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Oriented immobilization of [NiFeSe] hydrogenases on covalently- and noncovalently-functionalized carbon nanotubes for H2/air enzymatic fuel cells Solène Gentil, Syamim Muhamad Che Mansor, Hélène Jamet, Serge Cosnier, Christine Cavazza, and Alan Le Goff ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b00708 • Publication Date (Web): 28 Mar 2018 Downloaded from http://pubs.acs.org on March 28, 2018

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ACS Catalysis

Oriented immobilization of [NiFeSe] hydrogenases on covalentlyand noncovalently-functionalized carbon nanotubes for H2/air enzymatic fuel cells Solène Gentil†‡, Syamim Muhamad Che Mansor†, Hélène Jamet†, Serge Cosnier†, Christine Cavazza‡ and Alan Le Goff†* † ‡

Univ. Grenoble Alpes, CNRS, DCM, 38000 Grenoble, France Univ. Grenoble Alpes, CEA, CNRS, BIG-LCBM, 38000 Grenoble, France

ABSTRACT: We report the oriented immobilization of [NiFeSe] hydrogenases on both covalently- and non-covalently modified carbon nanotubes (CNTs) electrodes. A specific interaction of the [NiFeSe] hydrogenase from Desulfomicrobium baculatum with hydrophobic organic molecules was probed by electrochemistry, QCM-D and theoretical calculations. Taking advantages of these hydrophobic interactions, the enzyme was efficiently wired on anthraquinone and adamantane-modified CNTs. Owing to rational immobilization onto functionalized CNTs, the O2-tolerant [NiFeSe]-hydrogenase is able to efficiently operate in a H2/air gas diffusion enzymatic fuel cell.

KEYWORDS: hydrogenases, carbon nanotubes, diazonium, biofuel cells, hydrogen oxidation

INTRODUCTION The use of hydrogenases instead of precious metal catalysts in hydrogen fuel cells provide a renewable alternative to the design of biodegradable and biocompatible energy sources 1–7. In addition to high catalytic turnovers and minimal overpotentials, the biodiversity of enzymes provide a wide library of base metal catalysts in terms of operating conditions, substrates specificity and catalytic bias. However, hydrogenases are often multimeric large proteins, possessing a complex multimetallic active site. The latter is buried within the protein, requiring gas tunnels and pathways for transferring electrons and protons between the catalytic center and the molecular surface. This is the reason why the wiring of enzymes at electrode surface requires rational strategies in order to achieve fast electron transfer rates and maximized catalyst loading. In this respect, we and other have especially developed the use and functionalization of carbon nanomaterials in order to enhance direct electron transfer (DET) between enzyme active sites and electrodes while providing high conductive and high-surface-concentration electrocatalytic layers.8–12 In particular, the specific functionalization of surfaces have taken advantages of different types of interactions for the orientation of redox enzymes and the establishment of an optimal DET: electrostatic interactions taking advantage of enzyme dipolar moment,8,13–16 hydrophobic or hydrophilic interactions10,12,17–19, covalent attachment20,21 or specific protein surface amino-acid binding.9 Enzymatic hydrogen fuel cells rely on nickel- and ironbased hydrogenases to oxidize H2 and multicopper enzymes to reduce oxygen from air .14,22–26 Both types of enzymes have proven to be competitive in terms of catalytic turnovers as compared to Pt/C catalysts 27–29. However, while many types of strategies has been developed to design efficient biocath-

