Md Estak Ahmed

Suzanne Adam

Dibyajyoti Saha

Jennifer Fize

Vincent Artero

Abhishek Dey

Carole Duboc

Repurposing a Bio-Inspired NiFe Hydrogenase Model for CO2 Reduction with Selective Production of Methane as the Unique C-Based Product

scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Main text

The exponential increase in energy demand with an overwhelming dependence on fossil fuels leads to an uncontrolled growth in the CO2 concentration in the atmosphere. Although CO2 is the greenhouse gas that contributes most to global warming, it also represents a potential feedstock for the synthesis of value-added fine chemicals and fuels thanks to its high abundance and low cost. [START_REF] Lim | How to make the most of carbon dioxide[END_REF] However, chemical conversions of CO2 are difficult because of its high kinetic inertness. Consequently, the development of efficient, and stable catalysts for CO2 reduction remains one of the key challenges in the energy conversion domain, especially when multielectron processes involving more than two electrons are involved. [START_REF] Francke | Homogeneously Catalyzed Electroreduction of Carbon Dioxide-Methods, Mechanisms, and Catalysts[END_REF] In this context, selectivity becomes another crucial parameter, since diverse products can be generated as a function of the number of electrons implicated during the reduction process.

Homogeneous CO2 reduction electrocatalysts have so far demonstrated selectivity for the production of either CO or formate in aprotic solvents. Based on structure/function and mechanistic investigations carried out on series of first-row transition metal catalysts, including polypyridinyl Mn and N-based macrocyclic Fe and Co complexes, rational design of selective and efficient catalytic systems has become possible. [START_REF] Ahmed | Homogeneous Electrochemical Reduction of CO2 to CO by a Cobalt Pyridine Thiolate Complex[END_REF][START_REF] Rønne | Ligand-Controlled Product Selectivity in Electrochemical Carbon Dioxide Reduction Using Manganese Bipyridine Catalysts[END_REF][START_REF] Liu | Non-noble metal-based molecular complexes for CO2 reduction: From the ligand design perspective[END_REF][START_REF] Margarit | Tertiary Amine-Assisted Electroreduction of Carbon Dioxide to Formate Catalyzed by Iron Tetraphenylporphyrin[END_REF][START_REF] Fokin | The Impact of a Proton Relay in Binuclear α-Diimine-Mn(CO)3 Complexes on the CO2 Reduction Catalysis[END_REF][START_REF] Gotico | Recent advances in metalloporphyrin-based catalyst design towards carbon dioxide reduction: from bio-inspired second coordination sphere modifications to hierarchical architectures[END_REF][START_REF] Franco | Advances in the electrochemical catalytic reduction of CO2 with metal complexes[END_REF][START_REF] Fukuzumi | Mechanisms of catalytic reduction of CO2 with heme and nonheme metal complexes[END_REF][START_REF] Queyriaux | From noninnocent to guilty: on the role of redox-active ligands in the electro-assisted reduction of CO2 mediated by a cobalt(ii)-polypyridyl complex[END_REF] Their implementation in future devices now requires improving their stability and transitioning to aqueous solution. A promising strategy, developed by several groups, relies on surface immobilization to take full advantage of the benefits of heterogeneous catalysis. Overall, this approach has already demonstrated a considerable improvement to the stability of the electrocatalysts, and the modified electrodes can work in aqueous conditions at various pHs. [START_REF] Abdinejad | Electrocatalytic Reduction of CO2 to CH4 and CO in Aqueous Solution Using Pyridine-Porphyrins Immobilized onto Carbon Nanotubes[END_REF][START_REF] Torbensen | Emergence of CO2 electrolyzers including supported molecular catalysts[END_REF][START_REF] Zhang | CO2 Reduction: From Homogeneous to Heterogeneous Electrocatalysis[END_REF][START_REF] Sun | Electrocatalytic reduction of carbon dioxide: opportunities with heterogeneous molecular catalysts[END_REF][START_REF] Ren | Molecular electrocatalysts can mediate fast, selective CO2; reduction in a flow cell[END_REF][START_REF] Wang | A Hybrid Co Quaterpyridine Complex/Carbon Nanotube Catalytic Material for CO2 Reduction in Water[END_REF][START_REF] Wu | Domino electroreduction of CO2 to methanol on a molecular catalyst[END_REF][START_REF] Wang | CO2 electrochemical catalytic reduction with a highly active cobalt phthalocyanine[END_REF][START_REF] Weng | Active sites of copper-complex catalytic materials for electrochemical carbon dioxide reduction[END_REF][START_REF] Weng | Electrochemical CO2 Reduction to Hydrocarbons on a Heterogeneous Molecular Cu Catalyst in Aqueous Solution[END_REF] Intriguingly, in Nature, two enzymes with active sites displaying key structural analogies can either produce H2 or reduce CO2; both contain a heterobinuclear FeNi complex with sulfur-rich coordination spheres (Figure 1). While the [NiFe] hydrogenase generates H2 from the reversible reduction of H + , 22 the CO dehydrogenase reversibly reduces CO2 to CO. [START_REF] Can | Structure, Function, and Mechanism of the Nickel Metalloenzymes, CO Dehydrogenase, and Acetyl-CoA Synthase[END_REF][START_REF] Appel | Frontiers, Opportunities, and Challenges in Biochemical and Chemical Catalysis of CO2 Fixation[END_REF] Recently, we have reported an electrocatalytic system based on a NiFe model of the 1, right) physiadsorbed on a graphite electrode, active in acidic aqueous solutions for H2 