Direct Electron Transfer between a Site-Specific Pyrene-Modified

Feb 4, 2016 - Strategies to maximize direct electron transfer (DET) between redox enzymes and electrodes include the oriented immobilization of enzyme...
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Direct electron transfer between a site-specific pyrenemodified laccase and carbon nanotube/gold nanoparticle supramolecular assemblies for bioelectrocatalytic dioxygen reduction Noémie Lalaoui, Pierre Rousselot Pailley, Viviane Robert, Yasmina Mekmouche, Reynaldo Villalonga, Michael Holzinger, Serge Cosnier, Thierry Tron, and Alan Le Goff ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.5b02442 • Publication Date (Web): 04 Feb 2016 Downloaded from http://pubs.acs.org on February 5, 2016

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Direct electron transfer between a site-specific pyrenemodified laccase and carbon nanotube/gold nanoparticle supramolecular assemblies for bioelectrocatalytic dioxygen reduction Noémie Lalaoui†, Pierre Rousselot-Pailley‡, Viviane Robert‡, Yasmina Mekmouche‡, Reynaldo Villalonga§, Michael Holzinger†, Serge Cosnier†, Thierry Tron‡* and Alan Le Goff†*. †

Univ. Grenoble Alpes, DCM UMR 5250, F-38000 Grenoble, France CNRS, DCM UMR 5250, F-38000 Grenoble, France, ‡

Aix Marseille Université, CNRS, Centrale Marseille, ISM2 UMR 7313, 13397, Marseille, France

§

Department of Analytical Chemistry, Faculty of Chemistry, Complutense University of Madrid, 28040-Madrid, Spain

ABSTRACT: Strategies to maximize direct electron transfer (DET) between redox enzymes and electrodes include the oriented immobilization of enzymes onto an electroactive surface. Here, we present a strategy for achieving a controlled orientation of a fungal laccase on carbon nanotube-based electrodes. A homogeneous population of pyrene-modified laccase is obtained via the reductive amination of a unique surface accessible lysine residue engineered near the T1 copper centre of the enzyme. Immobilization of the site-specific functionalized enzyme is achieved either via π-stacking of pyrene on pristine CNT electrodes or through pyrene/β-cyclodextrin host guest interactions on β-cyclodextrin-modified gold nanoparticles (β-CD-AuNPs). Contrasting with unmodified and non-specifically modified (pyrene-NHS) laccaseelectrodes, an efficient DET is obtained at these nanostructured assembly. Modeling the direct bioelectrocatalysis of dioxygen reduction reveals an heterogeneity in ET rates on MWCNT electrodes wheras β-CD-AuNPs act as efficient electronic bridges, lowering ET rate dispersion and achieving a highly efficient reduction of O2 at low overpotential (≈ 80 mV) accompanied with high catalytic current densities of almost 3 mA cm-2.

KEYWORDS: Laccase, carbon nanotubes, dioxygen reduction, electrocatalysis, biofuel cells

Introduction Enzyme electrodes represent a class of bioelectrochemical systems with fascinating challenges for the design of electrocatalytic biomaterials for biosensing, bioreactors and bioenergy conversion. The electronic wiring of redox enzymes is an intensively studied subject with constant breakthroughs over the last decades, especially thanks to the development of nanotechnologies. In addition, the design of more and more efficient enzymatic fuel cells have driven the need for low-potential/high current bioelectrodes for oxidation of substrates such as dihydrogen or glucose and the reduction of dioxygen.1–8 In this matter, immobilization and wiring of multicopper oxidases (MCOs) have been widely investigated for their ability to reduce dioxygen at low-overpotential. In particular, laccases and bilirubin oxidases are almost exclusively employed at the biocathode of enzymatic biofuel cells.9,10 These MCOs possess a set of two copper-containing cen-

tres: a surface located T1 centre containing one CuII ion, responsible for the oxidation of substrates and the successive electron transfers to a buried trinuclear T2/T3 centre (TNC), structured between a type 3 pair of antiferromagnetically coupled CuII ions (T3) and a type 2 CuII ion (T2). The TNC achieves the four-electron reduction of dioxygen into water.11 Transferring efficiently electrons to an enzyme’s redox active site requires subtle adjustments. In particular, the direct electron transfer (DET) from an electrode to the biocatalyst is influenced by orientations of the protein that statistically increase the number of “plugged” enzymes. In this context, the direct wiring of laccases has evolved from original strategies to target a favorable DET to the high-redox-potential T1 copper centre which is supposed to be the electrochemical control centre in the reduction of dioxygen to water. In addition, the design of such enzyme electrodes has to preserve both the protein

