of nonspecific pyrene
Electron conduction pathway and laccase engineering
LAC3 from Trametes sp. C30 is a typical fungal laccase
3
4,35
obtained with high yield as a recombinant enzyme.
The
original LAC3 sequence contains naturally only two lysine
residues (K40 and K71) located at the surface of the amino-
terminus domain (GENEBANK AAR00925.1). These two
residues bearing primary amine groups are easily
36
functionalizable with chemical modules. We have recently
described a general strategy for obtaining single surface located
32
37
0
lysine variants of LAC3 (designed UNIK). From K (i.e. variant
devoid of lysine) we have already prepared UNIK161, a variant
II
which can be functionalized in the vicinity of the T1 Cu site at a
position almost diametrically opposed to positions K40 and K71
37
modifications
(through
pyrene-N-Hydrosuccinimide),
the
found in LAC3
.
immobilized enzyme exhibits high direct bioelectrocatalysis of O
2
Following our previous work on the orientation of pyrene-laccase
32
reduction on both MWCNT and gold nanoparticle-modified
electrodes. In order to evaluate the influence of the localization
of the anchoring point at the enzyme surface on electrocatalytic
properties, we are now exploring the covalent modification of
single lysine laccase variants. Here, we functionalized the native
LAC3 and two of its variants (UNIK71 and UNIK161, Fig. 1). We
used the well-known alkyne-azide Huisgen 1,3-dipolar
cycloaddition to efficiently attach these enzymes on MWCNT
electrodes covalently modified by 4-azidobenzene diazonium
salt . We show that such strategy is highly efficient both for the
immobilization and wiring of laccases on nanostructured
electrodes. Influence of the grafting conditions and that of the
location of the anchoring point at the enzyme’s surface on the
bioelectrocatalysis of oxygen reduction are discussed.
molecules at the MWCNT electrode surface, we initially
compared in silico the pyrene-UNIK161 and (pyrene) -LAC3
hybrid/MWCNT electrodes (Fig. SI1, see SI for computational
details). Modeling of the pyrene grafted K40,
2
K
71 and K161
3
6
positions was performed as previously published. Distances
II
from the anchor (pyrene) to the Cu ions and electron
conduction pathways in the protein matrix were evaluated for
each hybrid model (see Table 1).
Table 1: electron coupling (in eV) and distances (in Å) calculated between the
33
II
pyrene moieties and the Cu ions. Evaluation made with Pathways on the
laccase-pyrene/CNT models of Figure SI1.
Anchoring position
II
K
40
K
71
K
161
Cu
center
eV
d (Å)
eV
d (Å)
eV
d (Å)
-
12
10
10
10
-10
-7
T1
2 10
4 10
1 10
9 10
35
25
29
26
4 10
1 10
1 10
2 10
31
20
24
23
1 10
16
27
23
27
-
-
-
-8
-7
-8
-10
T2
1 10
-9
T3
T3
4 10
3 10
-9
Comparison of the electronic coupling values suggests that the
TNC of enzymes grafted at K40 and K71 positions might be
directly reducible by the electrode, lysine 71 being predicted to
be the position to graft for
a potential optimal electron
conduction (Fig. 1). The singulation of K40 and K71 positions was
therefore undertaken from LAC3 in order to be able to compare
DET performances from singly grafted variants. UNIK71 was
subsequently constructed and produced as previously
37
described. Positions 71 (UNIK71) and 161 (UNIK161) are almost
mirroring each other relative to the copper centers.
Figure 1. Model of the structure of LAC3 with the three possible locations of
lysine in either the native enzyme (K71 and K40) or its variants UNIK161 and
UNIK71. View generated with PyMol. In silico evaluated electronic coupling
values from an electron donating surface to the TNC (in eV) are added in red
LAC3 and its two variants were independently and covalently
grafted to probe the bioelectrocatalytic O reduction activity of
2
the enzyme as a function of a strictly controlled orientation at the
electrode surface. A commercial 4-ethynylbenzaldehyde was
used for the "alkynylation" (i.e. functionalization with an alkyne
group) of laccases in a reductive akylation reaction adapted from
(
see text for details).
