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Redox enzymes can work as efficient electrocatalysts. The coupling of redox enzymatic reactions with electrode reactions is called enzymatic bioelectrocatalysis, which imparts high reaction specificity to electrode reactions with nonspecific characteristics. The key factors required for bioelectrocatalysis are hydride ion/electron transfer characteristics and low specificity for either substrate in redox enzymes. Several theoretical features of steady-state responses are introduced to understand bioelectrocatalysis and to extend the performance of bioelectrocatalytic systems. Applications of the coupling concept to bioelectrochemical devices are also summarized with emphasis on the achievements recorded in the research group of the author. [ABSTRACT FROM AUTHOR]

Abstract: Bilirubin oxidase (BOD) was immobilized as a multiple layer in a cationic polymer (poly-l-lysine) matrix on an electrode surface. The BOD-modified electrode catalyzed four-electron reduction of dioxygen (O2) to water, without any mediator, to produce a diffusion-controlled voltammogram for the O2 reduction in a quiescent solution. The voltammogram was successfully analyzed by the theory of irreversible voltammograms. Under convective conditions where O2 depletion was negligible near the electrode surface, a steady-state voltammogram was obtained with a limiting current density of about 1 mAcm−2. The steady-state voltammogram was explained by an equation derived on the basis of a reaction layer model, in which BOD was considered to be diffusible in the immobilized layer. [Copyright &y& Elsevier]

Electrochemical studies on the applied aspects of quinoproteins are briefly reviewed. Catalytic reactions of quinoprotein enzymes can be connected to electrochemical reactions directly or by the mediation of molecules functioning as electron acceptors of the enzymes. Such an enzyme-electrochemical reaction is called bioelectrocatalysis. It provides a novel method of kinetic analysis of enzyme catalysis and even whole bacterial cell catalysis. The principle of bioelectrocatalysis is first described, then, the bioelectrocatalysis-based application of quinoproteins in biosensors is mentioned. Characteristics and performance of this type of biosensor is explained by citing our own work. Possible application in bioreactors and biofuel cells is also mentioned. [Copyright &y& Elsevier]

Porous gold (Au) electrodes prepared by anodization of Au electrodes in buffer solutions containing glucose were utilized as scaffolds for direct electron transfer (DET)-type bioelectrocatalysis of bilirubin oxidase (BOD). The three-dimensional porous structure appeared to promote DET-type bioelectrocatalysis of BOD. The current density of DET-type bioelectrocatalysis of BOD increased with the formation of a three-dimensional porous structure at the beginning of anodization; however, it showed saturation characteristics with a further increase in the non-Faradaic current. Moreover, the glucose concentration during anodization affected the activity of DET-type bioelectrocatalysis of BOD. Scanning electron microscopy of the prepared porous Au electrodes showed that the structure of the electrode surface depended on the glucose concentration. Achieving a pore size distribution suitable for substrate transport and enzyme orientation seemed to be dependent on the glucose concentration. Unlabelled Image • Anodization of gold electrodes in glucose solutions provides the porous structure. • The surface structure is affected by the glucose concentration during anodization. • Porous gold electrodes are effective scaffolds for bioelectrocatalysis. • Bioelectrocatalysis of bilirubin oxidase depends on the porous structure. [ABSTRACT FROM AUTHOR]

[Display omitted] Bilirubin oxidase (BOD) is a bioelectrocatalyst that reduces dioxygen (O 2) to water and is capable of direct electron transfer (DET)-type bioelectrocatalysis via its electrode-active site (T1 Cu). BOD from Myrothecium verrucaria (mBOD) has been widely studied and has strong DET activity. mBOD contains two N -linked glycans (N -glycans) with N472 and N482 binding sites distal to T1 Cu. We previously reported that different N -glycan compositions affect the enzymatic orientation on the electrode by using recombinant BOD expressed in Pichia pastoris and the deglycosylation method. However, the individual function of the two N -glycans and the effects of N -glycan composition (size, structure, and non-reducing termini) on DET-type reactions are still unclear. In this study, we utilize maleimide-functionalized polyethylene glycol (MAL-PEG) as an N -glycan mimic to evaluate the aforementioned effects. Site-specific enzyme-PEG crosslinking was carried out by specific binding of maleimide to Cys residues. Recombinant BOD expressed in Escherichia coli (eBOD), which does not have a glycosylation system, was used as a benchmark to evaluate the effect. Site-directed mutagenesis of Asn residue (N472 or N482) into Cys residue is utilized to realize site-specific glycan mimic modification to the original binding site. [ABSTRACT FROM AUTHOR]

