Electron Transfer Subunit Research Articles

Direct Electron Transfer-Type Oxidoreductases for Biomedical Applications.

Among the various types of enzyme-based biosensors, sensors utilizing enzymes capable of direct electron transfer (DET) are recognized as the most ideal. However, only a limited number of redox enzymes are capable of DET with electrodes, that is, dehydrogenases harboring a subunit or domain that functions specifically to accept electrons from the redox cofactor of the catalytic site and transfer the electrons to the external electron acceptor. Such subunits or domains act as built-in mediators for electron transfer between enzymes and electrodes; consequently, such enzymes enable direct electron transfer to electrodes and are designated as DET-type enzymes. DET-type enzymes fall into several categories, including redox cofactors of catalytic reactions, built-in mediators for DET with electrodes and by their protein hierarchic structures, DET-type oxidoreductases with oligomeric structures harboring electron transfer subunits, and monomeric DET-type oxidoreductases harboring electron transfer domains. In this review, we cover the science of DET-type oxidoreductases and their biomedical applications. First, we introduce the structural biology and current understanding of DET-type enzyme reactions. Next, we describe recent technological developments based on DET-type enzymes for biomedical applications, such as biosensors and biochemical energy harvesting for self-powered medical devices. Finally, after discussing how to further engineer and create DET-type enzymes, we address the future prospects for DET-type enzymes in biomedical engineering.

In silico simulations for prediction and modeling of protein engineering within catalytic subunit of direct electron transfer type FAD glucose dehydrogenase for increases in substrate specificity and structural stability.

The bacterial flavin adenine dinucleotide (FAD) glucose dehydrogenase (FADGDH) is composed of three subunits; 1) a catalytic subunit which belongs to the glucose-methanol-choline (GMC) oxidoreductase family contains an FAD cofactor and 3Fe-4S cluster in its redox center, 2) a hitchhiker protein subunit functioning in the bacterial TAT secretion system, and 3) a membrane-bound subunit with three heme c moieties, responsible for the transfer of electrons between the active-site cofactor and external electron acceptors. The presence of the electron transfer subunit makes this enzyme able to transfer electron directly to the electrode, thereby designates this enzyme as a direct electron transfer (DET) type GDH. The recent X-ray crystal structure elucidation of its catalytic subunit complexed with small subunit, revealed the unique substrate binding cavity of this enzyme. This enzyme was then mutated within its substrate pocket to increase substrate specificity towards glucose as a validation for later energy minimization and molecular dynamics simulations using AMBER MD. Modeling both the FAD and 3Fe-4S cluster elucidated a new methodology for modeling proteins involving covalent bonding of cofactors and namely metal ion cluster harboring proteins. From this modeling, the electron transfer pathway involving both cofactors and metal ion clusters can be further investigated in DET type enzymes. With these new modeling techniques, we can take a new approach in designing future mutational candidates by first evaluating them in silico then applying those results to our wet lab testing for higher throughput and a more targeted approach in designing and creating mutations.

Microgravity environment grown crystal structure information based engineering of direct electron transfer type glucose dehydrogenase

The heterotrimeric flavin adenine dinucleotide dependent glucose dehydrogenase is a promising enzyme for direct electron transfer (DET) principle-based glucose sensors within continuous glucose monitoring systems. We elucidate the structure of the subunit interface of this enzyme by preparing heterotrimer complex protein crystals grown under a space microgravity environment. Based on the proposed structure, we introduce inter-subunit disulfide bonds between the small and electron transfer subunits (5 pairs), as well as the catalytic and the electron transfer subunits (9 pairs). Without compromising the enzyme’s catalytic efficiency, a mutant enzyme harboring Pro205Cys in the catalytic subunit, Asp383Cys and Tyr349Cys in the electron transfer subunit, and Lys155Cys in the small subunit, is determined to be the most stable of the variants. The developed engineered enzyme demonstrate a higher catalytic activity and DET ability than the wild type. This mutant retains its full activity below 70 °C as well as after incubation at 75 °C for 15 min – much higher temperatures than the current gold standard enzyme, glucose oxidase, is capable of withstanding.

Thiosulfate oxidation in sulfur-reducing Shewanella oneidensis and its unexpected influences on the cytochrome c content.