Results and Discussion
38
Mc Farland and Francis (see Supplementary Information). As
previously evaluated, this functionalization strategy results in a ≈
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201905234
FULL PAPER
32,36
1:1 ratio between the enzyme and the functional group
. This
generation of aryl radicals followed by their reaction with the
unique anchor point in the UNIK-alkyne variant is designed to
allow only one possible orientation of the laccase molecules
after reaction (cycloaddition) at the azido-modified surface of
nanostructured electrodes. As previously mentioned, site-
specific modifications of the enzyme are a mean to probe a DET
between the electrode and the copper centers after enzyme
grafting (Fig. 1).
surface of MWCNTs and the subsequent formation of a
polyphenylene layer. The “click reaction” was then performed on
azido-modified MWCNT electrodes by soaking each of them in a
deaerated solution of the proper alkyne-modified derivative in
the presence of CuSO4, sodium ascorbate and tris-
hydroxypropyltriazolylmethylamine (THTPA) for 60 min at room
temperature. The modification of MWCNT electrodes was also
monitored by QCM-D experiments. The functionalization of
MWCNT-coated quartz crystal was monitored for both
spontaneous grafting of 4-azidobenzene diazonium and
immobilization of alkyne-modified laccases (Fig. S3-S5). A low
Sauerbrey mass uptakes was observed for the immobilization of
nonmodified LAC3 compared to alkyne-modified LAC3. This
underlines that weak interactions between the enzyme and
nonmodified crystals exist and corroborates the covalent
attachment of enzymes on azido-modified quartz crystals.
41
Considering a 50% degree of hydration for the protein layer,
final Sauerbrey mass uptakes would correspond to a surface
-
12
-2
coverage of 16 10
mol.cm for LAC3-modified MWCNT-
coated crystal. It is noteworthy that QCM-D performed on the
laccase variants did not show any striking difference in terms of
immobilization rates or mass uptakes. These QCM-D
experiments unambiguously demonstrate that the spontaneous
modification of electrodes with the 4-azidobenzene diazonium
results in the grafting of a thin polyphenylene layer at the surface
of MWCNTs. In turn, this active layer allows a rapid, efficient and
stable immobilization of clickable laccases.
Figure 2. Schematic representation of the covalent modification of CNT by
click chemistry of ferrocene-alkyne and alkyne modified LAC3 variants.
Covalent functionalization of MWCNT electrodes and
electrochemistry in non-turnover conditions
21,23
MWCNT electrodes were prepared as previously-described.
Figure illustrates the functionalization technique for the
2
immobilization of an alkyne-modified ferrocene used here as an
electrochemical probe and alkyne-modified LAC3 variants.
Electrografting of 4-azidobenzene diazonium tetrafluoroborate
was performed either by spontaneous or electrochemical
grafting of the 4-azidobenzene diazonium salt. Spontaneous
grafting corresponds to a simple soaking step in a solution of 4-
azidobenzene
diazonium
tetrafluoroborate
while
the
Figure 3. (A) CVs and (B) background-subtracted CVs of (a) alkyne-UNIK161
and (b)- ethynylferrocene functionalized MWCNT electrodes under argon at
pH = 5. The gray line corresponds to a non-modified MWCNT electrode (0.05
electrografting corresponds to CV scans performed below the
irreversible reduction of the 4-azidobenzene diazonium salt.
Spontaneous grafting corresponds to the ability of diazonium
salts to react with electrode surface, especially graphitized ones,
by spontaneous electron transfer from the surface to the
-
1
-1
mol L in acetate buffer solution, v = 10 mV s ).
Characterization of the UNIK161 electrode
39,40
diazonium salt.
This technique was compared to
Electrode functionalization with the alkyne-UNIK161 was
evaluated by comparison with an electrode modified with
ethynylferrocene (electrochemical probe). Figure 3 displays CV
performed under argon for MWCNT electrodes covalently-
modified by ethynylferrocene and alkyne-modified UNIK161 after
spontaneous grafting of the 4-azidobenzene diazonium salt.
electrografting as it allows to avoid undesirable thick
polyphenylene layer formation inevitably formed during the
electrografting process. Typical CV scans for the electrografting
of
a
2
mM solution of 4-azidobenzene diazonium
tetrafluoroborate in MeCN are given in Supplementary
Information (Fig. SI2). An irreversible reduction is observed at
E
red = +0.05 V vs. Ag/AgNO3. This reduction corresponds to the
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201905234
FULL PAPER
As expected for the ferrocene-modified electrode, a reversible
system characteristic of ferrocene moieties grafted at the
bioelectrocatalytic reduction of O
of -1.76 (+/-0.3) and -0.75(+/-0.2) mA cm were obtained after
spontaneous and electrografting respectively (fig. 4, black
2
. Maximum current densities
-
2
33
surface of MWCNTs
is observed at E1/2 = +0.57 (+/-0.01) V
(
E = 0 mV ) vs. SHE. For UNIK161-modified MWCNT electrodes, curves). It is noteworthy that a simple adsorption of the
a tiny reversible system is observed at E1/2 = +0.63 (+/-0.01) V enzyme on non-modified MWCNTs led to sluggish
E = 20 mV, pH 5) vs. SHE. This reversible system is likely irreversible reduction with a maximum current density of -
a
(
-
2
attributable to the electroactivity of the enzyme. It is noteworthy
2
0.52(+/-0.2) mA cm (Fig. SI7). Reduction of O was also
that the redox potential of the T1 center of LAC3 has been
investigated after addition of ABTS. This well-known laccase
redox mediator unravels the enzymatic activity of immobilized
UNIK161 which might not be in direct contact with the electrode.
