Does Acinetobacter calcoaceticus glucose dehydrogenase produce self-damaging H2O2?

The soluble glucose dehydrogenase (sGDH) from Acinetobacter calcoaceticus has been widely studied and is used, in biosensors, to detect the presence of glucose, taking advantage of its high turnover and insensitivity to molecular oxygen. This approach, however, presents two drawbacks: the enzyme has broad substrate specificity (leading to imprecise blood glucose measurements) and shows instability over time (inferior to other oxidizing glucose enzymes). We report the characterization of two sGDH mutants: the single mutant Y343F and the double mutant D143E/Y343F. The mutants present enzyme selectivity and specificity of 1.2 (Y343F) and 5.7 (D143E/Y343F) times higher for glucose compared with that of the wild-type. Crystallographic experiments, designed to characterize these mutants, surprisingly revealed that the prosthetic group PQQ (pyrroloquinoline quinone), essential for the enzymatic activity, is in a cleaved form for both wild-type and mutant structures. We provide evidence suggesting that the sGDH produces H2O2, the level of production depending on the mutation. In addition, spectroscopic experiments allowed us to follow the self-degradation of the prosthetic group and the disappearance of sGDH's glucose oxidation activity. These studies suggest that the enzyme is sensitive to its self-production of H2O2. We show that the premature aging of sGDH can be slowed down by adding catalase to consume the H2O2 produced, allowing the design of a more stable biosensor over time. Our research opens questions about the mechanism of H2O2 production and the physiological role of this activity by sGDH.

Bi-enzymatic chemo-mechanical feedback loop for continuous self-sustained actuation of conducting polymers.

Artificial actuators have been extensively studied due to their wide range of applications from soft robotics to biomedicine. Herein we introduce an autonomous bi-enzymatic system where reversible motion is triggered by the spontaneous oxidation and reduction of glucose and oxygen, respectively. This chemo-mechanical actuation is completely autonomous and does not require any external trigger to induce self-sustained motion. The device takes advantage of the asymmetric uptake and release of ions on the anisotropic surface of a conducting polymer strip, occurring during the operation of the enzymes glucose oxidase and bilirubin oxidase immobilized on its surface. Both enzymes are connected via a redox polymer at each extremity of the strip, but at the opposite faces of the polymer film. The time-asymmetric consumption of both fuels by the enzymatic reactions produces a double break of symmetry of the film, leading to autonomous actuation. An additional break of symmetry, introduced by the irreversible overoxidation of one extremity of the polymer film, leads to a crawling-type motion of the free-standing polymer film. These reactions occur in a virtually unlimited continuous loop, causing long-term autonomous actuation of the device.

Wiring of bilirubin oxidases with redox polymers on gas diffusion electrodes for increased stability of self-powered biofuel cells-based glucose sensing.

A new redox polymer/bilirubin oxidase (BOD)-based gas diffusion electrode was designed to be implemented as the non-current and non-stability limiting biocathode in a glucose/O2 biofuel cell that acts as a self-powered glucose biosensor. For the proof-of-concept, a bioanode comprising the Os-complex modified redox polymer P(VI-co-AA)-[Os(bpy)2Cl]Cl and FAD-dependent glucose dehydrogenase to oxidize the analyte was used. In order to develop an optimal O2-reducing biocathode for the biofuel cell Mv-BOD as well as Bp-BOD and Mo-BOD have been tested in gas diffusion electrodes in direct electron transfer as well as in mediated electron transfer immobilized in the Os-complex modified redox polymer P(VI-co-AA)-[Os(diCl-bpy)2]Cl2. The resulting biofuel cell exhibits a glucose-dependent current and power output in the concentration region between 1 and 10 mM. To create a more realistic test environment, the performance and long-term stability of the biofuel cell-based self-powered glucose biosensor has been investigated in a flow-through cell design.

Electron Transfer to the Trinuclear Copper Cluster in Electrocatalysis by the Multicopper Oxidases.

