Structural and Electrochemical Discussion on Direct Electron Transfer-Type Bioelectrocatalysis By Membrane-Bound Fructose Dehydrogenase from Gluconobacter Japonicus
Authors: Suzuki Yohei , Sowa Keisei , Kano Kenji , Kitazumi Yuki , Shirai Osamu 
Published: December 2024

Oxidoreductases have been utilized as bioelectrocatalysts to realize a variety of biotechnologies, such as biosensors, biofuel cells, solar fuel production, carbon dioxide capture and utilization, and cofactor-regeneration systems. These systems are based on bioelectrocatalysis which couples electrode and enzymatic reactions. Several metalloenzymes can communicate electronically with suitable electrodes without redox mediators. This phenomenon has been termed “direct electron transfer (DET)-type bioelectrocatalysis”. Owing to the mediator-less configuration, the reaction can offer the following benefits in future bioelectrochemical technologies: (i) minimized overvoltage, (ii) low cost, (iii) simple design, (iv) high degree of design freedom, and (v) nontoxic and environmentally friendly properties. However, there have been few reports of enzymes that can realize DET-type bioelectrocatalysis, thus the unique mechanism of the reaction has yet to be elucidated.
We focused on a d-fructose dehydrogenase (FDH) from Gluconobacter japonicus NBRC3260, which is known as a model enzyme for DET-type reactions. FDH is a unique enzyme with intense DET-type bioelectrocatalytic activity and has been extensively investigated from electrochemistry, protein engineering, and spectroscopy perspectives. FDH is a heterotrimeric membrane-bound protein with a molecular mass of ca. 138 kDa and is comprised of subunits I (67 kDa), II (51 kDa), and III (20 kDa). Subunit I contains a covalently bound flavin adenine dinucleotide (FAD), and subunit II carries three heme c moieties from its N-terminus called hemes 1c, 2c, and 3c. The redox potentials of hemes c in FDH and several variants were investigated using bioelectrochemical and spectroscopic methods. The DET pathway of FDH was examined with site-directed mutagenesis to replace the axial ligand of heme c or to delete the heme c moiety. These studies have shown that the electron is transferred from the reduced FAD through heme 3c to heme 2c and then to the electrode; heme 1c does not seem to be involved in the reaction. However, the entire three-dimensional (3D) structure of FDH and other DET-type membrane-bound quinohemoproteins, flavohemoproteins, and metallohemoproteins, remained unknown. Therefore, a quantitative discussion of their DET-type reaction was difficult.
In the present study, we clarified the 3D structure of FDH using cryo-electron microscopy and single-particle image analysis with a resolution of 2.5 Å (PDB ID: 8JEJ). This is the first study to report the entire structures of membrane-bound flavohemoproteins, quinohemoproteins, and metallohemoproteins capable of DET-type reactions. The structure has revealed the 3Fe-4S iron-sulfur cluster (3Fe4S) in subunit I. The electron transfer (ET) pathway during the catalytic oxidation of d-fructose through FAD, 3Fe4S, and hemes 3c, 2c, and 1c were examined based on Marcus’ theory. In addition, 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 the notion 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. We constructed variants in which each of the corresponding residues was replaced with alanine (W427A and F489A) by site-directed mutagenesis, and their effects on DET-type activity were investigated by electrochemical measurements. Kinetic analysis of steady-state catalytic waves has revealed that Trp427 plays an essential role in accelerating long-range ET and triples the standard rate constant of heterogeneous ET between enzymes and an electrode.
These groundbreaking findings provide vital information for searching for critical elements in DET-type reactions and a reasonable explanation for the outstanding DET-type activity of FDH. The appropriate mutation of aromatic residues to accelerate ET between an enzyme and an electrode will be a novel way to create new DET-type enzymes and innovative biomimetics.

Figure 1