Direct Electron Transfer-type Bioelectrocatalysis Research Articles

Construction of a protein-engineered variant of d-fructose dehydrogenase for direct electron transfer-type bioelectrocatalysis

d-Fructose dehydrogenase (FDH), a heterotrimeric membrane-bound enzyme, exhibits strong activity in direct electron transfer- (DET-) type bioelectrocatalysis. We constructed a variant (Δ1cFDH) that lacks 143 amino acid residues involving one heme c moiety (called heme 1c) on the N-terminus of subunit II, and characterized the bioelectrocatalytic properties of Δ1cFDH using cyclic voltammetry. A clear DET-type catalytic oxidation wave of d-fructose was observed at the Δ1cFDH-adsorbed Au electrodes. The result clearly indicates that the electrons accepted at the flavin adenine dinucleotide catalytic center in subunit I are transferred to electrodes via two of the three heme c moieties in subunit II without going through heme 1c. In addition, the limiting current density of Δ1cFDH was one and a half times larger than that of the native FDH in DET-type bioelectrocatalysis. The downsizing protein engineering causes an increase in the surface concentration of the electrochemically effective enzymes and an improvement in the heterogeneous electron transfer kinetics.

Effects of Mesoporous Structures on Direct Electron Transfer-Type Bioelectrocatalysis: Facts and Simulation on a Three-Dimensional Model of Random Orientation of Enzymes

Effects of Mesoporous Structures on Direct Electron Transfer-Type Bioelectrocatalysis: Facts and Simulation on a Three-Dimensional Model of Random Orientation of Enzymes

Analysis of factors governing direct electron transfer-type bioelectrocatalysis of bilirubin oxidase at modified electrodes

Direct electron transfer (DET)-type bioelectrocatalysis is an essential technique for constructing simple bioelectrochemical devices such as biosensors, bioreactors, and biofuel cells. Bilirubin oxidase (BOD), a biocatalyst for the four-electron reduction of dioxygen (O2) into water, is a promising enzyme for powerful DET-type biocathodes. Mesoporous carbon materials are often used in BOD-modified biocathodes. However, the supply of O2 becomes a key factor governing BOD-catalyzed current densities owing to its low solubility. In this study, we analyzed steady-state rotating disk voltammograms of a BOD-catalyzed reaction in a rigid manner by taking into account the mass-transport as well as the enzymatic and the interfacial electron transfer kinetics. Non-catalytic redox signals of adsorbed BOD were also analyzed to obtain the surface redox properties of BOD. The analysis revealed that modification of electrodes with bilirubin and/or negatively charged carbon nanotubes to improve the DET-type catalytic performance increased the amount of BOD molecules with the proper orientation for bioelectrocatalysis. The interfacial electron transfer kinetic characteristics remained almost unchanged.

Mutation of heme c axial ligands in d-fructose dehydrogenase for investigation of electron transfer pathways and reduction of overpotential in direct electron transfer-type bioelectrocatalysis

d-Fructose dehydrogenase (FDH), a flavoprotein-cytochrome c complex, exhibits high activity in direct electron transfer (DET)-type bioelectrocatalysis. One of the three types of heme c in FDH is the electron-donating site to the electrodes, and another heme c is presumed to not be involved in the catalytic cycle. In order to confirm the electron transfer pathway, we constructed three mutants in which the sixth axial methionine ligand (M301, M450, or M578) of one of the hemes was replaced with glutamine, which was selected with the expectation that it would shift the formal potential of the hemes in the negative direction. An M450Q mutant successfully reduced the overpotential by approximately 0.2V, giving a limiting current close to that of the native FDH. In contrast, an M301Q mutant remained almost unchanged and an M578Q mutant drastically decreased DET-type catalytic activity. The results indicate that the electron transfer in the native FDH occurs in sequence from the flavin, through the heme c with M578, to the heme c with M450 (as the electron-donating site to the electrodes), without going through the heme c with M301. The M450Q mutant will be useful for biofuel cells because of the decreased overpotential.