odes, the design of efficient bioanodes still requires to address major issues in terms of operational stability and oxygen sensitivity of hydrogenases. Several strategies have been employed to increase hydrogenase stability towards oxygen: (i) site-directed mutagenesis 30, (ii) the use of protective redox polymers 26,31–33 or redox bionanowires34 (iii) the use of oxygen-tolerant hydrogenases such as the membrane-bound hydrogenase from the hyperthermophilic bacterium Aquifex aeolicus 22,35,36or (iv) the design of particular fuel cell set-ups preventing oxygen to be in contact with hydrogenases. 14,37,38 [NiFeSe]-hydrogenases are a subclass of [NiFe]hydrogenases in which a nickel-bound cysteine is replaced by a selenocysteine and display unique properties. Protein film voltammetry performed on PGE has shown that NiFeSe hydrogenase from Desulfomicrobium baculatum (Db[NiFeSe]) exhibits high turnover frequencies towards H2 oxidation and a fast reactivation from O2 inactivation at low redox potentials.39 However, this is accompanied with fast deactivation process in both the presence of oxygen and at high potential. These characteristics have prevented the use of these enzymes as a bioanode for direct H2 oxidation for fuel cell applications. Reisner et al. have shown that these enzymes were much more efficient towards H2 production, in particular in H2 photoproduction at dye-sensitized TiO2 nanoparticles40 Only a recent work from Ruff and coworkers has demonstrated that a redox polymer was required to achieve mediated electrocatalytic oxidation of H2 while protecting [NiFeSe]-hydrogenase from Desulfovibrio vulgaris Hildenborough from oxidative deactivation.32 The interactions between this oxygen-resistant hydrogenase and modified electrodes have never been studied so far, especially at nanostructured electrodes. Here we report the functionalization of carbon nanotubes for the immobilization of Db[NiFeSe]-hydrogenase. We have studied the oriented im-

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mobilization of the enzyme at CNT covalently modified by electrografting of different types of aryldiazonium derivatives and noncovalently-modified with 1-pyrenebutyric acid adamantyl amide via pi-stacking interactions. Thanks to the comparison between each modification strategies of the nanostructured electrodes, we show that hydrophobic interactions via grafted adamantane or anthraquinone groups are able to promote the immobilization and orientation of this enzyme. Using electrochemistry, Quartz crystal microbalance with dissipation monitoring (QCM-D) and theoretical calculations, we demonstrate for the first time that Db[NiFeSe]-hydrogenase can be rationally immobilized for the achievement of efficient H2 oxidation. Recent works have shown that enzymes such as multicopper oxidases or hydrogenases could efficiently operate in gas diffusion electrodes (GDE). 37,41–44 GDE has the exceptional ability to operate catalysts at the interface between electrode, electrolyte and gas (air or H2), circumventing limitations coming from gas solubility and diffusion. We finally designed a fully-gas diffusion H2/air biofuel cell, demonstrating that Db[NiFeSe]-hydrogenase can efficiently operate in conventional fuel cell set-ups.

RESULTS AND DISCUSSION First, multiwall carbon nanotubes (MWCNT)-coated glassy carbon (GC) electrodes were made by drop-coating of a NMP dispersion of 10-nm-diameter MWCNTs. The MWCNT electrodes were covalently modified by electrografting. Three types of aryldiazonium salts were used:2amino-4-ethylphenyldiazonium,6carboxynaphthalenediazonium and 2-diazoniumanthraquinone tetrafluoroborate (Figure 1A). The purpose of this covalent modification is to modify surface properties of CNT sidewalls by providing negatively- or positively-charged molecules, or hydrophobic molecules such as anthraquinone. Using diazonium chemical or electrochemical functionalizations, we and others have already demonstrated that these types of molecules were able to interact and orientate metalloenzymes such as multicopper oxydases (MCOs)8,12,45,46 and hydrogenases14 on CNT sidewalls. Electrografting was performed by successive cyclic voltammogramms (CVs) scans below the typical irreversible reduction wave corresponding to the reduction of the aryldiazonium group. Figure 1B displays CV scans performed in a 2 mmol L-1 solution of each diazonium salts in acetonitrile (MeCN). An irreversible redox peak is observed at Epred = 0.05, 0.27 and 0.44 V vs Ag/AgNO3 for 4-(2aminoethyl)benzenediazonium (AE) 4carboxylatonaphtyldiazonium (CN) and 2diazoniumanthraquinone (AQ) respectively. This redox potential is correlated with the electronic contribution of the different aromatic groups. 47,48 On subsequent scans, the decrease of the reduction peak current is induced by the passivation of the as-formed polyphenylene layer at the surface of CNT sidewalls.