Thiosulfate, an important form of sulfur compounds, can serve as both electron donor and acceptor in various microorganisms. In Shewanella oneidensis, a bacterium renowned for respiratory versatility, thiosulfate reduction has long been recognized but whether it can catalyse thiosulfate oxidation remains elusive. In this study, we discovered that S. oneidensis is capable of thiosulfate oxidation, a process specifically catalysed by two periplasmic cytochrome c (cyt c) proteins, TsdA and TsdB, which act as the catalytic subunit and the electron transfer subunit respectively. In the presence of oxygen, oxidation of thiosulfate has priority over reduction. Intriguingly, thiosulfate oxidation negatively regulates the cyt c content in S. oneidensis cells, largely by reducing intracellular levels of cAMP, which as the cofactor modulates activity of global regulator Crp required for transcription of many cyt c genes. This unexpected finding provides an additional dimension to interplays between the respiration regulator and the respiratory pathways in S. oneidensis. Moreover, the data presented here identified S. oneidensis as the first bacterium known to date owning both functional thiosulfate reductase and dehydrogenase, and importantly, genomics analyses suggested that the number of bacterial species possessing this feature is rather limited.

Hydroxy-bridged resting states of a [NiFe]-hydrogenase unraveled by cryogenic vibrational spectroscopy and DFT computations.

The catalytic mechanism of [NiFe]-hydrogenases is a subject of extensive research. Apart from at least four reaction intermediates of H2/H+ cycling, there are also a number of resting states, which are formed under oxidizing conditions. Although not directly involved in the catalytic cycle, the knowledge of their molecular structures and reactivity is important, because these states usually accumulate in the course of hydrogenase purification and may also play a role in vivo during hydrogenase maturation. Here, we applied low-temperature infrared (cryo-IR) and nuclear resonance vibrational spectroscopy (NRVS) to the isolated catalytic subunit (HoxC) of the heterodimeric regulatory [NiFe]-hydrogenase (RH) from Ralstonia eutropha. Cryo-IR spectroscopy revealed that the HoxC protein can be enriched in almost pure resting redox states suitable for NRVS investigation. NRVS analysis of the hydrogenase catalytic center is usually hampered by strong spectral contributions of the FeS clusters of the small, electron-transferring subunit. Therefore, our approach to investigate the FeS cluster-free, 57Fe-labeled HoxC provided an unprecedented insight into the [NiFe] site modes, revealing their contributions in a spectral range otherwise superimposed by FeS cluster-derived bands. Rationalized by density functional theory (DFT) calculations, our data provide structural descriptions of the previously uncharacterized hydroxy- and water-containing resting states. Our work highlights the relevance of cryogenic vibrational spectroscopy and DFT to elucidate the structure of barely defined redox states of the [NiFe]-hydrogenase active site.

Construction of Super-Stabilized Direct Electron Transfer Type Glucose Dehydrogenase for Long Term Continuous Glucose Sensing System

Introduction: The development of super-stabilized flavin adenine dinucleotide glucose dehydrogenase (FADGDH) was achieved based on the rational biomolecular engineering.The FADGDH from Burkholderia cepacia (BcGDH) is the ideal enzyme for continuous glucose monitoring (CGM) sensor, because of its inherent DET ability (Yamazaki 2008, Lee 2018). BcGDH is composed of three subunits, which are the catalytic subunit harboring FAD and iron-sulfur (3Fe-4S) cluster, the electron transfer subunit harboring three heme c, and the small subunit which functions as a “hitchhiker” protein. We recently reported the BcGDH catalytic subunit complexed with small subunit (Yoshida et al., 2019, PDB 6a2u), thereby elucidated the intra-molecular electron transfer pathway, and inherent disulfide bond between catalytic subunit and small subunit.BcGDH shows two distinct temperature profiles due to variations in its quaternary structure (Sode 1996, Yamazaki 1999). BcGDH composed of three subunits showed an optimum reaction temperature of approximately 45 ̊C. By increasing temperature, enzyme activity gradually decreases, due to the dissociation of electron transfer subunit. However, at temperature above 60 ̊C, enzyme activity increases again with the increase the temperature and shows second peak at around 70 ̊C, which is derived from the complex composed of catalytic subunit and small subunit. This phenomenon suggested that the dissociation of the electron transfer subunit dominates the stability of BcGDH three subunit complex, which is the essential structure to show DET ability. Namely, to stabilize BcGDH enzyme complex, the stabilization of quaternary structure is the most rational strategy for this enzyme.We recently succeeded in the elucidation of crystal structure of BcGDH complex together with the electron transfer subunit. The availability of quaternary structure of this enzyme enabled us to design inter-subunit disulfide bond in BcGDH. In this study, the designing and characterization of super-stabilized BcGDH is presented. We also characterize the DET ability of the engineered BcGDH, which is the mandatory characteristic of this enzyme for the development of the future innovative biosensing systems. Methods: Amino acid substitution to cysteine residues were carried out at the residues existing on the surface of each subunit, to form inter-subunit disulfide bonds. Recombinantly prepared BcGDH mutant complexes were purified by affinity chromatography. The catalytic activity and thermal stability were investigated. Then DET-type sensors were constructed by immobilization on the gold electrode with self-assembled monolayer (SAM) layer, and investigated their stability by continuous operation. Results and Discussion: Based on the 3D structure of BcGDH complex, disulfide bonds between electron transfer subunit and either catalytic subunit or small subunit were designed. By the screening of the cystaine residue substituted mutants, some mutants showed remarkable high thermal stability as BcGDH complex without the decrease of the catalytic activity. Furthermore, multiple inter-subunit disulfide bonds made the enzyme as super-stabilized which showed high catalytic activity even by the incubation at 70 ̊C. Thus developed engineered enzyme revealed to show DET ability with electrode, indistinguishable to the wild type one. This super-stabilized enzyme will contribute the improvement of the operational stability of enzyme sensors. References Lee et al., Bioelectrochemistry. 121 (2018) 1–6Sode et al., Enzyme Microb. Technol. 19 (1996) 82–85Yamazaki et al., Appl. Biochem. Biotechnol. 77 (1999) 325–336Yamazaki et al., Anal. Lett. 41 (2008) 2363-2373.Yoshida et al., Acta Crystallogr. Sect. D Struct. Biol. 75 (2019) 841–851