Tungsten-containing formate dehydrogenase from Methylobacterium extorquens AM1 (FoDH1) catalyzes formate oxidation with NAD + . FoDH1 shows little direct communication with carbon electrodes, including mesoporous Ketjen Black-modified glassy carbon electrode (KB/GCE); however, it shows well-defined direct electron transfer (DET)-type bioelectrocatalysis of carbon dioxide reduction, formate oxidation, NAD + reduction, and NADH oxidation on gold nanoparticle (AuNP)-embedded KB/GCE treated with 4-mercaptopyridine. Microscopic measurements reveal that the AuNPs ( d = 5 nm) embedded on the KB surface are uniformly dispersed. Electrochemical data indicate that the pyridine moiety on the AuNPs plays important roles in facilitating the interfacial electron transfer kinetics and increasing the probability of productive orientation of FoDH1. The formal potential of the electrochemical communication site, which is most probably an ion‑sulfur cluster, is evaluated as − 0.591 ± 0.005 V vs. Ag | AgCl | sat. KCl from Nernst analysis of the steady-state catalytic waves. [ABSTRACT FROM AUTHOR]

Tungsten-containing formate dehydrogenase (FoDH1) with a molecular mass of 170 kDa from Methylobacterium extorquens AM1 catalyzes the oxidation of formate (HCOO − ) to carbon dioxide (CO 2 ) with NAD + as a natural electron acceptor in solution. FoDH1 does not produce any direct electron transfer (DET)-type bioelectrocatalytic wave at planar electrodes, but can adsorb on and communicate with mesoporous carbon electrodes. The curvature effect of mesoporous structures seems to increase the number of enzymes with orientations suitable for electrochemical communication. However, adsorption proceeds slowly on Ketjen Black-modified electrode and the catalytic current density remains low. Most probably, the size of the mesopores is too small to effectively trap FoDH1. The adsorbed FoDH1 catalyzes DET-type bioelectrocatalytic interconversion of the CO 2 /HCOO − and NAD + /NADH redox couples. Most probably, one of the iron–sulfur clusters located near the enzyme surface communicates with mesoporous electrodes. When the communication proceeds effectively, FoDH1 behaves as a novel bidirectional catalyst for the substrates, since FoDH1 can realize fast uphill intramolecular electron transfer. The non-covalently bound flavin mononucleotide (FMN) cofactor in FoDH1 is dissociated from some FoDH1 molecules and adsorbs on the mesoporous electrode to give a symmetrical surface-confined redox wave. Although adsorbed FMN cannot participate in mediated electron transfer (MET)-type bioelectrocatalysis, dissociated FMN in solution works as a mediator for MET-type bioelectrocatalysis of the HCOO − oxidation at planar electrodes. [ABSTRACT FROM AUTHOR]

A membraneless direct electron transfer (DET)-type dihydrogen (H 2 )/air-breathing biofuel cell without any mediator was constructed wherein bilirubin oxidase from Myrothecium verrucaria (BOD) and membrane-bound [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F (MBH) were used as biocatalysts for the cathode and the anode, respectively, and Ketjen black-modified water proof carbon paper (KB/WPCC) was used as an electrode material. The KB/WPCC surface was modified with 2-aminobenzoic acid and p -phenylenediamine, respectively, to face the positively charged electron-accepting site of BOD and the negatively charged electron-donating site of MBH to the electrode surface. A gas-diffusion system was employed for the electrodes to realize high-speed substrate supply. As result, great improvement in the current density of O2 reduction with BOD and H 2 reduction with MBH were realized at negatively and postively charged surfaces, respectively. Gas diffusion system also suppressed the oxidative inactivation of MBH at high electrode potentials. Finally, based on the improved bioanode and biocathode, a dual gas-diffusion membrane- and mediatorless H 2 /air-breathing biofuel cell was constructed. The maximum power density reached 6.1 mW cm −2 (at 0.72 V), and the open circuit voltage was 1.12 V using 1 atm of H 2 gas as a fuel at room temperature and under passive and quiescent conditions. [ABSTRACT FROM AUTHOR]

