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.

Influence of distal glycan mimics on direct electron transfer performance for bilirubin oxidase bioelectrocatalysts.

Bilirubin oxidase (BOD) is a bioelectrocatalyst that reduces dioxygen (O2) 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.

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

Tungsten-containing formate dehydrogenase from Methylorubrum extroquens AM1 (FoDH1)-a promising biocatalyst for the interconversion of carbon dioxide/formate and nicotine adenine dinucleotide (NAD+)/NADH redox couples-was investigated using structural biology and bioelectrochemistry. FoDH1 is reported to be an enzyme that can realize "direct electron transfer (DET)-type bioelectrocatalysis." However, its 3-D structure, electrode-active sites, and electron transfer (ET) pathways remain unclear. The ET pathways were investigated using 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.

Quantification of leuco-indigo in indigo-dye-fermenting suspension by normal pulse voltammetry.

Quantification of leuco-indigo is most important for Aizome, Japanese indigo-dyeing; however, there has been no convenient quantitative method. This study demonstrated that normal pulse voltammetry under quiescent conditions can be used to detect leuco-indigo. As a result of quantification of leuco-indigo in the depth direction in fermenting suspensions, the steady-state concentrations of leuco-indigo showed sigmoidal profiles in the depth direction. The steady state is caused by competitive reactions of microbial reduction of indigo and autoxidation of leuco-indigo by O2 dissolved from the air interface of the suspension. In addition, we investigated the effects of stirring the suspension and adding some nutrients to the concentration profile. The weakened activity was partially recovered by the addition of ethanol and remarkably recovered by the addition of hipolypepton or glucose. Knowledge is essential for the proper management of indigo-dye-fermenting suspensions.

Effects of N-linked glycans of bilirubin oxidase on direct electron transfer-type bioelectrocatalysis.

Bilirubin oxidase from Myrothecium verrucaria (mBOD) is a promising enzyme for catalyzing the four-electron reduction of dioxygen into water and realizes direct electron transfer (DET)-type bioelectrocatalysis. It has two N-linked glycans (N-glycans), and N472 and N482 are known as binding sites. Both binding sites located on opposite side of the type I (T1) Cu, which is the electrode-active site of BOD. We investigated the effect of N-glycans on DET-type bioelectrocatalysis by performing electrochemical measurements using electrodes with controlled surface charges. Two types of BODs with different N-glycans, mBOD and recombinant BOD overexpressed in Pichia pastoris (pBOD), and their deglycosylated forms (dg-mBOD and dg-pBOD) were used in this study. Kinetic analysis of the steady-state catalytic waves revealed that both size and composition of N-glycans affected the orientation of adsorbed BODs on the electrodes. Interestingly, the most favorable orientation was achieved with pBOD, which has the largest N-glycans. Furthermore, the effect of the orientation control by the N-glycans is cooperative with electrostatic interaction.

Isolation and characterization of indigo-reducing bacteria and analysis of microbiota from indigo fermentation suspensions.

In natural indigo dyeing, the water-insoluble indigo included in the composted indigo leaves called sukumo is converted to water-soluble leuco-indigo through the reduction activities of microorganisms under alkaline conditions. To understand the relationship between indigo reduction and microorganisms in indigo-fermentation suspensions, we isolated and identified the microorganisms that reduce indigo and analyzed the microbiota in indigo-fermentation suspensions. Indigo-reducing microorganisms, which were not isolated by means of a conventional indigo carmine-reduction assay method, were isolated by using indigo as a direct substrate and further identified and characterized. We succeeded in isolating bacteria closely related to Corynebacterium glutamicum, Chryseomicrobium aureum, and Enterococcus sp. for the first time. Anthraquinone was found to be an effective mediator that facilitated the indigo-reduction activity of the isolated strains. On analysis of the microbiota in indigo-fermentation suspensions, the ratio of indigo-reducing bacteria and others was found to be important for maintaining the indigo-reduction activity.

