ITQB: IBN - Artigos em revista internacional com arbitragem científica
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- Crossing the wallPublication . Faustino, Marisa M.; Fonseca, Bruno M.; Costa, Nazua L.; Lousa, Diana; Louro, Ricardo O.; Paquete, Catarina M.; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); MDPI AGBioelectrochemical systems (BES) are emerging as a suite of versatile sustainable technologies to produce electricity and added‐value compounds from renewable and carbon‐neutral sources using electroactive organisms. The incomplete knowledge on the molecular processes that allow electroactive organisms to exchange electrons with electrodes has prevented their real‐world implementation. In this manuscript we investigate the extracellular electron transfer processes performed by the thermophilic Gram‐positive bacteria belonging to the Thermincola genus, which were found to produce higher levels of current and tolerate higher temperatures in BES than mesophilic Gram‐negative bacteria. In our study, three multiheme c‐type cytochromes, Tfer_0070, Tfer_0075, and Tfer_1887, proposed to be involved in the extracellular electron transfer pathway of T. ferri-acetica, were cloned and over‐expressed in E. coli. Tfer_0070 (ImdcA) and Tfer_1887 (PdcA) were purified and biochemically characterized. The electrochemical characterization of these proteins supports a pathway of extracellular electron transfer via these two proteins. By contrast, Tfer_0075 (CwcA) could not be stabilized in solution, in agreement with its proposed insertion in the pepti-doglycan wall. However, based on the homology with the outer‐membrane cytochrome OmcS, a structural model for CwcA was developed, providing a molecular perspective into the mechanisms of electron transfer across the peptidoglycan layer in Thermincola.
- Conjuring up a ghostPublication . Trindade, I B; Hernandez, G; Lebègue, E; Barrière, F; Cordeiro, T; Piccioli, M; Louro, R O; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); SpringerIron is a fundamental element for virtually all forms of life. Despite its abundance, its bioavailability is limited, and thus, microbes developed siderophores, small molecules, which are synthesized inside the cell and then released outside for iron scavenging. Once inside the cell, iron removal does not occur spontaneously, instead this process is mediated by siderophore-interacting proteins (SIP) and/or by ferric-siderophore reductases (FSR). In the past two decades, representatives of the SIP subfamily have been structurally and biochemically characterized; however, the same was not achieved for the FSR subfamily. Here, we initiate the structural and functional characterization of FhuF, the first and only FSR ever isolated. FhuF is a globular monomeric protein mainly composed by α-helices sheltering internal cavities in a fold resembling the "palm" domain found in siderophore biosynthetic enzymes. Paramagnetic NMR spectroscopy revealed that the core of the cluster has electronic properties in line with those of previously characterized 2Fe-2S ferredoxins and differences appear to be confined to the coordination of Fe(III) in the reduced protein. In particular, the two cysteines coordinating this iron appear to have substantially different bond strengths. In similarity with the proteins from the SIP subfamily, FhuF binds both the iron-loaded and the apo forms of ferrichrome in the micromolar range and cyclic voltammetry reveals the presence of redox-Bohr effect, which broadens the range of ferric-siderophore substrates that can be thermodynamically accessible for reduction. This study suggests that despite the structural differences between FSR and SIP proteins, mechanistic similarities exist between the two classes of proteins.
- Electrochemical properties of pH-dependent flavocytochrome c3 from Shewanella putrefaciens adsorbed onto unmodified and catechol-modified edge plane pyrolytic graphite electrodePublication . Lebègue, Estelle; Costa, Nazua L.; Fonseca, Bruno M.; Louro, Ricardo O.; Barrière, Frédéric; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); Elsevier Science B.V., Amsterdam.The electroactivity of adsorbed flavocytochrome c3, a tetraheme FAD-containing flavoenzyme isolated from the bacterium Shewanella putrefaciens, is investigated by cyclic voltammetry at an edge plane pyrolytic graphite electrode before and after modification with grafted catechol serving as an efficient pH sensor based on a redox readout. Flavocytochrome c3 adsorption onto the unmodified or modified electrode surface is successfully achieved by cyclic voltammetry (100 consecutive cycles) in a flavocytochrome c3 solution containing polymyxin as co-adsorbate. The immobilized flavocytochrome c3 retains its electrochemical activity and its catalytic fumarate reductase activity. The redox activity of the protein arises from its FAD and four hemes cofactors. The experiments evidence that the hemes' redox potential of flavocytochrome c3 from Shewanella putrefaciens, for which no crystal structure is yet available, depend on pH which is at variance with data from the other strains Shewanella frigidimarina or Shewanella oneidensis.
- Improvement of the electron transfer rate in Shewanella oneidensis MR-1 using a tailored periplasmic protein compositionPublication . Delgado, Veronica Palma; Paquete, Catarina M.; Sturm, Gunnar; Gescher, Johannes; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); Elsevier Science B.V., Amsterdam.Periplasmic c-type cytochromes are essential for the electron transport between the cytoplasmic membrane bound menquinol oxidase CymA and the terminal ferric iron reductase MtrABC in the outer membrane of Shewanella oneidensis cells. Either STC or FccA are necessary for periplasmic electron transfer. We followed the hypothesis that the elimination of potential competing reactions in the periplasm and the simultaneous overexpression of STC (cctA)could lead to an accelerated electron transfer to the cell surface. The genes nrfA, ccpA, napB and napA were replaced by cctA. This led to a 1.7-fold increased ferric iron reduction rate and a 23% higher current generation in a bioelectrochemical system. Moreover, the quadruple mutant had a higher periplasmic flavin content. Further deletion of fccA and its replacement by cctA resulted in a strain with ferric iron reduction rates similar to the wild type and a lower concentration of periplasmic flavin compared to the quadruple mutant. A transcriptomic analysis revealed that the quadruple mutant had a 3.7-fold higher cctA expression which could not be further increased by the replacement of fccA. This work indicates that a synthetic adaptation of Shewanella towards extracellular respiration holds potential for increased respiratory rates and consequently higher current densities.
