Direct Electron Transfer-type Bioelectrocatalysis Research Articles

Essential Insight of Direct Electron Transfer-Type Bioelectrocatalysis by Membrane-Bound d-Fructose Dehydrogenase with Structural Bioelectrochemistry

Essential Insight of Direct Electron Transfer-Type Bioelectrocatalysis by Membrane-Bound <scp>d</scp>-Fructose Dehydrogenase with Structural Bioelectrochemistry

Kinetic and thermodynamic analysis of Cu2+-dependent reductive inactivation in direct electron transfer-type bioelectrocatalysis by copper efflux oxidase

Copper efflux oxidases (CueOs) are key enzymes in copper homeostasis systems. The mechanisms involved are however largely unknown. CueO-type enzymes share a typical structural feature composed of Methionine-rich (Met-rich) domains that are proposed to be involved in copper homeostasis. Bioelectrocatalysis using CueO-type enzymes in the presence of Cu2+ recently highlighted a new Cu2+-dependent catalytic pathway related to a cuprous oxidase activity. In this work, we further investigated the effects of Cu2+ on direct electron transfer (DET)-type bioelectrocatalytic reduction of O2 by CueO at NH2-functionalized multi-walled carbon nanotubes. The DET-type bioelectrocatalytic activity of CueO decreased at low potential in the presence of Cu2+, showing unique peak-shaped voltammograms that we attribute to inactivation and reactivation processes. Chronoamperometry was used to kinetically analyze these processes, and the results suggested linear free energy relationships between the inactivation/reactivation rate constant and the electrode potential. Pseudo-steady-state analysis also indicated that Cu2+ uncompetitively inhibited the enzymatic activity. A detailed model for the Cu2+-dependent reductive inactivation of CueO was proposed to explain the electrochemical data, and the related thermodynamic and kinetic parameters. A CueO variant with truncated copper-binding α helices and bilirubin oxidase free of Met-rich domains also showed such reductive inactivation process, which suggests that multicopper oxidases contain copper-binding sites that lead to inactivation.

Effects of N-linked glycans of bilirubin oxidase on direct electron transfer-type bioelectrocatalysis

Bilirubin oxidase from Myrothecium verrucaria (mBOD) is a promising enzyme for catalyzing the four-electron reduction of dioxygen into water and realizes direct electron transfer (DET)-type bioelectrocatalysis. It has two N-linked glycans (N-glycans), and N472 and N482 are known as binding sites. Both binding sites located on opposite side of the type I (T1) Cu, which is the electrode-active site of BOD. We investigated the effect of N-glycans on DET-type bioelectrocatalysis by performing electrochemical measurements using electrodes with controlled surface charges. Two types of BODs with different N-glycans, mBOD and recombinant BOD overexpressed in Pichia pastoris (pBOD), and their deglycosylated forms (dg-mBOD and dg-pBOD) were used in this study. Kinetic analysis of the steady-state catalytic waves revealed that both size and composition of N-glycans affected the orientation of adsorbed BODs on the electrodes. Interestingly, the most favorable orientation was achieved with pBOD, which has the largest N-glycans. Furthermore, the effect of the orientation control by the N-glycans is cooperative with electrostatic interaction.

Multiple electron transfer pathways of tungsten-containing formate dehydrogenase in direct electron transfer-type bioelectrocatalysis.

Tungsten-containing formate dehydrogenase from Methylorubrum extroquens AM1 (FoDH1)-a promising biocatalyst for the interconversion of carbon dioxide/formate and nicotine adenine dinucleotide (NAD+)/NADH redox couples-was investigated using structural biology and bioelectrochemistry. FoDH1 is reported to be an enzyme that can realize "direct electron transfer (DET)-type bioelectrocatalysis." However, its 3-D structure, electrode-active sites, and electron transfer (ET) pathways remain unclear. The ET pathways were investigated using structural information, electrostatic interactions between the electrode and the enzyme, and the differences in the substrates. Two electrode-active sites and multiple ET pathways in FoDH1 were discovered.