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structure and its environment requires soft immobilization techniques to avoid any enzyme denaturation or unfolding. For example, the hydrophobic nature of the amino acids nearby the T1 copper centre of laccases from several Trametes fungi has been exploited to immobilize enzymes with favored orientation.12 Electrodes, functionalized with hydrophobic molecules, have shown to strongly interact with these amino acids.12–14

a control of the enzyme orientation on nanostructured electrodes. Immobilizations on CNT sidewalls by πstacking interactions or on β-CD-modified gold nanoparticles (β-CD-AuNPs) via host-guest interactions between β−CD and pyrene results in robust biocathodes with excellent bioelectrocatalytic dioxygen reduction properties.

In the functionalization process, the electrode material plays an essential role. Within few years, carbon nanotubes (CNT) have become a material of choice for building electrodes. CNTs represent a class of nanomaterials with an ideal combination of high specific surface, high conductivity and efficient ET properties for enzyme wiring.7,9 As a matter of fact, the use of CNT-based electrodes has substantially increased both the enzyme surface coverage and heterogeneous ET rates.14–19 Furthermore, the chemistry of CNT sidewalls has been deeply-investigated affording the access to a wide variety of functional CNT nanohybrids. Covalent functionalization of CNTs with hydrophobic laccase-substrate-like molecule such as anthracene, naphthalene or anthraquinone groups has been performed via different chemical processes (amide coupling with CNT defects,20 amination14 or diazonium chemistry21,22). Non-covalent methods have also been especially developed for the immobilization of laccases using for example modified pyrene molecules that form ππ interactions with the CNT sidewalls. We have recently characterized pyrene molecules bearing anthraquinone groups, that have shown excellent laccase immobilization properties and efficient bioelectocatalysis via DET.16 Covalent immobilizations of laccases on nano-objects have also shown to favour DET to the T1 copper centre of laccases.23–26 Advantages of such stable diffusion-less systems led to the development of laccase immobilizations through amide coupling via NHS-activated ester27–29 or imino bonds via amine-modified gold nanoparticles.23 A combination of non-covalent and covalent methods was also studied to efficiently immobilize MCOs while favoring enzyme orientation by a substrate-like molecule on graphite-based or CNT-based electrodes27,29–32.

Laccase engineering The enzyme used (LAC3 from Trametes sp. C30) is a typical fungal laccase, the recombinant form of which can be obtained with high yield after purification.33,34 We have already challenged the robustness of this enzyme in chemo-enzymatic catalysis experiments involving an electron transfer either from a transition-metal catalyst or photosensitizers.35–38 The LAC3 sequence is naturally poor in lysine residues as it contains only two residues (K40 and K71) located at the surface of the amino-terminus domain (GENEBANK AAR00925.1). This material is used to produce variants with a unique lysine residue located at the desired position on the protein surface.

Targeting generic chemical functions on the laccase surface (hydroxyls from sugars, carboxylic groups, amino groups or any other nucleophiles) one can obtain a covalent anchoring of the enzyme although via a random localization on the protein shell. This cannot results in a fully controlled and homogeneous orientation of the enzyme molecules on electrode materials. On the other hand, non-covalent techniques rely on the enzyme’s specific surface chemistry where a real specificity and efficiency is difficult to assess. Moreover, most of strategies developed for covalent or non-covalent immobilization have favoured a “bottom-up” approach in which the native enzyme is eventually reacted on pre-functionalized surfaces leaving space for undesired non-specific adsorption. In this work, we report on a “top-down” approach in which an engineered laccase bearing a single pyrene group specifically located near the T1 copper centre allows

Results and Discussion

Figure 1. Site-specific pyrene modified laccase UNIK (R161>K). The enzyme was reacted with 2,2,2-Trifluoro-N-(4-oxobutyl)-N-(1-pyren-4-ylmethyl-1H-[1,2,3]triazol-4-ylmethyl)acetamide.