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Figure 1. (A) Schematic representation of electrografting of MWCNTs by reduction of different aryldiazonium salts; (B) Cyclic voltammetry (CV) of a 2 mmol L−1 solution of (a) 4carboxylatonaphtyldiazonium tetrafluoroborate (CN), (b) 4-(2aminoethyl)benzenediazonium tetrafluoroborate (AE) and (c) 2diazoniumanthraquinone tetrafluoroborate (AQ) in MeCN–TBAP (0.1 mol L−1) at a MWCNT electrode (5 scans, v = 20 mV s−1)

AE-MWCNT, CN-MWCNT and AQ-MWCNT were then compared towards the immobilization and orientation of Db[NiFeSe]-hydrogenase. Hence, 10 µL of Db[NiFeSe]hydrogenase (1.5 mg mL-1, pH = 7.6) were drop-casted. The electrodes were left to dry for 1 hour at room temperature and then rinsed with the phosphate buffer at pH = 7.6. CVs under H2 of the Db[NiFeSe] hydrogenase-modified MWCNT electrodes are displayed in Figure 2A. For all types of modified electrodes, an irreversible electrocatalytic anodic wave is observed in the presence of H2. This observation corresponds to the oxidation of H2 into H+ catalyzed by the immobilized enzyme. In the case of AQ-MWCNT (curve c, blue), a reversible redox system is observed at E1/2 = -0.54 V vs Ag/AgCl and corresponds to the 2e-/2H+ reversible anthraquinone reduction in water. In a control experiment, where no enzyme was immobilized, no catalytic current is observed on pristine MWCNTs or AQ-MWCNTs (Figure S1A and S2), underlining the poor catalytic activity of MWCNTs towards H2 oxidation. It is worth noting that the electrocatalytic wave for AE- and CN-MWCNT exhibits a typical deactivation process at high potential.

Figure 2. (A) CVs of the Db[NiFeSe] hydrogenase-functionalized (a) CN-MWCNT, (b) AE-MWCNT, (c) AQ-MWCNT and (d) pristine MWCNT electrodes under H2 at pH = 7.6; (B) CVs of the Db[NiFeSe] hydrogenase-functionalized AQ-MWCNT (dotted line) under argon and (straight line) under H2 bubbling at pH = 7.6 (50 mmol L-1 sodium phosphate buffer solution at pH = 7.6, v = 10 mV s-1)

The origin of this process has been extensively studied and arises from the oxidative formation of an inactive form of the enzyme.39,49 For AQ-MWCNT electrode (Figure 1B), negligi-