Kinetic and Thermodynamic Analysis of Cu2+-Dependent Reductive Inactivation in Direct Electron Transfer-Type Bioelectrocatalysis by Copper Efflux Oxidase

Kinetic and Thermodynamic Analysis of Cu2+-Dependent Reductive Inactivation in Direct Electron Transfer-Type Bioelectrocatalysis by Copper Efflux Oxidase

Cyanide sensitivity in direct electron transfer-type bioelectrocatalysis by membrane-bound alcohol dehydrogenase from Gluconobacter oxydans

An overexpression system of membrane-bound alcohol dehydrogenase (ADH) from Gluconobacter oxydans was constructed to examine its bioelectrocatalytic characteristics. The effects of cyanide (CN−) addition on the kinetics of direct electron transfer (DET)-type bioelectrocatalysis by ADH were analyzed. CN− enhanced the bioelectrocatalytic activity, while the catalytic activity in the solution remained unchanged, even in the presence of CN−. Electrochemical methods and electron spin resonance spectroscopy showed the detailed electron transfer pathway in the DET-type bioelectrocatalysis by ADH. Briefly, ADH is suggested to communicate with an electrode via a CN−-insensitive and H+-sensitive heme c in DET. These characteristics of ADH with respect to CN− suggest the involvement of ADH in CN−-insensitive respiration in G. oxydans.

Direct Bioelectrocatalytic Oxidation of Glucose by Gluconobacter oxydans Membrane Fractions in PEDOT:PSS/TEG-Modified Biosensors.

Recent years have witnessed an ever-increasing interest in developing electrochemical biosensors based on direct electron transfer-type bioelectrocatalysis. This work investigates the bioelectrocatalytic oxidation of glucose by membrane fractions of Gluconobacter oxydans cells on screen-printed electrodes modified with thermally expanded graphite and poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS). Electrooxidation of glucose was shown to occur without the presence of electron transport mediators. Chronoamperometric and cyclic voltametric characteristics showed an increase of anodic currents at electrode potentials of 0–500 mV relative to the reference electrode (Ag/AgCl). The direct electron transfer effect was observed for non-modified PEDOT:PSS as well as for PEDOT:PSS linked with crosslinkers and conductive fillers such as polyethylene glycol diglycidyl or dimethyl sulfoxide. Bioelectrodes with this composite can be successfully used in fast reagent-free glucose biosensors.

Enhancement of the Direct Electron Transfer-type Bioelectrocatalysis of Bilirubin Oxidase at the Interface between Carbon Particles

Enhancement of the Direct Electron Transfer-type Bioelectrocatalysis of Bilirubin Oxidase at the Interface between Carbon Particles

Direct electron transfer-type bioelectrocatalysis by membrane-bound aldehyde dehydrogenase from Gluconobacter oxydans and cyanide effects on its bioelectrocatalytic properties

The bioelectrocatalytic properties of membrane-bound aldehyde dehydrogenase (AlDH) from Gluconobacter oxydans NBRC12528 were evaluated. AlDH exhibited direct electron transfer (DET)-type bioelectrocatalytic activity for acetaldehyde oxidation at several kinds of electrodes. The kinetic and thermodynamic parameters for bioelectrocatalytic acetaldehyde oxidation were estimated based on the partially random orientation model. Moreover, at the multi-walled carbon nanotube-modified electrode, the coordination of CN− to AlDH switched the direction of the DET-type bioelectrocatalysis to acetate reduction under acidic conditions. These phenomena were discussed from a thermodynamic viewpoint.

Rational Surface Modification of Carbon Nanomaterials for Improved Direct Electron Transfer-Type Bioelectrocatalysis of Redox Enzymes

Interfacial electron transfer between redox enzymes and electrodes is a key step for enzymatic bioelectrocatalysis in various bioelectrochemical devices. Although the use of carbon nanomaterials enables an increasing number of redox enzymes to carry out bioelectrocatalysis involving direct electron transfer (DET), the role of carbon nanomaterials in interfacial electron transfer remains unclear. Based on the recent progress reported in the literature, in this mini review, the significance of carbon nanomaterials on DET-type bioelectrocatalysis is discussed. Strategies for the oriented immobilization of redox enzymes in rationally modified carbon nanomaterials are also summarized and discussed. Furthermore, techniques to probe redox enzymes in carbon nanomaterials are introduced.