A novel dimethylsulfoxide reductase family of molybdenum enzyme, Idr, is involved in iodate respiration by Pseudomonas sp. SCT.

Pseudomonas sp. strain SCT is capable of using iodate (IO3 - ) as a terminal electron acceptor for anaerobic respiration. A possible key enzyme, periplasmic iodate reductase (Idr), was visualized by active staining on non-denaturing gel electrophoresis. Liquid chromatography-tandem mass spectrometry analysis revealed that at least four proteins, designated as IdrA, IdrB, IdrP1 , and IdrP2 , were involved in Idr. IdrA and IdrB were homologues of catalytic and electron transfer subunits of respiratory arsenite oxidase (Aio); however, IdrA defined a novel clade within the dimethylsulfoxide (DMSO) reductase family. IdrP1 and IdrP2 were closely related to each other and distantly related to cytochrome c peroxidase. The idr genes (idrABP 1 P 2 ) formed an operon-like structure, and their transcription was upregulated under iodate-respiring conditions. Comparative proteomic analysis also revealed that Idr proteins and high affinity terminal oxidases (Cbb3 and Cyd), various H2 O2 scavengers, and chlorite (ClO2 - ) dismutase-like proteins were expressed specifically or abundantly under iodate-respiring conditions. These results suggest that Idr is a respiratory iodate reductase, and that both O2 and H2 O2 are formed as by-products of iodate respiration. We propose an electron transport chain model of strain SCT, in which iodate, H2 O2 , and O2 are used as terminal electron acceptors.

A membrane-bound [NiFe]-hydrogenase large subunit precursor whose C-terminal extension is not essential for cofactor incorporation but guarantees optimal maturation.

[NiFe]‐hydrogenases catalyze the reversible conversion of molecular hydrogen into protons end electrons. This reaction takes place at a NiFe(CN)2(CO) cofactor located in the large subunit of the bipartite hydrogenase module. The corresponding apo‐protein carries usually a C‐terminal extension that is cleaved off by a specific endopeptidase as soon as the cofactor insertion has been accomplished by the maturation machinery. This process triggers complex formation with the small, electron‐transferring subunit of the hydrogenase module, revealing catalytically active enzyme. The role of the C‐terminal extension in cofactor insertion, however, remains elusive. We have addressed this problem by using genetic engineering to remove the entire C‐terminal extension from the apo‐form of the large subunit of the membrane‐bound [NiFe]‐hydrogenase (MBH) from Ralstonia eutropha. Unexpectedly, the MBH holoenzyme derived from this precleaved large subunit was targeted to the cytoplasmic membrane, conferred H2‐dependent growth of the host strain, and the purified protein showed exactly the same catalytic activity as native MBH. The only difference was a reduced hydrogenase content in the cytoplasmic membrane. These results suggest that in the case of the R. eutropha MBH, the C‐terminal extension is dispensable for cofactor insertion and seems to function only as a maturation facilitator.

X-ray structure of the direct electron transfer-type FAD glucose dehydrogenase catalytic subunit complexed with a hitchhiker protein.