Fundamental insight into redox enzyme-based bioelectrocatalysis.

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.

Cyanide sensitivity in direct electron transfer-type bioelectrocatalysis by membrane-bound alcohol dehydrogenase from Gluconobacter oxydans.

An overexpression system of membrane-bound alcohol dehydrogenase (ADH) from Gluconobacter oxydans was constructed to examine its bioelectrocatalytic characteristics. The effects of cyanide (CN-) addition on the kinetics of direct electron transfer (DET)-type bioelectrocatalysis by ADH were analyzed. CN- enhanced the bioelectrocatalytic activity, while the catalytic activity in the solution remained unchanged, even in the presence of CN-. Electrochemical methods and electron spin resonance spectroscopy showed the detailed electron transfer pathway in the DET-type bioelectrocatalysis by ADH. Briefly, ADH is suggested to communicate with an electrode via a CN--insensitive and H+-sensitive heme c in DET. These characteristics of ADH with respect to CN- suggest the involvement of ADH in CN--insensitive respiration in G. oxydans.

Fluoride Ion-Selective Electrode for Organic Solutions.

Fluoride ions are used in battery electrolytes in fluoride shuttle batteries. Since organic solvents are used in battery electrolytes, there is a growing demand to develop appropriate methods for quantifying fluoride ion concentration in organic solvents. In this study, a fluoride ion-selective electrode (ISE) for organic solutions is proposed as an electrode of the second kind. A Ag|AgF electrode was made via the anodization of a silver wire in propylene carbonate (PC) containing dissolved fluoride ions. The resultant electrode exhibits a stable linear response of the open circuit potential to the logarithm of the fluoride ion concentration in PC solutions over a range of 10-4-10-2 mol dm-3. The lower and upper limits of the linear response were interpreted in terms of the solubility and the formation of a silver fluoride complex. The use of this electrode of the second kind is suitable for the analysis of fluoride ions in organic solutions and is a promising concept for the development of ISEs for the detection of ions in organic solutions under highly restrictive conditions.

Voltammetric in-situ monitoring of leuco-indigo in indigo-fermenting suspensions.

Cyclic voltammetry was successfully applied to in-vivo monitoring of leuco-indigo in indigo-fermenting suspensions under quiescent conditions without deoxygenation; the working and counter electrodes were kept on the surface of each suspension by a polyethylene vinyl alcohol tube holder. The anodic peak current was used as a measure of the leuco-indigo concentration. The voltammetric wave shape suggested partial solubilization of the indigo with some macromolecules in the fermenting suspensions, which lead to an in-situ method without any electrode surface pretreatment. The anodic peak current well reflected the dyeing activity of a suspensions. The results obtained for laboratory-level fermentation systems clarified the number of days required for dye fermentation, the effectiveness of addition of old suspension as an additive for preparing fresh fermenting suspensions, and the importance of addition of a nitrogen-based nutrient as well as a glucose-based one to recover the indigo-reducing activity. The method can also be applied to determine the amounts of indigo in used dye suspensions and extracts of fermented indigo leaves (sukumo) by adding a chemical reduction pretreatment.

Highly sensitive and stable fructose self-powered biosensor based on a self-charging biosupercapacitor.

Herein, we present an alternative approach to obtain a highly sensitive and stable self-powered biosensor that was used to detect D-fructose as proof of concept.In this platform, we perform a two-step process, viz. self-charging the biosupercapacitor for a constant time by using D-fructose as fuel and using the stored charge to realize the detection of D-fructose by performing several polarization curves at different D-fructose concentrations. The proposed BSC shows an instantaneous power density release of 17.6 mW cm-2 and 3.8 mW cm-2 in pulse mode and at constant load, respectively. Moreover, the power density achieved for the self-charging BSC in pulse mode or under constant load allows for an enhancement of the sensitivity of the device up to 10 times (3.82 ± 0.01 mW cm-2 mM-1, charging time = 70 min) compared to the BSC in continuous operation mode and 100 times compared to the normal enzymatic fuel cell. The platform can potentially be employed as a self-powered biosensor in food or biomedical applications.