production and robust for hours. [START_REF] Ahmed | Hydrogen Evolution from Aqueous Solutions Mediated by a Heterogenized NiFe -Hydrogenase Model: Low pH Enables Catalysis through an Enzyme-Relevant Mechanism[END_REF] In this context, the capability of the same modified edge-plane graphite (EPG) electrode to reduce CO2 was evaluated. Remarkably, we found that this electrocatalytic system can activate CO2 and selectively generate CH4, as the unique carbon-based product. Production of H2 is still observed, and under optimal conditions, faradic yields (FY) of 70 and 13 % for H2 and CH4 production can be obtained, respectively. We have thus developed a molecular complex modeling common structural features of both the [NiFe] hydrogenase and the CO dehydrogenase, which functions as part of a unique electrocatalytic system that, in the presence of CO2 and protons, can generate a mixture containing only H2 and CH4. Modified electrodes have been prepared by physiadsorption by dropcasting a dichloromethane solution of the binuclear NiFeCp complex on EPG electrodes. A previous investigation based on X-ray photoelectron (XPS) and attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopic data performed on these electrodes, demonstrated that the structure of the heterobinuclear complex is preserved after physiadsorption. [START_REF] Ahmed | Hydrogen Evolution from Aqueous Solutions Mediated by a Heterogenized NiFe -Hydrogenase Model: Low pH Enables Catalysis through an Enzyme-Relevant Mechanism[END_REF] The cyclic voltammogram (CV) of the modified electrode in a 100 mM KPF6 aqueous solution (pH 5.5) displays an irreversible cathodic signal (mid-wave potential at -0.75 V vs. SHE), assigned to the reduction of [LNi II Fe II Cp] + to [LNi I Fe II Cp] and an irreversible anodic process (Epa = 0.45 V vs. SHE; Epc = 0.18 V vs. SHE) attributed to the one-electron oxidation of [LNi II Fe II Cp] + (Figure S1). The catalyst surface loading was determined to be 6×10 -12 mol•cm -2 . [START_REF] Ahmed | Hydrogen Evolution from Aqueous Solutions Mediated by a Heterogenized NiFe -Hydrogenase Model: Low pH Enables Catalysis through an Enzyme-Relevant Mechanism[END_REF] When the pH value is dropped to 4, the cathodic wave becomes catalytic in nature, this process corresponding to hydrogen evolution (HER). [START_REF] Ahmed | Hydrogen Evolution from Aqueous Solutions Mediated by a Heterogenized NiFe -Hydrogenase Model: Low pH Enables Catalysis through an Enzyme-Relevant Mechanism[END_REF] Interestingly, when the same solution is saturated with CO2 ([CO2] ~3.9 mM), the catalytic wave displays a clear 150 mV anodic shift indicative of the fact that a new electrocatalytic process occurs (Figure 2). Controlled potential electrolysis (CPE) has been carried out at -1.3 V vs. SHE at pH 4 to identify the products. The headspace gas of the CPE cell was then analyzed by gas chromatography (GC). Only two products, H2 and CH4, were detected with a H2/CH4 molar ratio of 23:1 (Table 1, entry 1); the corresponding FY for CH4 evolution is 12%. No other gaseous product is formed, and analysis of the liquid phase failed to detect methanol or formaldehyde by [START_REF] Lim | How to make the most of carbon dioxide[END_REF] H NMR, or formate by ion chromatography. In the absence of CO2 or when unmodified EPG electrodes are used, no CH4 is generated (Table 1, entry 2). CPE experiments conducted in the presence of various partial pressures of CO2 show that the CH4 production increases linearly as a function of the square root of the concentration of CO2 (Figure S4), consistent with the fact that CH4 originates from CO2. The H2/CH4 ratio is slightly affected, with a larger production of H2, when the CO2 partial pressure decreases (Table 1, entry 1, and Table S1, entry 1). This ratio is also sensitive to the pH at which the CPE is run (Table 1, entries 1, 6-7). While a ratio of 23:1 is obtained at pH 4, it rises notably when the pH is either increased or decreased, i.e., the H2/CH4 ratios are 30:1 and 50:1 at pH 5 and 3, respectively (Figure S2). This ratio is also slightly affected by the applied potential of the CPE, but to a lesser extent (Table 1, entries 1, 8-10). It remains around 20:1 in the range -0.8 V to -1.3 V vs. SHE and starts to increase at more negative potential (H2/CH4 ratio of 28:1 at -1.4 V vs. SHE) (Figure S3). Table 1. Faradic yields and H2/CH4 ratio as a function of the experimental conditions in CO2 saturated aqueous buffer.

[NiFe] hydrogenase, [L N2S2 Ni II Fe II Cp(CO)] + (L N2S2 = 2,2'-(2,2'-bipryridine-6,6'- diyl)bis(1,1'-diphenylethanethiolate), named NiFeCp (Figure
a) [NiL N2S2 ] was immobilized instead of NiFeCp, b) [CpFe(CO)(CH3CN)2] + was immobilized instead of NiFeCp, c) a negligible amount of CH4 was detected, which was below the quantification limit. S5), with a relatively constant H2/CH4 ratio during the first few hours. (Figure S6). The increase of the H2/CH4 ratio observed during a 6-hour experiment could be related to the slight increase in pH that was measured after 3 hours (0.3 pH unit). After a CPE experiment carried out under the reference conditions (Table 1, entry 9), faradaic yields of 12% and 66% for CH4 and H2, respectively, were measured (current density at -1.3 V vs. SHE with CO2 = 8.8 mA•cm -2 , and current density at -1.3 V vs. SHE with Ar = 0.95 mA•cm-2 . After ~1650s of CPE, TONCH4 ~3.5×10 5 and TONH2 ~8.2×10 6 ; TOFCH4 = 214 s -1 , TOFH2 ~5.1×10 3 s -1 ; see SI for detailed calculations, Figure S7).