In this case, maximum current densities of -2.39 (+/-0.5) mA
40
measured at + 0.68 V vs. NHE at pH 6. Control experiments
with non-modified MWCNTs do not show any redox system in
this region, confirming the immobilization of both ferrocene and
UNIK161 by click chemistry (Figure SI6). Unfortunately, redox
peaks associated to UNIK161 rapidly vanished when the
electrode was tested at different pHs or at different scan rates.
This is mostly caused by the high background capacitive current
-
2
-2
cm and -5.91 (+/-0.8) mA cm were respectively observed
after spontaneous and electrografting (Fig. 4, blue curves).
Ratio of mediated/unmediated electrocatalytic current values
suggests that spontaneous grafting induces the most efficient
DET. Therefore, although the electrografting mode allows to
reach a high density of azide groups at the surface of the
electrode eventually leading to a high amount of immobilized
enzymes, these enzymes appear poorly connected (Fig. 4B, ≈
15% DET). To the contrary, with a thinner polyphenylene layer
induced by the spontaneous grafting of the diazonium salt, this
electrode offers less azido groups to click the enzyme but
these enzymes are fairly well connected to the electrode (Fig.
4A, ≈75% DET). These experiments underline the fact that the
grafting of the functional layer has to be optimized in order to
prevent inhibition of electron tunneling while maximizing
enzyme surface coverage. This being said, the CV of the
1
3,43
generated by MWCNTs.
By integration of the charge under
-
12
the symmetrical peaks surface, coverages of 0.5 10 and 1.0
-
9
-2
1
0
mol cm were respectively estimated for UNIK161 and
ferrocene. If a large difference of surface coverages is expected
given the size difference between ferrocene and the enzyme,
note that the surface covered by the enzyme may be here
underestimated as electroactivity of copper active sites of
laccases under non-turnover conditions has been always highly
3
difficult to distinguish by CV. This is corroborated by the higher
surface coverage measured by QCM-D (Fig. S5).
2
Electrochemistry under O and influence of the grafting
conditions
alkyne-UNIK161 modified electrode under O
2
is very similar to
CV of UNIK161-modified MWCNT electrodes were performed
under constant O bubbling in the supporting electrolyte. We
that previously obtained with the pyrene-UNIK161 modified
electrode in similar conditions. Eventually, the stability of the
32
2
studied the influence of the mode of electrode surface
functionalization with 4-azidobenzene diazonium on the
electrodes
was
evaluated
performing
one-hour
bubbling
chronoamperometry at +0.4 V vs. SHE under O
2
electrocatalytic reduction of
electrodes modified either by spontaneous or electrografting
during 5 consecutive scans (Fig. 4).
O
2
by comparing MWCNT
(Figure SI8). After one hour, the catalytic current decreased
-
2
from 1.7 (+/-0.2) to 1.1 (+/-0.2) mA cm . This catalytic current
-
2
stabilized around 0.5 mA cm after 3 days. These experiments
did not reveal obvious differences linked to the use of different
32
immobilization techniques or enzyme variants.
Influence of the orientation of the enzyme
In order to unveil an influence of the location of the grafting
point on the DET obtained with laccase variants, both LAC3
and UNIK71 were further modified with alkyne groups and
immobilized at azide-modified MWCNT electrodes, as
described above for UNIK161. Figure 5 displays CVs performed
2
under O in the absence or presence of ABTS.
Figure 4. CV of alkyne-UNIK161 MWCNT electrodes after the spontaneous
(
A) or the electro grafting (5 CV scans) of 4-azidobenzene diazonium
tetrafluoroborate (B). CVs obtained (a) under Ar, (b) under O and (c) after
the addition of 10 mM ABTS (0.05 mol L acetate buffer solution pH 5, v =
2
-1
-
1
1
0 mV s ).
For all electrodes, an irreversible electrocatalytic wave for O
2
reduction is observed at high potentials. This confirms that a
DET is obtained for the clicked UNIK161 achieving the
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201905234
FULL PAPER
44–46
0
.99) using equations developed in ref
(see
Figure 5. CV of (A) (alkyne)
UNIK71 MWCNT electrodes; (a) under Ar, (b) under O
2
-LAC3 MWCNT electrodes and (B) alkyne-
and (c) after the
addition of 10 mM ABTS (0.05 mol L acetate buffer solution pH 5, v = 10
supplementary materials for details). The simulation allowed
us to estimate several constant parameters: the catalysis
redox potential (Ecat), the ratio between the catalytic rate
2
-
1
-1
mV s ).
constant (kcat) and the interfacial ET rate constant at minimal
max
0
distance dmin (k
0
) and the ET tunneling factor d which is
The general behavior of the two electrodes both possessing an
alkyne functionalized K71 as grafting point is globally similar.
