Cytochrome b562 fusion to formaldehyde dehydrogenase enables increased direct electron transfer

Effect of cysteine mutations on direct electron transfer of horseradish peroxidase on gold

The mechanism by which voltage-gated sodium channels are trafficked to the surface of neurons is not well understood. Our previous work implicated the cytoplasmic N terminus of the sodium channel Na(v)1.6 in this process. We report that the N terminus plus the first transmembrane segment (residues 1-153) is sufficient to direct a reporter to the cell surface. To identify proteins that interact with the 117-residue N-terminal domain, we carried out a yeast two-hybrid screen of a mouse brain cDNA library. Three clones containing overlapping portions of the light chain of microtubule-associated protein Map1b (Mtap1b) were recovered from the screen. Interaction between endogenous Na(v)1.6 channels and Map1b in mouse brain was confirmed by co-immunoprecipitation. Map1b did not interact with the N terminus of the related channel Na(v)1.1. Alanine-scanning mutagenesis of the Na(v)1.6 N terminus demonstrated that residues 77-80 (VAVP) contribute to interaction with Map1b. Co-expression of Na(v)1.6 with Map1b in neuronal cell line ND7/23 resulted in a 50% increase in current density, demonstrating a functional role for this interaction. Mutation of the Map1b binding site of Na(v)1.6 prevented generation of sodium current in transfected cells. The data indicate that Map1b facilitates trafficking of Na(v)1.6 to the neuronal cell surface.

The microbe electric: conversion of organic matter to electricity

Self-assembled molecular monolayers (SAMs) have long been recognized as crucial “bridges” between redox enzymes and solid electrode surfaces, on which the enzymes undergo direct electron transfer (DET)—for example, in enzymatic biofuel cells (EBFCs) and biosensors. SAMs possess a wide range of terminal groups that enable productive enzyme adsorption and fine-tuning in favorable orientations on the electrode. The tunneling distance and SAM chain length, and the contacting terminal SAM groups, are the most significant controlling factors in DET-type bioelectrocatalysis. In particular, SAM-modified nanostructured electrode materials have recently been extensively explored to improve the catalytic activity and stability of redox proteins immobilized on electrochemical surfaces. In this report, we present an overview of recent investigations of electrochemical enzyme DET processes on SAMs with a focus on single-crystal and nanoporous gold electrodes. Specifically, we consider the preparation and characterization methods of SAMs, as well as SAM applications in promoting interfacial electrochemical electron transfer of redox proteins and enzymes. The strategic selection of SAMs to accord with the properties of the core redox protein/enzymes is also highlighted.

