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Flavin adenine dinucleotide-dependent d-fructose dehydrogenase (FDH) from Gluconobacter japonicus NBRC3260, a membrane-bound heterotrimeric flavohemoprotein capable of direct electron transfer (DET)-type bioelectrocatalysis, was investigated from the perspective of structural biology, bioelectrochemistry, and protein engineering. DET-type reactions offer several benefits in biomimetics (e.g., biofuel cells, bioreactors, and biosensors) owing to their mediator-less configuration. FDH provides an intense DET-type catalytic signal; therefore, extensive research has been conducted on the fundamental principles and applications of biosensors. Structural analysis using cryo-electron microscopy and single-particle analysis has revealed the entire FDH structures with resolutions of 2.5 and 2.7 Å for the reduced and oxidized forms, respectively. The electron transfer (ET) pathway during the catalytic oxidation of d-fructose was investigated by using both thermodynamic and kinetic approaches. Structural analysis has shown the localization of the electrostatic surface charges around heme 2c in subunit II, and experiments using functionalized electrodes with a controlled surface charge support the notion that heme 2c is the electrode-active site. Furthermore, two aromatic amino acid residues (Trp427 and Phe489) were located in a possible long-range ET pathway between heme 2c and the electrode. Two variants (W427A and F489A) were obtained by site-directed mutagenesis, and their effects on DET-type activity were elucidated. The results have shown that Trp427 plays an essential role in accelerating long-range ET and triples the standard rate constant of heterogeneous ET according to bioelectrochemical analysis.

Formate de­hydrogenases (FDHs) catalyze the two-electron oxidation of formate to carbon dioxide. FDHs can be divided into several groups depending on their subunit composition and active-site metal ions. Metal-dependent (Mo- or W-containing) FDHs from prokaryotic organisms belong to the superfamily of molybdenum enzymes and are members of the di­methyl­sulfoxide reductase family. In this short review, recent progress in the structural analysis of FDHs together with their potential biotechnological applications are summarized.

The effect of mutation on the redox potentials (E degrees') of the heme moieties in the variants of D-fructose dehydrogenase (FDH) was investigated by mediated spectroelectrochemical titrations. The replacement of the axial ligand of heme from methionine to glutamine changes the E degrees' value more negatively than that of the corresponding heme moiety in the recombinant (native) FDH (rFDH). The determined E degrees' values of non-targeted heme moieties in the variants were also shifted in a negative direction from that in rFDH. Thus, enzyme modification changes E degrees' of the heme moieties in unmodified protein regions. (C) The Author(s) 2021. Published by ECSJ.

Aizome (Japanese indigo dyeing) is a unique dyeing method using microbial activity under anaerobic alkaline conditions. In indigo-dye fermenting suspensions; microorganisms reduce indigo into leuco-indigo with acetaldehyde as a reductant. In this study; we constructed a semi-microbial biofuel cell using an indigo-dye fermenting suspension. Carbon fiber and Pt mesh were used as the anode and cathode materials, respectively. The open-circuit voltage (OCV) was 0.6 V, and the maximum output power was 32 µW cm−2 (320 mW m−2). In addition, the continuous stability was evaluated under given conditions starting with the highest power density; the power density rapidly decreased in 0.5 h due to the degradation of the anode. Conversely, at the OCV, the anode potential exhibited high stability for two days. However, the OCV decreased by approximately 80 mV after 2 d compared with the initial value, which was attributed to the performance degradation of the gas-diffusion-cathode system caused by the evaporation of the dispersion solution. This is the first study to construct a semi-microbial biofuel cell using an indigo-dye fermenting suspension.