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ble deactivation was observed on the CV time scale and higher current densities of 1.60 mA cm-2 were measured. Negativelyand positively- charged modified CNTs exhibit maximum current densities of 1.42 and 1.04 mA cm-2 at -0.50 V vs Ag/AgCl, higher as compared to pristine MWCNT (maximum current density of 0.77 mA cm-2 at -0.36 V vs Ag/AgCl). The influence of the thickness of the electrografted poly[anthraquinone] layer over the electrocatalytic oxidation of H2 was investigated by varying the number of electrografting CV scans (from 0 to 5 scans). The anthraquinone surface coverage is estimated from the integration of the faradic current under the reversible anthraquinone redox system for each grafting conditions. The amount of grafted anthraquinone increases continuously from 128 to 176 nmol cm-2 with the number of scans It is noteworthy that a simple incubation of the MWCNT electrode in a solution of 2-diazoniumanthraquinone allows the stacking of a substantial amount of anthraquinone, i.e. 128 nmol cm-2. The spontaneous grafting of diazonium on CNTs has already demonstrated to be highly efficient towards carbon- or metal-based materials.50,51 Furthermore, the increase of the amount of grafted anthraquinone correlates with the increase of the electrocatalytic oxidation of H2 (Figure S3). An H2 oxidation plateau of 1.9 mA cm-2 is reached for a surface concentration of 176 nmol cm-2. At this value, MWCNTs are likely optimally covered with anthraquinone, maximizing the number of wired Db[NiFeSe]-hydrogenase. In order to confirm the effect of hydrophobic interactions in the immobilization and direct wiring of Db[NiFeSe]hydrogenase, MWCNTs were also modified with adamantane groups, using a previously-described procedure. 1pyrenebutyric acid adamantyl amide is adsorbed at the surface of MWCNTs via pi-pi interactions (Figure 3A).10 These adamantane-modified MWCNTs (ADA-pyrene-MWCNT) were incubated in 1.5 mg mL-1 enzyme solution. Figure 3B displays CV performed under argon and H2. ADA-pyrene-MWCNT film exhibits maximum current densities of 1.6 mA cm-2 at 0.19 V vs Ag/AgCl, closed to the values obtained for anthraquinone-modified MWCNTs. Furthermore, owing to the fact that adamantane is not electroactive (on the contrary of anthraquinone), a non-turnover small reversible system is observed at E1/2 = -0.54 V vs Ag/AgCl (∆E = 20 mV) under argon for ADA-pyrene-MWCNT electrode. It is noteworthy that the presence of a small irreversible oxidation under argon for both pristine and ADA-pyrene-MWCNT electrodes arises from oxidation of H2 formed during the forward scan through proton reduction. This induces an overlap between this catalytic residual signal and the nonturnover signal on the backward scan for ADA-pyrene-MWCNT electrodes under argon. The reversible redox system likely corresponds to the reversible Db[NiFeSe]-hydrogenase redox system (Figure 3C). By integration of the charge under the reduction peak, a surface coverage of 11 pmol cm-2 was estimated. For comparison, a tiny redox system have been observed for this enzyme at pyrolytic graphite edge electrode, corresponding to an enzyme coverage of less than 1 pmol cm-2.39 A Turnover frequency of 850 s-1 for immobilized Db[NiFeSe] was estimated from surface coverage and maximum current density. For comparison, this is one order of magnitude lower has compared to TOF values measured in solution (8700 s-1). This is in the same range as compared to other type of immobilized hydrogenases studied at electrodes.6 This is one order of magnitude higher as compared to TOF values measured for photo-induced H2 production by NiFeSe immobilized on TiO2 nanoparticles.40 By

subtracting capacitive currents and noncatalytic signals (Figure S4), a comparison of catalytic waveshapes for all modified MWCNTs indicates that both AQ- and ADA-pyreneMWCNTs exhibit a similar electrocatalytic waveshape with a greatly reduced apparent oxidative deactivation as compared to pristine, AE- and CN-MWCNT electrodes. On the contrary, AE- and CN-MWCNT exhibit a rapid and previouslyobserved oxidative deactivation It is noteworthy that AE- and CN-MWCNT electrodes exhibit near-zero overpotentials for reversible H2 oxidation. This is indicated by the presence of both H2 oxidation and H+ reduction, the electrocatalytic waveshape crossing the zero-current at the standard redox potential of the H+/H2 couple at pH 7.6 (0.645 V vs. Ag/AgCl). On the contrary, no apparent H+ reduction at this potential, accompanied with a higher overpotential for H2 oxidation of several tenths of millivolts, is indicative of the presence of a higher heterogenous electron transfer rate constant for AQ- and ADA-pyrene-MWCNTs.52 This is counterbalanced by the high affinity of Db[NiFeSe]-hydrogenase for AQ- and ADA-pyrene-MWCNTs.

Figure 3. (A) Schematic representation of the functionalization of MWCNT by 1-pyrenebutyric acid adamantyl amide ; (B) CVs of the Db[NiFeSe] hydrogenase-functionalized ADA-pyreneMWCNT (red) and non-modified MWCNTs (grey) under argon (dotted line) and under H2 bubbling (straight line) at pH = 7.6 ; (C) CV of the Db[NiFeSe] hydrogenase-functionalized ADA-pyreneMWCNT under argon (50 mmol L-1 sodium phosphate buffer solution, v = 10mV s-1, CVs were reproduced on at least 3 electrodes)

It is noteworthy that the non-turnover signal is rarely observed for most hydrogenases. These features unambiguously confirmed the efficient wiring of Db[NiFeSe]-hydrogenase on both AQ- and ADA-pyrene-MWCNTs. In order to characterize the hydrophobic interactions between Db[NiFeSe]-hydrogenase and functionalized MWCNTs, theoretical calculations were performed by docking simulation via Autodock program. A similar docking pose for both adamantane and anthraquinone was identified from the most recurrent scoring pose among all docking runs (Figure 4).

