Réduction bioélectrocatalytique du dioxygène par des enzymes à cuivres connectées sur des électrodes nanostructurées et fonctionnalisées : intégration aux biopiles enzymatiques

The reduction of oxygen is realized in nature by oxidoreductase enzymes. Currently, these highly specific and efficient proteins are considered as biocatalysts for the development of biofuel cells. In this context, optimizing the orientation and the connection of multicopper oxidase (MCOs) for the reduction of O2 on functionalized carbon nanotubes was studied. In the first part of this manuscript, direct electron transfer of laccase is assessed and optimized by the non-covalent functionalization of CNTs by various hydrophobic derivatives. Electrochemical modeling and molecular dynamics enabled the rationalization of the developed biocathodes efficiency. In a second approach, the specific modification by pyrene moieties of laccases surface modified by protein engineered has also been considered. Additionally, supramolecular functionalization of CNTs by modified graphene sheets and gold nanoparticles also helped to promote laccase connection. The second part presents the development of other types of biocathodes based on the direct connection of bilirubin oxidase. Several strategies of covalent and non-covalent CNTs functionalization have been considered. The different biocathodes developed by the supramolecular assembly of nanostructured materials and MCOs delivered current density of several mA cm-2 for oxygen reduction. These new bioelectrodes combined with a bioanode which catalyzes the glucose oxidation have enabled the development of glucose/O2 enzymatic biofuel cells; delivering maximum power densities from 250 µW cm-2 to 750 µW cm-2 depending on the experimental conditions. Finally a hyperthermophilic hydrogenase based bioanode was developed and associated with a bilirubin oxidase-based biocathode to form a new design of H2/O2 biofuel cell. Within this device, the gas diffusion biocathode directly reduces oxygen from the air, which eliminates the use of a separation membrane while protecting the hydrogenase from its deactivation in the presence oxygen. This new biofuel cell delivers a maximum power density of 750 µW cm-2.Dans la nature, la réduction du dioxygène est catalysée par des enzymes de la famille des oxydoréductases. A l’heure actuelle, ces protéines spécifiques et efficaces sont envisagés comme biocatalyseurs au sein de biopile enzymatique. Dans ce contexte, l’optimisation de l’orientation et de la connexion d’oxydases multi-cuivre (MCOs) pour la réduction d’O2 sur des matrices de nanotubes carbone (CNTs) fonctionnalisées a été étudiée. Dans un premier temps, le transfert électronique direct de la laccase est optimisé par la fonctionnalisation non covalente de CNTs par divers dérivés hydrophobes. La dynamique moléculaire ainsi que la modélisation électrochimique ont permis la rationalisation des performances des différentes biocathodes développées. Dans une seconde approche, la modification spécifique par des groupements pyrène de la surface de laccases modifiées par mutagénèse a également été envisagée. La fonctionnalisation supramoléculaire de CNTs par des feuillets de graphène fonctionnalisés d’une part, et par des nanoparticules d’or d’autre part, a également permis de favoriser la connexion de laccases. La seconde partie présente l’élaboration d’autres types de biocathodes basées sur la connexion directe de bilirubines oxydases. Plusieurs stratégies de fonctionnalisation covalente et non covalente de CNTs ont été envisagées. Les différentes biocathodes élaborées par l’assemblage supramoléculaire de MCOs et de matériaux nanostructurés délivrent des densités de courant de réduction du dioxygène de plusieurs mA cm-2. Ces nouvelles bioélectrodes combinées à une bioanode qui catalyse l’oxydation du glucose ont permis le développement de biopiles enzymatiques glucose/O2 délivrant des densités maximales de puissances allant de 250 µW cm-2 à 750 µW cm-2 selon les conditions expérimentales. Enfin une bioanode à base d’une hydrogénase hyperthermophile a été développée et associée à une biocathode à base de bilirubine oxydase pour former un nouveau design de biopile H2/O2. Au sein de ce dispositif, la biocathode à diffusion de gaz réduit directement l’oxygène provenant de l’air, ce qui permet de s’affranchir de l’utilisation d’une membrane séparatrice tout en protégeant l’hydrogénase de sa désactivation en présence d’oxygène. Cette nouvelle biopile délivre une densité maximale de puissance de 750 µW cm-2

