Yoshihide Tokunou2 Kazuhito Hashimoto1 Akihiro Okamoto1

2, The University of Tokyo, Tokyo, , Japan
1, National Institute for Materials Science, Tsukuba, , Japan

Bacterial electron transport to a solid substrate or electrode located extracellularly is accomplished by unidirectional electron flow via an array of more than twenty heme redox centers arranged in the outer membrane c-type cytochrome complex (OM c-Cyts). This interfacial electron transport between OM c-Cyts and solid substrates is termed extracellular electron transport (EET). The rate of EET is largely enhanced by self-secreted flavin molecules associated with the formation of semiquinone (Sq) state as a binding redox cofactor in the OM c-Cyts. However, the more negative redox potential of bound flavin Sq than the hemes in OM c-Cyts is energetically unfavorable for the kinetics of EET. Given the primary focus of related work in the recent past has been the electron carriers and the redox potential landscape of reaction centers, the importance of associated proton transport has not been widely investigated in EET. We,herein, show that proton transfer in the OM flavocytochromes limits the rate of EET in Shewanella oneidensis MR-1. Using an in vivoelectrochemical assay, we observed a large kinetic isotope effect (KIE) following D2O addition (< 4%), specifically when EET was the rate-determining step for the current production of lactate oxidation respiration. Replacing flavin cofactors with twelve analogous molecules, the rate of EET correlated not with their redox potential but with their pKa at the nitrogen atom at position-5 (N(5)) in the isoalloxazine ring calculated by a quantum chemical approach. Because higher pKa represents stronger proton acceptability in N(5), this correlation suggests that the protonation reaction at N(5) in flavin associates and limits the rate of EET. We will further discuss about the rate-determining step of proton transport coupled with the redox reaction of the bound flavin cofactor in OM c-Cyts, with dataset for solvent KIE with partial deletion of OM c-Cyts complex.