the product of the medium exponential decay constant and
, the interval of distances between the electrochemical relay
d
0
2
Both electrodes perform O reduction at a similar onset
center and the electrode allowing ET (Table 2).
potential (nearly + 0.85 V vs. SHE), that is itself comparable to
that already obtained for the alkyne-UNIK161 (Fig. 4A). The
UNIK71-modified electrode (Fig. 5B) exhibits a high current
Table 2. Fitting parameters of simulated curves from Figure 6 for alkyne-
modified native LAC3, UNIK161 and UNIK71
.
density under O
addition of ABTS. This indicates that all immobilized UNIK71
enzymes establish direct electronic contact with the
electrode. The second anchoring point present in the (alkyne)
LAC3 (Fig. 5A) does not seem to improve neither the grafting
ABTS trace) nor the DET. Note that in this case the signal
may be composite, potentially including contributions of
enzymes resulting from two possible single point (K40 or K71
2
, accompanied with a negligible increase after
LAC3
0.706
0.047
2.33
UNIK161
UNIK71
0.720
0.029
3.13
E
cat
0.701
0.034
1.82
a
2
-
max
k
cat/k
0
(
j
lim
d
0
10.3
10.6
8.4
)
and a double point (K40 and K71) functionalization of the
nanotube surface. It is therefore difficult to infer which situation
modulates the signal from the raw CV.
2
R
0.998
27%
0.997
34%
0.998
4%
[
a]
%ABTS
[
a] 100*(1-(jlim (-ABTS)/ jlim (+ABTS))
Modelization
The electrocatalytic response of the different electrodes was
simulated using the model developed by Armstrong and Léger.
The location chosen for the grafting positions at the surface of
the enzymes and distances estimated from the graft to the
copper centers point the Cu T1 and the TNC as
4
4–46
II
In particular, this model takes into account a distribution
of orientations of catalytically active immobilized enzymes.
Background-subtracted experimental curves and simulated
electrochemical relay centers respectively for UNIK161 and for
both UNIK71 and LAC (Fig. 1, Table 1). Our initial in silico
evaluation of the systems suggests that energies coupling the
electron donating surface to their closest electrochemical relay
centers are similar for UNIK161 and UNIK71 (Table 1). The
slightly higher performance of the UNIK71 based electrode is
therefore directly associated to a lower distribution of tunneling
2
curves obtained for (alkyne) -LAC3, alkyne-UNIK161 and
alkyne-UNIK71 are displayed on Figure 6.
distances. This is observed with the lowest d
0
product
corresponding to a decreasing effect of the dispersion of
tunneling distances which might arise from a shorter electronic
coupling distance between the electrochemical redox center
44,46
and the electrode.
0
Interestingly, the d value for the Unik71
electrode is also significantly lower than that of the LAC3
electrode whereas these enzymes are having both K71 as
grafting point. Beyond the simple ascertainment that grafting
the enzyme through the two surface positions (K40, K71
located ca. 14 Å away (C-C distance) might not reduce
heterogeneity this could alternatively reveal significant
)
a
proportion of the LAC3 enzyme actually connected through the
position K40 only.
Figure 6. Background-subtracted CV of (alkyne)
2
-LAC3 (a), alkyne-UNIK161
and
respective simulated CV scan (red trace) (0.05 mol L acetate buffer
Beyond the above noted differences it appears that the three
(
b) and alkyne-UNIK71 (c) functionalized MWCNTelectrodes under O
2
-
1
2
electrodes all perform the electrocatalytic reduction of O with
-
2
-
1
a relative efficiency (current densities > 1.8 mA cm and a
DET efficiency from 70% to nearly 100%) and that whatever
the orientation chosen for the enzyme at the CNTs surface.
solution pH 5 , v = 10 mV s . Background-subtracted CVs take into account
the average between the backward and forward CV scans.
3
Turnover frequencies (TOF) values are in the order of 5.0 10
s
Despite a potential inaccuracy linked to diffusion limitations
into the MWCNT film that are not specifically taken into
account by this model, CV curves were all well-fitted (R
-1
according to the enzyme surface coverage measured by
QCM-D; these are in good agreement with TOF values
2
>
43
47
measured for BOD or for laccases. The UNIK161 enzyme
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201905234
FULL PAPER
II
offers
electrochemical relay to the dioxygen reduction center
TNC).[5, 8] The UNIK71 enzyme (as well as the LAC3
a
“classical” orientation with the Cu T1 as
Health of Univ Grenoble Alpes CBH-EUR-GS (ANR-17-EURE-
0
003) and ANR Multiplet (ANR-15- CE07-0021-01). The authors
wish to acknowledge the platform Chimie NanoBio ICMG FR
607 (PCN-ICMG). This work was also supported by the
(
enzyme) offers an opposite orientation with no relay to the
TNC, a TNC that however can be apparently directly reached
by electron tunneling though the protein matrix. Orientation
dependent electron tunneling from the surface to the TNC has
been proposed for several MCO for which a marked difference
2
Ministère de l’Environnement, de l’Energie et de la Mer.