Bioelectrocatalytic reduction of dioxygen by multi-copper oxidases oriented and connected on functionalized nanostructured electrodes : application to enzymatic biofuel cells

Dans la nature, la réduction du dioxygène est catalysée par des enzymes de la famille des oxydoréductases. A l’heure actuelle, ces protéines spécifiques et efficaces sont envisagés comme biocatalyseurs au sein de biopile enzymatique. Dans ce contexte, l’optimisation de l’orientation et de la connexion d’oxydases multi-cuivre (MCOs) pour la réduction d’O2 sur des matrices de nanotubes carbone (CNTs) fonctionnalisées a été étudiée. Dans un premier temps, le transfert électronique direct de la laccase est optimisé par la fonctionnalisation non covalente de CNTs par divers dérivés hydrophobes. La dynamique moléculaire ainsi que la modélisation électrochimique ont permis la rationalisation des performances des différentes biocathodes développées. Dans une seconde approche, la modification spécifique par des groupements pyrène de la surface de laccases modifiées par mutagénèse a également été envisagée. La fonctionnalisation supramoléculaire de CNTs par des feuillets de graphène fonctionnalisés d’une part, et par des nanoparticules d’or d’autre part, a également permis de favoriser la connexion de laccases. La seconde partie présente l’élaboration d’autres types de biocathodes basées sur la connexion directe de bilirubines oxydases. Plusieurs stratégies de fonctionnalisation covalente et non covalente de CNTs ont été envisagées. Les différentes biocathodes élaborées par l’assemblage supramoléculaire de MCOs et de matériaux nanostructurés délivrent des densités de courant de réduction du dioxygène de plusieurs mA cm-2. Ces nouvelles bioélectrodes combinées à une bioanode qui catalyse l’oxydation du glucose ont permis le développement de biopiles enzymatiques glucose/O2 délivrant des densités maximales de puissances allant de 250 µW cm-2 à 750 µW cm-2 selon les conditions expérimentales. Enfin une bioanode à base d’une hydrogénase hyperthermophile a été développée et associée à une biocathode à base de bilirubine oxydase pour former un nouveau design de biopile H2/O2. Au sein de ce dispositif, la biocathode à diffusion de gaz réduit directement l’oxygène provenant de l’air, ce qui permet de s’affranchir de l’utilisation d’une membrane séparatrice tout en protégeant l’hydrogénase de sa désactivation en présence d’oxygène. Cette nouvelle biopile délivre une densité maximale de puissance de 750 µW cm-2.The reduction of oxygen is realized in nature by oxidoreductase enzymes. Currently, these highly specific and efficient proteins are considered as biocatalysts for the development of biofuel cells. In this context, optimizing the orientation and the connection of multicopper oxidase (MCOs) for the reduction of O2 on functionalized carbon nanotubes was studied. In the first part of this manuscript, direct electron transfer of laccase is assessed and optimized by the non-covalent functionalization of CNTs by various hydrophobic derivatives. Electrochemical modeling and molecular dynamics enabled the rationalization of the developed biocathodes efficiency. In a second approach, the specific modification by pyrene moieties of laccases surface modified by protein engineered has also been considered. Additionally, supramolecular functionalization of CNTs by modified graphene sheets and gold nanoparticles also helped to promote laccase connection. The second part presents the development of other types of biocathodes based on the direct connection of bilirubin oxidase. Several strategies of covalent and non-covalent CNTs functionalization have been considered. The different biocathodes developed by the supramolecular assembly of nanostructured materials and MCOs delivered current density of several mA cm-2 for oxygen reduction. These new bioelectrodes combined with a bioanode which catalyzes the glucose oxidation have enabled the development of glucose/O2 enzymatic biofuel cells; delivering maximum power densities from 250 µW cm-2 to 750 µW cm-2 depending on the experimental conditions. Finally a hyperthermophilic hydrogenase based bioanode was developed and associated with a bilirubin oxidase-based biocathode to form a new design of H2/O2 biofuel cell. Within this device, the gas diffusion biocathode directly reduces oxygen from the air, which eliminates the use of a separation membrane while protecting the hydrogenase from its deactivation in the presence oxygen. This new biofuel cell delivers a maximum power density of 750 µW cm-2