In vivo, a variety of enzymatic cascade reactions are working to efficiently and rapidly metabolize biomolecules. Oxidoreductases especially play key roles in biochemical redox reactions such as fermentation, respiration, and photosynthesis, which might lead to efficient conversion of eco-friendly materials and production of useful compounds. Some enzymatic redox reactions can be artificially coupled with electrode reactions, which is called bioelectrocatalysis. Particularly, an electrical communication without any external electron mediators is called direct electron transfer (DET)-type bioelectrocatalysis, which can be an analytical system evaluating kinetic and thermodynamic characteristics of enzymes, and are expected to be applied to biomimetic devices such as biofuel cells, bioreactors, and biosensors.We focused on two DET-type enzymes: alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) from acetic acid bacteria (Gluconobacter oxydans). Both enzymes form heterotrimeric structures composed of the catalytic large subunit, the chaperonic small subunit, and the membrane-bound cytochrome c subunit. They physiologically play key roles in the respiration, and the electrons are transferred from substrates to ubiquinone in the periplasmic space. Although DET activities of ADH and ALDH have been already reported, their three-dimensional (3D) structures remain unknown, which prevents the elucidation of detailed electron transfer pathways and improved DET-type bioelectrocatalysis of ADH and ALDH. In this study, we attempted to establish the bienzymatic cascade for bioelectrochemical 4-electron oxidation of ethanol into acetate via acetaldehyde, using ADH and ALDH.To optimize the surface structure of electrodes for the two enzymes by considering their structural characteristics, we firstly elucidated the 3D structures of ADH and ALDH using cryo-electron microscopy analysis. The 3D structures were reconstructed with a 2.5 Å resolution for ADH and a 2.7 Å resolution for ALDH, respectively. Pyrroloquinoline quinone and four hemes c were resolved in ADH, while a molybdenum cofactor, two iron-sulfur clusters, and three hemes c were resolved in ALDH. Cyclic voltammograms were then recorded at multi-walled carbon nanotube-modified glassy carbon electrodes (CNT/GCs) functionalized with five pyrene derivatives and the enzyme (ADH or ALDH). Regardless of pyrene modification, all prepared electrodes performed clear DET-type ethanol oxidation by ADH and acetaldehyde oxidation by ALDH, respectively, and the 1-pyrenecarboxylic acid (PyCOOH)-functionalized CNT/GC showed the best catalytic properties for both ADH and ALDH.Based on the electrochemical results, PyCOOH-functionalized CNT/GC was selected as an electrode platform for bienzymatic electrochemical cascade. The electrodes were functionalized with a mixture of ADH and ALDH. The molar concentrations of ADH and ALDH (c 1 and c 2, respectively) were variously controlled under the condition that c 1 + c 2 was constant. The apparent DET-type current density for ethanol oxidation reached a maximum around log (c 1 / c 2) = 0.5, which was higher than that at c 1 / c 2 = ∞ (only ADH). Such increase in the DET-type current density indicates that acetaldehyde produced by ADH was subsequently oxidized by ALDH, which means a bienzymatic cascade. A mathematical model for a bienzymatic DET-type cascade reaction was constructed using the experimental results. These calculations indicated that the ratio of the amounts and catalytic constants of the two enzymes was a key factor controlling the performance of the bienzymatic cascade. In addition, the nanostructure of the enzyme-electrode interface which seems to affect diffusion of the intermediate product was also important for improving cascade efficiency.An ethanol/air biofuel cell was finally constructed with a bienzyme (ADH and ALDH)-functionalized bioanode and a bilirubin oxidase (BOD)-functionalized biocathode. The open-circuit voltage, the maximum current density, and the maximum power density were 0.75 ± 0.02 V, 2.69 ± 0.09 mA cm–2, and 0.48 ± 0.01 mW cm–2, respectively. Comparing these characteristics with those of a biofuel cell without ALDH, the performances were improved by ALDH co-adsorption. The ethanol/air biofuel cell worked much better than any other ethanol biofuel cells reported to date. The Faraday efficiency for acetate production of the ethanol/air biofuel cell reached 100 ± 4%, which indicates that the bienzymatic DET-type cascade using ADH and ALDH performed highly efficient 4-electron oxidation of ethanol into acetate. A conceptual diagram of the system is shown in the attached figure.These works will enhance the utilization of biomass fuels and lead to a low-carbon society. These discussions also suggest that the design of the nanostructured interface between the catalysts and electrodes are important for efficient turnover of the intermediate product in the multi-catalytic cascade system. Figure 1

Pulse plating of Pd-Ni alloys: Dependence on current density

This study aimed to address the stability limitations of third-generation biosensors using enzymes from mesophilic organisms, by engineering a stable direct electron transfer (DET)-type dehydrogenase capable of transferring electrons extracted from the substrate to the electrode. A fusion protein combining the mediated electron transfer (MET)-type aldose sugar dehydrogenase from the hyperthermophile Pyrobaculum aerophilum (PaeASD), which cannot transfer electrons generated by enzymatic reactions to the electrode without a mediator, and the natural electron transfer protein cytochrome b562 (cyt b562) was developed to investigate its potential for the DET reaction. A recombinant protein expression system was established in Escherichia coli to produce the PaeASD-cyt b562 fusion protein, which was purified from the soluble fraction of the host cells. Intramolecular electron transfer from pyrroloquinoline quinone (PQQ) to the heme group within the PaeASD-cyt b562 fusion protein was investigated using UV–Vis absorption spectroscopy. Upon the addition of glucose, an increase in absorption corresponding to reduced heme molecules was observed, indicating electron transfer from glucose to the heme group in the cyt b562 component via PQQ in the PaeASD component. The DET capability of the fusion protein was evaluated using cyclic voltammetry with screen-printed carbon electrodes. A glucose concentration-dependent increase in current response confirmed DET activity. Notably, the fusion protein retained over 80% of its initial current response even after 2 months of storage at 4 °C. The novel robust PaeASD-cyt b562 fusion protein demonstrated efficient DET capability, highlighting its high potential for application in the development of third-generation biosensors.