Gas chromatography coupled to mass spectrometry analysis (GC-MS) confirms that methane is generated during the electrolysis (m/z = 16 with expected mass profile). Interestingly, isotope-labeling experiments were performed with [START_REF] Torbensen | Emergence of CO2 electrolyzers including supported molecular catalysts[END_REF] CO2 that confirmed that [START_REF] Torbensen | Emergence of CO2 electrolyzers including supported molecular catalysts[END_REF] CH4 methane (m/z = 17 with expected mass profile) arises from CO2 reduction (Figure 3). The stable electrolysis current combined with a quasi-constant H2/CH4 ratio suggests that the NiFeCp catalyst is stable under CPE conditions. To provide additional support that the molecular nature of NiFeCp is retained during catalysis under such highly acidic conditions, XPS and ATR-FTIR experiments were performed on the modified EPG electrodes before and after electrolysis. The ATR-FTIR spectra are similar (Figure 4, left), both displaying a single CO ligand vibration at CO = 1916 cm -1 , previously assigned to the Fe-bound CO in immobilized NiFeCp (1926 cm -1 in an ATR-IR spectrum of the powder sample of NiFeCp). [START_REF] Ahmed | Hydrogen Evolution from Aqueous Solutions Mediated by a Heterogenized NiFe -Hydrogenase Model: Low pH Enables Catalysis through an Enzyme-Relevant Mechanism[END_REF] Additionally, both XPS spectra recorded before and after electrolysis are characterized by a single peak at 854.4 eV in the Ni 2p core region (Figure 5, top), which was previously unambiguously assigned to NiFeCp. [START_REF] Ahmed | Hydrogen Evolution from Aqueous Solutions Mediated by a Heterogenized NiFe -Hydrogenase Model: Low pH Enables Catalysis through an Enzyme-Relevant Mechanism[END_REF] In the same vein, the two XPS spectra recorded in the Fe 2p core region display a similar single peak at 708.9 eV, in agreement with the presence of a single Fe-based species (Figure 5, bottom). Finally, the CVs of the modified EPG electrode recorded before and after a CPE experiment under the reference conditions are comparable, indicating that the adsorbed complex still displays the redox properties of the molecular NiFeCp complex (Figure 4, right). The small current shift between the two CVs can be explained by the modification of the pH occurring during the CPE experiment.