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Figure 4. Binding modes for (A) anthraquinone- Db[NiFeSe]hydrogenase and (B) adamantane- Db[NiFeSe]-hydrogenase complexes in solution. Data were obtained by docking simulation.

Molecular dynamic simulations were then performed. Some variations were obtained between adamantane and anthraquinone. For anthraquinone, the tyrosine 209 orientates its position through π-stacking interactions (Figure S5). Adamantane interacts through hydrophobic interactions with the threonine 223 and the isoleucine 470 (Figure S6). Root mean-square deviation (RMSD) analysis along the trajectory were performed to check that the structures are stable (inset, Figures S2-3). Positions are not the same but both molecules remain in the same region, located at an average distance of around 9 Å from the distal [Fe4S4] cluster. This falls in the region of possible electron tunneling. While it is difficult to observe or characterize the real orientation or conformation of the enzyme on MWCNT sidewalls, these results are consistent with the ability of attached adamantane and anthraquinone to improve Db[NiFeSe] immobilization as well as its favorable orientation by decreasing the statistical distribution of distances between the distal [Fe4S4] cluster and the MWCNT sidewalls. Furthermore, CVs shows that electrocatalytic currents exhibits low oxidative deactivation process for both ADApyrene-MWCNT and AQ-MWCNT. It is unlikely that protection towards deactivation occurs via mediated electron transfer, as it as been seen for viologen-based hydrogel films.26,32,33,53 The main reason is that the rigidity of the poly[anthraquinone] layer might not be favorable for mediated electron transfer and efficient charge transport throughout the film.54 Furthermore, anthraquinone redox potential is similar to the redox potential of the enzyme (+0.54 V vs. Ag/AgCl). Despite the fact that an efficient mediated electron transfer is governed by many parameters, a negligible driving force is detrimental to a mediated electron transfer in most cases. This is corroborated by the fact that adamantane, a non-redoxactive group, exhibits similar electrocatalytic wave shape after background subtraction (Figure S4). The apparent resistance towards deactivation is likely caused by H2 diffusion limitations. This diffusion-limitation process arises from the favorable orientation of the enzyme on the surface of the electrode and associated high electrocatalytic activity and from the large porosity of MWCNT films. The effect of mass transport limitations have been already observed for hydrogenases from

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Aquifex aoelicus immobilized on carbon nanofibers55 or on modified MWCNTs14 and hydrogenases from Hydrogenovibrio marinus on GDE43 QCM-D experiments were performed in order to study and quantify the immobilization of Db[NiFeSe]-hydrogenase on functionalized MWCNTs. A pristine MWCNT film was first deposited on gold-coated piezoelectric quartz crystals. A membrane transfer process ensures a highly reproducible and stable deposition of the film on the quartz sample. MWCNTcoated quartz crystals were soaked either in a 2mM solution of 2-diazoniumanthraquinone tetrafluoroborate or 2 mM solution of 1-pyrenebutyric acid adamantyl amide and then rinsed. Typical QCM-D profiles for the immobilization of Db[NiFeSe]-hydrogenase on non-modified, AQ- and ADApyrene-MWCNT-coated quartz crystals are displayed in Figure 5. The frequency and dissipation shift are first stabilized by incubation of 300 µL of 50 mM phosphate buffer solution at pH = 7.6. After removal of 150 µL of buffer, 150µL of a 0.2 mg mL-1 enzyme solution were added. After stabilization, the quartz surface was rinsed by successive removal/addition steps of 50 µL buffer solution for 3 times. The injection of the enzyme solution triggers a frequency decrease of 118 Hz and 203 Hz for AQ-MWCNT-coated and ADA-pyrene-MWCNTcoated quartz crystals respectively. It is noteworthy that a control experiment performed on pristine MWCNT-coated quartz crystals exhibited negligible frequency decrease, underlining the weak interactions between non-modified MWCNTs and Db[NiFeSe] under these conditions and corroborating the fact that AQ and pyrene-ADA interact with specific domains of the Db[NiFeSe]hydrogenase. Furthermore, the three washing steps do not induce any frequency increase. Hence, the frequence shift for both AQ-MWCNT and ADA-pyrene-MWCNT films confirms the stable interaction between Db[NiFeSe]-hydrogenase and functionalized MWCNTs. In parallel, the increase of the dissipation factor for both AQ-MWCNT and ADA-pyreneMWCNT-coated quartz crystals reflects a decrease in the rigidity of the functionalized MWCNT surface, which underlines the stable immobilization of proteins.