The bacterial flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase complex derived from Burkholderia cepacia (BcGDH) is a representative molecule of direct electron transfer-type FAD-dependent dehydrogenase complexes. In this study, the X-ray structure of BcGDHγα, the catalytic subunit (α-subunit) of BcGDH complexed with a hitchhiker protein (γ-subunit), was determined. The most prominent feature of this enzyme is the presence of the 3Fe-4S cluster, which is located at the surface of the catalytic subunit and functions in intramolecular and intermolecular electron transfer from FAD to the electron-transfer subunit. The structure of the complex revealed that these two molecules are connected through disulfide bonds and hydrophobic interactions, and that the formation of disulfide bonds is required to stabilize the catalytic subunit. The structure of the complex revealed the putative position of the electron-transfer subunit. A comparison of the structures of BcGDHγα and membrane-bound fumarate reductases suggested that the whole BcGDH complex, which also includes the membrane-bound β-subunit containing three heme c moieties, may form a similar overall structure to fumarate reductases, thus accomplishing effective electron transfer.

Direct Electron Transfer Type Faradaic Enzyme EIS

Introduction Faradaic electrochemical impedance spectroscopy (Faradaic EIS) has been studied as the principle of biosensors. Considering that Faradaic EIS is a powerful method to investigate the surface of electrodes in details with high sensitivity, variety of Faradaic EIS based biosensors, such as immunosensors or DNA sensors have been reported. These biosensors are based on measuring the charge transfer resistance (Rct) of redox probes at the surface of electrodes. The one of the inherent limitations of currently reported Faradaic EIS based biosensors is the presence of redox probes, which is necessary to monitor the binding of target molecules to ligand which alter the flux of redox probes to the electrode surface, consequently, result the change of Rct. However, the redox probes are generally chemically unstable, and the configuration of continuous monitoring and/or in vivo monitoring type biosensors using redox probe are complicated and difficult. Therefore, the developments of alternative EIS sensor platforms, which make the robust measurements, have been expected.Besides, enzyme-based electrochemical biosensors are categorized into three generations, based on the electron acceptors. In the 1st-generation, oxygen serves as the electron acceptor of the enzyme reaction of oxidase, and the decreasing oxygen concentration or liberating hydrogen peroxide is detected electrochemically. The 2nd-generation principle uses artificial electron acceptors (mediators) for the oxidases or dehydrogenases enzyme oxidative half reactions, instead of oxygen, and the reduced artificial electron acceptors are measured electrochemically. The 3rd-generation principle uses enzymes with the ability of direct electron transfer (DET). Although, Faradaic enzyme EIS sensors have been reported by employing redox mediators or oxygen as the external electron acceptors, there have been no reports on sensors employing DET-type redox enzymes based on Faradaic EIS principle.In this study, we report the construction and characterization of direct electron transfer type Faradaic Enzyme EIS, as the novel biosensor employing DET-type redox enzymes without redox probe. The DET-type Faradaic Enzyme EIS employs flavin adenine dinucleotide (FAD) dependent glucose dehydrogenase (GDH) complex (FADGDH), which is consisted of three subunits: a catalytic subunit which has FAD and 3Fe-4S-type iron-sulfur cluster, an electron transfer subunit which has 3 hemes, and a small subunit as a hitchhiker protein. Thanks to the presence of the multi heme electron transfer subunit, FADGDH is capable of DET (ref.). Method DET-type FADGDH- modified or non-DET-type FADGDH (fungi derived FADGDH)- modified gold disk electrodes (GDEs) were constructed to elucidate its characteristic properties. FADGDHs were modified onto GDEs via self-assembled monolayer (SAM). FADGDHs modified GDEs were evaluated by EIS to analyse faradaic impedance towards different concentrations of glucose, with Ag/AgCl and Pt wire as the reference and counter electrodes, in the absence of redox probe. EIS measurements were performed in certain frequency range by AC voltage with an amplitude of 10 mV superimposed on DC potential. Obtained EIS data were analysed with an equivalent circuit to obtain electrochemical parameters. Result and Discussion When EIS was performed with DET-type FADGDH-modified GDE in the presence of glucose, the obtained semicircles on the Nyquist plots became smaller with increasing depending on glucose concentrations. In contrary, EIS analyses of the non-DET-type FADGDH-modified GDEs revealed that no impedance change was observed by changing glucose concentration. These results indicated that the impedance change with DET-type FADGDH-modified GDE was observed, which results the glucose concentration dependency of Rct values. Considering the observed high sensitivity toward glucose, DET-type Faradaic Enzyme EIS sensor would provide alternative platform for future impedimetric immunosensing system, which does not use redox probe. References Lee et al., Bioelectrochemistry 121, 1-6 (2018)Yamashita et al., Int. J. Mol. Sci. 19, 931 (2018)Shiota et al., Bioelectrochemistry 112, 178-183 (2016)Yamashita et al., Enzyme. Microb. Technol. 52, 123-128 (2013)Kakehi N et al., Biosens. Bioelectron., 22. 2250-2255 (2007)Tsuya et al., J. Biotechnol. 123, 127-136 (2006)Yamaoka et al., Biotechnol. Lett. 26, 1757-1761 (2004)Inose et al., Biochim. Biophys. Acta. 1645, 133-138 (2003) Figure 1

