Essential Insight of Direct Electron Transfer-Type Bioelectrocatalysis by Membrane-bound D-Fructose Dehydrogenase with Structural Bioelectrochemistry

25 July 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Flavin adenine dinucleotide-dependent D-fructose dehydrogenase (FDH) from Gluconobacter japonicus NBRC3260, a membrane-bound heterotrimeric flavohemoprotein capable of direct electron transfer (DET)-type bioelectrocatalysis, was investigated from the perspective of structural biology, bioelectrochemistry, and protein engineering. DET-type reactions offer several benefits in biomimetics (e.g., biofuel cells, bioreactors, and biosensors) owing to their mediator-less configuration. FDH provides an intense DET-type catalytic signal; therefore, extensive research has been conducted on the fundamental principles and applications of biosensors. Structural analysis using cryo-electron microscopy and single-particle analysis has revealed the entire FDH structures with resolutions of 2.5 and 2.7 Å for the reduced and oxidized forms, respectively. The electron transfer (ET) pathway during the catalytic oxidation of D-fructose was investigated using both thermodynamic and kinetic approaches. 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 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. Two variants (W427A and F489A) were obtained by site-directed mutagenesis, and their effects on DET-type activity were elucidated. The results have shown that Trp427 plays an essential role in accelerating long-range ET and triples the standard rate constant of heterogeneous ET based on bioelectrochemical analysis.


direct electron transfer
cryo-electron microscopy
membrane-bound D-fructose dehydrogenase

Supplementary materials

Supporting Information
Supporting information for the main manuscript


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