Enhanced direct electron transfer-type bioelectrocatalysis of bilirubin oxidase on negatively charged aromatic compound-modified carbon electrode

Effects of chemical modification of mesoporous Ketjen Black (KB) electrodes on direct electron transfer (DET)-type bioelectrocatalytic reduction of dioxygen by bilirubin oxidase (BOD) were investigated under air-saturated neutral conditions. Several amines were electrochemically oxidized at KB-modified electrode to generate nitrogen–carbon bond. The modification with negatively charged aromatic amines such as 4-aminobenzoic acid (4-ABA) drastically increased the catalytic current density compared with that by positively charged and non-charged aromatic compounds and negatively charged non-aromatic compound. Considering the basic amino acid residues around the type I site of BOD, it can be concluded that weakly negative charge on electrode surface induces a favorable orientation of BOD for the DET-type catalysis via the electrostatic interaction, while the π–π interaction is also essential for effective orientation of BOD on the electrode surface. The 4-ABA-modification leads to an increase in the heterogeneous electron transfer rate constant and a decrease in the randomness of the orientation as well as a slight increase in the surface concentration of BOD.

Role of 2-mercaptoethanol in direct electron transfer-type bioelectrocatalysis of fructose dehydrogenase at Au electrodes

Effects of the electrode potential on a direct electron transfer (DET)-type bioelectrocatalysis of fructose dehydrogenase (FDH) at Au electrodes were investigated. Adsorbed FDH showed the highest DET activity at an adsorption potential (Ead) around the point of zero charge (Epzc). Since FDH stock solution contains 2-mercaptoethanol (ME) for stabilization, ME is partially bound to the Au electrode. However, the DET activity drastically decreased at Ead>>Epzc. Au oxide layer is formed at the positive potentials to hinder the interfacial electron transfer. In contrast, only slight decrease in the DET activity was observed at sufficiently negative Ead (<<Epzc), where ME is reductively desorbed from the Au electrode, but co-exists in the solution. In contrast, when FDH and ME were adsorbed on Au electrodes at an open circuit potential and the FDH- and ME-adsorbed Au electrode was held at such a negative hold potential (Eho) in the buffer without ME, the DET activity drastically decreased. An addition of ME in the test solution prevented the decrease in the DET activity at the negative Eho. These results indicate that ME close to adsorbed FDH plays a significant role in the stabilization of FDH adsorbed on Au electrodes.

Role of a non-ionic surfactant in direct electron transfer-type bioelectrocatalysis by fructose dehydrogenase

ABSTRACT A heterotrimeric membrane-bound fructose dehydrogenase (FDH) from Gluconobacter japonicus NBRC3260 contains FAD in subunit I and three heme C moieties in subunit II as the redox centers, and is one of the direct electron transfer (DET)-type redox enzymes. FDH-catalyzed current density of fructose oxidation at hydrophilic mercaptoethanol (MEtOH)-modified Au electrode is much larger than that at hydrophobic mercaptoethane (MEtn)-modified Au electrode. Addition of a non-ionic surfactant Triton® X-100 (1%) completely quenches the catalytic current at the MEtn-modified Au electrode, while only small competitive effect is observed at the MEtOH-modified Au electrode. Quartz crystal microbalance measurements support the adsorption of FDH and Triton® X-100 on both of the modified electrodes. We propose a model to explain the phenomenon as follows. The surfactant forms a monolayer on the hydrophobic MEtn-modified electrode with strong hydrophobic interaction, and FDH adsorbs on the surface of the surfactant monolayer. The monolayer inhibits the electron transfer from FDH to the electrode. On the other hand, the surfactant forms a bilayer on the hydrophilic MEtOH-modified electrode. The interaction between the surfactant bilayer and the hydrophilic electrode is relatively weak so that FDH replaces the surfactant and is embedded in the bilayer to communicate electrochemically with the hydrophilic electrode.

Electrostatic interaction between an enzyme and electrodes in the electric double layer examined in a view of direct electron transfer-type bioelectrocatalysis

Effects of the electrode poential on the activity of an adsorbed enzyme has been examined by using copper efflux oxidase (CueO) as a model enzyme and by monitoring direct electron transfer (DET)-type bioelectrocatalysis of oxygen reduction. CueO adsorbed on bare Au electrodes at around the point of zero charge (Epzc) shows the highest DET activity, and the activity decreases as the adsorption potential (Ead; at which the enzyme adsorbs) is far from Epzc. We propose a model to explain the phenomena in which the electrostatic interaction between the enzyme and electrodes in the electric double layer affects the orientation and the stability of the adsorbed enzyme. The self-assembled monolayer of butanethiol on Au electrodes decreases the electric field in the outside of the inner Helmholtz plane and drastically diminishes the Ead dependence of the DET activity of CueO. When CueO is adsorbed on bare Au electrodes under open circuit potential and then is held at hold potentials (Eho) more positive than Epzc, the DET activity of the CueO rapidly decreases with the hold time. The strong electric field with positive surface charge density on the metallic electrode (σM) leads to fatal denaturation of the adsorbed CueO. Such denaturation effect is not so serious at Eho<<Epzc, but the electric field with negative σM induces an orientation inconvenient for the DET reaction during the adsorption process. A positively charged neomycin shows a promoter ability to CueO adsorbed at Ead<<Epzc. The phenomenon is also explained on the proposed model.