We have also tested another known binuclear NiFe HER electrocatalyst, [(xbsms)Ni II Fe II Cp(CO)] + , [START_REF] Canaguier | A structural and functional mimic of the active site of NiFe hydrogenases[END_REF] which also contains a NiFeS2 core, but the Ni ion is in a S4 environment instead of an N2S2 one, as in NiFeCp. Interestingly, the corresponding modified EPG electrode displays only HER catalytic activity, but no product arising from CO2 reduction is generated (Table 1, entry 5). Taken together, these experiments confirm that the reactivity of NiFeCp is specific to its molecular structure, which is entirely retained after electrolysis when immobilized onto an EPG electrode. The molecular catalyst described here distinguishes itself from almost all known systems by producing CH4 as the only carbon-based product, rather than mixtures of CO, short chain alkanes, and/or HCOOH. In the literature, the number of reported catalytic systems capable of generating products such as long-chain (C2+) alkanes [START_REF] Centi | Electrocatalytic conversion of CO2 to long carbon-chain hydrocarbons[END_REF][START_REF] Vasileff | Electrochemical Reduction of CO2 to Ethane through Stabilization of an Ethoxy Intermediate[END_REF] or alcohols [START_REF] Wu | Domino electroreduction of CO2 to methanol on a molecular catalyst[END_REF][START_REF] Liu | Recent progress on electrochemical reduction of CO2 to methanol[END_REF] is limited, and they generally display modest efficiency. In the specific case of CH4 production, only two main families of catalysts have been described based on two different approaches: (i) molecular metal-based porphyrins and derivates either via photocatalytic processes in aprotic solvents or via electrocatalysis in aqueous solutions when immobilized on modified electrodes, [START_REF] Weng | Active sites of copper-complex catalytic materials for electrochemical carbon dioxide reduction[END_REF][START_REF] Weng | Electrochemical CO2 Reduction to Hydrocarbons on a Heterogeneous Molecular Cu Catalyst in Aqueous Solution[END_REF][START_REF] Rao | Visible-light-driven methane formation from CO2 with a molecular iron catalyst[END_REF][START_REF] Rao | Visible-Light-Driven Conversion of CO2 to CH4 with an Organic Sensitizer and an Iron Porphyrin Catalyst[END_REF][START_REF] Shen | Electrocatalytic reduction of carbon dioxide to carbon monoxide and methane at an immobilized cobalt protoporphyrin[END_REF] and (ii) metallic Cu-based materials working in aqueous solution. [START_REF] Kuhl | New insights into the electrochemical reduction of carbon dioxide on metallic copper surfaces[END_REF][START_REF] Zhang | Polymersupported CuPd nanoalloy as a synergistic catalyst for electrocatalytic reduction of carbon dioxide to methane[END_REF][START_REF] Varela | Tuning the Catalytic Activity and Selectivity of Cu for CO2 Electroreduction in the Presence of Halides[END_REF][START_REF] Pérez-Gallent | Structure-and Potential-Dependent Cation Effects on CO Reduction at Copper Single-Crystal Electrodes[END_REF][START_REF] Kim | Morphology-controlled Au nanostructures for efficient and selective electrochemical CO2 reduction[END_REF][START_REF] Iyengar | Size dependent selectivity of Cu nano-octahedra catalysts for the electrochemical reduction of CO2 to CH4[END_REF][START_REF] Pan | Electrochemical CO2 reduction to methane with remarkably high Faradaic efficiency in the presence of a proton permeable membrane[END_REF] In general, the electrochemical production of methane from aqueous CO2 is observed for overpotential values higher than 0.8 V. [START_REF] Larrazábal | Building Blocks for High Performance in Electrocatalytic CO2 Reduction: Materials, Optimization Strategies, and Device Engineering[END_REF][START_REF] Note | Pt has been shown to be active for methane evolution with almost no overpotential requirement although with low faradic efficiency and low CO2 partial[END_REF] Physiadsorbed NiFeCp mediates CH4 evolution (apparent standard potential is 0.17 V vs. the Reversible Hydrogen Electrode) from -0.8 V vs. SHE in pH 4 aqueous buffer, corresponding to ~0.75 V onset overpotential requirement. At the mid-wave potential (-1.2 V vs. SHE) of the catalytic wave (Figure 2), for a more accurate description of the performance of a molecular electrocatalyst, [START_REF] Fourmond | H2 Evolution and Molecular Electrocatalysts: Determination of Overpotentials and Effect of Homoconjugation[END_REF] the overpotential for CH4 evolution is 1.13 V. Our molecular catalyst thus compares well with previously reported heterogeneous catalysts including immobilized molecular complexes. [START_REF] Weng | Active sites of copper-complex catalytic materials for electrochemical carbon dioxide reduction[END_REF][START_REF] Shen | Electrocatalytic reduction of carbon dioxide to carbon monoxide and methane at an immobilized cobalt protoporphyrin[END_REF] Electrochemical methane production on metallic (M) surfaces generally involves a M-CO intermediate, [START_REF] Nitopi | Progress and Perspectives of Electrochemical CO2 Reduction on Copper in Aqueous Electrolyte[END_REF][START_REF] Schreier | Competition between H and CO for Active Sites Governs Copper-Mediated Electrosynthesis of Hydrocarbon Fuels[END_REF][START_REF] Kortlever | Catalysts and Reaction Pathways for the Electrochemical Reduction of Carbon Dioxide[END_REF] although such an intermediate can be overcome for single-atom catalysts. [START_REF] Han | Stable and Efficient Single-Atom Zn Catalyst for CO2 Reduction to CH4[END_REF] In the case of physiadsorbed NiFeCp, CO2 reduction is inhibited in the presence of external CO, and only hydrogen is detected as the product (Table S1, entry 2). These observations indicate that CO is not involved as an intermediate during the CO2 reduction process. Based on experimental data and DFT calculations, it has been previously proposed that under homogeneous conditions, CO terminally binds at the Fe site of the reduced [LNi I Fe II Cp] complex. [START_REF] Brazzolotto | Tuning Reactivity of Bioinspired NiFe -Hydrogenase Models by Ligand Design and Modeling the CO Inhibition Process[END_REF] However, the electrocatalyst remains active for H2 production, though to a lesser extent, with hydride species being generated at the Ni site. [START_REF] Tang | Biomimetics of [NiFe]-Hydrogenase: Nickel-or Iron-Centered Proton Reduction Catalysis?[END_REF][START_REF] Brazzolotto | Nickel-centred proton reduction catalysis in a model of [NiFe] hydrogenase[END_REF] We can thus propose that a similar process takes place here, with CO blocking the Fe site, preventing the CO2 activation, but leaving the Ni site available for HER activity.

Overall our data suggest that production of CH4 proceeds through a mechanism involving two sites acting in a concerted manner, where CO2 is activated and transformed at one metal site, while the second metal site delivers hydride to the first site until CH4 is formed and released. Similar mechanisms have been proposed with Cubased electrodes, [START_REF] Schreier | Competition between H and CO for Active Sites Governs Copper-Mediated Electrosynthesis of Hydrocarbon Fuels[END_REF] and also for bimetallic nanoalloy catalysts, in which each metal plays a distinct role: one to activate the C-based intermediate products, and the second to stabilize the hydride species. [START_REF] Zhang | Polymersupported CuPd nanoalloy as a synergistic catalyst for electrocatalytic reduction of carbon dioxide to methane[END_REF] The selectivity of this present molecular catalyst, which can go beyond a 2-electron CO2 reduction process, is unique even when compared with the best metal-based materials described so far. Further studies are in progress in our laboratories to decipher the unique reaction mechanism underlying such a behavior. In parallel, ligand design is underway to tune the reactivity of physiadsorbed NiFeCp complexes and increase the selectivity for CH4 production over proton reduction.

Supporting Information Available: Experimental methods (preparation of the modified electrodes, description of the spectroscopic techniques and of the electrochemical techniques), additional data (redox properties of the modified electrodes, catalytic performance under different experimental conditions).

Figure 1 .

 1 Figure 1. Active site structures of [NiFe] hydrogenase (left) and Ni-CODH (middle), and the chemical structure of NiFeCp (right).

Figure 2 .

 2 Figure 2. CVs of NiFeCp-modified EPG electrodes (3.5 mm diameter) in aqueous (pH = 4, phosphate buffer 0.1 M) 100 mM KPF6 electrolyte at 50 mVs -1 under either Ar (blue line) or CO2 (black line) with an Ag/AgCl (satd. KCl) reference and Pt counter electrodes. CV

Figure 3 .

 3 Figure 3. GC-MS analysis of the gaseous headspace in the cell following CPE. The gas chromatogram (a), and the mass spectra of the methane product for solutions presaturated with 12 CO2 or 13 CO2 showing 12 CH4 (b) and 13 CH4 (c). The 13 CH4 mass spectrum has been corrected to eliminate water vapor contaminant, based on the NIST spectral database (m/z(H2O) = 18, 17) (See SI for details).