Keywords: laccase • diazonium • click chemistry • oxygen
46,
reduction • biofuel cells
of redox potential of the copper centers has been measured.
47
This is the case for MCOs for which experimental study
1
2
3
.
.
.
Mano, N., and Edembe, L. (2013). Bilirubin oxidases in
bioelectrochemistry: Features and recent findings. Biosens. Bioelectron.
50, 478–485.
conditions are apparently favoring the Alternative Resting (AR)
state of the enzyme, a form of TNC partly-reduced and fully
12–15
reducible only at low potential.
. This is also the case for
Le Goff, A., Holzinger, M., and Cosnier, S. (2015). Recent progress in
oxygen-reducing laccase biocathodes for enzymatic biofuel cells. Cell.
Mol. Life Sci. 72, 941–952.
II
50
low potential Cu T1 enzymes like CueO. In LAC3 and in its
variants (i.e. UNIK71 and UNIK161) the copper centers are
51
almost iso potential.
Apparently, none of our experimental
Mano, N., and de Poulpiquet, A. (2017). O2 Reduction in Enzymatic
Biofuel Cells. Chem. Rev. 118, 2392–2468.
conditions was favoring an AR state of the TNC. This shows
up in the CVs of the three different electrodes we tested as
they perform O reduction at the same onset whatever the
2
4.
Cracknell, J.A., Vincent, K.A., and Armstrong, F.A. (2008). Enzymes as
Working or Inspirational Electrocatalysts for Fuel Cells and Electrolysis.
Chem. Rev. 108, 2439–2461.
orientation of the enzyme at the CNTs surface. Moreover, the
similar Ecat obtained for all enzymes is probably related to the
5
.
.
Léger, C., and Bertrand, P. (2008). Direct Electrochemistry of Redox
Enzymes as a Tool for Mechanistic Studies. Chem. Rev. 108, 2379–
51
fact that TNC and T1 redox potentials are close. Overall, our
2438.
II
results establish that both the Cu T1 and the TNC centers of a
6
Elouarzaki, K., Cheng, D., Fisher, A.C., and Lee, J.-M. (2018). Coupling
orientation and mediation strategies for efficient electron transfer in
hybrid biofuel cells. Nat Energy 3, 574–581.
high potential laccase can be efficiently targeted from discrete
surface areas selected through the choice of single grafting
points.
7.
Walgama, C., Pathiranage, A., Akinwale, M., Montealegre, R., Niroula,
J., Echeverria, E., McIlroy, D.N., Harriman, T.A., Lucca, D.A., and
Krishnan, S. (2019). Buckypaper–Bilirubin Oxidase Biointerface for
Electrocatalytic Applications: Buckypaper Thickness. ACS Appl. Bio
Mater. 2, 2229–2236.
Conclusions
8
.
.
Le Goff, A., and Holzinger, M. (2018). Molecular engineering of the
bio/nano-interface for enzymatic electrocatalysis in fuel cells. Sustain.
Energ. Fuels 2, 2555–2566.
We achieved an efficient DET based electrocatalytic reduction
of dioxygen using diazonium modified MWCNTs easily
functionalized through a simple alkyne-azide Huisgen 1,3-
dipolar cycloaddition of alkynylated enzymes. Targeting
specific modifications of the surface of MCOs through single
surface exposed reactive variants, uniform enzyme/electrode
interfaces were generated. Hence, each enzyme molecule is
bound through the same single point. With this strategy, we
established that each of the two catalytic centers of a high
9
Shleev, S., Tkac, J., Christenson, A., Ruzgas, T., Yaropolov, A.I.,
Whittaker, J.W., and Gorton, L. (2005). Direct electron transfer between
copper-containing proteins and electrodes. Biosens. Bioelectron. 20,
2
517–2554.
1
0.
Shleev, S., El Kasmi, A., Ruzgas, T., and Gorton, L. (2004). Direct
heterogeneous electron transfer reactions of bilirubin oxidase at a
spectrographic graphite electrode. Electrochem. Comm. 6, 934–939.
Shleev, S., Jarosz-Wilkolazka, A., Khalunina, A., Morozova, O.,
Yaropolov, A., Ruzgas, T., and Gorton, L. (2005). Direct electron
transfer reactions of laccases from different origins on carbon
electrodes. Bioelectrochemistry 67, 115–124.
11.