A new gas-diffusion-type biocathode was constructed for carbon dioxide (CO 2 ) reduction. In this work, tungsten-containing formate dehydrogenase (FoDH1), which is a promising enzyme for interconversion of formate and CO 2 , was used as a catalyst and was absorbed on a Ketjen Black (KB)-modified electrode. We used 1,1′-trimethylene-2,2′-bipyridinium dibromide as a mediator, and the hydrophobicity of the FoDH1-absorbed electrode was optimized according to the weight ratio of the polytetrafluoroethylene binder to KB. We achieved cathodic current densities of about 20 mA cm − 2 under mild and quiescent conditions (neutral pH, atmospheric pressure, and room temperature). [ABSTRACT FROM AUTHOR]

d -Fructose dehydrogenase (FDH) from Gluconobacter japonicus NBRC3260 catalyzes the two-electron oxidation of d -fructose to 5-keto- d -fructose, and it is widely used in biofuel cells and biosensors. In this study, methoxy-substituent-functionalized carbon electrodes are constructed by electrochemical oxidation of methoxy-aniline derivatives on Ketjen Black (KB)-modified electrodes to improve the immobilization and bioelectrocatalysis of FDH. It is proposed that the specific interaction between FDH, especially the heme c moiety, and methoxy substituent(s) of amines on carbon electrode increases the proportion of the productively oriented FDH molecules to the total FDHs. Consequently, the limiting catalytic current density of the d -fructose oxidation increases to as much as 23 ± 2 mA cm −2 in FDH/2,4-dimethoxyaniline/KB/glassy carbon electrode, for example. [ABSTRACT FROM AUTHOR]

A reversible surface-confined system for potentiometric coulometry studies was fabricated by the deposition of a liquid film on an electrode surface, and the system was used to detect charge accumulation due to the peroxidase (POD)-catalyzed reduction of H 2 O 2 . The components of the liquid film were 1-ethyl-3-methylimidazolium bis(nonafluorobutanesulfonyl)imide as the electrolyte, dibutyl phthalate as the medium, and ferrocene as the redox-active mediator. When the thickness of the liquid film was less than that of the diffusion layer in the minimum potential step of the apparatus, a symmetric voltammogram, according to the Nernst equation, was recorded. The reduction of H 2 O 2 by POD was carried out on the liquid-film-modified electrode and studied by potentiometric coulometry. The addition of H 2 O 2 shifts the electrode potential positively, due to the oxidation of ferrocene. The change in the potential is in agreement with the Nernst equation. Thus, potentiometric coulometry, carried out on the liquid-film-modified electrode, enables the determination of the substrate without the requirement of a calibration curve. [ABSTRACT FROM AUTHOR]

d -Fructose dehydrogenase (FDH) is a heterotrimeric-membrane-bound enzyme capable of direct electron transfer (DET)-type bioelectrocatalysis. Subunit II contains three heme C moieties and is presumed to play a key role in the DET reaction because the subunit I/III subcomplex (without subunit II) lacks DET-type activity. We constructed an expression system for subunit II. A non-turnover signal from subunit II was not observed on voltammograms, and a subunit II-adsorbed electrode did not exhibit DET-type activity in the presence of the subunit I/III subcomplex and fructose. Gel filtration column chromatography indicated that subunit II formed multimeric complexes with four or more subunits. The aggregation of subunit II seemed to interfere with its direct communication with the electrodes. In contrast, when subunit II was mixed with the subunit I/III complex in a solution, they formed a 1:1 full complex, and the complex recovered DET-type bioelectrocatalytic activity. These results strongly suggest that the heme C in subunit II is the electron transfer site for the DET-type bioelectrocatalytic activity of FDH. [ABSTRACT FROM AUTHOR]

A heterotrimeric membrane-bound fructose dehydrogenase (FDH) from Gluconobacter japonicus NBRC3260 contains FAD in subunit I and three heme C moieties in subunit II as the redox centers, and is one of the direct electron transfer (DET)-type redox enzymes. FDH-catalyzed current density of fructose oxidation at hydrophilic mercaptoethanol (MEtOH)-modified Au electrode is much larger than that at hydrophobic mercaptoethane (MEtn)-modified Au electrode. Addition of a non-ionic surfactant Triton ® X-100 (1%) completely quenches the catalytic current at the MEtn-modified Au electrode, while only small competitive effect is observed at the MEtOH-modified Au electrode. Quartz crystal microbalance measurements support the adsorption of FDH and Triton ® X-100 on both of the modified electrodes. We propose a model to explain the phenomenon as follows. The surfactant forms a monolayer on the hydrophobic MEtn-modified electrode with strong hydrophobic interaction, and FDH adsorbs on the surface of the surfactant monolayer. The monolayer inhibits the electron transfer from FDH to the electrode. On the other hand, the surfactant forms a bilayer on the hydrophilic MEtOH-modified electrode. The interaction between the surfactant bilayer and the hydrophilic electrode is relatively weak so that FDH replaces the surfactant and is embedded in the bilayer to communicate electrochemically with the hydrophilic electrode. [ABSTRACT FROM AUTHOR]