(Invited) Current Understanding and Future Aspects of Direct Electron Transfer Mechanism of Fad Dependent Glucose Dehydrogenase Complex

Direct electron transfer (DET) between enzyme molecule and electrode provides the ideal principle for the enzyme based sensing system. In DET principle the electrons are transferred from enzyme active site to electrode directly in the absence of any additional electron acceptors, or mediators. This intriguing reaction is useful for various biodevices such as biosensors and biofuel-cells, especially by focusing anodic reactions, such as the oxidation of glucose, fructose, and ethanol. The absence of redox mediators in the system enables the operation of the sensor in redox potential closer to the co-factor in the enzyme, reducing the effect of interfering reactions by electroactive compounds in the sample. However, only a limited number of redox enzymes are capable of DET-reaction with electrodes. Among these, several dehydrogenases harboring a redox center in the catalytic subunit/domain and haem which is not involved in catalysis but act as a built-in mediator for electron transfer between enzyme and electrode are reported. One of the representative groups of DET enzymes is the oligomeric dehydrogenases composed of catalytic subunit harboring FAD as a co-factor, and an electron transfer subunit with haem c, the FAD dependent dehydrogenase complexes. Our research group have been engaged in the isolation, characterization, application and engineering of one of the representative FAD-dependent dehydrogenase complexes. FAD glucose dehydrogenase complex (FADGDH). FADGDH is comprised the three distinct subunits: the catalytic subunit (α subunit) that has an FAD cofactor in its redox center, shows catalytic activity, and oxidizes the first hydroxyl group of glucose; the small subunit (γ subunit), a hitch-hiker protein of the bacterial TAT secretion system, which is necessary for the proper folding and secretion of the α subunit; and the three heme-cytochrome c subunit (β subunit) that is in responsible for the transfer of electrons between the active-site cofactor and external electron acceptors. Thanks to the presence of β subunit, FADGDH is capable to transfer electrons directly to an electrode, making FADGDH as the ideal molecule for the 3rd Gen glucose sensors.This paper focuses on biomolecular aspects on DET mechanism of FAD dependent dehydrogenase complexes, considering current increasing number of applicational researches, and recent remarkable progress in understanding their DET mechanism, especially about the recent progress in understanding inter- and intra-molecular electron transfer of FADGDH are introduced. These studies included protein engineering approaches of these molecules, thereby led to construct engineered GDH. Finally, the future prospect of the FAD dependent dehydrogenase complexes will be addressed.

Direct electron transfer (DET) mechanism of FAD dependent dehydrogenase complexes ∼from the elucidation of intra- and inter-molecular electron transfer pathway to the construction of engineered DET enzyme complexes∼

Summary Direct electron transfer (DET) reaction is intriguing for application to biodevices such as biosensors and biofuel-cells. However, for improvement of DET for application to electrochemical devices, the understanding of its mechanism is essential. This review focuses on the recent progress on studies of biomolecular aspects of DET mechanism of flavin adenine dinucleotide (FAD) dependent dehydrogenase complex, especially, glucose dehydrogenase (GDH) and fructose dehydrogenase (FDH). These enzymes are hetero-trimeric membrane bound dehydrogenases, comprised of three distinctive subunits: a catalytic subunit, a small subunit, and an three haem c containing electron transfer subunit. The catalytic subunit was recently elucidated to harbor 3Fe-4S cluster as the FAD primary electron acceptor, and presumably to transfer electron from catalytic subunit to electron transfer subunit. The small subunit is a hitch-hiker protein, which is essential for proper folding and secretion of catalytic subunit into the periplasmic space. The electron transfer subunit has three haem binding motif, which make these enzyme complexes possible for DET. First, application studies of these enzymes to DET-based electrochemical devices are overviewed to address the challenges faced. Next, the recent progress in elucidation of inter- and intra-molecular electron transfer pathway of these enzymes were summarized. Finally, the future prospect of the application of FAD dependent dehydrogenase complexes was addressed.

Construction of Uniform Monolayer- and Orientation-Tunable Enzyme Electrode by a Synthetic Glucose Dehydrogenase without Electron-Transfer Subunit via Optimized Site-Specific Gold-Binding Peptide Capable of Direct Electron Transfer.

