Outstanding Superoxide Dismutase Catalytic Activity Of Simple Peptide-Based Nickel(II) Complexes.

We present here the most active synthetic Ni superoxide dismutase (NiSOD) mimic reported to date. Reactive oxygen species are aggressive compounds, which concentrations are tightly regulated in vivo. Among them, the superoxide anion, O2⸱-, is controlled by superoxide dismutases. Capitalizing on the versatility of the Amino-Terminal CuII- and NiII-binding (ATCUN) peptide motif, we introduced positive charges around the NiII center to favor the interaction with the superoxide radical anion. At physiological pH, the pentapeptide H-Cys-His-Cys-Arg-Arg-NH2 coordinates NiII after the deprotonation of one thiol, two amides, and either the second thiol or the N-terminal ammonium, leading to an equilibrium between the two N3S1 and N2S2 coordination modes. Under catalytic conditions, a kcat value of 8.6(4) x 106 L.mol-1.s-1 was measured. Within the first second, the catalyst remained undegraded with quantitative consumption of O2⸱- (completed up to 37 catalytic cycles). An extra arginine (Arg) was introduced at the peptide C-terminus to increase the global charge of the NiII complex from +1 to + 2. This had no effect on the catalytic performance, highlighting the critical role of charge distribution in space as a determining factor influencing the reactivity.

Optimising Electrical Interfacing between the Trimeric Copper Nitrite Reductase and Carbon Nanotubes.

The immobilization of copper-containing nitrite reductase (NiR) from Alcaligenes faecalis on functionalised multi-walled carbon nanotube (MWCNT) electrodes is reported. It is demonstrated that this immobilization is mainly driven by hydrophobic interactions, promoted by the modification of MWCNTs with adamantyl groups. Direct electrochemistry shows high bioelectrochemical reduction of nitrite at the redox potential of NiR with high current density of 1.41 mA cm-2 . Furthermore, the desymmetrization of the trimer upon immobilization induces an independent electrocatalytic behavior for each of the three enzyme subunits, corroborated by an electron-tunneling distance dependence.

A Series of Ni Complexes Based on a Versatile ATCUN-Like Tripeptide Scaffold to Decipher Key Parameters for Superoxide Dismutase Activity.

The cellular level of reactive oxygen species (ROS) has to be controlled to avoid some pathologies, especially those linked to oxidative stress. One strategy for designing antioxidants consists of modeling natural enzymes involved in ROS degradation. Among them, nickel superoxide dismutase (NiSOD) catalyzes the dismutation of the superoxide radical anion, O2•-, into O2 and H2O2. We report here Ni complexes with tripeptides derived from the amino-terminal CuII- and NiII-binding (ATCUN) motif that mimics some structural features found in the active site of the NiSOD. A series of six mononuclear NiII complexes were investigated in water at physiological pH with different first coordination spheres, from compounds with a N3S to N2S2 set, and also complexes that are in equilibrium between the N-coordination (N3S) and S-coordination (N2S2). They were fully characterized by a combination of spectroscopic techniques, including 1H NMR, UV-vis, circular dichroism, and X-ray absorption spectroscopy, together with theoretical calculations and their redox properties studied by cyclic voltammetry. They all display SOD-like activity, with a kcat ranging between 0.5 and 2.0 × 106 M-1 s-1. The complexes in which the two coordination modes are in equilibrium are the most efficient, suggesting a beneficial effect of a nearby proton relay.

Augmenting the Performance of Hydrogenase for Aerobic Photocatalytic Hydrogen Evolution via Solvent Tuning.

This work showcases the performance of [NiFeSe] hydrogenase from Desulfomicrobium baculatum for solar-driven hydrogen generation in a variety of organic-based deep eutectic solvents. Despite its well-known sensitivity towards air and organic solvents, the hydrogenase shows remarkable performance under an aerobic atmosphere in these solvents when paired with a TiO2 photocatalyst. Tuning the water content further increases hydrogen evolution activity to a TOF of 60±3 s-1 and quantum yield to 2.3±0.4 % under aerobic conditions, compared to a TOF of 4 s-1 in a purely aqueous solvent. Contrary to common belief, this work therefore demonstrates that placing natural hydrogenases into non-natural environments can enhance their intrinsic activity beyond their natural performance, paving the way for full water splitting using hydrogenases.

The laccase mediator system at carbon nanotubes for anthracene oxidation and femtomolar electrochemical biosensing.