Electrochemically‐driven reduction of carbon dioxide mediated by mono‐reduced Mo‐diimine tetracarbonyl complexes: electrochemical, spectroelectrochemical and theoretical studies

International audienceThe activation of carbon dioxide by three different Mo‐diimine complexes [Mo(CO) 4 (L)] (L = bipyridine (bpy), 1,10‐phenantroline (phen) or pyridylindoziline (py‐indz)) has been investigated by electrochemistry and spectroelectrochemistry. Under inert atmosphere, monoreduction of the complexes is ligand‐centred and leads to tetracarbonyl [Mo(CO) 4 (L)] •− species, whereas double reduction induces CO release. Under CO 2 , [Mo(CO) 4 (L)] complexes undergo unexpected coupled chemical‐electrochemical reactions at the first reduction step, leading to the formation of reduced CO 2 derivatives. The experimental results obtained from IR, NIR and UV‐Vis spectroelectrochemistry, as well as DFT calculations, demonstrate an electron‐transfer reaction whose rate is ligand‐dependent

Osmium(II) Complexes Bearing Chelating N‑Heterocyclic Carbene and Pyrene-Modified Ligands: Surface Electrochemistry and Electron Transfer Mediation of Oxygen Reduction by Multicopper Enzymes

We report the synthesis of original osmium­(II) complexes bearing chelating N-heterocyclic (NHC) and bipyridine ligands. The pincer ligand 1,1′-dimethyl-3,3′-methylenediimidazole-2,2′-diylidene was used to tune the redox properties of osmium complexes. Bipyridine ligands modified with pyrene groups were chosen to study the electrosynthesis of Os^II-NHC-based metallopolymers as well as the noncovalent immobilization of these complexes on carbon-nanotube (CNT) electrodes. Poly-[Os^II-NHC] polypyrene polymer was electrogenerated on a GC electrode, whereas the pyrene-modified [Os^II-NHC] could interact with the CNTs’ sidewalls through π–π interactions, allowing the immobilization of the NHC complexes at the surface of π-extended nanostructured electrodes. Furthermore, an Os^II-NHC complex was studied in water, showing electron transfer mediation with multicopper enzymes. UV–visible and electrochemical experiments demonstrate that redox properties of the Os^II-NHC complex provide sufficient driving force for electron transfer with bilirubin oxidase from <i>Myrothecium verrucaria</i> while achieving high potential electroenzymatic oxygen reduction at <i>E</i> = +0.45 V vs Ag/AgCl at pH 6.5

Osmium(II) Complexes Bearing Chelating N‑Heterocyclic Carbene and Pyrene-Modified Ligands: Surface Electrochemistry and Electron Transfer Mediation of Oxygen Reduction by Multicopper Enzymes

We report the synthesis of original osmium­(II) complexes bearing chelating N-heterocyclic (NHC) and bipyridine ligands. The pincer ligand 1,1′-dimethyl-3,3′-methylenediimidazole-2,2′-diylidene was used to tune the redox properties of osmium complexes. Bipyridine ligands modified with pyrene groups were chosen to study the electrosynthesis of Os^II-NHC-based metallopolymers as well as the noncovalent immobilization of these complexes on carbon-nanotube (CNT) electrodes. Poly-[Os^II-NHC] polypyrene polymer was electrogenerated on a GC electrode, whereas the pyrene-modified [Os^II-NHC] could interact with the CNTs’ sidewalls through π–π interactions, allowing the immobilization of the NHC complexes at the surface of π-extended nanostructured electrodes. Furthermore, an Os^II-NHC complex was studied in water, showing electron transfer mediation with multicopper enzymes. UV–visible and electrochemical experiments demonstrate that redox properties of the Os^II-NHC complex provide sufficient driving force for electron transfer with bilirubin oxidase from <i>Myrothecium verrucaria</i> while achieving high potential electroenzymatic oxygen reduction at <i>E</i> = +0.45 V vs Ag/AgCl at pH 6.5