II
potential laccase, namely the Cu T1 and the TNC centers, can
be efficiently reached from discrete surface areas selected
through the choice of single grafting points. The next step
towards improvements on the immobilization of our laccase
variants at the surface of MWCNTs requires to get more
insights into the structural characterization of the grafted
electrodes. In particular, an in situ evaluation of both active-
site-electrode distances and of the structural integrity (i.e.
identifying a partial deformation or unfolding of the enzyme
upon immobilization) is prioritized in order to better understand
which parameters influence the electrochemical behavior of a
copper enzyme to another.
1
2.
Dagys, M., Laurynėnas, A., Ratautas, D., Kulys, J., Vidžiūnaitė, R.,
Talaikis, M., Niaura, G., Marcinkevičienė, L., Meškys, R., and Shleev, S.
(
2017). Oxygen electroreduction catalysed by laccase wired to gold
nanoparticles via the trinuclear copper cluster. Energy & Environmental
Science 10, 498–502.
13.
Lalaoui, N., Holzinger, M., Le Goff, A., and Cosnier, S. (2016).
Diazonium Functionalisation of Carbon Nanotubes for Specific
Orientation of Multicopper Oxidases: Controlling Electron Entry Points
and Oxygen Diffusion to the Enzyme. Chem. Eur. J. 22, 10494–10500.
1
4. de Poulpiquet, A., Kjaergaard, C.H., Rouhana, J., Mazurenko, I., Infossi,
P., Gounel, S., Gadiou, R., Giudici-Orticoni, M.T., Solomon, E.I., Mano,
N., et al. (2017). Mechanism of Chloride Inhibition of Bilirubin Oxidases
and Its Dependence on Potential and pH. ACS Catal. 7, 3916–3923.
1
5.
Kjaergaard, C.H., Durand, F., Tasca, F., Qayyum, M.F., Kauffmann, B.,
Gounel, S., Suraniti, E., Hodgson, K.O., Hedman, B., Mano, N., et al.
Acknowledgements
(
2012). Spectroscopic and Crystallographic Characterization of
Alternative Resting” and “Resting Oxidized” Enzyme Forms of Bilirubin
This work was supported by the Labex ARCANE ((ANR-11-
LABX-0003-01), the Graduate School on Chemistry, Biology and
“
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201905234
FULL PAPER
Oxidase: Implications for Activity and Electrochemical Behavior of
32.
Lalaoui, N., Rousselot-Pailley, P., Robert, V., Mekmouche, Y.,
Villalonga, R., Holzinger, M., Cosnier, S., Tron, T., and Le Goff, A.
Multicopper Oxidases. J. Am. Chem. Soc. 134, 5548–5551.
1
6.
7.
Yates, N.D.J., Fascione, M.A., and Parkin, A. (2018). Methodologies for
Wiring” Redox Proteins/Enzymes to Electrode Surfaces. Chemistry – A
(2016). Direct Electron Transfer between a Site-Specific Pyrene-
“
Modified Laccase and Carbon Nanotube/Gold Nanoparticle
Supramolecular Assemblies for Bioelectrocatalytic Dioxygen Reduction.
ACS Catal. 6, 1894–1900.
European Journal 24, 12164–12182.
1
Blanford, C.F., Heath, R.S., and Armstrong, F.A. (2007). A stable
electrode for high-potential, electrocatalytic O2 reduction based on
rational attachment of a blue copper oxidase to a graphite surface.
Chem. Commun., 1710–1712.
33. Evrard, D., Lambert, F., Policar, C., Balland, V., and Limoges, B. (2008).
Electrochemical Functionalization of Carbon Surfaces by Aromatic
Azide or Alkyne Molecules: A Versatile Platform for Click Chemistry.
Chem. Eur. J. 14, 9286–9291.
18.
Blanford, C.F., Foster, C.E., Heath, R.S., and Armstrong, F.A. (2008).
Efficient electrocatalytic oxygen reduction by the ‘blue’ copper oxidase,
laccase, directly attached to chemically modified carbons. Faraday
Discussions 140, 319–335.
34. Klonowska, A., Gaudin, C., Asso, M., Fournel, A., Réglier, M., and Tron,
T. (2005). LAC3, a new low redox potential laccase from Trametes sp.
strain C30 obtained as a recombinant protein in yeast. Enzyme and
Microbial Technology 36, 34–41.
1
9.
0.
Giroud, F., and Minteer, S.D. (2013). Anthracene-modified pyrenes
immobilized on carbon nanotubes for direct electroreduction of O2 by
laccase. Electrochemistry Communications 34, 157–160.
35.
Mekmouche, Y., Zhou, S., Cusano, A.M., Record, E., Lomascolo, A.,
Robert, V., Simaan, A.J., Rousselot-Pailley, P., Ullah, S., Chaspoul, F.,
et al. (2014). Gram-scale production of a basidiomycetous laccase in
Aspergillus niger. Journal of Bioscience and Bioengineering 117, 25–27.