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Figure 5. (A) QCM-D profile (shifts in resonant frequency (black curves) and in dissipation (gray curves) vs. time) for the immobilization of Db[NiFeSe]-hydrogenase on non-modified MWCNT (dotted lines), AQ-MWCNT (dashed lines), ADApyrene-MWCNT (full lines) (3rd overtone data) before and (a) after injection of 0.2 mg mL-1 enzyme solution in 50 mM sodium phosphate buffer at pH = 7.6 for 60 minutes, (b) 3 washing steps using phosphate buffer ; (B) Plot of Db[NiFeSe]-hydrogenase mass uptake vs. time calculated from the Sauerbray equation56 for the 3rd overtone

Figure 5B displays the plot of the Sauerbrey mass uptake of Db[NiFeSe]-hydrogenase immobilized at the surface of both AQ-MWCNT and ADA-pyrene-MWCNT film as a function of time.56 Curves for each overtone show an average mass of adsorbed enzyme of 1.7 (+/-0.3) and 2.8 (+/-0.5) µg cm-2 for AQ-MWCNT and ADA-pyrene-MWCNT respectively. Considering a 50% degree of hydration for the enzyme layer,57 this would corresponds to a Db[NiFeSe]-hydrogenase surface coverage of 14 and 23 pmol cm-2 for AQ-MWCNT and ADApyrene-MWCNT respectively. It is worth comparing this value with the surfacic concentration of 11 pmol cm-2 obtained from direct electrochemistry (Figure 3C). This indicates that approximately half of the immobilized Db[NiFeSe]-hydrogenase exhibit DET with the functionalized-MWCNT sidewalls.

Figure 6. (A) Schematic representation of the H2/air enzymatic fuel cell; (B) CV of a AQ-MWCNT/ Db[NiFeSe]hydrogenase bioanode under argon and H2 (v = 5mV s-1) and a CN-MWCNT/MvBOD biocathode under argon and air (v = 10 mV s-1, pH 7.6, 25°C) ; (C) polarization (▲, red) and power (, blue) curves for Db[NiFeSe]-hydrogenase/MvBOD MWCNT fuel cell (phosphate buffer pH = 7.6, 25°C). The Db[NiFeSe]-hydrogenase-functionalized MWCNTs were finally integrated in an unprecedented enzymatic fuel cell design. First, MWCNT-coated Gas diffusion electrodes (GDE) were fabricated and modified with anthraquinone and Db[NiFeSe]-hydrogenase. The three-phase boundaries brought by GDE overcome gas solubility and diffusion limitations, while separating O2 and H2 supply. The Db[NiFeSe] hydrogenase -functionalized GDE exhibits excellent electrocatalytic oxidation of gaseous H2, without any apparent deactivation on the CV time scale (Figure 6). It is noteworthy that the reversible redox system attributed to AQ exhibits a non-ideal CV shape which is commonly observed on such porous MWCNTcoated GDE. An O2-reducing biocathode was designed according to our previous work.8 The bilirubin oxidase from Myrothecium verrucaria (MvBOD) was immobilized and oriented on naphthoate-modified MWCNTs. Both Gas diffu-