A heterotrimeric membrane-bound fructose dehydrogenase (FDH) complex from Gluconobacter japonicus NBRC3260 catalyzes oxidation of d-fructose into 2-keto-d-fructose and is one of typical enzymes allowing a direct electron transfer (DET)-type bioelectrocatalysis. Subunits I and II have a covalently bound flavin adenine dinucleotide and three heme C moieties, respectively. We have constructed subunit I/III subcomplex (ΔcFDH) lacking of the heme C subunit. ΔcFDH catalyzes the oxidation of d-fructose with several artificial electron acceptors, but loses the DET ability. The formal potentials (E°′) of the three heme C moieties of FDH have been determined to be −10±4, 60±8 and 150±4mV (vs. Ag|AgCl|sat. KCl) at pH5.0, while the onset potential of FDH-catalyzed DET-type bioelectrocatalytic wave is −100mV. Judging from these results, we conclude that FDH communicates electrochemically with electrodes via the heme C, and discuss the pathway of the electron transfer in the catalytic process. [ABSTRACT FROM AUTHOR]

Abstract: The mediated electro-enzymatic electrolysis systems based on the tricarboxylic acid (TCA) cycle reaction were examined on a micro-bulk electrolytic system. A series of the enzyme-catalyzed reactions in the TCA cycle was coupled with electrode reaction. Electrochemical oxidation of NADH was catalyzed by diaphorase with an aid of a redox mediator with a formal potential of −0.15V vs. Ag|AgCl. The mediator was also able to shuttle electrons between succinate dehydrogenase and electrode. The charge during the electrolysis increased on each addition of dehydrogenase reaction in a cascade of the TCA cycle. However, the electrolysis efficiencies were close to or less than 90% because of the product inhibition. Lactate oxidation to acetyl-CoA catalyzed by two NAD-dependent dehydrogenases was coupled with the bioelectrochemical TCA cycle reaction to achieve the 12-electron oxidation of lactate to CO2. The charge passed in the bioelectrocatalytic oxidation of 5nmol of lactate was 4mC, which corresponds to 70% of the electrolysis efficiency. [Copyright &y& Elsevier]

Abstract: Highly reproducible bioelectrocatalytic endpoint assays are described. The method is based on a complete redox conversion of a substrate to a redox mediator with a corresponding redox enzyme and an amperometric detection of the reduced mediator on a diffusionally independent microelectrode array. The current reaches a steady state within a few seconds and is proportional to the number of the integrated microelectrodes. The method has successfully been applied to histamine detection at micro-molar level and glucose detection at milli-molar level. [Copyright &y& Elsevier]

Abstract: Histamine dehydrogenase from Nocardioides simplex (HmDH) belongs to the family of soluble iron–sulfur flavoproteins having one [4Fe–4S] cluster and one 6-S-cysteinyl flavin mononucleotide per monomer. Direct electrochemistry of HmDH was studied using several carbon particle-modified glassy carbon electrodes (GCEs) and indium tin oxide (ITO) electrodes. HmDH gave a clear catalytic wave of the histamine oxidation without any mediator at a GCE modified with Ketjen Black (KB) with an average particle diameter of 39.5nm, although redox signal of the cofactors itself was not clearly recognized. Experimental data supported the irreversible adsorption of HmDH on the KB particles and the importance of the size of the carbon particle as well as the surface area as factors determining the current density of the direct electron transfer (DET)-type bioelectrocatalysis. On the other hand, HmDH was adsorbed on ITO electrodes as a monolayer, as evidenced by quartz crystal microbalance measurement, and showed a clear redox wave ascribed to the [4Fe–4S] cluster. However, some mediators were required to observe catalytic wave of histamine oxidation at ITO electrodes, indicating that the interaction between the two redox cofactors seems to be disrupted in the monomer HmDH adsorbed on the planar and hydrophilic surfaces of ITO electrodes. Surface properties of electrodes favorable for DET-type bioelectrocatalysis of HmDH are discussed. [Copyright &y& Elsevier]

