Characterization of CbcS from Geobacter sulfurreducens' Cbc4 complex: a putative novel respiratory pathway

Electroactive bacteria mediate electron exchange with external compounds through a process known as extracellular electron transfer (EET). A key step in EET is the transfer of electrons from the menaquinone pool to inner membrane-associated quinol-cytochrome c oxidoreductase complexes, which subsequently relay electrons to periplasmic redox partners. Gene-knockout and proteomic analyses have identified several critical components involved in EET in Geobacter sulfurreducens, including six inner membrane oxidoreductase gene clusters. Of these, three - CbcL, ImcH, and CbcBA - have been linked to specific respiratory pathways depending on the redox potential of the terminal electron acceptor. Cbc4 is one of the other inner membrane oxidoreductase complexes and is composed by three subunits: a membrane-anchored tetraheme c-type cytochrome (CbcS), an iron-sulfur protein containing four [4Fe-4S] clusters (CbcT), and an integral membrane protein (CbcU). In this study, the sequence and AlphaFold model of CbcS were analyzed and its cytochrome domain was produced, and structurally and functionally characterized using Nuclear Magnetic Resonance spectroscopy. CbcS has four bis-histidine low-spin hemes and the structure of its hemecore is homologous to CymA and NrfH from Shewanella and Desulfovibrio species, respectively, despite differences on its axial ligands. Potentiometric titrations showed that the redox active window of CbcS overlaps with those of the triheme periplasmic cytochrome family (PpcA-E), its putative redox partners. Nevertheless, NMR-monitored electron transfer experiments revealed that CbcS transfers electrons to PpcA through the heme group closer to the C-terminal (heme IV). Together, these findings provide insights on a putative new respiratory pathway in G. sulfurreducens.