Direct electron transfer (DET) between enzymes and electrodes is a key issue for practical use of bioelectrocatalytic devices as a bioenergy process, such as enzymatic electrosynthesis, biosensors, and enzyme biofuel cells. To date, based on the DET of bioelectrocatalysis, less than 1% of the calculated theoretical current was transferred to final electron acceptor due to energy loss at enzyme-electrode interface. This study describes the design and construction of a synthetic glucose dehydrogenase (GDH; α and γ subunits) combined with a gold-binding peptide at its amino or carboxy terminus for direct contact between enzyme and electrode. The fused gold-binding peptide facilitated stable immobilization of GDH and constructed uniform monolayer of GDH onto a Au electrode. Depending on the fused site of binding peptide to the enzyme complex, nine combinations of recombinant GDH proteins on the electrode show significantly different direct electron-transfer efficiency across the enzyme-electrode interface. The fusion of site-specific binding peptide to the catalytic subunit (α subunit, carboxy terminus) of the enzyme complex enabled apparent direct electron transfer (DET) across the enzyme-electrode interface even in the absence of the electron-transfer subunit (i.e., β subunit having cytochrome domain). The catalytic glucose oxidation current at an onset potential of ca. (-)0.46 V vs Ag/AgCl was associated with the appearance of an flavin adenine dinucleotide (FAD)/FADH2 redox wave and a stabilized bioelectrocatalytic current of more than 100 μA, determined from chronoamperometric analysis. Electron recovery was 7.64%, and the catalytic current generation was 249 μA per GDH enzyme loading unit (U), several orders of magnitude higher than the values reported previously. These observations corroborated that the last electron donor facing to electrode was controlled to be in close proximity without electron-transfer intermediates and the native affinity for glucose was preserved. The design and construction of the site-specific "sticky-ended" proteins without loss of catalytic activity could be applied to other redox enzymes having a buried active site.

Construction and characterization of flavin adenine dinucleotide glucose dehydrogenase complex harboring a truncated electron transfer subunit

One of the most prominent glucose dehydrogenases (GDHs) capable of direct electron transfer with electrodes is the FADGDH complex derived from Burkholderia cepacia. This FADGDH complex comprises the following three distinct subunits: the catalytic subunit (α subunit) that has an FAD cofactor in its redox center, a hitch-hiker protein from the bacterial TAT secretion system (γ subunit), and the electron transfer subunit (β subunit). The electron transfer subunit (β subunit) of the FADGDH complex is a three-heme c containing cytochrome c like molecule (heme 1, heme 2 and heme 3 from the N-terminal). In this study, an FADGDH complex harboring a truncated electron transfer subunit composed of only heme 3 was constructed, and its enzymatic activity and electrochemical properties were investigated to elucidate the role of heme 3 and its region. A truncated electron transfer subunit, trβ subunit, was designed using the 3D structures of homologous cytochrome c proteins. The designed trβ subunit was expressed as soluble and functional heme c molecules forming complexes with γα catalytic complexes. Thus, the formed FADGDH complex has inter-molecular electron transfers from the FAD to the trβ subunit, and from the trβ subunit to the external electron acceptor, showing electron transfer subunit-mediated characteristic dye-mediated dehydrogenase activity with a Ru-complex. Therefore, heme 3 in the electron transfer subunit is responsible for accepting the electron from the γα catalytic complex. Moreover, the FADGDH complex harboring the trβ subunit showed DET activity toward the electrode. Spectroelectrochemical observations revealed that the trβ subunit possessed a lower formal potential than any of the 3 hemes in the whole electron transfer subunit. These unexpected electrochemical properties of the heme in the trβ subunit may potentially result in the construction of a DET principle-based glucose sensor, which can be operated at a much lower potential than those achieved using the FADGDH complex with a whole electron transfer subunit.

The electrochemical behavior of a FAD dependent glucose dehydrogenase with direct electron transfer subunit by immobilization on self-assembled monolayers

Continuous glucose monitoring (CGM) is a vital technology for diabetes patients by providing tight glycemic control. Currently, many commercially available CGM sensors use glucose oxidase (GOD) as sensor element, but this enzyme is not able to transfer electrons directly to the electrode without oxygen or an electronic mediator. We previously reported a mutated FAD dependent glucose dehydrogenase complex (FADGDH) capable of direct electron transfer (DET) via an electron transfer subunit without involving oxygen or a mediator. In this study, we investigated the electrochemical response of DET by controlling the immobilization of DET-FADGDH using 3 types of self-assembled monolayers (SAMs) with varying lengths. With the employment of DET-FADGDH and SAM, high current densities were achieved without being affected by interfering substances such as acetaminophen and ascorbic acid. Additionally, the current generated from DET-FADGDH electrodes decreased with increasing length of SAM, suggesting that the DET ability can be affected by the distance between the enzyme and the electrode. These results indicate the feasibility of controlling the immobilization state of the enzymes on the electrode surface.

Involvement of formate dehydrogenases in stationary phase oxidative stress tolerance in Escherichia coli.

