Structure-guided electrostatic tuning of subunit interfaces to enhance direct electron transfer and stability in d-fructose dehydrogenase

Oxidoreductases have been utilized as bioelectrocatalysts to realize a variety of biotechnologies, such as biosensors, biofuel cells, solar fuel production, carbon dioxide capture and utilization, and cofactor-regeneration systems. These systems are based on bioelectrocatalysis which couples electrode and enzymatic reactions. Several metalloenzymes can communicate electronically with suitable electrodes without redox mediators. This phenomenon has been termed “direct electron transfer (DET)-type bioelectrocatalysis”. Owing to the mediator-less configuration, the reaction can offer the following benefits in future bioelectrochemical technologies: (i) minimized overvoltage, (ii) low cost, (iii) simple design, (iv) high degree of design freedom, and (v) nontoxic and environmentally friendly properties. However, there have been few reports of enzymes that can realize DET-type bioelectrocatalysis, thus the unique mechanism of the reaction has yet to be elucidated.We focused on a d-fructose dehydrogenase (FDH) from Gluconobacter japonicus NBRC3260, which is known as a model enzyme for DET-type reactions. FDH is a unique enzyme with intense DET-type bioelectrocatalytic activity and has been extensively investigated from electrochemistry, protein engineering, and spectroscopy perspectives. FDH is a heterotrimeric membrane-bound protein with a molecular mass of ca. 138 kDa and is comprised of subunits I (67 kDa), II (51 kDa), and III (20 kDa). Subunit I contains a covalently bound flavin adenine dinucleotide (FAD), and subunit II carries three heme c moieties from its N-terminus called hemes 1c, 2c, and 3c. The redox potentials of hemes c in FDH and several variants were investigated using bioelectrochemical and spectroscopic methods. The DET pathway of FDH was examined with site-directed mutagenesis to replace the axial ligand of heme c or to delete the heme c moiety. These studies have shown that the electron is transferred from the reduced FAD through heme 3c to heme 2c and then to the electrode; heme 1c does not seem to be involved in the reaction. However, the entire three-dimensional (3D) structure of FDH and other DET-type membrane-bound quinohemoproteins, flavohemoproteins, and metallohemoproteins, remained unknown. Therefore, a quantitative discussion of their DET-type reaction was difficult.In the present study, we clarified the 3D structure of FDH using cryo-electron microscopy and single-particle image analysis with a resolution of 2.5 Å (PDB ID: 8JEJ). This is the first study to report the entire structures of membrane-bound flavohemoproteins, quinohemoproteins, and metallohemoproteins capable of DET-type reactions. The structure has revealed the 3Fe-4S iron-sulfur cluster (3Fe4S) in subunit I. The electron transfer (ET) pathway during the catalytic oxidation of d-fructose through FAD, 3Fe4S, and hemes 3c, 2c, and 1c were examined based on Marcus’ theory. In addition, 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. We constructed variants in which each of the corresponding residues was replaced with alanine (W427A and F489A) by site-directed mutagenesis, and their effects on DET-type activity were investigated by electrochemical measurements. Kinetic analysis of steady-state catalytic waves has revealed that Trp427 plays an essential role in accelerating long-range ET and triples the standard rate constant of heterogeneous ET between enzymes and an electrode.These groundbreaking findings provide vital information for searching for critical elements in DET-type reactions and a reasonable explanation for the outstanding DET-type activity of FDH. The appropriate mutation of aromatic residues to accelerate ET between an enzyme and an electrode will be a novel way to create new DET-type enzymes and innovative biomimetics. Figure 1

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

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

Multi-walled and single-walled carbon nanotubes were synthesized on platinum plate (MWCNTs/pt) and gold wire (SWCNTs/Au) electrodes, respectively, using a chemical vapor deposition (CVD) method. These carbon nanotube-modified electrodes were immersed into solutions of glucose oxidase (GOX) and D-fructose dehydrogenase (FDH) to immobilize these enzymes onto the electrode surfaces. After GOX was immobilized onto the MWCNT/Pt electrode, the well-defined catalytic oxidation current was increased from ca. -0.45 V (vs. Ag/AgCl/saturated KCl), which was close to the redox potential of flavin adenine dinucleotide as a prosthetic group of GOX under physiological pH values. Furthermore, catalytic oxidation currents of fructose based on direct heterogeneous electron transfer reactions between FDH and the SWCNT/Pt electrode were observed.

Enhancement of direct electron transfer by aromatic thiol modification with truncated d-fructose dehydrogenase

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.

Bioelectrochemical characterization of the reconstruction of heterotrimeric fructose dehydrogenase from its subunits

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.