High-potential multicopper oxidases (MCOs) are excellent catalysts able to perform the oxygen reduction reaction (ORR) at remarkably low overpotentials. Moreover, MCOs are able to interact directly with the electrode surfaces via direct electron transfer (DET), that makes them the most commonly used electrocatalysts for oxygen reduction in biofuel cells. The central question in MCO electrocatalysis is whether the type 1 (T1) Cu is the primary electron acceptor site from the electrode, or whether electrons can be transferred directly to the trinuclear copper cluster (TNC), bypassing the rate-limiting intramolecular electron transfer step from the T1 site. Here, using site-directed mutagenesis and electrochemical methods combined with data modeling of electrode kinetics, we have found that there is no preferential superexchange pathway for DET to the T1 site. However, due to the high reorganization energy of the fully oxidized TNC, electron transfer from the electrode to the TNC does occur primarily through the T1 site. We have further demonstrated that the lower reorganization energy of the TNC in its two-electron reduced, alternative resting, form enables DET to the TNC, but this only occurs in the first turnover. This study provides insight into the factors that control the kinetics of electrocatalysis by the MCOs and a guide for the design of more efficient biocathodes for the ORR.

Direct dynamic read-out of molecular chirality with autonomous enzyme-driven swimmers.

A key approach for designing bioinspired machines is to transfer concepts from nature to man-made structures by integrating biomolecules into artificial mechanical systems. This strategy allows the conversion of molecular information into macroscopic action. Here, we describe the design and dynamic behaviour of hybrid bioelectrochemical swimmers that move spontaneously at the air-water interface. Their motion is governed by the diastereomeric interactions between immobilized enantiopure oligomers and the enantiomers of a chiral probe molecule present in solution. These dynamic bipolar systems are able to convert chiral information present at the molecular level into enantiospecific macroscopic trajectories. Depending on the enantiomer in solution, the swimmers will move clockwise or anticlockwise; the concept can also be used for the direct visualization of the degree of enantiomeric excess by analysing the curvature of the trajectories. Deciphering in such a straightforward way the enantiomeric ratio could be useful for biomedical applications, for the read-out of food quality or as a more general analogue of polarimetric measurements.

Redox-Polymer-Wired [NiFeSe] Hydrogenase Variants with Enhanced O2 Stability for Triple-Protected High-Current-Density H2 -Oxidation Bioanodes.

Variants of the highly active [NiFeSe] hydrogenase from D. vulgaris Hildenborough that exhibit enhanced O2 tolerance were used as H2 -oxidation catalysts in H2 /O2 biofuel cells. Two [NiFeSe] variants were electrically wired by means of low-potential viologen-modified redox polymers and evaluated with respect to H2 -oxidation and stability against O2 in the immobilized state. The two variants showed maximum current densities of (450±84) µA cm-2 for G491A and (476±172) µA cm-2 for variant G941S on glassy carbon electrodes and a higher O2 tolerance than the wild type. In addition, the polymer protected the enzyme from O2 damage and high-potential inactivation, establishing a triple protection for the bioanode. The use of gas-diffusion bioanodes provided current densities for H2 -oxidation of up to 6.3 mA cm-2 . Combination of the gas-diffusion bioanode with a bilirubin oxidase-based gas-diffusion O2 -reducing biocathode in a membrane-free biofuel cell under anode-limiting conditions showed unprecedented benchmark power densities of 4.4 mW cm-2 at 0.7 V and an open-circuit voltage of 1.14 V even at moderate catalyst loadings, outperforming the previously reported system obtained with the [NiFeSe] wild type and the [NiFe] hydrogenase from D. vulgaris Miyazaki F.

Mechanism of Reconstitution/Activation of the Soluble PQQ-Dependent Glucose Dehydrogenase from Acinetobacter calcoaceticus: A Comprehensive Study.

The ability to switch on the activity of an enzyme through its spontaneous reconstitution has proven to be a valuable tool in fundamental studies of enzyme structure/reactivity relationships or in the design of artificial signal transduction systems in bioelectronics, synthetic biology, or bioanalytical applications. In particular, those based on the spontaneous reconstitution/activation of the apo-PQQ-dependent soluble glucose dehydrogenase (sGDH) from Acinetobacter calcoaceticus were widely developed. However, the reconstitution mechanism of sGDH with its two cofactors, i.e., pyrroloquinoline quinone (PQQ) and Ca2+, remains unknown. The objective here is to elucidate this mechanism by stopped-flow kinetics under single-turnover conditions. The reconstitution of sGDH exhibited biphasic kinetics, characteristic of a square reaction scheme associated with two activation pathways. From a complete kinetic analysis, we were able to fully predict the reconstitution dynamics and also to demonstrate that when PQQ first binds to apo-sGDH, it strongly impedes the access of Ca2+ to its enclosed position at the bottom of the enzyme binding site, thereby greatly slowing down the reconstitution rate of sGDH. This slow calcium insertion may purposely be accelerated by providing more flexibility to the Ca2+ binding loop through the specific mutation of the calcium-coordinating P248 proline residue, reducing thus the kinetic barrier to calcium ion insertion. The dynamic nature of the reconstitution process is also supported by the observation of a clear loop shift and a reorganization of the hydrogen-bonding network and van der Waals interactions observed in both active sites of the apo and holo forms, a structural change modulation that was revealed from the refined X-ray structure of apo-sGDH (PDB: 5MIN).

