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Abstract We constructed a bioelectrochemical formate generating system without external power supply. CO 2 was reduced with tungsten-containing formate dehydrogenase adsorbed on a biocathode on which benzyl viologen was adsorbed as a mediator, while the electron for the CO 2 reduction was supplied by the oxidation of H 2 with [NiFe]‑hydrogenase adsorbed on a bioanode functionalized with p -phenylenediamine (PDA2+) to adsorb H 2 ase in orientations suitable for direct communication with the electrode. Gas-diffusion-system was employed for high-speed supply of both of the gaseous substrates. The two electrodes were short-circuited through an ammeter and a switch. In this system, the formate generation rate of the biocathode was 290 ± 20 pmol cm−2 s−1 at pH 6.5 and 25 °C. On the other hand, the formate oxidation by H+ as the reversed reaction was also confirmed in the cell. Graphical abstract Unlabelled Image Highlights • A formate generating system from CO 2 and H 2 without external power supply was constructed. • Formate dehydrogenase and hydrogenase were used for CO 2 reduction and H 2 oxidation. • The rate of the formate generation reached 290 ± 20 pmol cm−2 s−1. • The reversed reaction (H 2 production from formate and H+) was also realized in this system. • The system was discussed in view of thermodynamics of bioelectrochemistry. [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) 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]

We show a great possibility of mediated enzymatic bioelectrocatalysis in the formate oxidation and the carbon dioxide (CO 2 ) reduction at high current densities and low overpotentials. Tungsten-containing formate dehydrogenase (FoDH1) from Methylobacterium extorquens AM1 was used as a catalyst and immobilized on a Ketjen Black-modified electrode. For the formate oxidation, a high limiting current density ( j lim ) of ca. 24 mA cm − 2 was realized with a half wave potential ( E 1/2 ) of only 0.12 V more positive than the formal potential of the formate/CO 2 couple ( E °′ CO2 ) at 30 °C in the presence of methyl viologen (MV 2 + ) as a mediator, and j lim reached ca. 145 mA cm − 2 at 60 °C. Even when a viologen-functionalized polymer was co-immobilized with FoDH1 on the porous electrode, j lim of ca. 30 mA cm − 2 was attained at 60 °C with E 1/2 = E °′ CO2 + 0.13 V. On the other hand, the CO 2 reduction was also realized with j lim ≈ 15 mA cm − 2 and E 1/2 = E °′ CO2 − 0.04 V at pH 6.6 and 60 °C in the presence of MV 2 + . [ABSTRACT FROM AUTHOR]

Effects of the electrode potential on a direct electron transfer (DET)-type bioelectrocatalysis of fructose dehydrogenase (FDH) at Au electrodes were investigated. Adsorbed FDH showed the highest DET activity at an adsorption potential ( E ad ) around the point of zero charge ( E pzc ). Since FDH stock solution contains 2-mercaptoethanol (ME) for stabilization, ME is partially bound to the Au electrode. However, the DET activity drastically decreased at E ad >> E pzc . Au oxide layer is formed at the positive potentials to hinder the interfacial electron transfer. In contrast, only slight decrease in the DET activity was observed at sufficiently negative E ad (<< E pzc ), where ME is reductively desorbed from the Au electrode, but co-exists in the solution. In contrast, when FDH and ME were adsorbed on Au electrodes at an open circuit potential and the FDH- and ME-adsorbed Au electrode was held at such a negative hold potential ( E ho ) in the buffer without ME, the DET activity drastically decreased. An addition of ME in the test solution prevented the decrease in the DET activity at the negative E ho . These results indicate that ME close to adsorbed FDH plays a significant role in the stabilization of FDH adsorbed on Au electrodes. [ABSTRACT FROM AUTHOR]

