Salgueiro, C. A. | Search bioRxiv

bioRxiv2025

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

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.

biochemistry↗

bioRxiv2023

Role of the inner membrane cytochrome ImcH in Geobacter extracellular electron transfer and energy conservation

Electroactive bacteria combine the oxidation of carbon substrates with an extracellular electron transfer (EET) process that discharges electrons to an electron acceptor outside the cell. This process involves electron transfer through consecutive redox proteins that efficiently connect the inner membrane to the cell exterior. In this study, we isolated and characterized the quinone-interacting membrane cytochrome c ImcH from Geobacter sulfurreducens, which is involved in the EET process to high redox potential acceptors. Our work provides evidence that ImcH is electroneutral, as it transfers electrons and protons to the same side of the membrane, contributing to the maintenance of a proton motive force, and plays a central role in recycling the menaquinone pool. ImportanceGeobacter sulfurreducens is a model electroactive bacterium, widespread in the environment and of significant interest for biotechnological applications. Its ability to form thick and conductive biofilms on top of conducting surfaces makes this microbe very useful in bioelectrochemical systems for the production of energy or added value products. To explore Geobacter spp. as a biocatalyst it is essential to understand its metabolism, particularly the molecular mechanisms for extracellular electron transfer and energy conservation. Our results reveal the importance of ImcH in both processes, identifying this protein as a major player on Geobacter metabolism.

biochemistry↗