We investigated the use of POXA1b laccase from Pleurotus ostreatus for the oxidation of anthracene into anthraquinone. We show that different pathways can occur depending on the nature of the redox mediator combined to laccase, leading to different structural isomers. The laccase combined with 2,2'-azine-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) leads to the formation of 1,4-anthraquinone and/or 1,2-anthraquinone. The unprecedented role of carbon nanotubes (CNTs) as redox mediators for oxidation of anthracene into 9,10-anthraquinone is shown and corroborated by density-functional theory (DFT) calculations. Owing to the efficient adsorption of anthraquinones at CNT electrodes, anthracene can be detected with low limit-of-detection using either laccase in solution, CNT-supported laccase or laccase immobilized at magnetic beads exploiting the adhesive property of a chimeric hydrophobin-laccase.

Functionalizing Carbon Nanotubes with Bis(2,9-dialkyl-1,10-phenanthroline)copper(II) Complexes for the Oxygen Reduction Reaction.

A new ligand, namely, 2-(5-(pyren-1-yl)pentyl)-9-methyl-1,10-phenanthroline, as well as new bis(2,9-dialkyl-1,10-phenanthroline)copper(II) complexes were designed, which were immobilized on multiwalled carbon nanotube (MWCNT) electrodes. These complexes show a high tendency of autoreduction into their copper(I) form according to electrochemical and EPR experiments. These complexes exhibit strong interactions with MWCNT sidewalls either with or without anchor functions such as the pyrene moiety. The pyrene-modified derivative can be electropolymerized on glassy carbon and MWCNT electrodes to form a poly-[bis(2-(5-(pyren-1-yl)pentyl)-9-methyl-1,10-phenanthroline)copper(II)] metallopolymer film. Furthermore, these MWCNT-supported bis(2,9-dialkyl-1,10-phenanthroline)copper complexes demonstrate a low overpotential for a 4H+/4e- oxygen reduction reaction at pH 5 with an onset potential of 0.86 V versus RHE. Integration of these functionalized MWCNTs at gas-diffusion electrodes of H2/air fuel cells led to a high open-circuit voltage of 0.84 V and a maximum current density of 1.77 mW cm-2 using a Pt/C anode.

A Pyrene-Triazacyclononane Anchor Affords High Operational Stability for CO2 RR by a CNT-Supported Histidine-Tagged CODH.

An original 1-acetato-4-(1-pyrenyl)-1,4,7-triazacyclononane (AcPyTACN) was synthesized for the immobilization of a His-tagged recombinant CODH from Rhodospirillum rubrum (RrCODH) on carbon-nanotube electrodes. The strong binding of the enzyme at the Ni-AcPyTACN complex affords a high current density of 4.9 mA cm-2 towards electroenzymatic CO2 reduction and a high stability of more than 6×106  TON when integrated on a gas-diffusion bioelectrode.

Clicked Bifunctional Dendrimeric and Cyclopeptidic Addressable Redox Scaffolds for the Functionalization of Carbon Nanotubes with Redox Molecules and Enzymes.

Carbon nanotube electrodes were modified with ferrocene and laccase using two different click reactions strategies and taking advantage of bifunctional dendrimers and cyclopeptides. Using diazonium functionalization and the efficiency of oxime ligation, the combination of both multiwalled carbon nanotube surfaces and modified dendrimers or cyclopeptides allows the access to a high surface coverage of ferrocene in the order of 50 nmol cm-2, a 50-fold increase compared to a classic click reaction without oxime ligation of these highly branched macromolecules. Furthermore, this original immobilization strategy allows the immobilization of mono- and bi-functionalized active multicopper enzymes, laccases, via copper(I)-catalyzed azide-alkyne cycloaddition. Electrochemical studies underline the high efficiency of the oxime-ligated dendrimers or cyclopeptides for the immobilization of redox entities on surfaces while being detrimental to electron tunneling with enzyme active sites despite controlled orientation.

A Bioinspired NiII Superoxide Dismutase Catalyst Designed on an ATCUN-like Binding Motif.

Nickel superoxide dismutase (NiSOD) is an enzyme that protects cells against O2·-. While the structure of its active site is known, the mechanism of the catalytic cycle is still not elucidated. Its active site displays a square planar NiII center with two thiolates, the terminal amine and an amidate. We report here a bioinspired NiII complex built on an ATCUN-like binding motif modulated with one cysteine, which demonstrates catalytic SOD activity in water (kcat = 8.4(2) × 105 M-1 s-1 at pH = 8.1). Its reactivity with O2·- was also studied in acetonitrile allowing trapping two different short-lived species that were characterized by electron paramagnetic resonance or spectroelectrochemistry and a combination of density functional theory (DFT) and time-dependent DFT calculations. Based on these observations, we propose that O2·- interacts first with the complex outer sphere through a H-bond with the peptide scaffold in a [NiIIO2·-] species. This first species could then evolve into a NiIII hydroperoxo inner sphere species through a reaction driven by protonation that is thermodynamically highly favored according to DFT calculations.