Previously, we constructed a series of reduced-genome strains of Escherichia coli by combining large-scale chromosome deletions and then tested the sensitivity of these strains to the redox-cycling drug menadione. In this study, we analyzed a deletion that increased menadione sensitivity and discovered that loss of selenocysteine synthase genes was responsible for the strain's reduced tolerance to oxidative stress. Mutants of formate dehydrogenases, which are selenocysteine-containing enzymes, were also sensitive to menadione, indicating that these enzymes are involved in oxidative stress during stationary phase, specifically under microaerobic conditions in the presence of glucose. Among three formate dehydrogenases encoded by the E. coli genome, two were responsible for the observed phenotypes: formate dehydrogenase-H and -O. In a mutant of fdhD, which encodes a sulfur transferase that is essential for formate dehydrogenase activity, formate dehydrogenase-O could still contribute to oxidative stress tolerance, revealing a novel role for this protein. Consistent with this, overproduction of the electron transfer subunits of this enzyme, FdoH and FdoI, increased menadione tolerance and supported survival in stationary phase. These results suggested that formate dehydrogenase-O serves as an electron transfer element in glucose metabolism to promote oxidative stress tolerance and survival in stationary phase.

The dual-function chaperone HycH improves assembly of the formate hydrogenlyase complex

The assembly of multi-protein complexes requires the concerted synthesis and maturation of its components and subsequently their co-ordinated interaction. The membrane-bound formate hydrogenlyase (FHL) complex is the primary hydrogen-producing enzyme in Escherichia coli and is composed of seven subunits mostly encoded within the hycA-I operon for [NiFe]-hydrogenase-3 (Hyd-3). The HycH protein is predicted to have an accessory function and is not part of the final structural FHL complex. In this work, a mutant strain devoid of HycH was characterised and found to have significantly reduced FHL activity due to the instability of the electron transfer subunits. HycH was shown to interact specifically with the unprocessed species of HycE, the catalytic hydrogenase subunit of the FHL complex, at different stages during the maturation and assembly of the complex. Variants of HycH were generated with the aim of identifying interacting residues and those that influence activity. The R70/71/K72, the Y79, the E81 and the Y128 variant exchanges interrupt the interaction with HycE without influencing the FHL activity. In contrast, FHL activity, but not the interaction with HycE, was negatively influenced by H37 exchanges with polar residues. Finally, a HycH Y30 variant was unstable. Surprisingly, an overlapping function between HycH with its homologous counterpart HyfJ from the operon encoding [NiFe]-hydrogenase-4 (Hyd-4) was identified and this is the first example of sharing maturation machinery components between Hyd-3 and Hyd-4 complexes. The data presented here show that HycH has a novel dual role as an assembly chaperone for a cytoplasmic [NiFe]-hydrogenase.

Investigation of Intra- or Inter-Electron Transfer Pathway of Multiheme Electron Transfer Subunit of Iron Sulfur Flavo Cytochrome Type Glucose Dehydrogenase Complex

【Introduction】 With the increase demand for more accurate and precise monitoring of blood glucose, further development of novel principles and biosensing molecules are expected, to be dedicated for the diabetes patient care. Especially, the development of novel continuous glucose monitoring (CGM) system is inevitable in order to realize the future artificial pancreas. We have been engaged in the study and application of the bacterial FAD dependent glucose dehydrogenase (FADGDH) complex isolated from Burkholderia cepacia. FADGDH complex is composed of three subunits: a catalytic subunit (α-subunit), a hitch hiker protein of α-subunit (γ-subunit), and an electron transfer subunit (β-subunit).1-4 FADGDH complex represents several bacterial FAD dependent dehydrogenase complexes, which are also composed of the three subunits. These enzyme complexes are capable of direct electron transfer to an electrode because of the presence of electron transfer multiheme cytochrome c subunit. Thanks to this property, direct electron transfer principle based CGM is being developed using FADGDH complex. Recently, we have reported that catalytic subunit of such FAD dehydrogenase complexes harbor an Fe-S cluster, and the Fe-S cluster is involved in the intra electron transfer from FAD in the catalytic subunit, and also in the inter- electron transfer between catalytic subunit and electron transfer subunit5. These enzyme complexes are now categorized as the iron sulfur flavo cytochrome type dehydrogenase complexes However, the electron transfer pathway in the electron transfer subunit, as well as the the mechanisms of the electron transfer to the external electron acceptors are yet to be elucidated. In this paper, we report the site direct mutagenesis studies of the heme ligands of electron transfer subunit of FADGDH complex, the β-subunit, and attempt to elucidate the intra- and inter-electron transfer pathway of β-subunit. 【Materials and Methods】 The recombinant FADGDH complex wild type and those harboring mutant β-subunit were prepared using Escherichia coliBL21(DE3). The recombinant FADGDH complexes were recovered from the membrane fraction. The purification of FADGDH complexes was carried out by the combination of hydrophobic interaction chromatography. Dehydrogenase activity was measured using either Hexaammine ruthenium(III)chloride or phenazine methosulfate as an electron acceptor. Also, wild type FADGDH complex or mutant complex was immobilized on the working electrode of a glassy carbon electrode, and investigated direct electron transfer properties. 【Results and Discussion】 The results of recombinant expression of wild type FADGDH complex and all mutant complexes were successful, and the soluble enzyme complexes were prepared. The dye mediated dehydrogenase activity of mutant complexes showed different preference of electron acceptors compared with those of wild type complex. Also, the chrono-amperometric measurement of mutant FADGDH complexes showed different properties, depend on introduced mutation. Considering the our previous results of estimation of redox potential of three hemes of β-subunit, we propose the order of intra-molecular electron transfer in the β-subunit, and also the heme responsible for the electron transfer to the external electron acceptors. (1) K. Sode, et al., Enzyme Microbiol. Technol. 19, 82-85(1996) (2) T. Yamazaki, et al., Appl.Biochm.Biotechnol. 77, 325-335(1999) (3) T. Tsuya, et al., J. Biotechnol. 123, 127-136(2006) (4) H. Yamaoka, et al., Biotechnol Lett. 26, 1757-1761(2004) (5) M. Shiota, et al., Bioelectrochemistry. (2016)

