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Projeto de investigação
The way forward: optimization of respiratory electron transfer chains toward sustainable microbial electricity production.
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LIVING CAPACITORS: FUNCTIONAL CHARACTERIZATION OF A NOVEL CYTOCHROME ACTING AS A NANOWIRE
Publication . Fernandes, Ana Paula Nascimento; Salgueiro, Carlos; Paquete, Catarina
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.
Structural and functional characterization of the periplasmic cytochrome PpcA from Geobacter metallireducens
Publication . Portela, Maria do Pilar Castillo; Salgueiro, Carlos
The bacterium Geobacter metallireducens (Gm) is capable of transferring electrons to the cell’s
exterior and thus reduce extracellular electron acceptors. Because of this, Gm has been used for
electricity harvesting upon its association with electrodes and for bioremediation of contaminated
waters with Cr(VI) and U(VI), for example, which serve as extracellular acceptors. The triheme
c-type cytochrome PpcA from Gm is abundant in the periplasm and crucial for bridging the
electron transfer between the cytoplasm and the cell’s exterior. It shares 80% of identity with the
well-characterized PpcA from Geobacter sulfurreducens (Gs) but the functional properties,
namely the reduction potential and the hemes’ order of oxidation, are markedly different. In this
work, we have used nuclear magnetic resonance spectroscopy to structurally characterize PpcA
from Gm and to probe pH-linked conformational changes in the reduced and oxidized states. The
structural role of the highly conserved residue Val13 in the PpcA family of Gm and Gs was probed
by replacing it with alanine, isoleucine, serine and threonine. Replacement of Phe6 and Trp45 in
PpcA from Gm with the correspondent amino acids in PpcA from Gs – leucine and methionine
– has been achieved to probe their influence in the reduction potential. The obtained results
suggest that the structure in the reduced and oxidized states is conserved and similar to that of
PpcA from Gs, with localized differences in the polypeptide segments near hemes I and III. Val13
has been shown to be essential for the maintenance of a single heme core conformation. The
substitution of Phe6 and Trp45 yielded opposite effects on the cytochrome’s reduction potential
values, suggesting that these residues play different roles in the modulation of this property. These
observations emphasize the preponderant role of key residues in the structure of PpcA from Gm
and in the fine-tuning of its reduction potential values.
Characterization of extracellular electron transfer networks in Geobacter sulfurreducens, a key bacterium for bioremediation and bioenergy applications
Publication . Dantas, Joana Margarida Franco; Salgueiro, Carlos
Geobacter bacteria have awakened significantly attention because of their impact on natural environments and biotechnological applications that include the bioremediation of organic and inorganic contaminants, bioenergy production and bioelectronics. In addition to electron transfer towards extracellular terminal acceptors, Geobacter cells can also accept electrons from electrodes, in currentconsuming biofilms, a process that is currently explored in microbial electrosynthesis. These practical applications rely on an efficient transfer of electrons between the cell and its exterior, a process
designated extracellular electron transfer (EET). However, the precise mechanisms underlying EET processes are still under debate. Genetic and proteomics studies have identified several c-type cytochromes as key components for EET in G. sulfurreducens. These proteins are located at the innermembrane (IM), periplasm and outer-membrane (OM). Examples of such cytochromes include the IMassociated cytochrome MacA, periplasmic cytochromes PpcA-E and PccH, as well as, the OM cytochrome OmcF, which were studied in this Thesis.
Molecular interactions between PpcA-E and their putative redox partners, including a humic substance analogue molecule, MacA or PccH, were probed by NMR spectroscopy, stopped-flow kinetics and molecular docking. For the interacting pairs, their binding affinity was also determined by NMR chemical shift perturbation experiments. The results obtained showed that the interacting molecules establish reversible low-binding affinity complexes in specific regions of the proteins to warrant a rapid and selective electron transfer, a typical feature observed for electron transfer reactions between redox partners.
In addition, NMR spectroscopy was also used to determine the solution structure of OmcF in the reduced state, its pH-dependent conformational changes and backbone dynamics.
A biochemical and structural characterization of the cytochrome PccH was also carried out using circular dichroism, UV-visible and NMR spectroscopic techniques. The structure of PccH determined by X-ray crystallography showed that it is unique among the monoheme c-type cytochromes. The reduction potentials determined for PccH at different pH values by visible redox titrations are unusually low compared to those reported for other monoheme c-type cytochromes. Considering the structural and
functional features of PccH it was proposed that this protein represents a first characterized example of a new subclass of monoheme c-type cytochromes.
Overall, the results obtained constitute an important contribute to the current understanding of the G. sulfurreducens extracellular electron transfer mechanisms.
Characterization and interaction studies of triheme cytochromes from Geobacter: a contribution to the elucidation of extracellular electron transfer pathways
Publication . Ferreira, Marisa Raquel Martins de Brito; Salgueiro, Carlos
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.
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
3599-PPCDT
Número da atribuição
PTDC/BBB-BQB/3554/2014