Engineering glucose oxidase for bioelectrochemical applications.

There is still a growing interest in developing glucose sensors using glucose oxidase. Since 2012, over 1000 papers are published every year, while efficient commercial sensors exist on the market. Among those glucose sensors, few have been thought and well-engineered and do not solve the problems associated with glucose oxidase; among which the O2 sensitivity of the enzyme or the competition between O2 and redox mediators for GOx's electrons. Enzyme engineering has been employed to solve those issues but screening GOx in homogeneous solution with O2 as an electron acceptor is not suitable. Very few reports describe the specific reengineering of GOx for electrochemical applications and are the subject of this review. It starts with a brief presentation of glucose oxidase and presents the recent progress in glucose oxidase reengineering by highlighting the kind of engineering/mutations performed to increase its electron transfer rate to electrode surfaces and, to decrease its O2 sensitivity. In addition, the review highlights the need to develop new screening methods involving electrochemical probing, essential to develop the next generation of glucose sensors; specific to glucose, O2 independent, biocompatible and stable over 2 weeks.

Bilirubin oxidase-based silica macrocellular robust catalyst for on line dyes degradation.

We present a new heterogeneous biocatalyst based on the grafting of Bilirubin Oxidase from Bacillus pumilus into macrocellular Si(HIPE) materials dedicated to water treatment. Due to the host intrinsic high porosity and monolithic character, on-line catalytic process is reached. We thus used this biocatalyst toward uni-axial flux decolorizations of Congo Red and Remazol Brilliant Blue (RBBR) at two different pH (4 and 8.2), both in presence or absence of redox mediator. In absence of redox mediators, 40% decolorization efficiency was reached within 24 h at pH 4 for Congo Red and 80% for RBBR at pH 8.2 in 24 h. In presence of 10µM ABTS, it respectively attained 100% efficiency after 2h and 12h. We have also demonstrated that non-toxic species were generated upon dyes decolorization. These results show that unlike laccases, this new biocatalyst exhibits excellent decolorization properties over a wide range of pH. Beyond, this enzymatic-based heterogeneous catalyst can be reused during two months being simply stored at room temperature while maintaining its decolorization efficiency. This study shows that this biocatalyst is a promising and robust candidate toward wastewater treatments, both in acidic and alkaline conditions where in the latter efficient decolorization strategies were still missing.

Wireless Electromechanical Readout of Chemical Information.

Collecting electrochemical information concerning the presence of molecules in a solution is usually achieved by measuring current, potential, resistance, or impedance via connection to a power supply. Here, we suggest wireless electromechanical actuation as a straightforward readout of chemical information. This can be achieved based on the concept of bipolar electrochemistry, which allows measuring the presence of different model species in a quantitative way. We validate the concept by using a free-standing polypyrrole film. Its positively polarized extremity participates in an oxidation of the analyte and delivers electrons to the opposite extremity for the reduction of the polymer. This reduction is accompanied by the insertion of counterions and thus leads to partial swelling of the film, inducing its bending. The resulting actuation is found to be a linear function of the analyte concentration, and also a Michaelis-Menten type correlation is obtained for biochemical analytes. This electromechanical transduction allows an easy optical readout and opens up very interesting perspectives not only in the field of sensing but also far beyond, such as for the elaboration of self-regulating biomimetic systems.

Uphill production of dihydrogen by enzymatic oxidation of glucose without an external energy source.