Figure 4 .

 4 Figure 4. ATR-FTIR spectra (left) and CVs (right) of electrodes drop-cast with [LNi II Fe II Cp] + as prepared (green traces) and after 2 h electrolysis at -1.3 V vs. SHE (red traces), in degassed pH 4 phosphate buffer 0.1 M with 100 mM KPF6 electrolyte.

Figure 5 .

 5 Figure 5. XPS spectra at the Ni 2p and Fe 2p core level of electrodes drop-cast with [LNi II Fe II Cp] + as prepared (left) and after 2 h electrolysis at -1.3 V vs. SHE in degassed pH 4 phosphate buffer with 100 mM KPF6 electrolyte (right).

  d) a NiFeCp complex with

	a different N2S2 ligand was used: [(xbsms)Ni II Fe II Cp(CO)] + , H2xbsms = 1,2-bis(4-mercapto-3,3-dimethyl-2-
	thiabutyl)benzene. 26
	CPE run at -1.3 V vs. SHE in pH 4 phosphate buffer 0.1 M displays constant catalytic
	current at a value of ~9 mA/cm 2 (Figure
	entry	Catalyst	Applied potential (vs. SHE)	pH	Time (h)	H2/CH4 ratio	Faradic yield (H2)	Faradic yield (CH4)
	NiFeCp-EPG	-1.3	4	3	23	62	11
	EPG		-1.3	4	3	only H2	46	N/A
	Ni-EPG a	-1.3	4	3	only H2	36	N/A
	Fe-EPG b	-1.3	4	3	only H2 c	35	N/A
	(S4)NiFeCp-EPG d	-1.3	4	3	only H2 c	34	N/A
	NiFeCp-EPG	-1.3	3	3	50	62	6
	NiFeCp-EPG	-1.3	5	3	30	64	8
	NiFeCp-EPG	-0.8	4	3	19	64	13
	NiFeCp-EPG	-1.0	4	3	22	66	12
	NiFeCp-EPG	-1.4	4	3	28	61	8

Acknowledgments. Financial support for this work was provided by the Agence National de la Recherche in the framework of the "Investissements d'avenir" Program (ANR-15-IDEX-02), the Labex ARCANE (ANR-11-LABX-003), and the CBH-EUR-GS(ANR-17-EURE-0003), and ANR-DFG (ANR-16-CE92_0012_01 NiFeMim), the Indo-French Center for the Promotion of Advanced Research (IFCPAR/CEFIPRA grant no 5405-1).


Ilaria Sorrentino

Solène Gentil

Yannig Nedellec

Serge Cosnier

Alessandra Piscitelli

Paola Giardina

Alan Le Goff

POXC laccase from Pleurotus ostreatus : a high-performance multicopper enzyme for direct Oxygen Reduction Reaction operating in a proton-exchange membrane fuel cell

In this work, POXC, a laccase from Pleurotus ostreatus was immobilized on carbon nanotubes (CNTs) modified by electrografting of anthraquinone and naphthoate diazonium salts. Thanks to a favorable interaction between laccase and covalentlymodified CNTs, this laccase exhibits high-potential/high current ORR performances, surpassing the performances of the well-known laccase from Trametes versicolor (TvLAC) immobilized on the same nanostructured electrodes. Furthermore, immobilized POXC demonstrates high ORR activity over a wide range of pH (2 to 8), being also highly active at gas-diffusion electrode (GDE). Finally, thanks to these unique performances, this enzyme was able to operate at the interface of the microporous layer, humidified air and a polymer electrolyte, ie nafion ® , in a conventional H2/air protonexchange membrane fuel.

