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

Hibino, Yuya; Kawai, Shigeyuki; Kitazumi, Yuki; Shirai, Osamu; Kano, Kenji

doi:10.1016/j.elecom.2016.03.013

Cited by 38 publications

(38 citation statements)

References 19 publications

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“…These suggestions were proved by investigating FDH variants (M450Q and Δ1c_FDH). In the M450Q variant, M450, which is the sixth axial ligand of heme 2c, was replaced with glutamine in order to shift the formal potential of heme 2c in the negative direction, and the halfwave potential of the steady-state catalytic wave of the M450Q variant shifted negatively as expected [57]. The Δ1c_FDH variant lacking the heme 1c moiety showed an increase in the limiting catalytic current density, which was probably thanks to the downsizing of the enzyme [58].…”

Section: Protein Engineering Methods For the Improvement Of Det-type mentioning

confidence: 83%

Direct Electron Transfer-Type Bioelectrocatalysis of Redox Enzymes at Nanostructured Electrodes

et al. 2020

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Direct electron transfer (DET)-type bioelectrocatalysis, which couples the electrode reactions and catalytic functions of redox enzymes without any redox mediator, is one of the most intriguing subjects that has been studied over the past few decades in the field of bioelectrochemistry. In order to realize the DET-type bioelectrocatalysis and improve the performance, nanostructures of the electrode surface have to be carefully tuned for each enzyme. In addition, enzymes can also be tuned by the protein engineering approach for the DET-type reaction. This review summarizes the recent progresses in this field of the research while considering the importance of nanostructure of electrodes as well as redox enzymes. This review also describes the basic concepts and theoretical aspects of DET-type bioelectrocatalysis, the significance of nanostructures as scaffolds for DET-type reactions, protein engineering approaches for DET-type reactions, and concepts and facts of bidirectional DET-type reactions from a cross-disciplinary viewpoint.

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Section: Protein Engineering Methods For the Improvement Of Det-type mentioning

confidence: 83%

Direct Electron Transfer-Type Bioelectrocatalysis of Redox Enzymes at Nanostructured Electrodes

et al. 2020

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“…At potentials more positive than 0.5 V, the DET-type reaction is inhibited by gold oxide formed at such high potentials. 17,39 It can be considered that r_FDH and ¦1cFDH adsorbed randomly on the planar Au electrode. 31,32 Unfortunately, we could not find clear explanation on a question why the M450Q mutation could improve the orientation.…”

Section: Resultsmentioning

confidence: 99%

“…Especially, DET-type bioelectrocatalysis, in which the electrode and enzymatic reactions are directly coupled, plays an significant role in the construction of mediatorfree and simple bioelectrochemical-devices for biosensing (biosensors) and biochemical electricity production (biofuel cells) with minimum overpotential in theory. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] In order to improve or investigate the DET-type bioelectrocatalysis, several protein-engineering methods have been examined: the point mutation around the active site to change its catalytic characteristics, [17][18][19][20] the deglycosylation to shorten the distance between an electrode and the redox center buried in the enzyme and improve the interfacial electron transfer kinetics, 18,21,22 and the insertion of a tag sequence or cysteine residue(s) to control the orientation of the redox enzyme on gold (Au) electrodes. [23][24][25] D-Fructose dehydrogenase from Gluconobacter japonicus NBRC3260 (FDH; EC 1.1.99.11) is a heterotrimeric membrane protein consisting of subunits I (67 kDa), II (51 kDa), and III (20 kDa).…”

Section: Introductionmentioning

confidence: 99%

“…29 In previous works by our group, it has been proposed that electrons are transferred from the substrate, to FAD, heme 3c, heme 2c, and an electrode, and that heme 1c is not involved in the electron transfer of the DET-type bioelectrocatalysis; heme 2c is the electrode-active site in the DET-type bioelectrocatalytic reaction of FDH. 17 In contrast, however, heme 1c is the electron-donating site to UQs in the living cell and its presence is essential to complete the respiratory chain ( Fig. S1), since the cells expressing an FDH variant in which 143 amino acid residues involving the heme 1c binding site was largely deleted (¦1cFDH (Fig.…”