The general strategy to obtain single surface located lysine variants of LAC3 (designed UNIK) is based on the replacement of a codon encoding a surface located residue, by an AAA/G codon (encoding a K residue) in a library of lac3 sequences representing the natural variations (based on sequence alignment) found at positions 40 and 71.39 Functional variants can then be selected upon expression in the yeast Saccharomyces cerevisiae.40 Briefly, for the UNIK variant, used in this study, a new lysine residue has been introduced by substitution of the enzyme’s arginine R161 (R->K161), found in molecular models,

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at the surface nearby the T1 copper ion (see Fig. 1). Lysine residues, at position forty and seventy one, of the peptide chain (K40 and K71) have been found to be substituted respectively by a methionine M40 and a histidine H71 in the UNIK161 variant (see SI for construction details). Highly selective modifications of primary amino groups present at the surface of proteins can be obtained in mild conditions.41 An original 2,2,2-Trifluoro-N-(4-oxo-butyl)N-(1-pyren-4-ylmethyl-1H-[1,2,3]triazol-4-ylmethyl)acetamide reactant was synthesized and used for the "pyrenization" (i.e. functionnalization with a pyrene group) of laccases in a reductive amination reaction (see Supplementary Information). The efficiency of the functionalization was evaluated from the ratio of the relative absorbance of the pyrene group (ε345 = 37500 mol-1.L.cm-1) and that of the enzyme (ε610 = 5600 mol-1.L.cm-1) (figure S1). Typically, this functionalization strategy results in ≈ 1 pyrene group/UNIK161. With a unique anchor point in the vicinity of the T1 copper center, the UNIK161-pyrene is designed to allow only one orientation of the laccase molecules on the surface of nanostructured electrodes with extended π-systems. This orientation should, in principle, favour DET between the electrode and the T1 copper centre (Fig. 1).

Laccase electrodes We designed two types of CNT-based electrodes. Glassy carbon (GC) electrodes were modified with a pristine MWCNT film obtained by drop-coating of a MWCNT dispersion in N-methylpyrrolidone (NMP). This allows the deposition of a homogenous 5-µm-thick MWCNT film on the GC electrode. A second type of nanostructured electrode was designed by the modification of the MWCNT film with βCD-AuNPs. As previously described for β-CD-modified proteins42–44, MWCNTs were first functionalized with 1pyrenebutyric acid adamantyl amide (pyreneadamantane). A simple incubation of MWCNT electrodes in a solution of pyrene-adamantane affords the coverage of MWCNTs with adamantane groups (see scheme Fig. 2A). Thanks to the strong host-guest interaction between adamantane and β-CD in water, β-CD-AuNPs can be immobilized by supramolecular interactions on the surface of the MWCNT sidewalls. Figure 2 displays the Scanning Electron Microscopy (SEM) images of a pristine MWCNT electrode (Fig. 2B) and a β-CD-AuNP/MWCNT electrode (Fig. 2C) obtained after successive incubations of the MWCNT electrode in a DMF solution of pyreneadamantane and an aqueous solution of β-CD-AuNPs. SEM images underline the homogenous deposition of a MWCNT (9.5-nm-diameter) film. The β-CD-AuNPs have a diameter of approximately 3 nm and are easily visualized using SEM. Subsequent to the functionalization of MWCNTs with adamantane groups, the homogenous coverage of β-CD-AuNPs is confirmed by the presence of bright spots along MWCNT sidewalls (Fig. 2B). Characterization of MWCNT/UNIK161 electrode Pyrene-modified UNIK161 was drop-casted on MWCNT electrodes. A schematic representation of the functionalized electrodes and the associated CV recorded under argon and oxygen are displayed in Figure 3.