The development of hydrogen fuel cells, in particular protonexchange membrane fuel (PEMFC), at an industrial scale requires to tackle major challenges. The 4H + /4e -Oxygen Reduction Reaction (ORR) occurs at the cathode side. For now, ORR requires a high amount of platinum catalysts to be efficient. And no competitive, base-metal catalysts have been proven to represent a viable alternative yet. [1,2] One of this alternative is the development of ORR catalysts based on multicopper oxidases (MCOs) [3][4][5] or their synthetic models. [6][7][8] In enzymatic fuel cells, a subcategory of fuel cells in which electrocatalytic reactions are performed by immobilized enzymes, ORR mostly relies on MCOs. [3,5,[9][10][11] Thanks to high potential ORR, laccases and bilirubin oxidases have been most exclusively used in enzymatic fuel cells. The MCO active site is based on a set of two coppercontaining centres: a Type 1 (T1) copper centre located at the surface of the protein and at which the phenolic substrate is activated and a trinuclear copper centre (TNC) at which O2 is activated and reduced into H2O. In most cases where MCOs are immobilized on an electrode, electrons are transferred to the TNC via the T1 relay centre, which has to be at electrontunneling distance of the surface of the electrode in order to achieve a so-called Direct Electron Transfer (DET). In particular, different strategies have been studied in order to maximize amount of electroactive enzymes and minimize distance between the T1 centre and the surface of the electrode. Several studies have been performed on laccase from Trametes versicolor (TvLAC), which exhibits one the lowest overpotential towards ORR in the MCO family and comparable to Pt/C, around 100 mV. F. A. Armstrong and colleagues have shown that grafted polycyclic aromatics were able to promote the favorable immobilization of TvLAC on modified graphitic electrodes. [12,13] In order to increase the enzyme surface coverage, this strategy was transferred to nanostructured electrodes. Carbon nanotubes (CNTs) or graphene sheets were modified by non-covalent [14][15][16][17] and covalent [18][19][20][21] methods to introduce anthraquinone, anthracene or naphthalene molecules to favour TvLAC orientation. This strategy has led to the design of highly-efficient oxygen-reducing biocathodes for fuel cell applications. However, the ORR studies of these enzymes have been exclusively limited to near-neutral-pH buffered solutions, at which the enzymatic activity is optimal. However, a functional PEMFC has to rely on solid, "unbuffered" and acidic polymer electrolytes such as nafion ® . The enzymatic activity of most enzymes is strongly inhibited in such acidic and low-water content medium. The only example of such enzyme operating in a PEMFC has been shown by Minteer and colleagues using laccase from Rhus vernificera entrapped in neutralized tetrabutylammonium-bromide-modified-Nafion ® and operating in H2/O2 fuel cell. [22] Unfortunately, the fuel cell exhibited low cell voltages of 0.6 V despite high current densities, which were reached at low voltages under O2. In this work, we studied the direct electrochemistry of POXC, a laccase from Pleurotus ostreatus, which has not been investigated on electrodes yet. POXC is the most abundantly secreted laccase under all growth conditions examined until now, and seems to play a major role during vegetative growth. [23] The enzyme has been extensively characterized in solution: it is a high redox potential laccase with an acidic pI. [24,25] Multi-walled carbon-nanotube (MWCNT)-coated glassy carbon and gasdiffusion electrodes (GCE and GDE respectively) were modified by electrografting of 6-carboxynaphthalenediazonium and 2diazoniumanthraquinone tetrafluoroborate salts according to previously-described procedures. [26] We have already demonstrated that such functionalization strategy leads to the formation of a thin organic layer on the surface of CNT sidewalls. The synergistic effect of grafted functional groups and CNT graphene sidewalls induces a favorable and specific interaction with metalloenzymes such as bilirubin oxidases [26,27] , promoting direct electron transfer between MWCNTs and the enzyme active site or electron relay. Here, we show that this covalent functionalization strategy favors both POXC immobilization and DET. The behavior of this enzyme was, for the first time, exhaustively investigated towards ORR by Cyclic Voltammetry (CV) and electrochemical models.

Furthermore, the comparison between POXC and TvLAC at the same functionalized electrodes was investigated, underlining the similar overpotentials towards ORR for both laccases and the higher current densities obtained for POXC as compared to TvLAC. Finally, high current densities over a wide range of pH (between 2 and pH 8) make POXC, a promising enzyme able to operate at the triple phase boundary between a polymer electrolyte, i.e. nafion ® , the MWCNT-coated GDE and the gas phase in a conventional PEMFC.

Direct

Electrochemistry of POXC on covalentlyfunctionalized MWCNTs POXC was immobilized on pristine MWCNTs and MWCNTs modified with 6-carboxynaphthalenediazonium and 2diazoniumanthraquinone tetrafluoroborate salts according to a previously-described procedure. [26] Figure 1 For all MWCNT electrodes, an irreversible electrocatalytic reduction wave is unambiguously attributed to the electroenzymatic reduction of O2 into H2O by immobilized POXC. For naphthoate-and anthraquinone-modified MWCNT, a respective 23-fold and 35-fold increase, as compared to pristine MWCNTs, are observed in terms of current density, reaching 2 mA cm -2 . As it has been demonstrated for TvLAC in the case of anthraquinone-modified electrodes [13] , this can be attributed to the favorable hydrophobic interactions between anthraquinone and the hydrophobic substrate cavity of POXC. Furthermore, this is the first time that a similar observation can be made with naphtoate groups for these enzymes. However, this type of functional groups has already been shown to improve DET for bilirubin oxidases from Myrothecium verrucaria, owing to a combination of hydrophobic interactions and electrostatic interactions via negatively-charged carboxylate groups. [26,28] In order to confirm these hydrophobic interactions and to directly compare POXC with TvLAC in the same conditions, the same experiments were performed by using commercially-available TvLAC. As expected, modification of MWCNTs with hydrophobic molecules such as anthraquinone or naphthoate groups also led to an important increase in current density for TvLAC, reaching 0.7 mA cm -2 at pH 5 (figure S1). According to previous studies, [13,14,21] this is in good agreement with the presence of a favorable interaction between the cavity of both POXC or TvLAC and naphthoate-and anthraquinone-modified MWCNTs. Long-term stability experiments were performed by one-hour chronoamperometric discharge at 0.2 V almost every day during 20 days (figure S2). POXC immobilized on both anthraquinoneor naphthoate-modified MWCNTs shows similar and excellent activity over weeks. The bioelectrodes still achieve 65% of their activity after 7 days and 38 % after 20 days, namely 0.4 mA cm -2 . This stability is superior to bioelectrodes based on entrapment of laccases in redox hydrogels (66 % of the activity is lost after 5 days for laccase from Coriolus hirsutus) [29,30] and falls in the same range as previously-described nanostructured electrodes based on laccases from Trametes sp. [14,31] Trametes hirsuta laccases immobilized at gold nanoparticle-based electrodes exhibited maximum current densities of 1 mA cm -2 , with a 40% loss of activity after 5 days of continuous operation. [32] The same laccase was immobilized at anthraquinone-modified CNTs which exhibited current densities up to 3.5 mA cm -2 with 21 % loss of activity after 7 days. [18] Pyrene-modified laccases from Trametes sp C30 immobilized at both MWCNT and gold-nanoparticlebased electrodes exhibited maximum current density of 3 mA cm -2 , losing 60 % of its activity after one month. [31] TvLac immobilized at adamantane-or pyrene-modified MWCNTs [14,17] exhibited maximum current densities of 2 mA cm -2 keeping 66% and 50 % of its initial activity after one month. TvLAC immobilized at Anthracene-modified MWCNTs exhibited maximum current density of 140 µA cm -2 accompanied with loss of activity of 25 % after 24 h. [19]

