LIVING CAPACITORS: FUNCTIONAL CHARACTERIZATION OF A NOVEL CYTOCHROME ACTING AS A NANOWIRE

In an Era where environmental issues are a growing concern, microorganisms that have remarkable features, such as extracellular electron transfer (EET) ability, present major opportunities in diverse biotechnological fields. Geobacter bacteria have shown an extraordinary respiratory flexibility, with its dissimilatory metal reduction ability and EET to electrode surfaces, and numerous c-type cytochromes were pointed as key players. However, the understanding of the mechanisms involved and hence, the advances in practical applications, are still in its early days and it is crucial to move further and unveil not only the components involved, but also their roles and partners in electron transfer. The dodecaheme GSU1996, composed of four similar triheme domains (A–D), was proposed to work as a natural nanowire, owing to its linear structure and large number of hemes. In this work, the in vitro functional characterization of the GSU1996 was attempted, in a modular characterization based strategy. Here, the triheme domains C and D assisted in the characterization of the C-terminal end of GSU1996, the hexaheme fragment CD. The first step encompassed the assignment of the heme groups signals in the nuclear magnetic resonance spectra of the triheme domains and of the hexaheme fragment, which is the protein with the highest number of hemes assigned to date. The second step comprised the determination of the microscopic thermodynamic parameters of fragment CD. This provided mechanistic information on the dominant microstates and included the determination of the reduction potentials of the hemes, redox interactions between hemes and ionizable centers and among neighboring hemes. The third and final step consisted in the determination of the microscopic kinetic parameters of fragment CD. This unveiled details about the reactivity of the heme groups and included the calculation of the reference rate constants for each heme in the reduction/oxidation process. All combined, the data revealed that a heme located at the end of the C-terminal edge of GSU1996 shows the necessary skills to accept electrons from redox partners. In vitro interaction studies performed between GSU1996 and the periplasmic cytochrome PpcA and its homologues (PpcC–E), revealed that it is possible that GSU1996 and PpcA may be redox partners in G. sulfurreducens, as they form a transient redox complex that involves the C-terminal fragment of GSU1996. Work has also been started to disclose other electron transfer components of G. sulfurreducens, namely, the outer membrane tetraheme cytochrome OmcE; the hexaheme OmcS and the nanowire cytochrome GSU2210. New constructs and expression systems were tested, based in the pBAD vector, albeit none of the attempts have been successful. Although in vitro studies provide information and allow the evaluation of the functional properties of these proteins, in vivo studies are essential to assess the actual roles and interacting partners in the cells. Therefore, a novel approach was also tested towards the in vivo labeling of c-type cytochromes, based in the attachment of a tetracysteine tag that is fluorescent upon binding with commercially available biarsenical dyes. However, no expression of the model tagged protein was accomplished