Heterologous Overexpression and Characterization of a Flavoprotein-Cytochrome c Complex Fructose Dehydrogenase of Gluconobacter japonicus NBRC3260

A heterotrimeric flavoprotein-cytochrome c complex fructose dehydrogenase (FDH) of Gluconobacter japonicus NBRC3260 catalyzes the oxidation of d-fructose to produce 5-keto-d-fructose and is used for diagnosis and basic research purposes as a direct electron transfer-type bioelectrocatalysis. The fdhSCL genes encoding the FDH complex of G. japonicus NBRC3260 were isolated by a PCR-based gene amplification method with degenerate primers designed from the amino-terminal amino acid sequence of the large subunit and sequenced. Three open reading frames for fdhSCL encoding the small, cytochrome c, and large subunits, respectively, were found and were presumably in a polycistronic transcriptional unit. Heterologous overexpression of fdhSCL was conducted using a broad-host-range plasmid vector, pBBR1MCS-4, carrying a DNA fragment containing the putative promoter region of the membrane-bound alcohol dehydrogenase gene of Gluconobacter oxydans and a G. oxydans strain as the expression host. We also constructed derivatives modified in the translational initiation codon to ATG from TTG, designated (TTG)FDH and (ATG)FDH. Membranes of the cells producing recombinant (TTG)FDH and (ATG)FDH showed approximately 20 times and 100 times higher specific activity than those of G. japonicus NBRC3260, respectively. The cells producing only FdhS and FdhL had no fructose-oxidizing activity, but showed significantly high d-fructose:ferricyanide oxidoreductase activity in the soluble fraction of cell extracts, whereas the cells producing the FDH complex showed activity in the membrane fraction. It is reasonable to conclude that the cytochrome c subunit is responsible not only for membrane anchoring but also for ubiquinone reduction.

Fructose/dioxygen biofuel cell based on direct electron transfer-type bioelectrocatalysis

One-compartment biofuel cells without separators have been constructed, in which d-fructose dehydrogenase (FDH) from Gluconobacter sp. and laccase from Trametes sp. (TsLAC) work as catalysts of direct electron transfer (DET)-type bioelectrocatalysis in the two-electron oxidation of d-fructose and four-electron reduction of dioxygen as fuels, respectively. FDH adsorbs strongly and stably on Ketjen black (KB) particles that have been modified on carbon papers (CP) and produces the catalytic current with the maximum density of about 4 mA cm(-2) without mediators at pH 5. The catalytic wave of the d-fructose oxidation is controlled by the enzyme kinetics. The location and the shape of the catalytic waves suggest strongly that the electron is directly transferred to the KB particles from the heme c site in FDH, of which the formal potential has been determined to be 39 mV vs. Ag|AgCl|sat. KCl. Electrochemistry of three kinds of multi-copper oxidases has also been investigated and TsLAC has been selected as the best one of the DET-type bioelectrocatalyst for the four-electron reduction of dioxygen in view of the thermodynamics and kinetics at pH 5. In the DET-type bioelectrocatalysis, the electron from electrodes seems to be transferred to the type I copper site of multi-copper oxidases. TsLAC adsorbed on carbon aerogel (CG) particles with an average pore size of 22 nm, that have been modified on CP electrodes, produces the catalytic reduction current of dioxygen with a density of about 4 mA cm(-2), which is governed by the mass transfer of the dissolved dioxygen. The FDH-adsorbed KB-modified CP electrodes and the TsLAC-adsorbed CG-modified CP electrodes have been combined to construct one-compartment biofuel cells without separators. The open-circuit voltage was 790 mV. The maximum current density of 2.8 mA cm(-2) and the maximum power density of 850 microW cm(-2) have been achieved at 410 mV of the cell voltage under stirring.