Hosting Adamantane in the Substrate Pocket of Laccase: Direct Bioelectrocatalytic Reduction of O₂ on Functionalized Carbon Nanotubes

We report the efficient immobilization and orientation of laccase from <i>Trametes versicolor</i> on MWCNT electrodes using 1-pyrenebutyric acid adamantyl amide as a supramolecular linker. We demonstrate the ability of adamantane to specifically interact with the hydrophobic cavity of laccase, while pyrene interacts with MWCNT sidewalls by π–π interactions. Adamantane allows the oriented immobilization of laccases on MWCNT electrodes. Using an anthraquinone-modified pyrene derivative for comparison, adamantane-modified MWCNTs achieve the stable immobilization and orientation of a higher number of enzymes per surface units, as confirmed by electrochemistry, theoretical calculations, and quartz crystal microbalance experiments. Furthermore, the efficient direct electron transfer ensures bioelectrocatalytic oxygen reduction at high half-wave potential of 0.55 V vs SCE accompanied by no kinetic limitation by the heterogeneous electron transfer and maximum current densities of 2.4 mA cm^–2

Electron-Rich, Diiron Bis(monothiolato) Carbonyls: C–S Bond Homolysis in a Mixed Valence Diiron Dithiolate

The synthesis and redox properties are presented for the electron-rich bis­(monothiolate)­s Fe₂(SR)₂­(CO)₂­(dppv)₂ for R = Me ([<b>1</b>]⁰), Ph ([<b>2</b>]⁰), CH₂Ph ([<b>3</b>]⁰). Whereas related derivatives adopt <i>C</i>₂-symmetric Fe₂(CO)₂P₄ cores, [<b>1</b>]⁰–[<b>3</b>]⁰ have <i>C</i>_s symmetry resulting from the unsymmetrical steric properties of the axial vs equatorial R groups. Complexes [<b>1</b>]⁰–[<b>3</b>]⁰ undergo 1e^– oxidation upon treatment with ferrocenium salts to give the mixed valence cations [Fe₂(SR)₂­(CO)₂­(dppv)₂]⁺. As established crystallographically, [<b>3</b>]⁺ adopts a rotated structure, characteristic of related mixed valence diiron complexes. Unlike [<b>1</b>]⁺ and [<b>2</b>]⁺ and many other [Fe₂­(SR)₂L₆]⁺ derivatives, [<b>3</b>]⁺ undergoes C–S bond homolysis, affording the diferrous sulfido-thiolate [Fe₂­(SCH₂Ph)­(S)­(CO)₂­(dppv)₂]⁺ ([<b>4</b>]⁺). According to X-ray crystallography, the first coordination spheres of [<b>3</b>]⁺ and [<b>4</b>]⁺ are similar, but the Fe–sulfido bonds are short in [<b>4</b>]⁺. The conversion of [<b>3</b>]⁺ to [<b>4</b>]⁺ follows first-order kinetics, with <i>k</i> = 2.3 × 10^–6 s^–1 (30 °C). When the conversion is conducted in THF, the organic products are toluene and dibenzyl. In the presence of TEMPO, the conversion of [<b>3</b>]⁺ to [<b>4</b>]⁺ is accelerated about 10×, the main organic product being TEMPO-CH₂Ph. DFT calculations predict that the homolysis of a C–S bond is exergonic for [Fe₂­(SCH₂Ph)₂­(CO)₂­(PR₃)₄]⁺ but endergonic for the neutral complex as well as less substituted cations. The unsaturated character of [<b>4</b>]⁺ is indicated by its double carbonylation to give [Fe₂­(SCH₂Ph)­(S)­(CO)₄­(dppv)₂]⁺ ([<b>5</b>]⁺), which adopts a bioctahedral structure