Chemical systems do not allow the coupling of energy from several simple reactions to drive a subsequent reaction, which takes place in the same medium and leads to a product with a higher energy than the one released during the first reaction. Gibbs energy considerations thus are not favorable to drive e.g., water splitting by the direct oxidation of glucose as a model reaction. Here, we show that it is nevertheless possible to carry out such an energetically uphill reaction, if the electrons released in the oxidation reaction are temporarily stored in an electromagnetic system, which is then used to raise the electrons' potential energy so that they can power the electrolysis of water in a second step. We thereby demonstrate the general concept that lower energy delivering chemical reactions can be used to enable the formation of higher energy consuming reaction products in a closed system.

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).

O2 Reduction in Enzymatic Biofuel Cells.

Catalytic four-electron reduction of O2 to water is one of the most extensively studied electrochemical reactions due to O2 exceptional availability and high O2/H2O redox potential, which may in particular allow highly energetic reactions in fuel cells. To circumvent the use of expensive and inefficient Pt catalysts, multicopper oxidases (MCOs) have been envisioned because they provide efficient O2 reduction with almost no overpotential. MCOs have been used to elaborate enzymatic biofuel cells (EBFCs), a subclass of fuel cells in which enzymes replace the conventional catalysts. A glucose/O2 EBFC, with a glucose oxidizing anode and a O2 reducing MCO cathode, could become the in vivo source of electricity that would power sometimes in the future integrated medical devices. This review covers the challenges and advances in the electrochemistry of MCOs and their use in EBFCs with a particular emphasis on the last 6 years. First basic features of MCOs and EBFCs are presented. Clues provided by electrochemistry to understand these enzymes and how they behave once connected at electrodes are described. Progresses realized in the development of efficient biocathodes for O2 reduction relying both on direct and mediated electron transfer mechanism are then discussed. Some implementations in EBFCs are finally presented.

Halides inhibition of multicopper oxidases studied by FTIR spectroelectrochemistry using azide as an active infrared probe.

An infrared spectroelectrochemical study of Trametes hirsuta laccase and Magnaporthe oryzae bilirubin oxidase has been performed using azide, an inhibitor of multicopper oxidases, as an active infrared probe incorporated into the T2/T3 copper cluster of the enzymes. The redox potential-controlled measurements indicate that N3- stretching IR bands of azide ion bound to the T2/T3 cluster are only detected for the oxidized enzymes, confirming that azide only binds to Cu2+. Moreover, the process of binding/dissociation of azide ion is shown to be reversible. The interaction of halide anions, which also inhibit multicopper oxidases, with the active site of the enzymes was studied by measuring the changes in the azide FTIR bands. Enzymes inhibited by azide respond differently upon addition of fluoride or chloride ions to the sample solution inhibited by azide. Fluoride ions compete with azide for binding at one of the T2/T3 Cu ions, whereas competition from chloride ions is much less evident.

Carbon nanotube fiber mats for microbial fuel cell electrodes.

Novel carbon nanotube based electrodes of microbial fuel cells (MFC) have been developed. MFC is a promising technology for the wastewater treatment and the production of electrical energy from redox reactions of natural substrates. Performances of such bio-electrochemical systems depend critically on the structure and properties of the electrodes. The presently developed materials are made by weaving fibers solely comprised of carbon nanotubes. They exhibit a large scale porosity controlled by the weaving process. This porosity allows an easy colonization by electroactive bacteria. In addition, the fibers display a nanostructuration that promotes excellent growth and adhesion of the bacteria at the surface of the electrodes. This unique combination of large scale porosity and nanostructuration allows the present electrodes to perform better than carbon reference. When used as anode in a bioelectrochemical reactor in presence of Geobacter sulfurreducens bacteria, the present electrodes show a maximal current density of about 7.5mA/cm2.

On-chip enzymatic microbiofuel cell-powered integrated circuits.

A variety of diagnostic and therapeutic medical technologies rely on long term implantation of an electronic device to monitor or regulate a patient's condition. One proposed approach to powering these devices is to use a biofuel cell to convert the chemical energy from blood nutrients into electrical current to supply the electronics. We present here an enzymatic microbiofuel cell whose electrodes are directly integrated into a digital electronic circuit. Glucose oxidizing and oxygen reducing enzymes are immobilized on microelectrodes of an application specific integrated circuit (ASIC) using redox hydrogels to produce an enzymatic biofuel cell, capable of harvesting electrical power from just a single droplet of 5 mM glucose solution. Optimisation of the fuel cell voltage and power to match the requirements of the electronics allow self-powered operation of the on-board digital circuitry. This study represents a step towards implantable self-powered electronic devices that gather their energy from physiological fluids.

