Direct Electron Transfer Activity Research Articles

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.

Direct Electron Transfer Reaction of Cytochrome c Immobilized on a Bare ITO Electrode

Abstract To measure the direct electron transfer (DET) reaction of cytochrome c (Cytc) immobilized on a bare ITO electrode after removing the adsorbed molecules, automated solution exchange (ASE) processes were performed to induce their desorption. By fitting the absorbance decay curve observed at the Soret band peak position of Cytc at around 408 nm during the ASE processes with a double exponential equation, the final immobilized fraction was estimated to be 58.6% of the Cytc adsorbed on bare ITO electrodes under the experimental conditions. Cyclic voltammograms (CVs) of Cytc adsorbed on the bare ITO electrodes were measured for 60 min to elucidate the DET activity of immobilized Cytc. After repeated CV measurements, approximately 90% of immobilized Cytc was found to remain from the evaluation based on the coulombic amount of reduction and oxidation peaks. The scan rate dependent peak separation data from the immobilized Cytc between reduction and oxidation peaks in CVs produced 2.7 times larger DET reaction rate constant than that previously reported for the Cytc adsorbed on the bare ITO electrode.

Direct Electron Transfer of Hemoglobin on Indium-Tin Oxide Electrodes Synthesized By Dip-Coating Methods Under Different Sintering Temperature

The direct electron transfer (DET) between electrodes and redox proteins is important when considering practical applications such as body implantable devices for cardiac resynchronization therapy (CRT) and deep brain stimulation (DBS), since such electrical devices should require biocompatibility, simplicity of electrodes, potential difference in electrochemical cells, etc. We have investigated the DET properties of hemoglobin (Hb) molecules on indium-tin-oxide (ITO) electrodes [1-3]. In our previous study, the ITO electrodes were synthesized on glass plates by using dip-coating solution with different Sn/(Sn+In) molar ratio, and Hb molecules showed the DET activity on the synthesized ITO electrodes [3]. It was found that the DET activities of Hb molecules were different at ITO electrodes synthesized from different Sn ratio. The largest DET current was observed on the ITO electrode synthesized from a Sn ratio of 0.50. The results also suggested from previous studies with different Sn ratio that the active sites and electrical properties of ITO might be related with the DET behavior with Hb molecules. To understand the relationship between crystallinity of ITO and DET of Hb molecules on electrode surfaces, in this study, ITO electrodes were prepared on quartz plates under different sintering temperature from 350°C to 950°C for 15 min by using dip-coating solution with the Sn ratio of 0.50. The coating procedure was repeated ten times. X-ray diffraction (XRD) peaks attributed to Sn-doped In2O3 were observed, with the peak intensity increasing with increasing sintering temperature. Cyclic voltammetry (CV) was conducted with a three-electrode system in 39 mM phosphate buffered saline (PBS; pH 7.4) with or without 100 μM Hb solution. ITO sintered at 350°C and 950°C behaved as poor electrodes due to their high electrical resistance. Hb molecules showed good DET activities on the ITO electrodes sintered at 500°C, 650°C, and 800°C. The results indicated that there were optimum crystallinity of the ITO electrodes for showing DET activity to Hb molecules.

Function of C-terminal hydrophobic region in fructose dehydrogenase

Fructose dehydrogenase (FDH) catalyzes oxidation of d-fructose into 2-keto-d-fructose and is one of the enzymes allowing a direct electron transfer (DET)-type bioelectrocatalysis. FDH is a heterotrimeric membrane-bound enzyme (subunit I, II, and III) and subunit II has a C terminal hydrophobic region (CHR), which was expected to play a role in anchoring to membranes from the amino acid sequence. We have constructed a mutated FDH lacking of CHR (ΔchrFDH). Contrary to the expected function of CHR, ΔchrFDH is expressed in the membrane fraction, and subunit I/III subcomplex (ΔcFDH) is also expressed in a similar activity level but in the soluble fraction. In addition, the enzyme activity of the purified ΔchrFDH is about one twentieth of the native FDH. These results indicate that CHR is concerned with the binding between subunit I(/III) and subunit II and then with the enzyme activity. ΔchrFDH has clear DET activity that is larger than that expected from the solution activity, and the characteristics of the catalytic wave of ΔchrFDH are very similar to those of FDH. The deletion of CHR seems to increase the amounts of the enzyme with the proper orientation for the DET reaction at electrode surfaces. Gel filtration chromatography coupled with urea treatment shows that the binding in ΔchrFDH is stronger than that in FDH. It can be considered that the rigid binding between subunit I(/III) and II without CHR results in a conformation different from the native one, which leads to the decrease in the enzyme activity in solution.

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.

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.

The Expression and Electroochemical Characterization of Fructose Dehydrogenase

Redox enzymes and proteins are related to the many vital processes, such as tricarboxylic cycle and respiratory chain. There are only a limited number of redox enzymes and proteins that enable to couple enzyme reaction and electrode reaction without any mediators. In other words, they can transfer the electron to (or from) electrodes directly. This reaction is called direct electron transfer (DET)-type bioelectrocatalysis reaction. DET reaction has received considerable attention for construction of electrochemical applications, such as biosensors and biofuel cells. There must be several factors governing the DET reaction, but the details have not yet clearly elucidated.D-Fructose dehydrogenase (FDH; EC 1.1.99.11) from Gluconobacter japonicus NCBR 3260 is a heterotrimeric membrane-bound enzyme. Subunits I and II have covalently bound flavin adenine dinucleotide (FAD) and three heme C moieties, respectively. FDH shows strict substrate specificity to D-fructose and is used in diagnosis and food analysis. FDH is one of the DET-type enzymes. FDH has high DET activity. DET reaction of FDH occurs at a large variety of electrodes. The FAD in subunit I is the catalytic site to accept electrons form the substrate, and the electrons are transferred to the electrode through the heme C in subunit II.For the first step to explore the mechanisms of the DET reaction of FDH, we sequenced the genes encoding each subunit of the FDH complex from G. japonicus NBRC3260 and constructed an expression system to highly produce FDH in a G. oxydans strain. We also constructed derivatives modified in subunit I/III subcomplex (ΔcFDH) lacking of the heme C subunit. The recombinant FDH reacts with electrode directly. However, ΔcFDH catalyzes the oxidation of D-fructose with several artificial electron acceptors, but loses the DET ability. The formal potentials (E˚') of the three heme C moieties of FDH have been determined to be –10 ± 4, 60 ± 8 and 150 ± 4 mV (vs. Ag|AgCl|sat. KCl) at pH 5.0, while the onset potential of FDH-catalyzed DET-type bioelectrocatalytic wave is –100 mV. Judging from these results, we conclude that FDH communicates electrochemically with electrodes via the heme C.

The pressure dependence of the lattice parameters of MnSb and MnTe

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.