Molecular Design for Catalysts of Microbial Extracellular Electron Transfer: Redox Active Molecules Bound with Outer-Membrane Cytochrome C in Shewanella Oneidensis MR-1
Authors: Tokunou Yoshihide , Hashimoto Kazuhito , Okamoto Akihiro 
Published: February 2020

1. Introduction
Some microbial strains have an ability to transport respiratory electrons generated from cell inside to extracellular solid substrates via electron transfer proteins, c-type cytochromes, located at outer membrane (OM c-Cyts).[1] This interfacial electron transport between microbe and solid substrate is called extracellular electron transport, EET. Since EET can be coupled with intracellular enzymatic reactions, to understand and control the EET kinetics have impacts for development of bioelectrochemical technologies, such as microbial fuel cells[2] and electrode biosynthesis[3] as well as geochemical mineral cycling[4] and bioremediation. Recently, we found that in model EET microbe, Shewanella oneidensis MR-1, the rate of EET is largely enhanced by self-secreted flavin (Flavin mononucleotide: FMN, Riboflavin: RF) working as a binding redox active center, cofactor, in OM c-Cyts.[5] In other words, bound flavin cofactor in OM c-Cyts has catalytic activity for electron transfer from OM c-Cyts to electrode. In order to understand the EET acceleration mechanism and control the rate of EET, here, we identified and characterized alternative cofactor molecules.[6] Our data demonstrate that heterocyclic compounds containing nitrogen atom at 5-position in isoalloxazine ring (N(5)) (Figure) largely enhance current production from S. oneidensis MR-1 as efficient as the bound flavin, indicating that heterocyclic compounds with N(5) act as cofactors. Furthermore, we found that the pKa at N(5) site essentially affects the EET kinetics, providing us novel strategy for designing small molecule for regulating the EET rate to control pKa at N(5).
2. Experimental
A single chamber, three electrode system was used for electrochemical measurement of intact S. oneidensis MR-1, which was inoculated for 12 hours in defined-media (DM) after 24 hours cultivation in LB. A tin-doped indium oxide (ITO) substrate, Ag/AgCl (KCl saturated) and a platinum wire were used as working, reference and counter electrode, respectively. 4.0 mL DM containing 2.0 µM flavin-like molecules (Figure) was deaerated by N2 bubbling. Cell suspensions with OD600 = 0.1 were inoculated in the reactor with the electrode poised at +0.4V vs. SHE in the presence of 10 mM lactate as a sole electron donor at 303K. To compare the EET rate for each heterocyclic compounds, the concentration of the bound cofactor was normalized by dissociation constant estimated from the data of differential pulse voltammetry.[6]

3. Results and Discussion
To identify the molecules working as alternative cofactor in OM c-Cyts, we observed current generation from S. oneidensis MR-1 with several molecules possessing flavin-like polycyclic backbone (Figure). All the compounds containing N(5) as well as flavin increased current production in the range of 10-20 times compared with the absent of small molecules, while α-AQS, the molecule without N(5) in polycyclic backbone showed much less enhancement. These data strongly suggest that the N(5) compounds can operate as the cofactor to enhance the rate of EET as the flavin molecules. Differential pulse voltammetry revealed that the N(5) molecules have more positive redox potential in bound state compared with the bound flavin semiquinone, which is more favorable for receiving electrons from heme redox centers in OM c-Cyts. However, among these molecules, the larger enhancement factor did not follow more positive redox potential. On the other hand, the cofactors that have higher pKa at N(5) showed higher maximum current production. Given that the redox reaction of flavin semiquinone couples with the protonation/deprotonation reaction at N(5),[7] these results may indicate that protonation reaction at N(5) site associated with redox reaction in cofactors strongly affects the rate of EET.
4. Conclusion
Our study revealed that heterocyclic compounds containing N(5) can enhance the EET rate as an alternative cofactor in OM c-Cyts. Furthermore, we found that pKa at N(5) strongly affects the rate of EET, which enables us to regulate the rate of EET by a structural modification of heterocyclic compounds containing N(5) and pKa control. These novel mechanistic insights would largely impact EET-utilizing bioelectrochemical technologies as well as general understanding for electrical interaction between microbe and extracellular environments.
5. References
[1] H. A. Liu et al, Angew. Chem. Int. Ed., 2010, 49, 6596-6599. [2] D. R. Lovley, Nat. Rev. Microbiol., 2006, 4, 497-508 [3] Rabaey, K. and Rozendal, R. A, Nat. Rev. Microbiol. 2010, 8, 706−716. [4] K. H. Nealson and D. Saffarini, Annu. Rev. Microbiol., 1994, 48, 311-343 [5] A. Okamoto et al,
Proc. Natl. Acad. Sci. USA, 2013, 110, 7856-7861. [6] Y. Tokunou et al,
J. Phys. Chem. C, in press [7] R. D. Drapper et al, BIOCHEM AND BIOPHYSICS, 1968, 125, 802

Figure 1