Electrochemical Properties of pH-Dependent Flavocytochrome C
3 from Shewanella Putrefaciens Adsorbed Onto Catechol-Modified Carbon Electrode
Authors: Lebègue Estelle , Costa Nazua L , Fonseca Bruno M , Louro Ricardo O , Barrière Frédéric 
Published: May 2020

A century after the demonstration that electrons from microbial metabolism can be harvested with electrodes, electron transfer processes in biofilms of microorganisms performing respiration on extracellular solids are now being studied in order to increase the performance of microbial electrochemical technologies such as microbial fuel cells. Extracellular electron transfer occurring at the microorganism/electrode interface stimulated a wide range of fundamental and applied studies because of important implications in biotechnology, bioenergetics and bioremediation. Most studies focus on two Gram-negative mesophilic bacteria able to transfer electrons from their respiratory metabolism to extracellular solids, namely Shewanella oneidensis MR-1 and Geobacter sulfurreducens. In these model electroactive microorganisms, multiheme c-type cytochromes transfer electrons from cytoplasmic and inner-membrane oxidizing enzymes towards cell surface redox proteins that are responsible for the reduction of solid phase electron acceptors. It is also known that electron transfer is usually coupled to proton transfer which causes acidification in the anodic biofilms and in the anolyte with deleterious consequences for the biofilm metabolism and stability. The fundamental understanding of these processes that involve large proteins (>70 kDa) often with complex redox properties (with ≥10 heme centers in some multihemes cytochromes) is still in its infancy and precludes the optimization of microbial electrochemical technologies. In Gram-negative electroactive bacteria such as Shewanella, several redox proteins have pH-dependent potentials, such as flavocytochrome c
3 (M
r = 63.8 kDa) which is one of the most abundant proteins in the periplasm and the only functional fumarate reductase in this microorganism.

This work focuses on the design and the development of an efficient and versatile electrochemical platform able to probe charge (electron/proton) transfer properties of proteins from Gram-negative electroactive bacteria on modified carbon electrodes.[1] The electrodes are first modified with pH-responsive electrophores such as quinone units and then proteins are immobilized for electrochemical studies. If relevant, the protein electroactivity can be probed directly at the modified electrode while proton transport, if any, performed by the protein can be detected thanks to the grafted pH sensor.The electrochemical detection of a pH-dependent redox protein from electroactive bacteria at an electrode modified to act additionally as an efficient pH sensor based on a redox readout is demonstrated. The pH sensing electrode was previously designed and showed to allow the immobilization and study of pH-independent and redox active cytochrome c.[1] Here we extend this work to flavocytochrome c
3, a tetraheme FAD-containing periplasmic flavoenzyme isolated from the bacterium Shewanella putrefaciens, taken as a model pH-dependent redox protein from electroactive bacteria [2]. The modification of the electrode surface with the pH sensing modifier (catechol) stems from our previous experience in the tailoring of bio-interfaces of carbon electrodes with covalent electrografting. Flavocytochrome c
3 adsorption onto the modified electrode surface is successfully achieved by cyclic voltammetry in a flavocytochrome c
3 solution containing polymyxin as co-adsorbate. The electrochemical activity of the immobilized flavocytochrome c
3 is detected without alteration of its native structure and by keeping intact its electrochemical properties and its catalytic fumarate reductase activity.[2] The redox activity of the protein arises from its FAD and four hemes cofactors. The experiments evidence that the hemes’ redox potentials of flavocytochrome c
3 from Shewanella putrefaciens, for which no crystal structure is yet available, depend on pH which is at variance with data from the other strains Shewanella frigidimarina or Shewanella oneidensis.

[1] Lebègue, E.; Louro, R. O.; Barrière, F. Electrochemical Detection of pH-Responsive Grafted Catechol and Immobilized Cytochrome c onto Lipid Deposit-Modified Glassy Carbon Surface. ACS Omega
2018, 3 (8), 9035–9042. https://doi.org/10.1021/acsomega.8b01425.

[2] Lebègue, E.; Costa, N. L.; Fonseca, B. M.; Louro, R. O.; Barrière, F. Electrochemical Properties of pH-Dependent Flavocytochrome C3 from Shewanella Putrefaciens Adsorbed onto Unmodified and Catechol-Modified Edge Plane Pyrolytic Graphite Electrode. Journal of Electroanalytical Chemistry
2019, 847, 113232. https://doi.org/10.1016/j.jelechem.2019.113232.

Figure 1