Physiology and bioenergetics of [NiFe]-hydrogenase 2-catalyzed H2-consuming and H2-producing reactions in Escherichia coli.

Escherichia coli uptake hydrogenase 2 (Hyd-2) catalyzes the reversible oxidation of H2 to protons and electrons. Hyd-2 synthesis is strongly upregulated during growth on glycerol or on glycerol-fumarate. Membrane-associated Hyd-2 is an unusual heterotetrameric [NiFe]-hydrogenase that lacks a typical cytochrome b membrane anchor subunit, which transfers electrons to the quinone pool. Instead, Hyd-2 has an additional electron transfer subunit, termed HybA, with four predicted iron-sulfur clusters. Here, we examined the physiological role of the HybA subunit. During respiratory growth with glycerol and fumarate, Hyd-2 used menaquinone/demethylmenaquinone (MQ/DMQ) to couple hydrogen oxidation to fumarate reduction. HybA was essential for electron transfer from Hyd-2 to MQ/DMQ. H2 evolution catalyzed by Hyd-2 during fermentation of glycerol in the presence of Casamino Acids or in a fumarate reductase-negative strain growing with glycerol-fumarate was also shown to be dependent on both HybA and MQ/DMQ. The uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited Hyd-2-dependent H2 evolution from glycerol, indicating the requirement for a proton gradient. In contrast, CCCP failed to inhibit H2-coupled fumarate reduction. Although a Hyd-2 enzyme lacking HybA could not catalyze Hyd-2-dependent H2 oxidation or H2 evolution in whole cells, reversible H2-dependent reduction of viologen dyes still occurred. Finally, hydrogen-dependent dye reduction by Hyd-2 was reversibly inhibited in extracts derived from cells grown in H2 evolution mode. Our findings suggest that Hyd-2 switches between H2-consuming and H2-producing modes in response to the redox status of the quinone pool. Hyd-2-dependent H2 evolution from glycerol requires reverse electron transport.

Connection between the membrane electron transport system and Hyn hydrogenase in the purple sulfur bacterium, Thiocapsa roseopersicina BBS

Thiocapsa. roseopersicina BBS has four active [NiFe] hydrogenases, providing an excellent opportunity to examine their metabolic linkages to the cellular redox processes. Hyn is a periplasmic membrane-associated hydrogenase harboring two additional electron transfer subunits: Isp1 is a transmembrane protein, while Isp2 is located on the cytoplasmic side of the membrane. In this work, the connection of HynSL to various electron transport pathways is studied. During photoautotrophic growth, electrons, generated from the oxidation of thiosulfate and sulfur, are donated to the photosynthetic electron transport chain via cytochromes. Electrons formed from thiosulfate and sulfur oxidation might also be also used for Hyn-dependent hydrogen evolution which was shown to be light and proton motive force driven. Hyn-linked hydrogen uptake can be promoted by both sulfur and nitrate. The electron flow from/to HynSL requires the presence of Isp2 in both directions. Hydrogenase-linked sulfur reduction could be inhibited by a QB site competitive inhibitor, terbutryne, suggesting a redox coupling between the Hyn hydrogenase and the photosynthetic electron transport chain. Based on these findings, redox linkages of Hyn hydrogenase are modeled.