- How thermophilic gram-positive organisms perform extracellular electron transferPublication . Costa, N. L.; Hermann, B.; Fourmond, V.; Faustino, M. M.; Teixeira, M.; Einsle, O.; Paquete, C. M.; Louro, R. O.; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); American Society for MicrobiologyExtracellular electron transfer is the key process underpinning the development of bioelectrochemical systems for the production of energy or addedvalue compounds. Thermincola potens JR is a promising Gram-positive bacterium to be used in these systems because it is thermophilic. In this paper, we describe the structural and functional properties of the nonaheme cytochrome OcwA, which is the terminal reductase of this organism. The structure of OcwA, determined at 2.2-Å resolution, shows that the overall fold and organization of the hemes are not related to other metal reductases and instead are similar to those of multiheme cytochromes involved in the biogeochemical cycles of nitrogen and sulfur. We show that, in addition to solid electron acceptors, OcwA can also reduce soluble electron shuttles and oxyanions. These data reveal that OcwA can work as a multipurpose respiratory enzyme allowing this organism to grow in environments with rapidly changing availability of terminal electron acceptors without the need for transcriptional regulation and protein synthesis.
- Optimizing Electroactive OrganismsPublication . Fonseca, Bruno M.; Silva, Luis; Trindade, Ines B.; Moe, Elin; Matias, Pedro M.; Louro, Ricardo O.; Paquete, Catarina M.; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); Frontiers MediaExtracellular electron transfer pathways allow bacteria to transfer electrons from the cellmetabolism to extracellular substrates, such as metal oxides in natural environmentsand electrodes in microbial electrochemical technologies (MET). Studies of electroactivemicroorganisms and mainly of Shewanella oneidensis MR-1 have demonstrated thatextracellular electron transfer pathways relies on several multiheme c-type cytochromes.The small tetraheme cytochrome c (STC) is highly conserved among Shewanellaspecies and is one of the most abundant cytochromes in the periplasmic space. Ittransfers electrons from the cell metabolism delivered by the inner-membrane tetrahemecytochrome CymA, to the porin-cytochrome complex MtrCAB in the outer-membrane,to reduce solid electron acceptors outside the cell, or electrodes in the case of MET.In this work knock-out strains of STC of S. oneidensis MR-1, expressing STC fromdistinct Shewanella species were tested for their ability to perform extracellular electrontransfer, allowing to explore the effect of protein mutations in living organisms. Thesestudies, complemented by a biochemical evaluation of the electron transfer properties ofthe individual proteins, revealed a considerable plasticity in the molecular componentsinvolved in extracellular electron transfer. The results of this work are pioneering andof significant relevance for future rational design of cytochromes in order to enhanceextracellular electron transfer and thus contribute to the practical implementation of MET.
- Structure and reactivity of a siderophore-interacting protein from the marine bacterium Shewanella reveals unanticipated functional versatilityPublication . Trindade, Inês B.; Silva, José M.; Fonseca, Bruno M.; Catarino, Teresa; Fujita, Masaki; Matias, Pedro M.; Moe, Elin; Louro, Ricardo O.; Instituto de Tecnologia Química e Biológica António Xavier (ITQB); DQ - Departamento de Química; ASBMB - American Society for Biochemistry and Molecular BiologySiderophores make iron accessible under iron-limited conditions and play a crucial role in the survival of microorganisms. Because of their remarkable metal-scavenging properties and ease in crossing cellular envelopes, siderophores hold great potential in biotechnological applications, raising the need for a deeper knowledge of the molecular mechanisms underpinning the siderophore pathway. Here, we report the structural and functional characterization of a siderophore-interacting protein from the marine bacterium Shewanella frigidimarina NCIBM400 (SfSIP). SfSIP is a flavin-containing ferric-siderophore reductase with FAD- and NAD(P)H-binding domains that have high homology with other characterized SIPs. However, we found here that it mechanistically departs from what has been described for this family of proteins. Unlike other FAD-containing SIPs, SfSIP did not discriminate between NADH and NADPH. Furthermore, SfSIP required the presence of the Fe 2+ -scavenger, ferrozine, to use NAD(P)H to drive the reduction of Shewanella-produced hydroxamate ferric-siderophores. Additionally, this is the first SIP reported that also uses a ferredoxin as electron donor, and in contrast to NAD(P)H, its utilization did not require the mediation of ferrozine, and electron transfer occurred at fast rates. Finally, FAD oxidation was thermodynamically coupled to deprotonation at physiological pH values, enhancing the solubility of ferrous iron. On the basis of these results and the location of the SfSIP gene downstream of a sequence for putative binding of aerobic respiration control protein A (ArcA), we propose that SfSIP contributes an additional layer of regulation that maintains cellular iron homeostasis according to environmental cues of oxygen availability and cellular iron demand.