Characterization and interaction studies of triheme cytochromes from Geobacter: a contribution to the elucidation of extracellular electron transfer pathways

Geobacter species are frequently the most abundant Fe(III)-reducing microorganism in soils and sediments. They can also reduce other metals in the same type of environments and, in addition, make electrical connections with electrodes to produce electricity from waste organic matter or to drive anaerobic process with electrical energy. Proteomic and genetic studies have identified several multiheme cytochromes as essential for Fe(III) reduction. From all the cytochromes that were shown to be involved in the reduction of Fe(III), the best characterized to date are five periplasmic triheme cytochromes from Geobacter sulfurreducens, which constitute the so-called PpcA-family. The members of this family are designated PpcA, PpcB, PpcC, PpcD, PpcE. A similar family was found in Geobacter metallireducens (PpcA, PpcB, PpcC, PpcE and PpcF) but none of these proteins was characterized to date. When compared to the other homologs found in G. sulfurreducens, PpcF differs the most and for this reason was targeted in the present work. To characterize this cytochrome, PpcF was firstly expressed and purified. The yield obtained was approximately 1 mg/L of cell culture. The molecular mass of the protein was confirmed by mass spectroscopy (9737.13 Da). The molar extinction coefficient was determined (87.4 mM-1cm-1). The UV-visible spectral characteristics of PpcF are consistent with low-spin heme groups with His-His axial coordination, a feature that was further confirmed by Nuclear Magnetic Resonance spectroscopy. The assignment of the heme substituent signals of PpcF in both reduced and oxidized states together with the analysis of their NOE connectivities showed that the heme core structure is similar to those of the PpcA family cytochromes in G. sulfurreducens. The reduction potentials of PpcF were determined at pH 7 and 8 (-56 mV and – 64 mV versus the standard hydrogen electrode, respectively). Lastly 2D-1H NMR exchange spectroscopy was used to determine the order of oxidation of the heme groups in PpcF: IV-I-III. In the second part of this thesis it was analyzed the possible molecular interaction between cytochromes PpcA, PpcB and PpcE from G. sulfurreducens and Fe(III) citrate. This molecule can be utilized as terminal electron acceptor by this bacterium and PpcA, PpcB and PpcE were shown to be crucial in this electron transfer pathway. For these purpose isotopic 15N-labeled cytochromes were expressed and purified. NMR spectroscopy enabled us to assign the protein NH backbone and heme methyl proton signals, as well as to probe the interaction regions between each cytochrome and Fe(III) citrate. The chemical shift perturbation studies showed that in all cytochromes the interaction region is located in the vicinity of heme IV