Bioelectrochemical Research on Direct Electron Transfer-type Bioelectrocatalysis and Their Applications

Direct electron transfer (DET)-type bioelectrocatalysis, in which enzymatic reactions proceed without the involvement of redox mediators, is a powerful approach for investigating intrinsic enzymatic features relevant to the development of advanced biomimetic systems.However, because numerous DET-capable enzymes are associated with cellular membranes, determining their full three-dimensional conformations is challenging when relying solely on conventional X-ray crystallography.The advent of cryo-electron microscopy coupled with single-particle reconstruction techniques has recently made it feasible to quickly resolve their structures.A notable interdisciplinary advancement termed "structural bioelectrochemistry" has emerged, integrating structural biology with electrochemical analysis to accelerate functional determination through enzyme modification and electrochemical probing.This review highlights recent fundamental research on DET-type enzymes and summarizes their bioelectrochemical applications, such as biosensors, biofuel cells, and bioreactors. IntroductionRedox enzymes facilitate electron transfer (ET) between electron donors and acceptors.The coupling of catalytic and electrode reactions is called bioelectrocatalysis. 1- 14 This interdisciplinary approach not only advances the fundamental understanding of enzymes but also underpins the development of bioinspired electrochemical systems.In direct electron transfer (DET)-type reactions, enzymes shuttle electrons to an electrode without using redox mediators. [15][16]6][17][18][19][20][21][22][23][24][25][26][27][28][29][30] This mediator-free configuration confers several practical benefits, including system simplification, cost reduction, design versatility, environmental compatibility, and improved biocompatibility. ACCEPTED MANUSCRIPT3 Multimeric dehydrogenases such as fructose dehydrogenase (FDH), alcohol dehydrogenase (ADH), and aldehyde dehydrogenase (ALDH) from Gluconobacter sp, and formate dehydrogenase 1 (FoDH1) from Methylorubrum extorquens are promising DET-type enzymes. [31][32]2][33][34] They can catalyze the oxidation of organic substrates (e.g., sugars, alcohols, aldehydes, and organic acids) or the reduction of carbon dioxide.These enzymes demonstrate substantial DET-type activity and have been intensively studied from three core methodological perspectives.First, electrochemical measurements were employed to evaluate the catalytic properties by analyzing thermodynamic (e.g., redox potential) and kinetic (e.g., catalytic current) parameters.Secondly, protein engineering has enabled the generation of various enzyme variants to probe the roles of cofactors and specific residues involved in catalysis, intramolecular ET, and interfacial ET with electrodes.Third, structural biology provides critical insights into the three-dimensional (3D) architectures of enzymes, leading to rational design strategies and deepening our understanding of their electrochemical functions.Although electrochemical measurements and protein engineering provide detailed information on the bioelectrochemical properties of enzymes, structural analysis using conventional X-ray crystallography is both time-consuming and unsuitable for some enzymes (membrane-bound proteins).Many DET-type multimeric dehydrogenases are membrane-bound proteins, and the overall steric structures of these enzymes have remained largely unknown for many decades.This limitation has recently been addressed by breakthroughs in cryo-electron microscopy (cryo-EM) and single-particle image analysis technologies. 35Cryo-EM enables high-resolution and rapid structural determination of biomolecules from small sample amounts without the need for crystallization, thereby revealing the structures of a wide range of enzymes and their