Direct Bioelectrocatalysis of Fungal Pyrroloquinoline Quinone-Dependent Pyranose Dehydrogenase Depending on the Alkyl Chain Lengths of Self-Assembled Monolayers

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Redox enzyme catalysis coupled with electrode reaction is called bioelectrocatalysis and has become a key technology applicable to bioelectrochemical devices such as biosensors, biofuel cells and bioreactors. Since electron transfer between the enzyme and the electrode is the most important phenomenon, discussion of it has dominated the study of bioelectrocatalysis. Direct electron transfer (DET) between oxidoreductases and electrodes is important not only for understanding the fundamental properties of redox proteins, but also for developing mediator-free bioelectronic devices. Among enzymes with different coenzymes, pyrroloquinoline quinone (PQQ)-dependent dehydrogenases are promising biocatalysts for both biosensors and biofuel cells. However, only a limited number of studies have reported DET between PQQ in the enzyme and the electrode. Fungal PQQ-dependent pyranose dehydrogenase from Coprinopsis cinerea (CcPDH)is a multifactor-containing enzyme with superior DET ability [1].The enzyme has a three-domain structure, an N-terminal heme b-binding cytochrome domain, a central catalytic domain with PQQ as a cofactor, and a C-terminal cellulose-binding domain. The substrate undergoes oxidation in the PQQ domain followed by interdomain electron transfer (IET) from the reduced PQQ cofactor to heme b in the cytochrome domain. CcPDHis the attractive quinohemoprotein with a PQQ domain and a cytochrome domain, both of which are possible domains for DET. Previous work has shown that the PQQ domain can engage in direct bioelectrocatalysis without the cytochrome domain. In addition, the DET of the PQQ domain was investigated using self-assembled monolayer (SAM)-coated electrodes. Importantly, a high catalytic current density of 1.6 mA/cm2 was achieved for the oxidation of L-fucose under optimised conditions [2]. These results indicate a highly efficient DET to PQQ in the active site of the fungal PQQ-dependent dehydrogenase. On the other hand, it is unclear how DET proceeds at the electrode for the full-length enzyme. The study of DET and IET by an electron transfer protein linked via a linker to a catalytic protein, such as CcPDH, is useful for incorporating these processes into biosensors and biofuel cells based on direct bioelectrocatalysis, as well as for creating fusion proteins that enable DET capability.In the present study, we demonstrated that this process distinguishes direct bioelectrocatalysis via the cytochrome domain and direct bioelectrocatalysis from the PQQ domain by regulating the distance from the electrode to CcPDH using various alkyl chains of SAMs [3]. Catalytic currents by the full-length enzyme were obtained on SAM with chain alkyl lengths of C6 or more, whereas no catalytic currents were obtained by the isolated PQQ domain. The results indicated that direct bioelectrocatalysis occurred through the cytochrome domain of CcPDH for chain lengths of 6 or more. The heme-to-electrode distance is shorter than the PQQ-to-electrode distance when the redox center of each domain is closest to the SAM layer. The PQQ-to-electrode distance exceeds 15 Å for C6-OH-SAM (16.9 Å), but the heme-to-electrode distance is less than 15 Å even for C11-OH-SAM (14.2 Å). Thus, in full-length CcPDH, DET of the PQQ domain does not occur at distances greater than 15 Å, and direct bioelectrocatalysis proceeds through the heme in the cytochrome domain when chain lengths are 6 or greater. Although the optimum pH in the PQQ domain is pH 6.0, the optimal pH is approximately 8.5 for electron transfer of the full-length enzyme through the cytochrome domain. It has been suggested that IET is the rate-limiting step in the pH range 6.0 to 8.5 for the enzyme activity in aqueous solution. The pH dependence of the catalytic current for L-fucose oxidation was examined on the enzyme electrode with C6-OH-SAM, in which DET proceeds only from the cytochrome domain. An enzymatic turnover rate (k cat) at a limiting catalytic current was obtained using the electroactive coverage of the enzyme on the electrode. In direct bioelectrocatalysis through interdomain electron transfer of the cytochrome domain, k cat was found to be pH dependent with an optimal pH of 8.5; therefore, the rate-limiting step that governs pH dependence is likely the IET process.[1] K. Takeda, et al., Curr Opin Chem Biol, 49 (2019) 113-121, [2] K. Takeda, et al., Electrochim Acta, 359 (2020) 136982, [3] K. Takeda, et al., Electrochemistry, 92 (2024) 022011.