The ability to control direct electron transfer can facilitate the development of new molecular electronics, light-harvesting materials, and photocatalysis. However, control of direct electron transfer has been rarely reported, and the molecular conformation-electron dynamics relationships remain unclear. We describe direct electron transfer at buried interfaces between an organic polymer semiconductor film and a gold substrate by observing the first dynamical electric field-induced vibrational sum frequency generation (VSFG). In transient electric field-induced VSFG measurements on this system, we observe dynamical responses (<150 fs) that depend on photon energy and polarization, demonstrating that electrons are directly transferred from the Fermi level of gold to the lowest unoccupied molecular orbital of organic semiconductor. Transient spectra further reveal that, although the interfaces are prepared without deliberate alignment control, a subensemble of surface molecules can adopt conformations for direct electron transfer. Density functional theory calculations support the experimental results and ascribe the observed electron transfer to a flat-lying polymer configuration in which electronic orbitals are found to be delocalized across the interface. The present observation of direct electron transfer at complex interfaces and the insights gained into the relationship between molecular conformations and electron dynamics will have implications for implementing novel direct electron transfer in energy materials.

Reactive electrochemical ceramic membrane for effective removal of high concentration humic acid: Insights of different performance and mechanisms

Large deformation analysis of diffusion-induced buckling of nanowires in lithium-ion batteries

Redox enzyme catalysis coupled with electrode reaction is called bioelectrocatalysis and has become a key technology applicable to bioelectrochemical devices such as biosensors, biofuel cells and bioreactors. Since electron transfer between the enzyme and the electrode is the most important phenomenon, discussion of it has dominated the study of bioelectrocatalysis. Direct electron transfer (DET) between oxidoreductases and electrodes is important not only for understanding the fundamental properties of redox proteins, but also for developing mediator-free bioelectronic devices. Among enzymes with different coenzymes, pyrroloquinoline quinone (PQQ)-dependent dehydrogenases are promising biocatalysts for both biosensors and biofuel cells. However, only a limited number of studies have reported DET between PQQ in the enzyme and the electrode. Fungal PQQ-dependent pyranose dehydrogenase from Coprinopsis cinerea (CcPDH)is a multifactor-containing enzyme with superior DET ability [1].The enzyme has a three-domain structure, an N-terminal heme b-binding cytochrome domain, a central catalytic domain with PQQ as a cofactor, and a C-terminal cellulose-binding domain. The substrate undergoes oxidation in the PQQ domain followed by interdomain electron transfer (IET) from the reduced PQQ cofactor to heme b in the cytochrome domain. CcPDHis the attractive quinohemoprotein with a PQQ domain and a cytochrome domain, both of which are possible domains for DET. Previous work has shown that the PQQ domain can engage in direct bioelectrocatalysis without the cytochrome domain. In addition, the DET of the PQQ domain was investigated using self-assembled monolayer (SAM)-coated electrodes. Importantly, a high catalytic current density of 1.6 mA/cm2 was achieved for the oxidation of L-fucose under optimised conditions [2]. These results indicate a highly efficient DET to PQQ in the active site of the fungal PQQ-dependent dehydrogenase. On the other hand, it is unclear how DET proceeds at the electrode for the full-length enzyme. The study of DET and IET by an electron transfer protein linked via a linker to a catalytic protein, such as CcPDH, is useful for incorporating these processes into biosensors and biofuel cells based on direct bioelectrocatalysis, as well as for creating fusion proteins that enable DET capability.In the present study, we demonstrated that this process distinguishes direct bioelectrocatalysis via the cytochrome domain and direct bioelectrocatalysis from the PQQ domain by regulating the distance from the electrode to CcPDH using various alkyl chains of SAMs [3]. Catalytic currents by the full-length enzyme were obtained on SAM with chain alkyl lengths of C6 or more, whereas no catalytic currents were obtained by the isolated PQQ domain. The results indicated that direct bioelectrocatalysis occurred through the cytochrome domain of CcPDH for chain lengths of 6 or more. The heme-to-electrode distance is shorter than the PQQ-to-electrode distance when the redox center of each domain is closest to the SAM layer. The PQQ-to-electrode distance exceeds 15 Å for C6-OH-SAM (16.9 Å), but the heme-to-electrode distance is less than 15 Å even for C11-OH-SAM (14.2 Å). Thus, in full-length CcPDH, DET of the PQQ domain does not occur at distances greater than 15 Å, and direct bioelectrocatalysis proceeds through the heme in the cytochrome domain when chain lengths are 6 or greater. Although the optimum pH in the PQQ domain is pH 6.0, the optimal pH is approximately 8.5 for electron transfer of the full-length enzyme through the cytochrome domain. It has been suggested that IET is the rate-limiting step in the pH range 6.0 to 8.5 for the enzyme activity in aqueous solution. The pH dependence of the catalytic current for L-fucose oxidation was examined on the enzyme electrode with C6-OH-SAM, in which DET proceeds only from the cytochrome domain. An enzymatic turnover rate (k cat) at a limiting catalytic current was obtained using the electroactive coverage of the enzyme on the electrode. In direct bioelectrocatalysis through interdomain electron transfer of the cytochrome domain, k cat was found to be pH dependent with an optimal pH of 8.5; therefore, the rate-limiting step that governs pH dependence is likely the IET process.[1] K. Takeda, et al., Curr Opin Chem Biol, 49 (2019) 113-121, [2] K. Takeda, et al., Electrochim Acta, 359 (2020) 136982, [3] K. Takeda, et al., Electrochemistry, 92 (2024) 022011.