Schneider, L., Mekmouche, Y., Rousselot-Pailley, P., Simaan, A.J.,
Robert, V., Reglier, M., Aukauloo, A., and Tron, T. (2015). Visible-Light-
Driven Oxidation of Organic Substrates with Dioxygen Mediated by a
[Ru(bpy)(3)](2+)/Laccase System. ChemSusChem 8, 3048–3051.
2
Meredith, M.T., Minson, M., Hickey, D., Artyushkova, K., Glatzhofer,
D.T., and Minteer, S.D. (2011). Anthracene-Modified Multi-Walled
Carbon Nanotubes as Direct Electron Transfer Scaffolds for Enzymatic
Oxygen Reduction. ACS Catal. 1, 1683–1690.
36.
2
1.
Bourourou, M., Elouarzaki, K., Lalaoui, N., Agnès, C., Le Goff, A.,
Holzinger, M., Maaref, A., and Cosnier, S. (2013). Supramolecular
Immobilization of Laccase on Carbon Nanotube Electrodes
Functionalized with (Methylpyrenylaminomethyl)anthraquinone for
Direct Electron Reduction of Oxygen. Chem. Eur. J. 19, 9371–9375.
Lalaoui, N., Le Goff, A., Holzinger, M., Mermoux, M., and Cosnier, S.
37. Robert, V., Monza, E., Tarrago, L., Sancho, F., De Falco, A., Schneider,
L., Ngoutane, E.N., Mekmouche, Y., Pailley, P.R., Simaan, A.J., et al.
(2018). Probing the Surface of a Laccase for Clues towards the Design
of Chemo-Enzymatic Catalysts (vol 82, pg 607, 2017). ChemPlusChem
83, 831–831.
22.
23.
24.
25.
26.
(
2015). Wiring Laccase on Covalently Modified Graphene: Carbon
Nanotube Assemblies for the Direct Bio-electrocatalytic Reduction of
Oxygen. Chem. Eur. J. 21, 3198–3201.
38.
McFarland, J.M., and Francis, M.B. (2005). Reductive Alkylation of
Proteins Using Iridium Catalyzed Transfer Hydrogenation. J. Am. Chem.
Soc. 127, 13490–13491.
Lalaoui, N., Elouarzaki, K., Le Goff, A., Holzinger, M., and Cosnier, S.
(
2013). Efficient direct oxygen reduction by laccases attached and
39. Mesnage, A., Lefèvre, X., Jégou, P., Deniau, G., and Palacin, S. (2012).
Spontaneous Grafting of Diazonium Salts: Chemical Mechanism on
Metallic Surfaces. Langmuir 28, 11767–11778.
oriented on pyrene-functionalized polypyrrole/carbon nanotube
electrodes. Chem. Commun. 49, 9281–9283.
Lalaoui, N., David, R., Jamet, H., Holzinger, M., Le Goff, A., and
Cosnier, S. (2016). Hosting Adamantane in the Substrate Pocket of
Laccase: Direct Bioelectrocatalytic Reduction of O2 on Functionalized
Carbon Nanotubes. ACS Catal. 6, 4259–4264.
40.
41.
42.
43.
44.
Gentil, S., Che Mansor, S.M., Jamet, H., Cosnier, S., Cavazza, C., and
Le Goff, A. (2018). Oriented Immobilization of [NiFeSe] Hydrogenases
on Covalently and Noncovalently Functionalized Carbon Nanotubes for
H2/Air Enzymatic Fuel Cells. ACS Catal., 3957–3964.
Nazaruk, E., Sadowska, K., Biernat, J.F., Rogalski, J., Ginalska, G.,
and Bilewicz, R. (2010). Enzymatic electrodes nanostructured with
functionalized carbon nanotubes for biofuel cell applications. Anal.
Bioanal. Chem. 398, 1651–1660.
Seifert, M., Rinke, M.T., and Galla, H.-J. (2010). Characterization of
Streptavidin Binding to Biotinylated, Binary Self-Assembled Thiol
Monolayers—Influence of Component Ratio and Solvent. Langmuir 26,
6386–6393.
Sosna, M., Chrétien, J.-M., Kilburn, J.D., and Bartlett, P.N. (2010).
Monolayer anthracene and anthraquinone modified electrodes as
platforms for Trametes hirsuta laccase immobilisation. Phys. Chem.
Chem. Phys. 12, 10018–10026.
Balland, V., Hureau, C., Cusano, A.M., Liu, Y., Tron, T., and Limoges,
B. (2008). Oriented immobilization of
a fully active monolayer of
histidine-tagged recombinant laccase on modified gold electrodes.
Chem. Eur. J. 14, 7186–7192.
2
2
2
7.
Bahr, J.L., and Tour, J.M. (2001). Highly Functionalized Carbon
Nanotubes Using in Situ Generated Diazonium Compounds. Chem.