Abstract d -Fructose dehydrogenase (FDH), a membrane-bound heterotrimeric enzyme, shows strong activity in direct electron transfer (DET)-type bioelectrocatalysis. An FDH variant (Δ1 c 2 c FDH) 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 Δ1 c 2 c FDH-adsorbed Au electrodes. Detailed analysis of the steady-state catalytic current indicated that Δ1 c 2 c FDH transports the electrons to the electrode via heme 3 c at a more negative potential and at more improved kinetics than the recombinant (native) FDH. Graphical abstract Unlabelled Image Highlights • An FDH variant which lacks heme 1 c and heme 2 c moieties (Δ1 c 2 c FDH) was constructed. • Δ1 c 2 c FDH showed high activity of the direct electron transfer-type bioelectrocatalysis. • Δ1 c 2 c FDH transferred the electrons at potentials more negative than the native FDH. • The energy loss in the DET-type bioelectrocatalysis of FDH has decreased. • The interfacial electron transfer kinetics has been improved. [ABSTRACT FROM AUTHOR]

d -Fructose dehydrogenase (FDH), a heterotrimeric membrane-bound enzyme, exhibits strong activity in direct electron transfer- (DET-) type bioelectrocatalysis. We constructed a variant (Δ1 c FDH) that lacks 143 amino acid residues involving one heme c moiety (called heme 1 c ) on the N-terminus of subunit II, and characterized the bioelectrocatalytic properties of Δ1 c FDH using cyclic voltammetry. A clear DET-type catalytic oxidation wave of d -fructose was observed at the Δ1 c FDH-adsorbed Au electrodes. The result clearly indicates that the electrons accepted at the flavin adenine dinucleotide catalytic center in subunit I are transferred to electrodes via two of the three heme c moieties in subunit II without going through heme 1 c . In addition, the limiting current density of Δ1 c FDH was one and a half times larger than that of the native FDH in DET-type bioelectrocatalysis. The downsizing protein engineering causes an increase in the surface concentration of the electrochemically effective enzymes and an improvement in the heterogeneous electron transfer kinetics. [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]

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]

d -Fructose dehydrogenase (FDH), a flavoprotein-cytochrome c complex, exhibits high activity in direct electron transfer (DET)-type bioelectrocatalysis. One of the three types of heme c in FDH is the electron-donating site to the electrodes, and another heme c is presumed to not be involved in the catalytic cycle. In order to confirm the electron transfer pathway, we constructed three mutants in which the sixth axial methionine ligand (M301, M450, or M578) of one of the hemes was replaced with glutamine, which was selected with the expectation that it would shift the formal potential of the hemes in the negative direction. An M450Q mutant successfully reduced the overpotential by approximately 0.2 V, giving a limiting current close to that of the native FDH. In contrast, an M301Q mutant remained almost unchanged and an M578Q mutant drastically decreased DET-type catalytic activity. The results indicate that the electron transfer in the native FDH occurs in sequence from the flavin, through the heme c with M578, to the heme c with M450 (as the electron-donating site to the electrodes), without going through the heme c with M301. The M450Q mutant will be useful for biofuel cells because of the decreased overpotential. [ABSTRACT FROM AUTHOR]

Fructose dehydrogenase (FDH) catalyzes oxidation of d -fructose into 2-keto- d -fructose and is one of the enzymes allowing a direct electron transfer (DET)-type bioelectrocatalysis. FDH is a heterotrimeric membrane-bound enzyme (subunit I, II, and III) and subunit II has a C terminal hydrophobic region (CHR), which was expected to play a role in anchoring to membranes from the amino acid sequence. We have constructed a mutated FDH lacking of CHR (ΔchrFDH). Contrary to the expected function of CHR, ΔchrFDH is expressed in the membrane fraction, and subunit I/III subcomplex (ΔcFDH) is also expressed in a similar activity level but in the soluble fraction. In addition, the enzyme activity of the purified ΔchrFDH is about one twentieth of the native FDH. These results indicate that CHR is concerned with the binding between subunit I(/III) and subunit II and then with the enzyme activity. ΔchrFDH has clear DET activity that is larger than that expected from the solution activity, and the characteristics of the catalytic wave of ΔchrFDH are very similar to those of FDH. The deletion of CHR seems to increase the amounts of the enzyme with the proper orientation for the DET reaction at electrode surfaces. Gel filtration chromatography coupled with urea treatment shows that the binding in ΔchrFDH is stronger than that in FDH. It can be considered that the rigid binding between subunit I(/III) and II without CHR results in a conformation different from the native one, which leads to the decrease in the enzyme activity in solution. [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]