Pollution Control of Nitrate-selective Membrane by the Inner Solution and On-site Monitoring of Nitrate Concentration in Soil.

A liquid-membrane type nitrate-selective electrode was improved to lower the influence of contaminants by modifying its inner electrode system from Ag | AgCl | Cl- to Ag | Ag+. The NO3--selective electrode displayed a linear response to the concentration of NO3- with a Nernstian slope of -53 ± 1 mV decade-1, in the concentration region between 10-5 and 2 mol dm-3 (M). The NO3- detection limit was about 10-5 M. The electrochemical response of this electrode was stable for more than 30 days. The deterioration in responding characteristics due to the coexistence of Cl- was suppressed by use of the Ag | Ag+ redox couple in the absence of Cl- inside the NO3--selective electrode.

Electrical cell-to-cell communication using aggregates of model cells.

Cell-to-cell communication via a local current caused by ion transport is elucidated using a model-cell system. To imitate tissues such as smooth muscles and cardiac muscles, liquid-membrane cells mimicking the function of K+ and Na+ channels were made. Connecting these channel-mimicking cells (K+ channel and voltage-gated Na+ channel) in parallel, model cells imitating living cell functions were constructed. Action-potential propagation within the cell aggregate model constructed by multiple model cells was investigated. When an action potential was generated at one cell, the cell behaved as an electric power source. Since a circulating current flowed around the cell, it flowed through neighboring model cells. Influx and efflux currents caused negative and positive shifts of the membrane potential, respectively, on the surface of neighboring model cells. The action potential was generated at the depolarized domain when the membrane potential exceeded the threshold of the voltage-gated Na+ channels. Thus, the action potential spread all over the cell system. When an external electric stimulus was applied to the layered cell-aggregate model system, propagation of the action potential was facilitated as if they were synchronized.

Development Perspective of Bioelectrocatalysis-Based Biosensors.

Bioelectrocatalysis provides the intrinsic catalytic functions of redox enzymes to nonspecific electrode reactions and is the most important and basic concept for electrochemical biosensors. This review starts by describing fundamental characteristics of bioelectrocatalytic reactions in mediated and direct electron transfer types from a theoretical viewpoint and summarizes amperometric biosensors based on multi-enzymatic cascades and for multianalyte detection. The review also introduces prospective aspects of two new concepts of biosensors: mass-transfer-controlled (pseudo)steady-state amperometry at microelectrodes with enhanced enzymatic activity without calibration curves and potentiometric coulometry at enzyme/mediator-immobilized biosensors for absolute determination.

Automatic Management of Nutrient Solution for Hydroponics-Construction of Multi-ion Stat.

In order to improve plant factories, an appropriate control system on fertilization is urgently required. An automatic management system to control nutrient concentration was constructed using a programmable logic controller (PLC) and ion selective electrodes (ISEs) of nitrate, phosphate, and potassium ion. The concentration of nutrient components in a culture solution was monitored using these ISEs. When the concentration of the nutrient components diminished to the threshold set as an optimum condition (0.1 - 2.0 mM), an appropriate amount of a concentrated solution of each nutrient component was added to the culture solution using solenoid valves connected with the PLC. The present cultivation system was simply constructed without any computers and pumps. Three kinds of automatic control systems simultaneously worked and did not influence each other.

Direct electron transfer-type bioelectrocatalysis of FAD-dependent glucose dehydrogenase using porous gold electrodes and enzymatically implanted platinum nanoclusters.

The direct electron transfer (DET)-type bioelectrocatalysis of flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (GDH) from Aspergillus terreus (AtGDH) 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 AtGDH 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 AtGDH-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 AtGDH (=FAD) and the conductive material, and improved the heterogeneous electron transfer kinetics between them.

Fabrication of a Phosphate Ion Selective Electrode Based on Modified Molybdenum Metal.