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sion anode and cathode were integrated in a conventional PEMFC-type fuel cell, electrodes being separated by a liquid electrolyte holding in a teflon chamber (pH 7.6). Figure 6C displays the polarization and power curves, resulting from successive galvanostatic discharges performed during 30 seconds. The enzymatic fuel cell was operated at 25°C with humidified streams of H2 and air at atmospheric pressure. The fuel cell delivers a maximum power density of 0.89 mW cm-2 at 0.80 V and is characterized by an open-circuit voltage (OCV) of 1.1 V. The latter value is in good agreement with the sum of the open-circuit potentials measured at both GDE. Owing to the Db[NiFeSe]-hydrogenase properties and its efficient direct wiring on functionalized MWCNTs, exceptional OCV of 1.1 V is reached accompanied with excellent high current/high potential shape of the polarization curve. It is noteworthy that decreased power output was observed below pH 7.0 (Figure S4), underlining the fact that the fuel cell is limited by the bioanode performances. In addition, the enzymatic fuel cell achieved high stability towards high current discharge of 0.70 mA cm-2 during 2500 s (Figure S5). As already mentioned, Db[NiFeSe]-hydrogenase is protected from oxidative deactivation, owing to its efficient immobilization on functionalized MWCNTs. Furthermore, Db[NiFeSe]hydrogenase is also protected from O2 deactivation, owing to the gas-diffusion system. This system prevents oxygen from air to diffuse to the anode, enabling Db[NiFeSe]-hydrogenase to oxidize H2 without being inactivated by O2. This work underlines the fact that oxygen-resistant hydrogenases such as Db[NiFeSe] can be successfully operated in conventional fuel cells if the fuel cell is specifically designed. Stability experiments show that the fuel cell exhibits a 20% power output decrease after one hour of continuous operation. This is caused by slow deactivation of Db[NiFeSe] under continuous discharge. After reactivation of the anode, the fuel cell power output can still be fully recovered after one day of storage. For comparison, the performances of this Db[NiFeSe]hydrogenase-based fuel cell is closed to the performances of the best enzymatic fuel cells. Biofuel cells based on hydrogenases from thermophilic microorganisms exhibit lower opencircuit voltages of 1.0 V, which is caused by higher overpotential for H2 oxidation and O2 reduction performed by H2ase from Aquifex aoelicus and BOD from Bacillus pumilus respectively. Owing to high electrocatalytic activity at high temperature, the biofuel cell reaches 1.5 mW cm-2 at 60°C and 0.65 mW cm-2 at 30°C with a separating membrane.35 Using a gasdiffusion cathode, this biofuel cell delivers 0.72 mW cm-2 at 40 °C.14 A recently-developped biofuel cell based on a gasdiffusion system and a [NiFe] hydrogenase from Desulfovibrio vulgaris MF immobilized on Ketjen Black delivers 6.1 mW cm-2 at 25 °C with an open-circuit voltage of 1.12V.38 The recent biofuel cell based on the [NiFe] hydrogenase from Desulfovibrio vulgaris MF immobilized in protective methylviologen-based hydrogels delivers 0.2 mW cm-2 at 40°C with an open-circuit voltage of 0.95 V.26

CONCLUSIONS In conclusion, the oriented immobilization of Db[NiFeSe]hydrogenase was achieved via the rational interactions between the enzyme and grafted hydrophobic molecules such as anthraquinone and adamantane. Both QCM-D and docking simulations confirmed the stable binding of adamantane and anthraquinone at the surface of the enzyme, close to the distal [Fe4S4] cluster. Owing to the excellent direct wiring of

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Db[NiFeSe]-hydrogenase, high amount of wired enzymes, accompanied with high current density for H2 oxidation were achieved with negligible deactivation at high potential. These findings allow Db[NiFeSe]-hydrogenase to operate in an efficient H2-gas-oxidizing bioanode integrated in a functional enzymatic H2/air fuel cell. Such particular enzymes, which are not considered in the first place to be able to operate in functional systems such as fuel cells, can turn out to be a relevant alternative if their rational immobilization and integration is specifically undertaken. This work underlines the fact that a combination of molecular engineering of surfaces and engineering of functional devices can open up new possibility for both Db[NiFeSe]-hydrogenases and related models58,59 by taking into account the specificity of these catalysts.