Ultimate downsizing of d-fructose dehydrogenase for improving the performance of direct electron transfer-type bioelectrocatalysis

Porous carbon materials, including carbon nanotubes and carbon blacks, and mesoporous carbons, carbon cryogel and carbon aerogel, have attracted attention for improving the performance of enzymatic electrode. Carbon nanotubes and carbon blacks aggregate and create mesospaces suitable for enzymatic reactions. However, much of the mesospaces is unused because the pore size is random and controlling the pore structure and pore size is difficult. In contrast, carbon cryogel or aerogel has a well-controlled mesopore size distribution with an average pore diameter ranging from 10 to 100 nm. However, the procedure of carbon gel production is time-consuming and not suitable for mass production. We focused on a new mesoporous carbon material, MgO-templated carbon (MgOC) as an alternative to carbon gels. The mesopore size can be controlled by alternating the MgO template size[1]. One of the striking advantages of MgOC over the previously reported mesoporous carbons is its simple and cost-effective production procedure. The effects of the pore structure of MgOCs on the direct electron transfer (DET) reaction of redox enzyme, D-fructose dehydrogenase, and bilirubin oxidase, were investigated[2]. We found two effects of optimizing pore structure. First, the macropores of MgOC can enhance current production. When the pore size of MgOC was much larger than the size of enzyme, the MgOC could adsorb a sufficient amount of enzyme even on the pores formed inside the MgOC particle structure, and the enzymes adsorbed inside the MgOC worked well as an electrocatalyst. In contrast, when the pore size of MgOC was comparable to the size of enzymes, the enzymes could not penetrate the MgOC particles through the inter-connecting pores, and only the enzyme adsorbed on the mesopores formed at the surface of the carbon particles produced catalytic current. Second, the mesopores which were close to the size of enzymes improve thermal stability of electrode. When enzymes molecules were embedded in the carbon mesopores with pore size comparable to that of enzymes, an enhanced contact area between enzymes and the MgOC surface led to strong adsorption of enzymes on the MgOC surface as well as to enzyme stabilization by preventing the denaturation, aggregation and collision of enzyme molecules. Furthermore, we developed the novel MgOC which have hierarchical structure composed by meso-macro pores. This material improved current density and thermal stability of enzyme modified electrodes. Reference [1] T. Morishita, T. Tsumura, M. Toyoda, J. Przepiórski, A. W. Morawski, H. Kanno, and M. Inagaki, Carbon, 48, 2690 (2010) [2] H. Funabashi, K. Murata, S. Tsujimura, Electrochemistry, 83(5), 372 (2015).

Structural and electrochemical elucidation of biocatalytic mechanisms in direct electron transfer-type D-fructose dehydrogenase

Membrane-bound heterotrimeric d-fructose dehydrogenase (FDH) from Gluconobacter japonicus exhibits distinct direct electron transfer (DET)-type bioelectrocatalytic activity. FDH contains 3 heme c moieties (heme 1c, 2c, and 3c), and the two downsized variants lacking heme 1c moiety (Δ1c FDH) or heme 1c and 2c moieties (Δ1c2c FDH) were constructed in our previous research. Recently, we elucidated the entire structure of FDH, enabling a structural perspective in mechanistic discussions. In this study, we quantitatively analyzed the downsizing effect of Δ1c- and Δ1c2c-deletion on DET-type bioelectrocatalysis. Non-catalytic redox signals of adsorbed enzymes were successfully obtained, providing a basis for independently evaluating kinetic parameters from DET-type catalytic waves. Analytical results revealed that the downsizing enhanced the electron transfer rate constant by 7-fold and 4-fold for Δ1c FDH and Δ1c2c FDH, respectively. Furthermore, the acceleration factors were discussed using structural predictions of the variants.

The present work reports the preparation of binderless carbon-coated porous films and the study of their performance as monolithic bioanodes. The films were prepared by coating anodic aluminum oxide (AAO) films with a thin layer of nitrogen-doped carbon by chemical vapor deposition. The films have cylindrical straight pores with controllable diameter and length. These monolithic films were used directly as bioelectrodes by loading the films with D-fructose dehydrogenase (FDH), an oxidoreductase enzyme that catalyzes the oxidation of D-fructose to 5-keto-D-fructose. The immobilization of the enzymes was carried out by physical adsorption in liquid phase and with an electrostatic attraction method. The latter method takes advantage of the fact that FDH is negatively charged during the catalytic oxidation of fructose. Thus the immobilization was performed under the application of a positive voltage to the CAAO film in a FDH-fructose solution in McIlvaine buffer (pH 5) at 25 oC. As a result, the FDH modified electrodes with the latter method show much better electrochemical response than that with the conventional physical adsorption method. Due to the singular porous structure of the monolithic films, which consists of an array of straight and parallel nanochannels, it is possible to rule out the effect of the diffusion of the D-fructose into the pores. Thus the improvement in the performance upon using the electrostatic attraction method can be ascribed not only to a higher uptake, but also to a more appropriate molecule orientation of the enzyme units on the surface of the electrodes.

<i>Harvesting Energy using Biocatalysts</i>

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