Investigation of Intra- or Inter-Electron Transfer Pathway of Multiheme Electron Transfer Subunit of Iron Sulfur Flavo Cytochrome Type Glucose Dehydrogenase Complex (2016)

【Introduction】 With the increase demand for more accurate and precise monitoring of blood glucose, further development of novel principles and biosensing molecules are expected, to be dedicated for the diabetes patient care. Especially, the development of novel continuous glucose monitoring (CGM) system is inevitable in order to realize the future artificial pancreas. We have been engaged in the study and application of the bacterial FAD dependent glucose dehydrogenase (FADGDH) complex isolated from Burkholderia cepacia . FADGDH complex is composed of three subunits: a catalytic subunit (α-subunit), a hitch hiker protein of α-subunit (γ-subunit), and an electron transfer subunit (β-subunit). 1-4 FADGDH complex represents several bacterial FAD dependent dehydrogenase complexes, which are also composed of the three subunits. These enzyme complexes are capable of direct electron transfer to an electrode because of the presence of electron transfer multiheme cytochrome c subunit. Thanks to this property, direct electron transfer principle based CGM is being developed using FADGDH complex. Recently, we have reported that catalytic subunit of such FAD dehydrogenase complexes harbor an Fe-S cluster, and the Fe-S cluster is involved in the intra electron transfer from FAD in the catalytic subunit, and also in the inter- electron transfer between catalytic subunit and electron transfer subunit 5 . These enzyme complexes are now categorized as the iron sulfur flavo cytochrome type dehydrogenase complexes However, the electron transfer pathway in the electron transfer subunit, as well as the the mechanisms of the electron transfer to the external electron acceptors are yet to be elucidated. In this paper, we report the site direct mutagenesis studies of the heme ligands of electron transfer subunit of FADGDH complex, the β-subunit, and attempt to elucidate the intra- and inter-electron transfer pathway of β-subunit. 【Materials and Methods】 The recombinant FADGDH complex wild type and those harboring mutant β-subunit were prepared using Escherichia coli BL21(DE3). The recombinant FADGDH complexes were recovered from the membrane fraction. The purification of FADGDH complexes was carried out by the combination of hydrophobic interaction chromatography. Dehydrogenase activity was measured using either Hexaammine ruthenium(III)chloride or phenazine methosulfate as an electron acceptor. Also, wild type FADGDH complex or mutant complex was immobilized on the working electrode of a glassy carbon electrode, and investigated direct electron transfer properties. 【Results and Discussion】 The results of recombinant expression of wild type FADGDH complex and all mutant complexes were successful, and the soluble enzyme complexes were prepared. The dye mediated dehydrogenase activity of mutant complexes showed different preference of electron acceptors compared with those of wild type complex. Also, the chrono-amperometric measurement of mutant FADGDH complexes showed different properties, depend on introduced mutation. Considering the our previous results of estimation of redox potential of three hemes of β-subunit, we propose the order of intra-molecular electron transfer in the β-subunit, and also the heme responsible for the electron transfer to the external electron acceptors. (1) K. Sode, et al., Enzyme Microbiol. Technol. 19, 82-85(1996) (2) T. Yamazaki, et al., Appl.Biochm.Biotechnol. 77, 325-335(1999) (3) T. Tsuya, et al., J. Biotechnol. 123, 127-136(2006) (4) H. Yamaoka, et al., Biotechnol Lett. 26, 1757-1761(2004) (5) M. Shiota, et al., Bioelectrochemistry. (2016)