Substituent Effects in Carbon-Nanotube-Supported Copper Phenolato Complexes for Oxygen Reduction Reaction.

Unprotected mononuclear pyrene-modified (bispyridylaminomethyl)methylphenol copper complexes were designed to be immobilized at multiwalled carbon nanotube (MWCNT) electrodes and form dinuclear bis(µ-phenolato) complexes on the surface. These complexes exhibit a high oxygen reduction reaction activity of 12.7 mA cm-2 and an onset potential of 0.78 V versus reversible hydrogen electrode. The higher activity of these complexes compared to that of mononuclear complexes with bulkier groups is induced by the favorable early formation of a dinuclear catalytic species on MWCNT.

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

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

From Graphite to Laccase Biofunctionalized Few-Layer Graphene: A "One Pot" Approach Using a Chimeric Enzyme.

A chimeric enzyme based on the genetic fusion of a laccase with a hydrophobin domain was employed to functionalize few-layer graphene, previously exfoliated from graphite in the presence of the hydrophobin. The as-produced, biofunctionalized few-layer graphene was characterized by electrochemistry and Raman spectroscopy, and finally employed in the biosensing of phenols such as catechol and dopamine. This strategy paves the way for the functionalization of nanomaterials by hydrophobin domains of chimeric enzymes and their use in a variety of electrochemical applications.

Mononuclear Ni(II) Complexes with a S3O Coordination Sphere Based on a Tripodal Cysteine-Rich Ligand: pH Tuning of the Superoxide Dismutase Activity.

The superoxide dismutase (SOD) activity of mononuclear NiII complexes, whose structures are inspired by the NiSOD, has been investigated. They have been designed with a sulfur-rich pseudopeptide ligand, derived from nitrilotriacetic acid (NTA), where the three acid functions are grafted with cysteines (L3S). Two mononuclear complexes, which exist in pH-dependent proportions, have been fully characterized by a combination of spectroscopic techniques including 1H NMR, UV-vis, circular dichroism, and X-ray absorption spectroscopy, together with theoretical calculations. They display similar square-planar S3O coordination, with the three thiolates of the three cysteine moieties from L3S coordinated to the NiII ion, together with either a water molecule at physiological pH, as [NiL3S(OH2)]-, or a hydroxo ion in more basic conditions, as [NiL3S(OH)]2-. The 1H NMR study has revealed that contrary to the hydroxo ligand, the bound water molecule is labile. The cyclic voltammogram of both complexes displays an irreversible one-electron oxidation process assigned to the NiII/NiIII redox system with Epa = 0.48 and 0.31 V versus SCE for NiL3S(OH2) and NiL3S(OH), respectively. The SOD activity of both complexes has been tested. On the basis of the xanthine oxidase assay, an IC50 of about 1 µM has been measured at pH 7.4, where NiL3S(OH2) is mainly present (93% of the NiII species), while the IC50 is larger than 100 µM at pH 9.6, where NiL3S(OH) is the major species (92% of the NiII species). Interestingly, only NiL3S(OH2) displays SOD activity, suggesting that the presence of a labile ligand is required. The SOD activity has been also evaluated under catalytic conditions at pH 7.75, where the ratio between NiL3S(OH2)/ NiL3S(OH) is about (86:14), and a rate constant, kcat = 1.8 × 105 M-1 s-1, has been measured. NiL3S(OH2) is thus the first low-molecular weight, synthetic, bioinspired Ni complex that displays catalytic SOD activity in water at physiological pH, although it does not contain any N-donor ligand in its first coordination sphere, as in the NiSOD. Overall, the data show that a key structural feature is the presence of a labile ligand in the coordination sphere of the NiII ion.

Direct Electrochemistry of Bilirubin Oxidase from Magnaporthe orizae on Covalently-Functionalized MWCNT for the Design of High-Performance Oxygen-Reducing Biocathodes.

Herein, the direct electrochemistry of bilirubin oxidase from Magnaporthe orizae (MoBOD) was studied on CNTs functionalized by electrografting several types of diazonium salts. The functionalization induces favorable or unfavorable orientation of MoBOD, the latter being compared to the well-known BOD from Myrothecium verrucaria (MvBOD). On the same nanostructured electrodes, MoBOD can surpass MvBOD in terms of both current densities and minimal overpotentials. Added to the fact that MoBOD is also highly active at the gas-diffusion electrode (GDE), these findings make MoBOD one of the MCOs with the highest catalytic activity towards the oxygen reduction reaction (ORR).

