Essential Insight of Direct Electron Transfer-Type Bioelectrocatalysis by Membrane-Bound <scp>d</scp> -Fructose Dehydrogenase with Structural Bioelectrochemistry

Abstract

High Resolution Image Download MS PowerPoint Slide 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 by using both thermodynamic and kinetic approaches. Structural analysis has shown the localization of the electrostatic surface charges around heme 2 c in subunit II, and experiments using functionalized electrodes with a controlled surface charge support the notion that heme 2 c 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 2 c 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 according to bioelectrochemical analysis.