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• Electrochemical property of FDH lacking a cytochrome subunit (ΔC FDH) was studied. • DET of ΔC FDH was observed at porous Au electrodes modified with thiols. • Electrode-active site of ΔC FDH was estimated by kinetic analysis of DET signals. • Uncharged aromatic thiols on porous Au electrodes enhance DET of ΔC FDH. • An enhancement mechanism for DET by aromatic thiols was proposed. A heterotrimeric membrane-bound d -fructose dehydrogenase (FDH) from Gluconobacter japonicus , composed of subunits L, C, and S, shows strong direct electron transfer (DET)-type bioelectrocatalytic performance in the two-electron oxidation of d -fructose. The electrode-active site of the enzyme is the heme c moiety in the subunit C. A previous study reported the construction of the subunit L/S subcomplex (ΔC FDH) lacking the subunit C. In this work, we attempted to realize the DET-type reaction of ΔC FDH using porous gold electrodes functionalized with several thiols, and investigated the influence of the thiols on DET. Kinetic analysis of the steady-state catalytic waves revealed that the electrode-active site of ΔC FDH is the [3Fe-4S] iron-sulfur cluster in the L subunit. The experimental results demonstrated that uncharged aromatic thiols on the electrode enhance the DET-type reaction of ΔC FDH. Based on the pH-dependent profile of the DET-type activity and the surface conditions of the modified thiols evaluated by electrochemical reductive desorption, we suggest an enhancement mechanism for DET by aromatic thiols. [ABSTRACT FROM AUTHOR]

Bilayer lipid membranes (BLM) are widely employed to study the electrochemical behavior across cell membranes, particularly the ion transport in the presence of hydrophobic ions or ionophores. However, owing to the physical fragility of BLMs, they are readily broken down by external shocks, such as the applications of a potential difference, current, and vibration. Thus, in this study, a facile method for enhancing the physical stability of BLMs was developed using a track-etched membrane (TM). Physically stable BLMs were rapidly fabricated within the apertures of TM by dropping an n -decane solution containing lecithin and cholesterol on TM. Therefore, BLM formation was confirmed by capacitance measurement, and the estimated membrane thickness and mean capacitance were 3.5 ± 0.4 nm and 0.51 ± 0.05 μF cm−2, respectively. The applied membrane potential could be extended to ≥1.5 V, which is approximately 10 times higher than those of conventional BLMs, and the lifetime exceeded 40 min. The applicability of the fabricated BLM was verified by electrochemical measurements during ion transport. [Display omitted] [ABSTRACT FROM AUTHOR]

• Homologous expression system was established in M. extorquence AM1. • Recombinant FoDH1 and the β subunit variant (FoDH1B) were obtained. • Their bioelectrochemical properties and the electron transfer pathways have been elucidated. • Coupling of an efficient DET-type NAD+/NADH regenerating system with NAD-dependent enzymes was demonstrated. Nicotinamide adenine dinucleotide redox couple (NAD+/NADH) is a significant coenzyme in the catabolism of organisms. Coupling NAD-dependent enzymatic reaction and NAD+/NADH regenerating systems can be utilized for biomimetics-associated applications, such as the production of bioreactors, biofuel cells, and biosensors. Direct electron transfer (DET)-type bioelectrocatalysis, in which an enzyme directly exchanges electrons with the electrode, can produce an efficient, simple, and biocompatible system owing to a mediator-free configuration. Formate dehydrogenase 1 from Methylorubrum extorquens AM1 (FoDH1) has been reported to have diaphorase and DET-type activity. The β subunit (diaphorase subunit) of FoDH1 is an attractive variant for constructing an efficient regenerating system due to downsizing effects and efficient interfacial electron transfer. In this study, we attempt to construct a homologous expression system to establish a recombinant FoDH1 (rFoDH1) expression system and to obtain the β subunit variant (FoDH1B). Their bioelectrochemical properties and the ET pathways of DET-type reactions have been elucidated. Finally, we demonstrated the coupling of an efficient DET-type NAD+/NADH regenerating system with NAD-dependent enzymes. [Display omitted] [ABSTRACT FROM AUTHOR]

• Critical amino acid residues of FDH in catalysis were predicted. • Four FDH variants were evaluated using electrochemistry and structural biology. • Biocatalytic mechanisms of FDH have been elucidated. • Structures of in-silico variants were validated by comparing them to cryo-EM structures. • Relative activity of variants with D-tagatose was improved over that of rFDH. D-Fructose dehydrogenase (FDH) from Gluconobacter japonicus NBRC3260, a membrane-bound heterotrimeric flavohemoprotein capable of intense direct electron transfer (DET)-type bioelectrocatalysis, was investigated using protein engineering, electrochemistry, structural biology, and bioinformatics. DET-type reactions have led to an increase in energy efficiency, biocompatibility, and design freedom in bioelectrochemical devices, such as biofuel cells, biosensors, biosupercapacitors, and bioreactors. However, one of the greatest challenges for this reaction is the limited number of the applicable enzymes. The creation of variants with altered substrate specificities using template enzymes is a promising solution to address this problem. For creating variants using FDH as a template, it is important to understand the biocatalytic mechanisms. Recently, the structure of FDH has been analyzed using cryo-electron microscopy (cryo-EM) single particle analysis, and the three amino acid residues (N1146, H1147, and N1190) may be critical for substrate recognition by FDH. In this study, we aimed to understand the mechanisms of the biocatalytic reaction of FDH and discuss the roles of these residues. In addition, we validated the structures of the in-silico variants by comparing them to the cryo-EM structures. Furthermore, for the variants of N1146 involved in D-fructose recognition, we have verified the changes in specificity for other substrates. [ABSTRACT FROM AUTHOR]

[Display omitted] • Effects of Cu2+ on bioelectrocatalysis of copper efflux oxidase were investigated. • Cu2+ electrochemically induced reductive inactivation of multicopper oxidases. • Kinetic and thermodynamic parameters were refined using electrochemical data. • Uncompetitive inhibition appeared reasonable for the Cu2+ effect on the enzyme. • A detailed model for Cu2+-dependent inactivation/reactivation was proposed. 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 O 2 by CueO at NH 2 -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. [ABSTRACT FROM AUTHOR]