Direct Electron Transfer of Fungal Pyrroloquinoline Quinone-Dependent Dehydrogenase Lacking the Cytochrome Domain
Authors: Takeda Kouta , Nakamura Nobuhumi 
Published: December 2020

Bioelectrocatalysis is the use of biomaterials as catalysts for redox reactions combined with electrode reactions. Electrodes modified with enzymes have been extensively studied in the field of bioelectrochemistry. Since electron transfer between the enzyme and the electrode is the most important phenomenon, the discussion about it has dominated the study of bioelectrocatalysis. Direct electron transfer (DET) between oxidoreductases and electrodes is important not only for understanding the fundamental features of redox proteins, but also for developing mediator-free bioelectronics devices. Among the enzymes with various coenzymes, the pyrroloquinoline quinone (PQQ)-dependent dehydrogenases are promising biocatalysts for both biosensors and biofuel cells. However, a limited number of studies have reported DET between PQQ in the enzyme and the electrode.
We discovered a fungal PQQ-dependent pyranose dehydrogenase from the basidiomycete Coprinopsis cinerea (CcPDH) as the first example of a eukaryotic PQQ-dependent enzyme [1-3]. This enzyme comprises three domains: an N-terminal cytochrome b domain, a central PQQ dependent catalytic domain (PQQ domain), and a C-terminal family 1 carbohydrate-binding module (CBM1), and thus, it is regarded as quinohemoprotein. A previous study demonstrated that full-length CcPDH exhibited the direct bioelectrocatalysis based on DET on a glassy carbon (GC) electrode [4]. The catalytic currents were observed at a lower potential than the redox potential of heme b in the cytochrome domain, suggesting that both PQQ in the catalytic domain and heme b in the cytochrome domain are capable of DET. In the present study, the PQQ domain (containing only the PQQ cofactor) which was prepared through genetic engineering, was studied in DET with various electrodes.
A 2-mercaptoethanol self-assembled monolayer (SAM)-coated polycrystalline gold electrode was found to be superior for the DET of PQQ in the domain, and the catalytic current density was higher than that of the bare GC electrode. The catalytic current density has increased 10 times. The amount of immobilized PQQ domain was determined to be 8.2 ± 0.4 pmol/cm2 by using a 27 MHz quartz-crystal microbalance (QCM), suggesting an approximate protein monolayer formation on the SAM modified gold surface. To improve current density, gold nanoparticles (AuNPs) were modified on top of polycrystalline gold electrodes. Importantly, a high catalytic current density of 1.6 mA/cm2 for the oxidation of L-fucose was achieved under optimized conditions. These results show highly efficient DET to PQQ in the active site of the fungal PQQ-dependent dehydrogenase. Thus, the PQQ-domain/SAM/AuNP coated polycrystalline gold electrode could serve as a highly sensitive biosensor and a highly active sugar oxidizing anode for a biofuel cell.
Cyclic voltammetry using a AuNP modified electrode in the absence of substrate showed two pairs of redox peaks with midpoint potentials of E
1 = -45 mV and E
2 = -216 mV, indicating the redox reaction of the bound PQQ in the catalytic domain is composed of two one-electron steps via a semiquinone radical state. This was confirmed by the detection of an EPR signal with a g-value of 2.0065, which was assigned to a PQQ semiquinone radical. The details of DET of the PQQ domain will be discussed.
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[2] K. Takeda, et al., PLoS One, 10 (2015) e0115722.
[3] K. Takeda, et al., Curr Opin Chem Biol, 49 (2019) 113-121.
[4] K. Takeda, et al., Biochem Biophys Res Commun, 477 (2016) 369-373.