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Projeto de investigação
Cabos de alimentação bióticos– Explorando novas vias de transferência de electrões por filamentos bacterianos condutores nas áreas da bioenergia e da bioelectrónica
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A Biochemical Deconstruction-Based Strategy to Assist the Characterization of Bacterial Electric Conductive Filaments
Publication . Silva, Marta A.; Fernandes, Ana P.; Turner, David L.; Salgueiro, Carlos A.; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); MDPI - Multidisciplinary Digital Publishing Institute
Periplasmic nanowires and electric conductive filaments made of the polymeric assembly of c-type cytochromes from Geobacter sulfurreducens bacterium are crucial for electron storage and/or extracellular electron transfer. The elucidation of the redox properties of each heme is fundamental to the understanding of the electron transfer mechanisms in these systems, which first requires the specific assignment of the heme NMR signals. The high number of hemes and the molecular weight of the nanowires dramatically decrease the spectral resolution and make this assignment extremely complex or unattainable. The nanowire cytochrome GSU1996 (~42 kDa) is composed of four domains (A to D) each containing three c-type heme groups. In this work, the individual domains (A to D), bi-domains (AB, CD) and full-length nanowire were separately produced at natural abundance. Sufficient protein expression was obtained for domains C (~11 kDa/three hemes) and D (~10 kDa/three hemes), as well as for bi-domain CD (~21 kDa/six hemes). Using 2D-NMR experiments, the assignment of the heme proton NMR signals for domains C and D was obtained and then used to guide the assignment of the corresponding signals in the hexaheme bi-domain CD. This new biochemical deconstruction-based procedure, using nanowire GSU1996 as a model, establishes a new strategy to functionally characterize large multiheme cytochromes.
Widespread extracellular electron transfer pathways for charging microbial cytochrome OmcS nanowires via periplasmic cytochromes PpcABCDE
Publication . Portela, Pilar C.; Shipps, Catharine C.; Shen, Cong; Srikanth, Vishok; Salgueiro, Carlos A.; Malvankar, Nikhil S.; Faculdade de Ciências e Tecnologia (FCT); UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; Nature Portfolio
Extracellular electron transfer (EET) via microbial nanowires drives globally-important environmental processes and biotechnological applications for bioenergy, bioremediation, and bioelectronics. Due to highly-redundant and complex EET pathways, it is unclear how microbes wire electrons rapidly (>106s−1) from the inner-membrane through outer-surface nanowires directly to an external environment despite a crowded periplasm and slow (<105s−1) electron diffusion among periplasmic cytochromes. Here, we show that Geobacter sulfurreducens periplasmic cytochromes PpcABCDE inject electrons directly into OmcS nanowires by binding transiently with differing efficiencies, with the least-abundant cytochrome (PpcC) showing the highest efficiency. Remarkably, this defined nanowire-charging pathway is evolutionarily conserved in phylogenetically-diverse bacteria capable of EET. OmcS heme reduction potentials are within 200 mV of each other, with a midpoint 82 mV-higher than reported previously. This could explain efficient EET over micrometres at ultrafast (<200 fs) rates with negligible energy loss. Engineering this minimal nanowire-charging pathway may yield microbial chassis with improved performance.
Tethered heme domains in a triheme cytochrome allow for increased electron transport distances
Publication . Nash, Benjamin W.; Fernandes, Tomás M.; Burton, Joshua A.J.; Morgado, Leonor; van Wonderen, Jessica H.; Svistunenko, Dimitri A.; Edwards, Marcus J.; Salgueiro, Carlos A.; Butt, Julea N.; Clarke, Thomas A.; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; Faculdade de Ciências e Tecnologia (FCT); Wiley-Blackwell
Decades of research describe myriad redox enzymes that contain cofactors arranged in tightly packed chains facilitating rapid and controlled intra-protein electron transfer. Many such enzymes participate in extracellular electron transfer (EET), a process which allows microorganisms to conserve energy in anoxic environments by exploiting mineral oxides and other extracellular substrates as terminal electron acceptors. In this work, we describe the properties of the triheme cytochrome PgcA from Geobacter sulfurreducens. PgcA has been shown to play an important role in EET but is unusual in containing three CXXCH heme binding motifs that are separated by repeated (PT)x motifs, suggested to enhance binding to mineral surfaces. Using a combination of structural, electrochemical, and biophysical techniques, we experimentally demonstrate that PgcA adopts numerous conformations stretching as far as 180 Å between the ends of domains I and III, without a tightly packed cofactor chain. Furthermore, we demonstrate a distinct role for its domain III as a mineral reductase that is recharged by domains I and II. These findings show PgcA to be the first of a new class of electron transfer proteins, with redox centers separated by some nanometers but tethered together by flexible linkers, facilitating electron transfer through a tethered diffusion mechanism rather than a fixed, closely packed electron transfer chain.
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Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
Concurso para Financiamento de Projetos de Investigação Científica e Desenvolvimento Tecnológico em Todos os Domínios Científicos - 2020
Número da atribuição
PTDC/BIA-BQM/4967/2020