Achieving efficient electrochemical communication between redox enzymes and various electrode materials is one of the main challenges in bioelectrochemistry and is of great importance for developing electronic applications. Cellobiose dehydrogenase (CDH) is an extracellular flavocytochrome composed of a catalytic FAD containing dehydrogenase domain (DH(CDH)), a heme b containing cytochrome domain (CYT(CDH)), and a flexible linker region connecting the two domains. Efficient direct electron transfer (DET) of CDH from the basidiomycete Phanerochaete chrysosporium (PcCDH) covalently attached to mixed self-assembled monolayer (SAM) modified gold nanoparticle (AuNP) electrode is presented. The thiols used were as follows: 4-aminothiophenol (4-ATP), 4-mercaptobenzoic acid (4-MBA), 4-mercaptophenol (4-MP), 11-mercapto-1-undecanamine (MUNH(2)), 11-mercapto-1-undecanoic acid (MUCOOH), and 11-mercapto-1-undecanol (MUOH). A covalent linkage between PcCDH and 4-ATP or MUNH(2) in the mixed SAMs was formed using glutaraldehyde as cross-linker. The covalent immobilization and the surface coverage of PcCDH were confirmed with surface plasmon resonance (SPR). To improve current density, AuNPs were cast on the top of polycrystalline gold electrodes. For all the immobilized PcCDH modified AuNPs electrodes, cyclic voltammetry exhibited clear electrochemical responses of the CYT(CDH) with fast electron transfer (ET) rates in the absence of substrate (lactose), and the formal potential was evaluated to be +162 mV vs NHE at pH 4.50. The standard ET rate constant (k(s)) was estimated for the first time for CDH and was found to be 52.1, 59.8, 112, and 154 s(-1) for 4-ATP/4-MBA, 4-ATP/4-MP, MUNH(2)/MUCOOH, and MUNH(2)/MUOH modified electrodes, respectively. At all the mixed SAM modified AuNP electrodes, PcCDH showed DET only via the CYT(CDH). No DET communication between the DH(CDH) domain and the electrode was found. The current density for lactose oxidation was remarkably increased by introduction of the AuNPs. The 4-ATP/4-MBA modified AuNPs exhibited a current density up to 30 μA cm(-2), which is ∼70 times higher than that obtained for a 4-ATP/4-MBA modified polycrystalline gold electrode. The results provide insight into fundamental electrochemical properties of CDH covalently immobilized on gold electrodes and promote further applications of CDHs for biosensors, biofuel cells, and bioelectrocatalysis.

The direct electrochemistry of redox proteins has been achieved at a variety of electrodes, including modified gold, pyrolytic graphite and metal oxides. Careful design of electrode surfaces and electrolyte conditions are required for the attainment of rapid and reversible protein-electrode interaction. The electron transfer reactions of more complex systems, such as redox enzymes, are now being examined. The 'well-behaved' electrochemistry of redox proteins can be usefully exploited by coupling the electrode reaction to enzymes for which the redox proteins act as cofactors. In systems where direct electron transfer is very slow, small electron carriers, or mediators, may be employed to enhance the rate of electron exchange with the electrode. The organometallic compound ferrocene and its derivatives have proved particularly effective in this role. A new generation of electrochemical biosensors employs ferrocene derivatives as mediators.

Creation of a novel DET type FAD glucose dehydrogenase harboring Escherichia coli derived cytochrome b562 as an electron transfer domain

Electron transfer (ET) is the fundamental redox process of life and element cycling. The ET distance is normally as short as nanometers or micrometers in the subsurface. However, the redox gradient in the subsurface is as long as centimeters or even meters. This gap triggers an intriguing question whether directional long-distance ET from reduced to oxidized zones exists along the redox gradient. By using electron-donating capacity variation as a proxy of ET, we show that ET can last over 10 cm along the redox gradient in sediment columns, through a directional long-distance ET chain from reduced to oxidized zones constituted by a series of short-distance electron hopping reactions. Microbial and chemical processes synergistically mediate the long-distance ET chain, with an estimated flux of 6.73 μmol e−/cm2 per day. This directional long-distance ET represents an overlooked but important “remote” source of electrons for local biogeochemical and environmental processes.