Section: Introductionmentioning

confidence: 99%

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Discussion on Direct Electron Transfer-Type Bioelectrocatalysis of Downsized and Axial-Ligand Exchanged Variants of d-Fructose Dehydrogenase

et al. 2020

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D-Fructose dehydrogenase (FDH) gives a clear direct electron transfer (DET)-type bioelectrocatalytic wave even at planar gold (Au) electrodes. The recombinant (native) FDH (r_FDH) has three hemes c in subunit II (1c, 2c, and 3c from N-terminus). With a view to downsize the enzyme and shorten the distance between an electrode-active site and an electrode, we constructed a variant that lacked 143 amino acid residues involving the heme 1c moiety (Δ1cFDH) and a variant that lacked 199 amino acid residues involving the heme 1c and 2c moieties (Δ1c2cFDH). In order to shift the redox potential of heme 2c of Δ1cFDH to the negative direction, the M450 residue as the axial 6th ligand of heme 2c was also replaced with glutamine (M450QΔ1cFDH). The DET-type catalytic properties of r_FDH and the three variants at planar Au electrodes were compared with each other, and the steady-state waves were analyzed on a random orientation model. The orientation of the enzymes on the electrode was also discussed. In addition, in order to examine the electron transfer pathway in the DETtype reaction of Δ1c2cFDH, ESR measurements and inhibition of DET-type reaction by cyanide ion were performed.

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“…Although very elegant, this strategy has been less employed so far, mainly because it can affect both stability and activity of the protein before and after immobilization. However, unlike nonspecific adsorption where multipoint connections between enzymes and electrodes are possible, site-specific attachment can provide an orientational immobilization that will also dictate the distance between the enzyme active site and the electrode surface [118][119][120]. The approach can thus maximize the rate of direct ET, with low distribution of ET rates.…”

Section: Enzyme Engineeringmentioning

confidence: 99%

Controlling Redox Enzyme Orientation at Planar Electrodes

et al. 2018

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Abstract:Redox enzymes, which catalyze reactions involving electron transfers in living organisms, are very promising components of biotechnological devices, and can be envisioned for sensing applications as well as for energy conversion. In this context, one of the most significant challenges is to achieve efficient direct electron transfer by tunneling between enzymes and conductive surfaces. Based on various examples of bioelectrochemical studies described in the recent literature, this review discusses the issue of enzyme immobilization at planar electrode interfaces. The fundamental importance of controlling enzyme orientation, how to obtain such orientation, and how it can be verified experimentally or by modeling are the three main directions explored. Since redox enzymes are sizable proteins with anisotropic properties, achieving their functional immobilization requires a specific and controlled orientation on the electrode surface. All the factors influenced by this orientation are described, ranging from electronic conductivity to efficiency of substrate supply. The specificities of the enzymatic molecule, surface properties, and dipole moment, which in turn influence the orientation, are introduced. Various ways of ensuring functional immobilization through tuning of both the enzyme and the electrode surface are then described. Finally, the review deals with analytical techniques that have enabled characterization and quantification of successful achievement of the desired orientation. The rich contributions of electrochemistry, spectroscopy (especially infrared spectroscopy), modeling, and microscopy are featured, along with their limitations.

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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

Cited by 38 publications

References 19 publications

Direct Electron Transfer-Type Bioelectrocatalysis of Redox Enzymes at Nanostructured Electrodes

Direct Electron Transfer-Type Bioelectrocatalysis of Redox Enzymes at Nanostructured Electrodes

Discussion on Direct Electron Transfer-Type Bioelectrocatalysis of Downsized and Axial-Ligand Exchanged Variants of d-Fructose Dehydrogenase

Controlling Redox Enzyme Orientation at Planar Electrodes

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