An amperometric biosensor of L-fucose in urine for the first screening test of cancer

Quantitative routine detection of fucose, which is a cancer marker, in urine is effective for the preliminary screening of cancer. Amperometric biosensing methods have the advantage of being simple, rapid, and precise for urinalysis. However, coexisting electroactive interferences such as ascorbic acid (AA), dopamine (DA), and uric acid (UA) prevent accurate measurements. In this work, an amperometric l-fucose biosensor unaffected by interferences was developed and utilizes direct electron transfer type bioelectrocatalysis of pyrroloquinoline quinone (PQQ)-dependent pyranose dehydrogenase from Coprinopsis cinerea (CcPDH). The isolated PQQ domain from CcPDH was immobilized on gold nanoparticle (AuNP)-modified electrodes, which obtained a catalytic current at a lower potential than the oxidation potential of the interfering compounds. Applying an operating potential of −0.1 V vs. Ag|AgCl (3 M NaCl) enabled the detection of l-fucose while completely eliminating the oxidation of AA, DA, and UA on the electrodes. The increase in the specific area of the electrodes by increasing the AuNP drop-casting time resulted in an improvement in the sensor performance. The biosensor exhibited a linear range for l-fucose detection between 0.1 mM and 1 mM (R2 = 0.9996), including a cut-off value, the sensitivity was 3.12 ± 0.05 μA mM−1 cm−2, and the detection limit was 13.6 μM at a signal-to-noise ratio of three. The biosensor can be used to quantify the concentration of l-fucose at physiological levels and does not require urine preprocessing, making it applicable to practical use for point-of-care testing with urine.

Diffusion-limited electrochemical d-fructose sensor based on direct electron transfer-type bioelectrocatalysis by a variant of d-fructose dehydrogenase at a porous gold microelectrode

Abstract An electrochemical d -fructose sensor was fabricated by immobilizing a variant of d -fructose dehydrogenase on a porous gold microelectrode. The enzyme-modified electrode bioelectrocatalytically oxidizes d -fructose via direct electron transfer. The catalytic current reaches a steady-state limiting value at 0.05 V vs. Ag|AgCl (sat. KCl). The temperature dependence of the sensor suggests that its response is limited by the diffusion of d -fructose. Therefore, the sensor requires no calibration. The sensitivity of the sensor, (2.0 ± 0.2) × 102 μA cm−2 mM−1, is reproducible. The upper limit of detection is ~2.0 mM and the kinetics of the bioelectrocatalytic reaction is determined the limitation. The fabricated sensors were applied in the determination of d -fructose in commercial beverages as well as honey, and compared favorably with a more commonly used photometric method.

Rapid Fabrication of Nanoporous Gold as a Suitable Platform for the Direct Electron Transfer-type Bioelectrocatalysis of Bilirubin Oxidase

Rapid Fabrication of Nanoporous Gold as a Suitable Platform for the Direct Electron Transfer-type Bioelectrocatalysis of Bilirubin Oxidase

Discussion on Direct Electron Transfer-Type Bioelectrocatalysis of Downsized and Axial-Ligand Exchanged Variants of d-Fructose Dehydrogenase

Discussion on Direct Electron Transfer-Type Bioelectrocatalysis of Downsized and Axial-Ligand Exchanged Variants of d-Fructose Dehydrogenase

Direct Electron Transfer-Type Bioelectrocatalysis of Redox Enzymes at Nanostructured Electrodes

Direct electron transfer (DET)-type bioelectrocatalysis, which couples the electrode reactions and catalytic functions of redox enzymes without any redox mediator, is one of the most intriguing subjects that has been studied over the past few decades in the field of bioelectrochemistry. In order to realize the DET-type bioelectrocatalysis and improve the performance, nanostructures of the electrode surface have to be carefully tuned for each enzyme. In addition, enzymes can also be tuned by the protein engineering approach for the DET-type reaction. This review summarizes the recent progresses in this field of the research while considering the importance of nanostructure of electrodes as well as redox enzymes. This review also describes the basic concepts and theoretical aspects of DET-type bioelectrocatalysis, the significance of nanostructures as scaffolds for DET-type reactions, protein engineering approaches for DET-type reactions, and concepts and facts of bidirectional DET-type reactions from a cross-disciplinary viewpoint.

Diffusion-limited biosensing of dissolved oxygen by direct electron transfer-type bioelectrocatalysis of multi-copper oxidases immobilized on porous gold microelectrodes

Diffusion-controlled amperometric biosensors for dissolved oxygen (O2) were constructed by immobilization of multi-copper oxidases (copper efflux oxidase and bilirubin oxidase) on porous gold microdisk electrodes fabricated by anodization in a glucose solution. The immobilized enzymes rapidly consumed O2 near the electrode at potentials more negative than 0.2 V vs. Ag|AgCl|sat. KCl via direct electron transfer-type bioelectrocatalysis and the reduction current reached the steady state limiting value under static conditions. The fabricated biosensor exhibited a linear response to dissolved O2 concentration and was almost identical to the theoretical sensor, based on nonlinear diffusion of O2 around the microdisk electrode. The biosensor response was fast enough to monitor the catalytic consumption of dissolved O2 by glucose oxidase and exhibited storage stability for more than six days.