Figure 2. (A) schematic representation of the β-CD-AuNP modification of MWCNTs, (B) SEM images of a pristine MWCNT film and (C) of the β-CD-AuNP/MWCNT modified electrodes

Figure 3. Left: CVs of the UNIK161-functionalized MWCNT electrode under argon or oxygen for (a) the bare UNIK161, (b) UNIK161-NHS-pyrene and (c) UNIK161-pyrene in 0.1 M phos-1 phate buffer (pH 5.0); scan rate v = 0.01V s . Right: scheme of the UNIK161-pyrene immobilized and oriented on MWCNTs.

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We first performed control experiments with the unmodified UNIK161 enzyme adsorbed on the MWCNT electrode under argon or oxygen. Under oxygen, a maximum current density of 0.27 mA cm-2 at 0V vs SCE was measured. This low catalytic currents for dioxygen reduction illustrates a poor DET for such adsorbed enzymes. In a striking contrast, UNIK161-pyrene exhibited excellent DET properties on pristine MWCNT (Fig. 3c). While the onset potential of 0.6 V vs SCE measured suggests that DET is achieved at low overpotential via the T1 copper of the favourably oriented UNIK161, an efficient electrocatalytic dioxygen reduction was observed with a maximum current density of 1.15 mA cm-2 at 0 V vs SCE. For the comparison, a UNIK161 modified with pyrene-NHS (noted UNIK161-NHS-pyrene) was used to functionalize the MWCNT electrode. Pyrene functions were randomly attached to UNIK161 via activated ester coupling. With non-homogeneously distributed pyrene functions on the surface of the enzyme (3 pyrene groups for one laccase according UV-visible measurements from figure S1), the UNIK161-NHS-pyrene exhibits an electrocatalytic activity towards dioxygen reduction with a maximum faradaic current density of 0.37 mA cm-2 at 0V vs SCE (Fig. 3b). These results clearly underlines that a substantial improvement in the electrode performance can be obtained through a site-specific functionalization near the T1 copper centre of the enzyme.

Characterization of MWCNT/β β -CD-AuNP/ UNIK161 electrode The UNIK161-pyrene was also immobilized on β-CDAuNP/MWCNT electrodes. The β-CD-AuNPs offer hostguest interactions between pyrene groups and β-CD moieties on the AuNPs surface. CVs obtained under argon or oxygen are shown in Figure 4.

Figure 4. Left: Cyclic voltammograms of the UNIK161pyrene/β-CD-AuNP/MWCNT electrode under argon (black dotted line) and oxygen (red line). The CV of the UNIK161pyrene/MWCNT electrode under oxygen (dashed line) is presented for the comparison. Conditions: 0.1 M phosphate -1 buffer (pH 5.0); scan rate v = 0.01V s . Right: scheme of the UNIK161-pyrene immobilized and oriented on β-CD-AuNPs modified MWCNTs.

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As observed for pristine MWCNT, an onset potential of 0.6 V was measured. On the other hand, CVs recorded with the UNIK161-pyrene/β-CD-AuNP/MWCNT electrode revealed a higher efficiency in electrocatalysis performed by the laccase accompanied with a more classic bioelectrocatalytic sigmoidal waveshape. Indeed, with this electrode setting, UNIK161 reaches a high electrocatalytic current of 2.75 mA cm-2 at 0 V vs SCE. It is noteworthy that the two electrodes (UNIK161pyrene/MWCNT and UNIK161-pyrene/β-CDAuNP/MWCNT electrodes) exhibit stable CV curves during several hours of continuous cycling. In chronoamperometric experiments, stable electrocatalytic currents were also obtained for at least one hour of continuous operation at 0 V vs SCE under oxygen saturation. After one week of storage, all the electrodes retained ca. 60% of their initial activity and kept these performances during at least one month (Figure S2). The DET achieved for the modified LAC3 variant (UNIK161-pyrene) on MWCNT electrodes is largely competitive with a previously-described setups based on a His-tagged LAC3 immobilized and oriented on a gold electrode surface and combined to the complex [Os(bpy) pyCl] as redox mediator in solution.45 Without the need for a mediator, the maximum current density obtained with the UNIK161-pyrene on MWCNT electrodes are up to two orders of magnitude higher than that obtained at the gold surface through a Mediated Electron Transfer (MET) pathway. Such differences in efficiencies for the same enzyme may be related to differences in the anchor points targeted on the enzyme surface, to the effective surface coverage of nanostructured electrode materials, or to a combination of these factors. 2+