Mechanism of chloride inhibition of bilirubin oxidases and its dependence on potential and pH.

Bilirubin oxidases (BODs) belong to the multi-copper oxidase (MCO) family and efficiently reduce O2 at neutral pH and in physiological conditions where chloride concentrations are over 100 mM. BODs were consequently considered to be Cl- resistant contrary to laccases. However, there has not been a detailed study on the related effect of chloride and pH on the redox state of immobilized BODs. Here, we investigate by electrochemistry the catalytic mechanism of O2 reduction by the thermostable Bacillus pumilus BOD immobilized on carbon nanofibers in the presence of NaCl. The addition of chloride results in the formation of a redox state of the enzyme, previously observed for different BODs and laccases, which is only active after a reductive step. This behavior has not been previously investigated. We show for the first time that the kinetics of formation of this state is strongly dependent on pH, temperature, Cl- concentration and on the applied redox potential. UV-visible spectroscopy allows us to correlate the inhibition process by chloride with the formation of the alternative resting form of the enzyme. We demonstrate that O2 is not required for its formation and show that the application of an oxidative potential is sufficient. In addition, our results suggest that the reactivation may proceed thought the T3 ß.

The nature of the rate-limiting step of blue multicopper oxidases: Homogeneous studies versus heterogeneous.

Multicopper oxidases (MCOs) catalyzed two half reactions (linked by an intramolecular electron transfer) through a Ping-Pong mechanism: the substrate oxidation followed by the O2 reduction. MCOs have been characterized in details in solution or immobilized on electrode surfaces. The nature of the rate-limiting steps, which is controversial in the literature, is discussed in this mini review for both cases. Deciphering such rate-limiting steps is of particular importance to efficiently use MCOs in any applications requiring the reduction of O2 to water.

Low-Molecular-Weight Hydrogels as New Supramolecular Materials for Bioelectrochemical Interfaces.

Controlling the interface between biological tissues and electrodes remains an important challenge for the development of implantable devices in terms of electroactivity, biocompatibility, and long-term stability. To engineer such a biocompatible interface a low molecular weight gel (LMWG) based on a glycosylated nucleoside fluorocarbon amphiphile (GNF) was employed for the first time to wrap gold electrodes via a noncovalent anchoring strategy, that is, self-assembly of GNF at the electrode surface. Scanning electron microscopy (SEM) studies indicate that the gold surface is coated with the GNF hydrogels. Electrochemical measurements using cyclic voltammetry (CV) clearly show that the electrode properties are not affected by the presence of the hydrogel. This coating layer of 1 to 2 µm does not significantly slow down the mass transport through the hydrogel. Voltammetry experiments with gel coated macroporous enzyme electrodes reveal that during continuous use their current is improved by 100% compared to the noncoated electrode. This demonstrates that the supramolecular hydrogel dramatically increases the stability of the bioelectrochemical interface. Therefore, such hybrid electrodes are promising candidates that will both offer the biocompatibility and stability needed for the development of more efficient biosensors and biofuel cells.

How the Intricate Interactions between Carbon Nanotubes and Two Bilirubin Oxidases Control Direct and Mediated O2 Reduction.

Due to the lack of a valid approach in the design of electrochemical interfaces modified with enzymes for efficient catalysis, many oxidoreductases are still not addressed by electrochemistry. We report in this work an in-depth study of the interactions between two different bilirubin oxidases, (from the fungus Myrothecium verrucaria and from the bacterium Bacillus pumilus), catalysts of oxygen reduction, and carbon nanotubes bearing various surface charges (pristine, carboxylic-, and pyrene-methylamine-functionalized). The surface charges and dipole moment of the enzymes as well as the surface state of the nanomaterials are characterized as a function of pH. An original electrochemical approach allows determination of the best interface for direct or mediated electron transfer processes as a function of enzyme, nanomaterial type, and adsorption conditions. We correlate these experimental results to theoric voltammetric curves. Such an integrative study suggests strategies for designing efficient bioelectrochemical interfaces toward the elaboration of biodevices such as enzymatic fuel cells for sustainable electricity production.