ASSOCIATED CONTENT Supporting information available Experimental part, Figure S1 to S8. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [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 This work was supported by the Ministère de l’Environnement, de l’Energie et de la Mer and LabEx ARCANE programme (ANR11-LABX-0003-01), the Université Grenoble Alpes (IDEX-IRS 2017, FUNBIOCO project), and the CNRS (Cellule energie 2017, COSYNBIO project) The authors acknowledge support from the plateforme de Chimie NanoBio ICMG FR 2607 (PCN-ICMG). Hugues Bonnet, Liliane Guerente and Laure Bar are acknowledged for valuable help and discussions on QCM-D experiments.

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Figure 1. (A) Schematic representation of electrografting of MWCNTs by reduction of different aryldiazonium salts; (B) Cyclic voltammetry (CV) of a 2 mmol L−1 solution of (a) 4-carboxylatonaphtyldiazonium tetrafluoroborate (CN), (b) 4-(2-aminoethyl)benzenediazonium tetrafluoroborate (AE) and (c) 2diazoniumanthraquinone tetrafluoroborate (AQ) in MeCN–TBAP (0.1 mol L−1) at a MWCNT electrode (5 scans, v = 20 mV s−1) 207x97mm (150 x 150 DPI)

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Figure 2. (A) CVs of the Db[NiFeSe] hydrogenase-functionalized (a) CN-MWCNT, (b) AE-MWCNT, (c) AQMWCNT and (d) pristine MWCNT electrodes under H2 at pH = 7.6; (B) CVs of the Db[NiFeSe] hydrogenasefunctionalized AQ-MWCNT (dotted line) under argon and (straight line) under H2 bubbling at pH = 7.6 (50 mmol L-1 sodium phosphate buffer solution at pH = 7.6, v = 10 mV s-1) 166x86mm (150 x 150 DPI)

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Figure 3. CVs of the Db[NiFeSe] hydrogenase-functionalized ADA-pyrene-MWCNT (red) and non-modified MWCNTs (grey) under argon (dotted line) and under H2 bubbling (straight line) at pH = 7.6 (50 mmol L-1 sodium phosphate buffer solution, v = 10mV s-1, CVs were reproduced on at least 3 electrodes) 118x107mm (150 x 150 DPI)

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Figure 4. Binding modes for (A) anthraquinone- Db[NiFeSe]-hydrogenase and (B) adamantane- Db[NiFeSe]hydrogenase complexes in solution. Data were obtained by docking simulation. 133x162mm (150 x 150 DPI)

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Figure 5. (A) QCM-D profile (shifts in resonant frequency (black curves) and in dissipation (gray curves) vs. time) for the immobilization of Db[NiFeSe]-hydrogenase on non-modified MWCNT (dotted lines), AQMWCNT (dashed lines), ADA-pyrene-MWCNT (full lines) (3th overtone data) before and (a) after injection of 0.2 mg mL-1 enzyme solution in 50 mM sodium phosphate buffer at pH = 7.6 for 60 minutes, (b) 3 washing steps using phosphate buffer ; (B) Plot of Db[NiFeSe]-hydrogenase mass uptake vs. time calculated from the Sauerbray equation55 for the 3rd overtone 130x164mm (150 x 150 DPI)

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Figure 6. (A) Schematic representation of the H2/air enzymatic fuel cell; (B) CV of a AQ-MWCNT/ Db[NiFeSe]-hydrogenase bioanode under argon and H2 (v = 5mV s-1) and a CN-MWCNT/MvBOD biocathode under argon and air (v = 10 mV s-1, pH 7.6, 25°C) ; (C) polarization (▲, red) and power (′, blue) curves for Db[NiFeSe]-hydrogenase/MvBOD MWCNT fuel cell (phosphate buffer pH = 7.6, 25°C). 211x383mm (150 x 150 DPI)

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