Synergetic Effects of Combined Nanomaterials for Biosensing Applications.

Nanomaterials have become essential components for the development of biosensors since such nanosized compounds were shown to clearly increase the analytical performance. The improvements are mainly related to an increased surface area, thus providing an enhanced accessibility for the analyte, the compound to be detected, to the receptor unit, the sensing element. Nanomaterials can also add value to biosensor devices due to their intrinsic physical or chemical properties and can even act as transducers for the signal capture. Among the vast amount of examples where nanomaterials demonstrate their superiority to bulk materials, the combination of different nano-objects with different characteristics can create phenomena which contribute to new or improved signal capture setups. These phenomena and their utility in biosensor devices are summarized in a non-exhaustive way where the principles behind these synergetic effects are emphasized.

Interprotein Electron Transfer between FeS-Protein Nanowires and Oxygen-Tolerant NiFe Hydrogenase.

Self-assembled redox protein nanowires have been exploited as efficient electron shuttles for an oxygen-tolerant hydrogenase. An intra/inter-protein electron transfer chain has been achieved between the iron-sulfur centers of rubredoxin and the FeS cluster of [NiFe] hydrogenases. [NiFe] Hydrogenases entrapped in the intricated matrix of metalloprotein nanowires achieve a stable, mediated bioelectrocatalytic oxidation of H2 at low-overpotential.

Carbon-Nanotube-Supported Bio-Inspired Nickel Catalyst and Its Integration in Hybrid Hydrogen/Air Fuel Cells.

A biomimetic nickel bis-diphosphine complex incorporating the amino acid arginine in the outer coordination sphere was immobilized on modified carbon nanotubes (CNTs) through electrostatic interactions. The functionalized redox nanomaterial exhibits reversible electrocatalytic activity for the H2 /2 H+ interconversion from pH 0 to 9, with catalytic preference for H2 oxidation at all pH values. The high activity of the complex over a wide pH range allows us to integrate this bio-inspired nanomaterial either in an enzymatic fuel cell together with a multicopper oxidase at the cathode, or in a proton exchange membrane fuel cell (PEMFC) using Pt/C at the cathode. The Ni-based PEMFC reaches 14 mW cm-2 , only six-times-less as compared to full-Pt conventional PEMFC. The Pt-free enzyme-based fuel cell delivers ≈2 mW cm-2 , a new efficiency record for a hydrogen biofuel cell with base metal catalysts.

Diazonium Functionalisation of Carbon Nanotubes for Specific Orientation of Multicopper Oxidases: Controlling Electron Entry Points and Oxygen Diffusion to the Enzyme.

We report the controlled orientation of bilirubin oxidases (BOD) from Myrothecium verrucaria on multiwalled carbon nanotubes (MWCNTs) functionalised by electrografting of 6-carboxynaphthalenediazonium and 4-(2-aminoethyl)benzenediazonium salts. On negatively charged naphthoate-modified MWCNTs, a high-potential (0.44 V vs. SCE) oxygen reduction electrocatalysis is observed, occurring via the T1 copper centre. On positively charged ammonium-modified MWCNTs, a low-potential (0.15 V) oxygen reduction electrocatalysis is observed, occurring through a partially oxidised state of the T2/T3 trinuclear copper cluster. Finally, chemically modified naphthoate MWCNTs exhibit high bioelectrocatalytic current densities of 3.9 mA cm(-2) under air at gas-diffusion electrode.

Recent progress in oxygen-reducing laccase biocathodes for enzymatic biofuel cells.

This review summarizes different approaches and breakthroughs in the development of laccase-based biocathodes for bioelectrocatalytic oxygen reduction. The use of advanced electrode materials, such as nanoparticles and nanowires is underlined. The applications of recently developed laccase electrodes for enzymatic biofuel cells are reviewed with an emphasis on in vivo application of biofuel cells.

Electrocatalytic O2 Reduction at a Bio-inspired Mononuclear Copper Phenolato Complex Immobilized on a Carbon Nanotube Electrode.

An original copper-phenolate complex, mimicking the active center of galactose oxidase, featuring a pyrene group was synthesized. Supramolecular pi-stacking allows its efficient and soft immobilization at the surface of a Multi-Walled Carbon Nanotube (MWCNT) electrode. This MWCNT-supported galactose oxidase model exhibits a 4 H(+)/4 e(-) electrocatalytic activity towards oxygen reduction at a redox potential of 0.60 V vs. RHE at pH 5.