2

To get further insights in the DET mechanism of UNIK161-pyrene on these nanostructured electrodes, we applied different theoretical models for DET based bioelectrocatalytic reduction of dioxygen.

Models for dioxygen reduction by laccases at nanostructured electrodes Different models have been studied to elucidate DET of multicopper oxidases on electrodes.46–49 The Armstrong’s group has greatly contributed to the modelling of thin protein films supported by voltammetric data on metalloenzymes. Their most advanced model describes the successive single ET between the electrode and the enzyme where a small substrate such as hydrogen is oxidized by taking into account a dispersion of ET rates related to different orientations of the enzyme molecules on the entire electrode surface.46,50,51 Originally developed for hydrogenases, this model has also been successfully applied to MCOs such as bilirubin oxidases and laccases in which the T1 copper centre acts as an electron relay ensuring the successive four single ET to the TNC where the dioxygen reduction takes place.23,46,47 While immobilizing the biocatalyst, minimizing mass transfer contributions, the model gives a good description of the kinetic limitation of the electrocatalytic wave. In our systems, maintaining the dioxygen concentration relatively constant

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through a continuous gas flow or rotating of the electrodes above 1000 rpm, no increase of catalytic currents (indicative of mass-transport limitations) was observed with any of the tested bioelectrodes. Therefore, we modeled the CVs from Figure 3 in which the Faradaic electrocatalytic currents follows the equation 1 (reproduced from ref 51): 

    1 

 1   1    1  exp  

where e1 = exp(nF/RT)(E-E°Cu(T1)), e2 = exp(4F/RT)(E°Cu(T1)-EO2/H2O), EO2/H2O = 0.69V vs SCE at pH5, α is the transfer coefficient, p is the ratio of the sum of catalytic constant for the reversible heterogeneous dioxygen catalysis over k0max ET rate constant at the minimal distance between the electrochemical relay centre and the electrode. Since laccase performs an irreversible electrocatalytic reduction of dioxygen, p can be written as kcat/k0max. βd0 is the ET tunneling factor accounting for ET rate dispersions. Since we are in the case of an irreversible electrocatalytic reduction, the limiting current density is defined in relation with the dioxygen reduction rate constant kcat.46 The electrocatalytic wave for the UNIK161-functionalized MWCNT electrode was well-fitted using n = 1. Experimental and fitted slopes for MWCNT electrodes are displayed in Figure 5A.

sonable to think that electrocatalysis is governed by two distinct populations of laccases molecules: one immobilized on MWCNTs and one immobilized on β-CD-AuNPs. Since equation 1 gives an excellent fit for UNIK161functionalized MWCNT electrode, we could hypothesize that this fitting curve is representative of the population of laccases directly immobilized on MWCNT sidewalls. Based on these two assumptions, we could be able to decipher the contribution of the population of laccases immobilized on AuNPs. The best fit for the experimental curve (red curve, Fig. 5B) was thus achieved by deconvolution into two theoretical curves describing i) the behavior of the laccase immobilized on MWCNTs (curve a, Fig. 5B) as previously used to model the laccase-MWCNT electrode (see Fig. 5A) and ii) a simple Nersntian electrocatalytic process corresponding to the contribution of AuNPs with only two parameters, E0 and jlim (curves b, Fig. 5B). The constant values obtained for the simulated curves are collected in Table 1. Summing both terms (curves a+b) allows obtaining a theoretical curve c that matches well the experimental data over a large range of potentials for the UNIK161-pyrene/β-CD-AuNPs/MWCNT electrode. Therefore, this model describes satisfactorily the β-CD-AuNP/MWCNT electrode response, consistent with a dual mode of immobilization of the UNIK161-pyrene molecules on β-CD-AuNPs and MWCNT surfaces. Table 1. Constants derived from simulated CVs of the laccase-pyrene β -CD-AuNP/MWCNT electrode* Contribution

