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

Abstract

Electroactive bacteria mediate electron exchange with external surfaces 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 quinone-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 a membrane-anchored tetraheme c-type cytochrome (CbcS), an iron-sulfur protein containing four [4Fe-4S] clusters (CbcT), and an integral membrane subunit (CbcU). In this study, the sequence and AlphaFold model of CbcS were analyzed and its cytochrome domain was produced, and structurally and functionally characterized. Nuclear Magnetic Resonance spectroscopy data validated the hemecore arrangement predicted by AlphaFold, showing that, despite the differences in axial ligands (CbcS has four bis-histidine low-spin hemes), CbcS hemecore is homologous to CymA and NrfH from Shewanella and Desulfovibrio species, respectively. Potentiometric titrations showed that CbcS redox active window superimposes with the ones of its putative redox partners from the triheme periplasmic cytochrome family PpcA-E; however, electron transfer reactions monitored by NMR revealed that CbcS is able to transfer electrons to PpcA. Furthermore, NMR redox titrations allowed to identify heme IV as the exit gate for electrons. Together, these findings contribute to the understanding of the molecular mechanisms of EET and provide insights on a putative new respiratory pathway in G. sulfurreducens.