Kinetic analysis

In order to get more insights into the difference between nonmodified MWCNTs, naphthoate-and anthraquinonemodified MWCNTS towards POXC and to extract kinetic parameters, the model developed by Armstrong and Léger was applied to the faradaic electrocatalytic wave. [33,[START_REF] Hexter | Proc. Natl. Acad. Sci[END_REF] This model takes into account the random distribution of redox-active enzyme on the electrode surface. This distribution of orientation is taking into account in the d0 tunneling factor. d0 is the product of the exponential decay constant and d0, the minimal distance between the enzyme electron entry point or electrochemical relay centre and the electrode. This factor accounts for the existence of an apparent cone of electron transfer rate distribution, arising from different statistical orientations of POXC. Figure 2 displays the experimental background-subtracted CV curve accompanied by its simulated counterpart after fitting of the CV curve for each MWCNT electrodes under O2 at pH = 3. It is noteworthy that, beyond immobilization of the enzyme, mass transfer contributions were minimized through stirring the solution and maintaining a constant oxygen flow. By increasing stirring speed (or by increasing Rotating Disk Electrode rate above 1000 rpm [31] ), no increase of electrocatalytic currents (indicative of mass-transport limitations) was observed with any of the tested bioelectrodes. It is noteworthy that this model takes into account that the electrocatalysis has to be limited by the kinetics of the electroenzymatic reaction. In this case, diffusion limitations, arising from the highly porous structure of the MWCNT film, might influence the electrocatalytic response, especially at high overpotentials. This underlines the fact that this model has to be considered as an approximation of the experimental response. However, models fit well with experimental curves for all tested conditions with R 2 of 0.99 in most cases (see supplementary materials for details). This fit gives access to constant parameters, i.e. the catalysis redox potential (Ecat), the ratio between catatylic rate constant kcat and heterogenous electron transfer rate constant at minimal distance k0 max and the electron transfer tunneling factor d0. High d0 value is observed for pristine MWCNTs (d0 = 26), arising from the high randomization of orientation on MWCNT sidewalls. As expected, much smaller values of 5.0 and 5.3 are observed for anthraquinone-modified and naphthoate-modified MWCNTS respectively, underlining that the orientation of POXC is favored at these modified surfaces. As previously mentioned, this effect has been already observed for TvLAC on different type of functionalized electrodes [13,14,19,[START_REF] Thorum | [END_REF] and can be extended to POXC. The electrocatalytic waveshape for ORR performed by POXC was also modeled between pH 2 and pH 8 for MWCNTs modified with anthraquinone and naphthoate groups (figure 3). First, these fits allow the access to Ecat between pH 2 and 8, allowing the calculation of the dependence of Ecat towards pH (figure 3C). This dependence was well fitted using eq. ( 1):

)
where R is the gas constant, T the temperature, F the Faraday constant and n the number of electron involved in the redox system. Ka is the proton dissociation constant for the oxidized form of the enzyme and Ecat(acid) is the limiting value of Ecat at pH below pKa. Using n = 1, the fit gives access to an Ecat(acid) of 0.57 ± 0.01 V and a pKa of 5.1 ± 0.2 (R 2 = 0.96). This equation accounts for a 1e -/1H + accompanied with an acid-base couple with a pKa of 5.1. First, the redox potential of Ecat of 0.55 V vs. Ag/AgCl (or 0.75 V vs. NHE) at pH 5 matches very well with the redox potential of POXC measured by redox titration (0.74 V at pH 5 [36] ). This unambiguously confirms that the electrochemical redox centre is controlled by DET to the T1 copper centre. Furthermore, this particular pH dependence has already been observed for other copper enzymes such as azurin [37] CueO [38] or BODs. [39] The monoelectronic T1 Cu(II)/Cu(I) proton-coupled electron transfer (PCET) is thus influenced by the acid/base couple of an amino acid such as histidine which induces a structural rearrangement of the T1 copper centre upon the reduction/oxidation process. [38,40] This also implies that this protonation causes an increase in ORR overpotentials below pH 5 when comparing with the evolution of the standard redox potential of the O2/H2O couple (gray line, figure 3C). This dependence was also compared with the evolution of the catalytic potential of TvLAC obtained by the same procedure. The Ecat for TvLAC follows a typical linear trend towards pH (slope of 52 mV, red line, figure 3C) corresponding to a typical 1e -/1H + . By comparing the two laccases, it is noteworthy that TvLAC and POXC exhibit closed redox potential above pH 5. This implies that TvLAC and POXC likely share the same electrocatalytic mechanism involving electron transfers at the T1 centre, the only striking difference being the involvement of an amino acid protonation step below pH 5 in the case of POXC. When looking at maximum current densities for both types of modified MWCNT electrodes, POXC shows an electrocatalytic activity over a wide pH range between pH 2 and pH 8, reaching maximum current density of 1. 97 mA cm -2 at pH 3. Naphthoate-or anthraquinone-modified MWCNTS exhibit similar behavior over this pH range. The decrease of current densities below pH 3 for naphthoate might be attributed to an influence of the protonation of naphthoate groups (pKa ~ 4) on a partial desorption of POXC or on an unfavorable orientation of the enzyme.

