Construction and characterization of flavin adenine dinucleotide glucose dehydrogenase complex harboring a truncated electron transfer subunit

Abstract One of the most prominent glucose dehydrogenases (GDHs) capable of direct electron transfer with electrodes is the FADGDH complex derived from Burkholderia cepacia . This FADGDH complex comprises the following three distinct subunits: the catalytic subunit (α subunit) that has an FAD cofactor in its redox center, a hitch-hiker protein from the bacterial TAT secretion system (γ subunit), and the electron transfer subunit (β subunit). The electron transfer subunit (β subunit) of the FADGDH complex is a three-heme c containing cytochrome c like molecule (heme 1, heme 2 and heme 3 from the N-terminal). In this study, an FADGDH complex harboring a truncated electron transfer subunit composed of only heme 3 was constructed, and its enzymatic activity and electrochemical properties were investigated to elucidate the role of heme 3 and its region. A truncated electron transfer subunit, trβ subunit, was designed using the 3D structures of homologous cytochrome c proteins. The designed trβ subunit was expressed as soluble and functional heme c molecules forming complexes with γα catalytic complexes. Thus, the formed FADGDH complex has inter-molecular electron transfers from the FAD to the trβ subunit, and from the trβ subunit to the external electron acceptor, showing electron transfer subunit-mediated characteristic dye-mediated dehydrogenase activity with a Ru-complex. Therefore, heme 3 in the electron transfer subunit is responsible for accepting the electron from the γα catalytic complex. Moreover, the FADGDH complex harboring the trβ subunit showed DET activity toward the electrode. Spectroelectrochemical observations revealed that the trβ subunit possessed a lower formal potential than any of the 3 hemes in the whole electron transfer subunit. These unexpected electrochemical properties of the heme in the trβ subunit may potentially result in the construction of a DET principle-based glucose sensor, which can be operated at a much lower potential than those achieved using the FADGDH complex with a whole electron transfer subunit.