The Redox Potential Measurements for Heme Moieties in Variants of d-Fructose Dehydrogenase Based on Mediator-assisted Potentiometric Titration

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

Improvement in the Power Output of a Reverse Electrodialysis System by the Addition of Poly(sodium 4-styrenesulfonate)

Salinity gradient energy generated by the contact between seawater and river water is one of the promising renewable energies. In the reverse electrodialysis (RED), salinity gradient energy is directly translated into the electricity. The representative problem is a large electrical resistance of river water or dilute solutions. The dilute solutions are poor electrically conductive. This results in a huge energy loss when an electrical current passes through it. In this study, sodium chloride (NaCl) or poly(sodium 4-styrenesulfonate) (NaPSS) was added to the dilute solutions to increase the conductivities and enhance the power outputs of the RED cells. When NaCl was added, the power output reached 11.4 ± 0.6 µW. On the other hand, when NaPSS was added, the power output increased up to 19.6 ± 0.6 µW

Indigo-Mediated Semi-Microbial Biofuel Cell Using an Indigo-Dye Fermenting Suspension

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

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

電極を基質認識できる酵素の反応メカニズムを解明 --次世代バイオセンシングにつながる基盤技術--. 京都大学プレスリリース. 2023-10-16.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

Structure and function relationship of formate dehydrogenases: an overview of recent progress

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

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

International audienceCopper 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

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

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 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 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 based on bioelectrochemical analysis

Experimental and Theoretical Insights into Bienzymatic Cascade for Mediatorless Bioelectrochemical Ethanol Oxidation with Alcohol and Aldehyde Dehydrogenases

The efficient utilization of biomass fuels is a critical component of a sustainable energy economy. Via respiration, acetic acid bacteria can oxidize biomass ethanol into acetic acid using membrane-bound alcohol and aldehyde dehydrogenases (ADH and AlDH, respectively). Focusing on the ability of these enzymes to interact directly and electrically with electrode materials, we constructed a mediatorless bioanode for ethanol oxidation based on a direct electron transfer (DET)-type bienzymatic cascade by ADH and AlDH. The three-dimensional structural data of ADH and AlDH elucidated by cryo-electron microscopy were valuable for effectively designing electrode platforms with multi-walled carbon nanotubes (MWCNTs) and pyrene derivatives. DET-type bioelectrocatalysis by ADH and AlDH was improved by using 1-pyrene carboxylic acid-functionalized MWCNT. The catalytic current densities for bienzymatic ethanol oxidation were recorded at the bioanodes modified by various ADH/AlDH ratios. The reaction model was constructed by focusing on the competitive ad-sorption of two enzymes on the electrode surface and the collection efficiency of the intermediately produced acetaldehyde. The power output of an ethanol/air biofuel cell using the bienzymatic bioanode reached 0.48 ± 0.01 mW cm–2, which is the highest value reported for ethanol biofuel cells. In addition, the Faraday efficiency of acetate production by the cell reached 100 ± 4%. This study will lead to efficient conversion of biomass fuels based on a multi-catalytic cascade system

Structural and Bioelectrochemical Elucidation of Direct Electron Transfer-type Membrane-bound Fructose Dehydrogenase

Flavin adenine dinucleotide (FAD)-dependent fructose dehydrogenase (FDH) from Gluconobacter japonicus NBRC3260, a membrane-bound flavohemoprotein capable of direct electron transfer (DET)-type bioelectrocatalysis, was investigated from the viewpoints of structural biology and bioelectrochemistry. As FDH provides a strong DET-type catalytic signal, extensive research has been conducted. Structural analysis using cryo-electron microscopy (cryo-EM) and single-particle analysis revealed the entire FDH structure. The electron transfer (ET) pathway during the catalytic oxidation of D-fructose was investigated using thermodynamic and kinetic approaches in bioelectrochemistry, as well as structural information. Key amino acid residues that play important roles in substrate specificity and ET acceleration have also been proposed

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

Tungsten-containing formate dehydrogenase from Methylorubrum extroquens AM1 (FoDH1) was investigated from structural biology and bioelectrochemistry. The electron transfer (ET) pathways were investigated by 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