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

Suzuki, Yohei Makino, Fumiaki Miyata, Tomoko Tanaka, Hideaki Namba, Keiichi Kano, Kenji Sowa, Keisei Kitazumi, Yuki Shirai, Osamu 京都大学 DOI:10.1021/acscatal.3c03769

2023.10.20

概要

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 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. 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.

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参考文献

This research was supported by the Platform Project for

Supporting Drug Discovery and Life Science Research (Basis

for Supporting Innovative Drug Discovery and Life Science

Research (BINDS)) from AMED under Grant

JP22ama121003 to K.N., JSPS KAKENHI Grant

JP21H01961 to Y.K., JSPS KAKENHI Grant JP22K14831 to

K.S., and FY 2022 Kusunoki 125 of Kyoto University 125th

Anniversary Fund to K.S. This study was supported in-part by

the Program for the Development of Next-generation Leading

Scientists with Global Insight (L-INSIGHT), sponsored by the

Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. We express our gratitude to Mr. Hirou

Kaku, Mr. Koryu Ou, and Mr. Yasuyuki Hamano for their

financial support. We thank Dr. Hideto Matsuoka of the

Graduate School of Science, Osaka City University for his

technical assistance with the EPR measurements. We would

also like to thank Editage (www.editage.com) for the English

language editing.

AUTHOR INFORMATION

Corresponding Author

Keisei Sowa − Division of Applied Life Sciences, Graduate

School of Agriculture, Kyoto University, Sakyo, Kyoto 6068502, Japan; orcid.org/0000-0001-9767-4922;

Email: sowa.keisei.2u@kyoto-u.ac.jp

Authors

Yohei Suzuki − Division of Applied Life Sciences, Graduate

School of Agriculture, Kyoto University, Sakyo, Kyoto 6068502, Japan

Fumiaki Makino − Graduate School of Frontier Biosciences,

Osaka University, Suita, Osaka 565-0871, Japan; JEOL Ltd.,

Akishima, Tokyo 196-8558, Japan

Tomoko Miyata − Graduate School of Frontier Biosciences,

Osaka University, Suita, Osaka 565-0871, Japan

Hideaki Tanaka − Institute for Protein Research, Osaka

University, Suita, Osaka 565-0871, Japan

Keiichi Namba − Graduate School of Frontier Biosciences,

Osaka University, Suita, Osaka 565-0871, Japan; RIKEN

Center for Biosystems Dynamics Research, Suita, Osaka 5650874, Japan; RIKEN SPring-8 Center, Sayo, Hyogo 679-

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