E0 (V)

MWCNT

0.51

β-CD-AuNP

0.51

jlim

p

βdo

1.29

0.04

13.3

1.76

-

-

-2

(mA cm )

* e2 values were fitted using E°O2/H2O = 0.69 V vs SCE. Large values of e2 are expected from the irreversible electrocatalytic reduction of O2 by laccase.

Figure 5. (A) Experimental (red) and simulated (black) CV scans for the UNIK161-pyrene/MWCNT electrode under oxygen; (B) Experimental (red) and simulated (black) CV scans for the UNIK161-pyrene/β-CD-AuNP/MWCNT electrode under oxygen. (a) Simulation of the UNIK161-pyrene/MWCNT contribution; (b) pure Nernstian contribution attributed to β-CD-AuNPs, (c) sum of simulations (a)+(b). The background was subtracted from experimental CVs to remove the capacitive current contribution and obtain an average of the back and forward scans.

Modelling the CVs of the UNIK161-pyrene/β-CDAuNP/MWCNT electrode did not provide a satisfactory fitting curve using equation (1). At this stage, it was rea-

Discussion With a single pyrene molecule grafted in the vicinity of the T1 copper centre, the UNIK161-pyrene is designed to favour DET between nanostructured electrodes and the enzyme’s primary electron acceptor site. In fact, UNIK161pyrene modified electrodes (MWCNT and β-CDAuNP/MWCNT) allow excellent electrocatalytic reduction of dioxygen at a low overpotential (≈80mV). For both, MWCNT and β-CD-AuNP electrocatalysis, the redox potential for the dioxygen reduction (E0= 0.51 V vs SCE from simulation of the curves) at pH 5 is fully consistent with the redox potential previously estimated by spectroelectrochemistry for the T1 copper ion of LAC3 from Trametes sp. C30, UNIK’s parental enzyme (0.68V vs NHE or 0.44 V vs SCE at pH 6).45 The ineffectiveness of electrodes functionalized with the unmodified enzyme underlines that the catalytic current is essentially related to enzymes wired to the electrode surface through pyrene

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functions. Based on the site-specific modification of the enzyme surface (i.e. K161), we conclude that the first layer of electroactive molecules at the electrode surface consists of a single enzyme population. In contrast, random chemical modification of UNIK161 by a pyrene derivative (e.g. pyrene-NHS) generates electrode surfaces covered with randomly-oriented laccases. This likely accounts for the 75% decrease in electroactivity consistently observed for electrodes functionalized with a pyrene-NHS reacted laccase as compared to those functionalized with the UNIK161-pyrene. The two types of used electrodes (MWCNT and β-CDAuNPs-MWCNT) offer different surfaces and supramolecular modes of interaction to the functionalization with the UNIK161-pyrene: π-stacking on MWCNT, and both, πstacking and host-guest interaction on β-CD-AuNPsMWCNT. Globally, the β-CD-AuNP/MWCNT electrode is 2-2.5 times more efficient than the unmodified MWCNT electrode. This superiority can be attributed to: i) an increased electrode surface coverage provided by β-CDAuNPs associated to an increase in catalytic currents by 130%. This is consistent with SEM images shown in figure 2 which underline the high surface coverage of β-CDAuNPs together with the already high surface area of the MWCNT deposit; and ii) the excellent DET properties of AuNPs. While electrocatalytic performances of the laccase immobilized on MWCNT is well-modelled taking into account a dispersion in non-homogeneous ET rates, electrocatalytic performances of the immobilized laccase on AuNPs (after subtraction of the contribution of laccases in direct contact with MWCNTs) is well-fitted using a simple Nernstian catalytic process. The fact that a dispersion of ET rates is observed on MWCNTs (βdo = 13.3) might indicate that, despite a favourable orientation of UNIK161, the heterogeneous rate constant is still influenced by the existing distance between the MWCNT surface and the T1 copper centre. On the contrary, a pure Nernstian behaviour is observed for AuNPs underlining the fact that, with high heterogeneous ET rate values, the distance between the T1 copper ion and the electrode is no longer limiting. Consequently, the bioelectrocatalysis is only limited by the enzymatic catalysis. Such effects of AuNPs (mixed DET behaviours, gain in bioelectroactivity) have been already observed with a bio-cathode made of AuNP-modified Low-Density-Graphite electrode and a laccase from Trametes Hirsuta.23 However, the authors of this study have observed a 100-mV potential shift between laccases wired on AuNPs and laccases probably wired on graphite. This shift has been attributed to a modification of the enzyme structure, more than to an ET pathway involving the TNC centre. In our work, as no shift in the redox potential of the enzyme is observed between MWCNTs and AuNPs contributions, we conclude that the site-specific targeting of the pyrene at the enzyme surface allows an homogeneous consequent orientation of the enzyme on the electrode with an excellent conservation of the enzyme structure and activity. In addition, maximum current densities as high as 2.3 mA cm-2 can be reached at high potential of 0.4 V (3 mA cm-2 at 0 V)

