A carregar...
Projeto de investigação
Sem título
Financiador
Autores
Publicações
Cytochromes in Geobacter: fostering sustainable sources of energy production
Publication . Castillo Portela, Maria do Pilar; Salgueiro, Carlos; Malvankar, Nikhil
Geobacter bacteria congregate interesting features for application in sustainability: they are able
to couple the oxidation of toxic organic compounds, such as toluene, to the reduction of Fe(III)
and toxic Mn(IV), Cr(III), U(VI). Furthermore, they can associate with electrodes in Microbial
Fuel Cells (MFC) to produce electrical energy. These features are possible because of the chain
of multiheme cytochromes poised at the different cellular compartments that relay the
electrons to the cell’s exterior – Extracellular Electron Transfer (EET). The applications are
promising but challenges still lay ahead, especially since the EET chain components and their
interactions are not well-characterized. This work focused on three main aspects of Geobacter
– exploration of long-range ET; assessment of redox partnership in the context of oxidative
stress; manipulation of the redox properties of cytochromes. In the long-range ET realm, we
studied the redox partnership of triheme periplasmic cytochromes PpcA-E with native OmcS
nanowires and determined for the first time that PpcA-E can directly provide reducing power
to OmcS, clarifying this ET route. We worked on the heterologous expression system in E. coli
for OmcZ and OmcS to, respectively, improve it and develop it to, in the future, facilitate
sample obtention for biochemical studies. In the context of oxidative stress, we determined
that PpcA-E provide reducing power to MacA peroxidase, specifically to its HP heme,
elucidating this redox partnership. The role of PpcA-family periplasmic cytochromes is pivotal
in various ET pathways and, as such, we studied how their redox properties can be
manipulated to fine tune the ET directionality of future Geobacter-engineered strains with
minimal components. Using protein engineering and PpcA from G. metallireducens as a
framework, we have shifted its midpoint reduction potential value 71% towards the one of
PpcA from G. sulfurreducens. These studies contributed to the elucidation of two redox
partnerships important in Geobacter’s ET chain, to the establishment of the foundations for the
heterologous overexpression of two nanowires, and to the creation of a mutants’ library of PpcA
periplasmic cytochromes, a pivotal component of the ET chain.
Biochemical characterization of the inner membrane cytochrome CbcL: a gate for extracellular electron transfer in Geobacter sulfurreducens
Publication . Antunes, Jorge Manuel Andrade; Morgado, Maria Leonor; Salgueiro, Carlos
Exoelectrogenic microorganisms are in the spotlight due to their particular respiratory mechanisms and potential application in several biotechnological fields, including bioremediation, bioenergy production and microbial electrosynthesis. All these emergent applications rely on the capability of exoelectrogens to perform extracellular electron transfer, a mechanism that allows the bacteria to transfer electrons to the cell’s exterior. These respiratory pathways encompass different multiheme cytochromes along the inner membrane, periplasmic space, and outer membrane. CbcL from Geobacter sulfurreducens is an inner membrane associated multiheme cytochrome that plays an essential role in the transfer of electrons to final electron acceptors with a low redox potential, as Fe(III) oxides and electrodes poised at -100 mV. CbcL has a membrane di-heme b-type cytochrome domain with six transmembrane helices, linked to a periplasmic cytochrome domain with nine c-type heme groups.
In this thesis, heterologous expression tests were performed for CbcL´s periplasmic domain. Its purification was also optimized by different types of chromatography and complementary spectroscopic techniques were used in its structural and functional characterization. Circular dichroism in the Far-UV region of the spectrum was used to determine the thermal stability of the cytochrome and predict its secondary structural elements. The protein was found to have a high percentage of disordered regions, as previously observed for other multiheme proteins. The reduction potentials of CbcL´s periplasmic domain were determined by potentiometric titrations followed by UV-visible spectroscopy at pH 7 and 8, and the obtained values allow a thermodynamically favorable transfer of electrons to its putative redox partners. Nuclear magnetic resonance was used to determine the spin state of the heme groups and to probe the biomolecular interactions between CbcL and the periplasmic triheme cytochrome PpcA. Crystallization tests were also carried out for future determination of CbcL´s periplasmic domain structure.
The results obtained in this thesis show for the first time how electrons are injected into the periplasm of Geobacter sulfurreducens, allowing their subsequent transfer to the cell’s exterior.
Molecular Mechanisms of Microbial Extracellular Electron Transfer
Publication . Paquete, Catarina M.; Morgado, Leonor; Salgueiro, Carlos A.; Louro, Ricardo O.; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; IMR Press Limited
Extracellular electron transfer is a key metabolic process of many organisms that enables them to exchange electrons with extracellular electron donors/acceptors. The discovery of organisms with these abilities and the understanding of their electron transfer processes has become a priority for the scientific and industrial community, given the growing interest on the use of these organisms in sustainable biotechnological processes. For example, in bioelectrochemical systems electrochemical active organisms can exchange electrons with an electrode, allowing the production of energy and added-value compounds, among other processes. In these systems, electrochemical active organisms exchange electrons with an electrode through direct or indirect mechanisms, using, in most cases, multiheme cytochromes. In numerous electroactive organisms, these proteins form a conductive pathway that allows electrons produced from cellular metabolism to be transferred across the cell surface for the reduction of an electrode, or vice-versa. Here, the mechanisms by which the most promising electroactive bacteria perform extracellular electron transfer will be reviewed, emphasizing the proteins involved in these pathways. The ability of some of the organisms to perform bidirectional electron transfer and the pathways used will also be highlighted.
Unidades organizacionais
Descrição
Palavras-chave
Contribuidores
Financiadores
Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
3599-PPCDT
Número da atribuição
PTDC/BIA-BQM/4967/2020