Mater., 3823–3824.
Lalaoui, N., Le Goff, A., Holzinger, M., and Cosnier, S. (2015). Fully
Oriented Bilirubin Oxidase on Porphyrin-Functionalized Carbon
Nanotube Electrodes for Electrocatalytic Oxygen Reduction. Chem. Eur.
J. 21, 16868–16873.
8.
Dyke, C.A., and Tour, J.M. (2003). Unbundled and Highly
Functionalized Carbon Nanotubes from Aqueous Reactions. Nano Lett.
Hexter, S.V., Grey, F., Happe, T., Climent, V., and Armstrong, F.A.
(2012). Electrocatalytic mechanism of reversible hydrogen cycling by
enzymes and distinctions between the major classes of hydrogenases.
Proc. Natl. Acad. Sci. 109, 11516–11521.
3, 1215–1218.
9. Le Goff, A., Moggia, F., Debou, N., Jegou, P., Artero, V., Fontecave, M.,
Jousselme, B., and Palacin, S. (2010). Facile and tunable
functionalization of carbon nanotube electrodes with ferrocene by
covalent coupling and π-stacking interactions and their relevance to
glucose bio-sensing. J. Electroanal. Chem., 57–63.
45.
46.
Hexter, S.V., Esterle, T.F., and Armstrong, F.A. (2014). A unified model
for surface electrocatalysis based on observations with enzymes. Phys.
Chem. Chem. Phys. 16, 11822–11833.
3
0. Mateljak, I., Monza, E., Lucas, M.F., Guallar, V., Aleksejeva, O., Ludwig,
R., Leech, D., Shleev, S., and Alcalde, M. (2019). Increasing Redox
Potential, Redox Mediator Activity, and Stability in a Fungal Laccase by
Computer-Guided Mutagenesis and Directed Evolution. ACS Catal.,
Léger, C., Jones, A.K., Albracht, S.P.J., and Armstrong, F.A. (2002).
Effect of a Dispersion of Interfacial Electron Transfer Rates on Steady
State Catalytic Electron Transport in [NiFe]-hydrogenase and Other
Enzymes. J. Phys. Chem. B 106, 13058–13063.
4
561–4572.
Al-Lolage, F.A., Bartlett, P.N., Gounel, S., Staigre, P., and Mano, N.
2019). Site-Directed Immobilization of Bilirubin Oxidase for
Electrocatalytic Oxygen Reduction. ACS Catal. 9, 2068–2078.
47.
48.
Gutiérrez-Sánchez, C., Pita, M., Vaz-Domínguez, C., Shleev, S., and
De Lacey, A.L. (2012). Gold Nanoparticles as Electronic Bridges for
Laccase-Based Biocathodes. J. Am. Chem. Soc. 134, 17212–17220.
Solomon, E.I., Heppner, D.E., Johnston, E.M., Ginsbach, J.W., Cirera,
J., Qayyum, M., Kieber-Emmons, M.T., Kjaergaard, C.H., Hadt, R.G.,
31.
(
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201905234
FULL PAPER
and Tian, L. (2014). Copper Active Sites in Biology. Chem. Rev. 114,
51.
Lazarides, T., Sazanovich, I.V., Simaan, A.J., Kafentzi, M.C., Delor, M.,
Mekmouche, Y., Faure, B., Réglier, M., Weinstein, J.A., Coutsolelos,
A.G., et al. (2013). Visible Light-Driven O2 Reduction by a Porphyrin–
Laccase System. J. Am. Chem. Soc. 135, 3095–3103.
3659–3853.
4
9.
0.
Jones, S.M., and Solomon, E.I. (2015). Electron transfer and reaction
mechanism of laccases. Cell. Mol. Life Sci. 72, 869–883.
5
Schlesinger, O., Pasi, M., Dandela, R., Meijler, M.M., and Alfonta, L.
(
2018). Electron transfer rate analysis of a site-specifically wired copper
oxidase. Phys. Chem. Chem. Phys. 20, 6159–6166.
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201905234
FULL PAPER
Entry for the Table of Contents (Please choose one layout)
Layout 1:
FULL PAPER
Text for Table of Contents
Solène Gentil, Pierre-Rousselot-Pailley,
Ferran Sancho, Viviane Robert,
Yasmina Mekmouche, Victor Guallar,
Thierry Tron* and Alan Le Goff*
Page No. – Page No.
Efficiency of site-specific clicked
laccase-carbon nanotubes
biocathodes towards O reduction
2
Layout 2:
FULL PAPER
Author(s), Corresponding Author(s)*
(
(Insert TOC Graphic here; max. width: 11.5 cm; max. height: 2.5 cm))
Page No. – Page No.
Title
Text for Table of Contents
This article is protected by copyright. All rights reserved.