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which is among the best performances for MCO-based dioxygen-reducing biocathodes.9,10 In conclusion, we achieved efficient DET based electrocatalytic reduction of dioxygen using nanostructured βCD-AuNPs and/or MWCNT electrodes with a site-specific surface modification of a fungal laccase with a single covalently bound pyrene group close to the T1 centre. Doing so, the majority of the of enzyme molecules making the first layer on the electrode surface consists in a single oriented enzyme population which makes the T1 copper centre of the enzyme readily available for DET. We also clearly highlighted the synergistic combination of CNTs with β-CD-AuNPs. While an efficient DET with MWCNTs is obtained with rate dispersions, AuNPs greatly narrows the ET pathways while evenly increasing the number of wired enzymes. At these electrodes, bioelectrocatalytic dioxygen reduction is only limited by the enzyme surface coverage and catalysis.

ASSOCIATED CONTENT SUPPORTING INFORMATION Chemicals and general procedures; Obtention of the UNIK161 variant; Preparation of MWCNT electrodes, Synthesis of the 2,2,2-Trifluoro-N-(4-oxo-butyl)-N-(1-pyren-4-ylmethyl-1H[1,2,3]triazol-4-ylmethyl)-acetamide; Functionalization of Unik161 by 2,2,2-Trifluoro-N-(4-oxo-butyl)-N-(1-pyren-4ylmethyl-1H-[1,2,3]triazol-4-ylmethyl)-acetamide; Functionalization of Unik161 by 1-Pyrenebutyric acid-Nhydroxysuccinimide ester; UV-visible spectrum of UNIK161, UNIK161-pyrene and UNIK161-NHS-pyrene in water (Fig. S1); One-hour chronoamperometry performed on UNIK161pyrene/β-CD-AuNP/MWCNT electrode under oxygen (Fig. S2). This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [email protected] ; [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT The authors wish to acknowledge the support from the platform Chimie NanoBio ICMG FR 2607 (PCN-ICMG) and from the LabEx ARCANE (ANR-11-LABX-0003-01). The authors also thank the ANR Investissements d'avenirNanobiotechnologies 10-IANN-0-02 program for financial support. They also thank the GDR CNRS 3540 “Biopiles” for partial financial support. Electron microscopy was performed at the CMTC characterization platform of Grenoble INP supported by the Centre of Excellence of Multifunctional Architectured Materials "CEMAM" n°AN-10-LABX-44-01 funded by the "Investments for the Future" Program. We would also like to thank ZEISS

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for valuable support and discussion in operating GeminiSEM 500.

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