A phosphate ion-selective electrode using molybdenum metal was constructed. The modified molybdenum electrode responded to HPO42- in the presence of molybdenum dioxide and molybdophosphate (PMo12O403-) on the surface. The electrode exhibited a linear response to HPO42- in the concentration range between 1.0 × 10-5 and 1.0 × 10-1 M (mol dm-3) in the pH range from 8.0 to 9.5 with a detection limit of 1.0 × 10-6 M. The sensor showed near Nernstian characteristics (27.8 ± 0.5 mV dec-1) at pH 9.0. Since the responding potential was attributed to the activity of HPO42-, the potential at a given concentration of phosphate depended on the pH. The electrode indicated a good selectivity with respect to other common anions such as NO3-, SO42-, Cl-, HCO3- and CH3COO-. The modified molybdenum electrode can be continuously used for over a 1 month with good reproducibility. The feasibility of the electrochemical sensor was proved by successful for the detection of phosphate in real samples.

The influence of the shape of Au nanoparticles on the catalytic current of fructose dehydrogenase.

Graphite electrodes were modified with triangular (AuNTrs) or spherical (AuNPs) nanoparticles and further modified with fructose dehydrogenase (FDH). The present study reports the effect of the shape of these nanoparticles (NPs) on the catalytic current of immobilized FDH pointing out the different contributions on the mass transfer-limited and kinetically limited currents. The influence of the shape of the NPs on the mass transfer-limited and the kinetically limited current has been proved by using two different methods: a rotating disk electrode (RDE) and an electrode mounted in a wall jet flow-through electrochemical cell attached to a flow system. The advantages of using the wall jet flow system compared with the RDE system for kinetic investigations are as follows: no need to account for substrate consumption, especially in the case of desorption of enzyme, and studies of product-inhibited enzymes. The comparison reveals that virtually identical results can be obtained using either of the two techniques. The heterogeneous electron transfer (ET) rate constants (kS) were found to be 3.8 ± 0.3 s-1 and 0.9 ± 0.1 s-1, for triangular and spherical NPs, respectively. The improvement observed for the electrode modified with AuNTrs suggests a more effective enzyme-NP interaction, which can allocate a higher number of enzyme molecules on the electrode surface. Graphical abstract The shape of gold nanoparticles has a crucial effect on the catalytic current related to the oxidation of D-(-)-fructose to 5-keto-D-(-)-fructose occurring at the FDH-modified electrode surface. In particular, AuNTrs have a higher effect compared with the spherical one.

Bioelectrocatalytic performance of d-fructose dehydrogenase.

This review summarizes the bioelectrocatalytic properties of d-fructose dehydrogenase (FDH), while taking into consideration its enzymatic characteristics. FDH is a membrane-bound flavohemo-protein with a molecular mass of 138 kDa, and it catalyzes the oxidation of d-fructose to 5-keto-d-fructose. The characteristic feature of FDH is its strong direct-electron-transfer (DET)-type bioelectrocatalytic activity. The pathway of the DET-type reaction is discussed. An overview of the application of FDH-based bioelectrocatalysis to biosensors and biofuel cells is also presented, and the benefits and problems associated with it are extensively discussed.

The origin of hyperpolarization based on the directional conduction of action potential using a model nerve cell system.

In nerve cells, changes in local membrane potentials are generated and propagated along a nerve axon mainly by the function of K+ and Na+ channels. Generally, concurrent monitoring of multi-points on an axon is performed based on the voltage-clamp method. As the respective membrane potentials have been evaluated by considering the relations between the applied potential, the local current, and conductance, experimental values are not directly evaluated. We directly measured the actual membrane potentials and local currents of the respective cells using a nerve-model system comprising liquid-membrane cells. It was then proven that the action potential spreads along the axon toward the axon terminal due to the function of both the channel-type receptors in the synapse and voltage-gated Na+ channels on the axon, and that hyperpolarization cannot be caused by only the operation of the delayed-K+ and the voltage-gated Na+ channels.