Abstract: Intermolecular interactions between water-soluble pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) and an Os complex-containing redox polymer coated on a glassy carbon electrode surface were investigated, especially by focusing on their electrostatic interaction, in which the electrostatic interaction was changed by varying the ionic strength in the solution. The redox polymer showed a strong repulsive interaction between the positively charged Os complexes, especially at low ionic strengths. The polymer worked as a good electron-shuttling mediator between PQQ-GDH and the electrode to produce a steady-state catalytic current ascribed to the d-glucose oxidation. The catalytic current drastically increased with an increase in the ionic strength, presumably due to the reduction of the repulsive electrostatic interaction between the active site of the enzyme and the Os complex in the polymer and between the Os complexes. The electrostatic interaction governs the electrostatic interaction between the redox site of PQQ-GDH and the Os complexes, and the dynamics of the Os complexes on the electrode surface. [Copyright &y& Elsevier]

Combinations of bilirubin oxidase and metal complexes: [W(CN)8]3−/4−, [Os(CN)6]3−/4− and [Mo(CN)8]3−/4− (the formal potentials, E0′(M), being 0.320, 0.448, and 0.584 V vs. Ag|AgCl, respectively, at pH 7.0), allowed bioelectrocatalytic reduction of O2 to water at their formal potentials near neutral pH. The O2 reduction current appeared even at the standard potential of the O2/H2O redox couple, E0′(O2/H2O), when [Mo(CN)8]3−/4− was used at pH 7.4, though the magnitude was small. The magnitude of the bioelectrocatalytic current systematically decreased with the decrease in the potential difference between E0′(O2/H2O) and E0′(M). A limiting current as large as 17 mA/cm2 of a projected electrode surface area was obtained at 0.25 V (−0.37 V vs. E0′(O2/H2O)) for the O2 reduction at pH 7.0 with a carbon felt electrode modified with electrostatically entrapped bilirubin oxidase and [W(CN)8]3−/4− at the electrode rotation rate of 4000 rpm. [Copyright &y& Elsevier]

• Membrane-bound aldehyde dehydrogenase (AlDH) was examined as a bioelectrocatalyst. • AlDH enabled the direct electron transfer (DET)-type acetaldehyde oxidation. • The bioelectrocatalysis of AlDH was kinetically and thermodynamically analyzed. • Cyanide (CN−) inhibition proved the involvement of hemes c in DET by AlDH. • CN−-coordinated AlDH catalyzed the DET-type acetate reduction under acidic conditions. 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. [ABSTRACT FROM AUTHOR]

Bioelectrocatalysis has become one of the most important research fields in electrochemistry and provided a firm base for the application of important technology in various bioelectrochemical devices, such as biosensors, biofuel cells, and biosupercapacitors. The understanding and technology of bioelectrocatalysis have greatly improved with the introduction of nanostructured electrode materials and protein-engineering methods over the last few decades. Recently, the electroenzymatic production of renewable energy resources and useful organic compounds (bioelectrosynthesis) has attracted worldwide attention. In this review, we summarize recent progress in the applications of enzymatic bioelectrocatalysis. [ABSTRACT FROM AUTHOR]

Abstract: The charge accumulation due to peroxidase (POD)-catalyzed reduction of H2O2 in a test solution (4μL) by Os(II) in a POD/PVI[Os(dmebpy)2Cl]-immobilized layer on an electrode (PVI=poly(1-vinylimidazole), dmebpy=4,4′-dimethyl-2,2′-bipyridine) was monitored potentiometrically for the detection of H2O2. Before potentiometry, the Os(II)/Os(III) ratio of the modified electrode was controlled by pre-electrolysis at a given potential in a separated electrolysis cell. The redox potential of the Os polymer film in the test solution shifted to the positive side on the addition of H2O2 and reached a constant value due to the accumulation of Os(III) in the film. The total amount of the accumulated charge was determined from the area of the portion corresponding to the redox potential shift on a reversible cyclic voltammogram recorded separately. The low detection limit (5pmol H2O2) was realized with 82–90% of the recovery percentage. [Copyright &y& Elsevier]

• The issue of "true or myth" of DET-type bioelectrocatalysis of flavoproteins was examined. • DET-bioelectrocatalysis of FAD-dependent GDH was observed at porous Au electrodes. • Enzymatically implanted Pt nanoclusters drastically enhanced DET-catalytic signals. • The catalytic waves were analyzed on a model of dispersed Pt nanocluster model. • All flavoproteins may do DET-type bioelectrocatalysis at tuned electrodes. The direct electron transfer (DET)-type bioelectrocatalysis of flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (GDH) from Aspergillus terreus (At GDH) 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 At GDH 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 At GDH-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 At GDH (=FAD) and the conductive material, and improved the heterogeneous electron transfer kinetics between them. [ABSTRACT FROM AUTHOR]