Crossing the Wall: Characterization of the Multiheme Cytochromes Involved in the Extracellular Electron Transfer Pathway of

Bioelectrochemical systems (BES) are emerging as a suite of versatile sustainable technologies to produce electricity and added-value compounds from renewable and carbon-neutral sources using electroactive organisms. The incomplete knowledge on the molecular processes that allow electroactive organisms to exchange electrons with electrodes has prevented their real-world implementation. In this manuscript we investigate the extracellular electron transfer processes performed by the thermophilic Gram-positive bacteria belonging to the genus, which were found to produce higher levels of current and tolerate higher temperatures in BES than mesophilic Gram-negative bacteria. In our study, three multiheme -type cytochromes, Tfer_0070, Tfer_0075, and Tfer_1887, proposed to be involved in the extracellular electron transfer pathway of , were cloned and over-expressed in . Tfer_0070 (ImdcA) and Tfer_1887 (PdcA) were purified and biochemically characterized. The electrochemical characterization of these proteins supports a pathway of extracellular electron transfer via these two proteins. By contrast, Tfer_0075 (CwcA) could not be stabilized in solution, in agreement with its proposed insertion in the peptidoglycan wall. However, based on the homology with the outer-membrane cytochrome OmcS, a structural model for CwcA was developed, providing a molecular perspective into the mechanisms of electron transfer across the peptidoglycan layer in .