Laccase-based PEMFC

POXC was integrated in a GDE. GDE is used in PEMFC technology, allowing the catalyst to operate at the interface between the catalytic layer, the humidified gas phase and the polymer electrolyte. We and others have successfully adapted this technology for the integration of enzymes in fuel cells. [41,42,[42][43][44][45][46][47] Anthraquinone was electrografted on a GDE modified with a thin layer of MWCNTs. Half-cell experiments were performed at pH 3 under Ar, humidified air and O2 (figure 4A). POXC exhibits similar behavior at both MWCNT-coated GDE and GCE. This confirms that POXC can operate at the interface between the electrolyte, the gas phase and the electrode. Maximum current densities of 1.2 and 1.9 mA cm -2 were obtained under air and O2 at MWCNT-coated GDE respectively in quiescent solution. Similar current densities between GCE and GDE indicate that electrocatalysis is likely limited by the kinetics of the enzymatic reaction. The performance of this POXC-based GDE is among the best performances for a laccase-based GDE, surpassing GDE based on laccases from Trametes hirsuta or Rhus vernificera, which delivers maximum current densities of 0.5 to 1 mA cm -2 under O2. [42,43] Since one of the main advantage of POXC is its ability to efficiently operate at acidic pH, this prompted us to integrate POXC in a conventional PEMFC. Figure 4C displays the polarization and power curves measured for this POXC/Pt PEMFC, obtained by successive constant current discharges. A commercial Pt/C GDE was used at the anode for H2 oxidation. The 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 0.12 mW cm -2 at 0.38 V, accompanied with an open-circuit voltage (OCV) of 0.90 V. High cell voltage of 0.90 V confirms that POXC is still active at the interface between the MWCNT layer, air and the polymer electrolyte. The presence of an important slope in the ohmic region of the polarization curve is indicative of the fact that proton conduction through the nafion ® membrane is hindered by the low amount of produced water. However, the OCV of 0.90 V of this POXC/Pt PEMFC is closed to the OCV (around 1.0 V) observed for conventional Pt/Pt PEMFC, despite lower power densities [1,48] . In a control experiment in which the cathode is based on pristine MWCNTs (without immobilization of POXC), low voltages of 0.7 V and power densities of 45 µW cm -2 underline the poor catalytic activity of MWCNTs (figure S3). For comparison, a full Pt/C PEMFC delivers 100 mW cm -2 approximately in the same test cell at 60°C [48] . Furthermore, this is the highest OCV reported so far for a fuel cell based on an enzyme operating in a nafion ®based PEMFC. A previous example has been shown by Minteer and colleagues with TvLAC. However, low OCV of 0.6 V were observed for this enzyme, despite high current densities of 10 mA cm -2 at low voltages of 0.2 V. Furthermore, we have recently shown that bilirubin oxidase from Myrothecium verrucaria was particularly efficient in a non-noble metal fuel cell running in a pH 5 phosphate buffer but was not able to operate in nafion ® -based PEMFC. [48] This work thus demonstrates that DET of POXC is promoted at naphthoate-and anthraquinone-modified MWCNTs. Thanks to its efficient wiring on MWCNT electrodes, direct electrochemistry of POXC can be exhaustively investigated at these electrodes. POXC demonstrates efficient ORR performances over a wide range of pH, surpassing performances of the most well-known laccases towards ORR at both GCE and GDE. This acidophilic laccase is able to work at a three-phase boundary between a solid electrolyte, CNT sidewalls and humidified air. This work demonstrated that the study of novel enzymes by direct electrochemistry can led to the discovery of highly-efficient biocatalyst able to operate in metal-catalyst-based conventional systems which, by design, are unconventional for enzymes This is the reason why its activity might be hindered by the strong acidic nature of sulfonated groups and the low water content at the interface, despite gas humidification. Therefore, there is still room for improvement, especially in order to design enzymespecific proton-exchange membranes that would increase water production and retention at the interface between the enzyme, the electrode and the humidified gas. Furthermore, such copper biocatalysts that perform low-overpotential ORR in a PEMFC also pave the way for the development of novel molecular copper catalysts that would perform low-overpotential ORR in a fuel cell. [6,8]

Figure 1 .

 1 Figure 1. 3D representation of POX and CV of POXC-functionalized MWCNT electrodes under O2 for (a) pristine MWCNTs, (b) naphthoate-modified MWCNTs and anthraquinone-modified MWCNTs (stirred and oxygen-purged citrate-phosphate buffer pH 3, v = 10 mV s-1).

Figure 2 .

 2 Figure 2. Background-subtracted CV of POXC-functionalized (a) MWCNT, (b) naphthoate-MWCNT and (c) anthraquinone-MWCNT electrodes under O2. The simulated red trace takes into account the average between the backward and forward scan.

Figure 3 .

 3 Figure 3. Background-subtrated CV of POXC-functionalized (A) anthraquinone-MWCNT and (B) naphthoate-MWCNT between pH 2 and pH 8. The simulated red traces take into account the average between the backward and forward scan. (C) Evolution of Ecat and (D)jlim estimated from simulated CV curves performed between pH 2.2 and 8.

Figure 4 .

 4 Figure 4. . CV of a POXC-functionalized AQ-MWCNT GDE under humidified Argon, air and O2 (v = 10 mV s-1, pH 3, 25 °C) ; (B) Schematic representation of the H2/air fuel cell; (C) polarization (▲, black) and power

Acknowledgements

This work was supported by the Ministère de l'Environnement, de l'Energie et de la Mer and LabEx ARCANE programme (ANR-11-LABX-0003-01). The authors acknowledge support from the plateforme de Chimie NanoBio ICMG FR 2607 (PCN-ICMG) and a grant from the University Federico II "Progetto di Ateneo IENA (Immobilization of ENzymes on hydrophobinfunctionalized NAnomaterials)".

Keywords: laccase • oxygen reduction • fuel cells • diazonium • carbon nanotubes