Direct electron transfer-type bioelectrocatalysis of FAD-dependent glucose dehydrogenase using porous gold electrodes and enzymatically implanted platinum nanoclusters

The direct electron transfer (DET)-type bioelectrocatalysis of flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (GDH) from Aspergillus terreus (AtGDH) was carried out using porous gold (Au) electrodes and enzymatically implanted platinum nanoclusters (PtNCs). The porous Au electrodes were prepared by anodization of planar Au electrodes in a phosphate buffer containing glucose as a reductant. Moreover, PtNCs were generated into AtGDH by an enzymatic reduction of hexachloroplatinate (IV) ion. The modification was confirmed by native polyacrylamide gel electrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses. The AtGDH-adsorbed porous Au electrode showed a DET-type bioelectrocatalytic wave both in the presence and absence of PtNCs; however, the current density with PtNCs (~1 mA cm−2 at 0 V vs. Ag|AgCl|sat. KCl) was considerably higher than that without PtNCs. The kinetic and thermodynamic analysis of the steady-state catalytic wave indicated that inner PtNCs shortened the distance between the catalytic center of AtGDH (=FAD) and the conductive material, and improved the heterogeneous electron transfer kinetics between them.

Ultimate downsizing of d-fructose dehydrogenase for improving the performance of direct electron transfer-type bioelectrocatalysis

d-Fructose dehydrogenase (FDH), a membrane-bound heterotrimeric enzyme, shows strong activity in direct electron transfer (DET)-type bioelectrocatalysis. An FDH variant (Δ1c2cFDH) which lacks 199 amino acid residues including two heme c moieties from N-terminus was constructed, and its DET-type bioelectrocatalytic performance was evaluated with cyclic voltammetry at Au planar electrodes. A DET-type catalytic current of d-fructose oxidation was clearly observed on Δ1c2cFDH-adsorbed Au electrodes. Detailed analysis of the steady-state catalytic current indicated that Δ1c2cFDH transports the electrons to the electrode via heme 3c at a more negative potential and at more improved kinetics than the recombinant (native) FDH.

Factors affecting the interaction between carbon nanotubes and redox enzymes in direct electron transfer-type bioelectrocatalysis

The effects of three types of water-soluble carbon nanotubes (CNTs) of different lengths on the direct electron transfer (DET)-type bioelectrocatalysis of redox enzymes were investigated. Bilirubin oxidase (BOD), copper efflux oxidase (CueO), and a membrane-bound NiFe hydrogenase (H2ase) were used as model redox enzymes for four-electron dioxygen (O2) reduction (in the case of BOD and CueO) and two-electron dihydrogen (H2) oxidation (in the case of H2ase). As a result, diffusion-controlled O2 reduction in an O2-saturated neutral buffer was realized by BOD on CNTs of a length of 1μm, but the catalytic current densities decreased as the length of CNTs increased. However, almost opposite trends were obtained when CueO and H2ase were utilized as the biocatalysts. Factors of the CNTs and the enzymes affecting the characteristics of the DET-type bioelectrocatalysis of the three enzymes were discussed. Finally, the electrostatic interaction between an enzyme (especially the portion near the redox active center) and CNTs is proposed as one of the most important factors governing the performance of DET-type bioelectrocatalysis.

Direct Electron Transfer-type Bioelectrocatalysis of Peroxidase at Mesoporous Carbon Electrodes and Its Application for Glucose Determination Based on Bienzyme System.

Non-catalytic direct electron transfer (DET) signal of Compound I of horseradish peroxidase (POD) was first detected at 0.7 V on POD/carbon nanotube mixture-modified electrodes. Excellent performance of DET-type bioelectrocatalysis was achieved with POD immobilized with glutaraldehyde on Ketjen Black (KB)-modified electrodes for H2O2 reduction with an onset potential of 0.65 V (vs. Ag | AgCl | sat. KCl) without any electrode surface modification. The POD-immobilized KB electrode was found to be suitable for detecting H2O2 with a low detection limit (0.1 μM at S/N = 3) at -0.1 V. By co-immobilizing glucose oxidase (GOD) and POD on the KB-modified electrode, a bienzyme electrode was constructed to couple the oxidase reaction of GOD with the DET-type bioelectrocatalytic reduction of H2O2 by POD. The amperometric detection of glucose was performed with a high sensitivity (0.33 ± 0.01 μA cm-2 μM-1) and a low detection limit (2 μM at S/N = 3).