Structural and Functional Insights of an Unprecedented Multiheme Cytochrome from Geobacter Sulfurreducens

Abstract

Geobacter bacteria have been inspiring the development of many biotechnological applications, which seek improvements in the fields of microbial energy production, bioremediation of contaminated waters and soils, development of sustainable electronic devices and materials, as well as biomedical devices. Geobacter sulfurreducens, the microorganism with the highest current production in microbial fuel cells known to date, possesses over 100 cytochromes that assure an effective electron transfer between the intracellular components and the cell exterior. The most abundant group of cytochromes in this microorganism is the PpcA-family, composed of five periplasmic c7 triheme cytochromes with high structural homology and identical heme coordination (His-His). GSU0105 is a periplasmic triheme cytochrome expressed by G. sulfurreducens in Fe(III) reducing conditions, but not expressed at all in cultures grown on fumarate, linking the cytochrome directly with extracellular electron transfer (EET). This cytochrome possesses a low sequence identity with the cytochromes from the PpcA-family and a inevitably different heme-coordination, based on the analysis of its amino acid sequence. This detail, together with the direct linkage with EET pathways, makes GSU0105 an interesting target for functional and structural studies. In this work, several biophysical techniques, including UV-visible, CD, EPR and NMR spectroscopies, were used to characterize the cytochrome. Initial evidences about the secondary structural elements and redox behavior of the cytochrome were probed. NMR and EPR were used to pinpoint the spin-states of the heme groups in different oxidation states. The results obtained, together with BLAST and structural predictions, support the fact that GSU0105 is a member of a new subclass of triheme cytochromes. Acknowledgements: This work was supported by Fundação para a Ciência e Tecnologia (FCT, Portugal) through grants SFRH/BD/145039 and PTDC/BIA-BQM/31981/2017 and by UCIBIO, which is financed by national funds from FCT (UIDB/04378/2020).