insights on the functional mechanisms of PgcA from Geobacter sulfurreducens

Funding Information: This work was supported by Fundação para a Ciência e Tecnologia (FCT) through the following grants: SFRH/BD/145039/2019 (T. M. F.), EXPL/BIA-BQM/0770/2021 (L. M.) and PTDC/BIABQM/4967/2020 (C. A. S.). This work was also supported by national funds from FCT in the scope of the projects (i) UIDP/04378/2020 and UIDB/04378/2020 (Research Unit on Applied Molecular Biosciences – UCIBIO) and (ii) LA/P/0140/2020 (Associate Laboratory Institute for Health and Bioeconomy – i4HB). The NMR spectrometers are part of the National NMR Network and are supported by FCT ( ROTEIRO/0031/2013 and PINFRA/22161/2016 ) cofounded by FEDER through COMPETE 2020, POCI, PORL and FCT through PIDDAC. Funding Information: Elisabete Ferreira (BioLab, UCIBIO, FCT-NOVA) is acknowledged for the technical assistance with the circular dichroism and differential scanning calorimetry experiments. T. M. F. L. M. and C. A. S. conceptualization; T. M. F. M. A. S. L. M. and C. A. S. investigation; T. M. F. M. A. S. L. M. formal analysis; T. M. F. writing–original draft; T. M. F. M. A. S. L. M. and C. A. S. writing–review & editing; T. M. F. visualization; L. M. and C. A. S. supervision; L. M. and C. A. S. funding acquisition; C. A. S. project administration. This work was supported by Fundação para a Ciência e Tecnologia (FCT) through the following grants: SFRH/BD/145039/2019 (T. M. F.), EXPL/BIA-BQM/0770/2021 (L. M.) and PTDC/BIABQM/4967/2020 (C. A. S.). This work was also supported by national funds from FCT in the scope of the projects (i) UIDP/04378/2020 and UIDB/04378/2020 (Research Unit on Applied Molecular Biosciences – UCIBIO) and (ii) LA/P/0140/2020 (Associate Laboratory Institute for Health and Bioeconomy – i4HB). The NMR spectrometers are part of the National NMR Network and are supported by FCT (ROTEIRO/0031/2013 and PINFRA/22161/2016) cofounded by FEDER through COMPETE 2020, POCI, PORL and FCT through PIDDAC. Publisher Copyright: © 2023 The AuthorsMicrobial extracellular reduction of insoluble compounds requires soluble electron shuttles that diffuse in the environment, freely diffusing cytochromes, or direct contact with cellular conductive appendages that release or harvest electrons to assure a continuous balance between cellular requirements and environmental conditions. In this work, we produced and characterized the three cytochrome domains of PgcA, an extracellular triheme cytochrome that contributes to Fe(III) and Mn(IV) oxides reduction in Geobacter sulfurreducens. The three monoheme domains are structurally homologous, but their heme groups show variable axial coordination and reduction potential values. Electron transfer experiments monitored by NMR and visible spectroscopy show the variable extent to which the domains promiscuously exchange electrons while reducing different electron acceptors. The results suggest that PgcA is part of a new class of cytochromes - microbial heme-tethered redox strings - that use low-complexity protein stretches to bind metals and promote intra- and intermolecular electron transfer events through its cytochrome domains.publishersversionpublishe

Biochemical Characterization of Cytochrome CbcL

project LA/P/0140/2020 (i4HB). Publisher Copyright: Copyright © 2022 Antunes, Silva, Salgueiro and Morgado.Exoelectrogenic microorganisms are in the spotlight due to their unique respiratory mechanisms and potential applications in distinct biotechnological fields, including bioremediation, bioenergy production and microbial electrosynthesis. These applications rely on the capability of these microorganisms to perform extracellular electron transfer, a mechanism that allows the bacteria to transfer electrons to the cell’s exterior by establishing functional interfaces between different multiheme cytochromes at the inner membrane, periplasmic space, and outer membrane. The multiheme cytochrome CbcL from Geobacter sulfurreducens is associated to the inner membrane and 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 transmembranar di-heme b-type cytochrome domain with six helices, linked to a periplasmic cytochrome domain with nine c-type heme groups. The complementary usage of ultraviolet-visible, circular dichroism and nuclear magnetic resonance permitted the structural and functional characterization of CbcL’s periplasmic domain. The protein was found to have a high percentage of disordered regions and its nine hemes are low-spin and all coordinated by two histidine residues. The apparent midpoint reduction potential of the CbcL periplasmic domain was determined, suggesting a thermodynamically favorable transfer of electrons to the putative redox partner in the periplasm − the triheme cytochrome PpcA. The establishment of a redox complex between the two proteins was confirmed by probing the electron transfer reaction and the molecular interactions between CbcL and PpcA. The results obtained show for the first time how electrons are injected into the periplasm of Geobacter sulfurreducens for subsequent transfer to the cell’s exterior.publishersversionpublishe

The Importance of Multiheme Cytochromes

Publisher Copyright: Copyright: © 2022 The Author(s)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.publishersversionpublishe

Protein engineering of electron transfer components from electroactive geobacter bacteria

SFRH/BD/145039/2019 SFRH/BPD/114848/2016 PTDC/BIA-BQM/31981/2017 UIDP/04378/2020 UIDB/04378/2020Electrogenic microorganisms possess unique redox biological features, being capable of transferring electrons to the cell exterior and converting highly toxic compounds into nonhazardous forms. These microorganisms have led to the development of Microbial Electrochemical Technologies (METs), which include applications in the fields of bioremediation and bioenergy production. The optimization of these technologies involves efforts from several different disciplines, ranging from microbiology to materials science. Geobacter bacteria have served as a model for understanding the mechanisms underlying the phenomenon of extracellular electron transfer, which is highly dependent on a multitude of multiheme cytochromes (MCs). MCs are, therefore, logical targets for rational protein engineering to improve the extracellular electron transfer rates of these bacteria. However, the presence of several heme groups complicates the detailed redox characterization of MCs. In this Review, the main characteristics of electroactive Geobacter bacteria, their potential to develop microbial electrochemical technologies and the main features of MCs are initially highlighted. This is followed by a detailed description of the current methodologies that assist the characterization of the functional redox networks in MCs. Finally, it is discussed how this information can be explored to design optimal Geobacter-mutated strains with improved capabilities in METs.publishersversionpublishe

Biomolecular Interaction Studies Between Cytochrome PpcA From Geobacter sulfurreducens and the Electron Acceptor Ferric Nitrilotriacetate (Fe-NTA)

Geobacter sulfurreducens bacterium exhibits an enormous respiratory versatility, including the utilization of several toxic and radioactive metals as electron acceptors. This versatility is also replicated in the capability of the most abundant cytochrome in G. sulfurreducens, the periplasmic triheme cytochrome PpcA, to reduce uranium, chromium and other metal ions. From all possible electron transfer pathways in G. sulfurreducens, those involved in the iron reduction are the best characterized to date. Previously, we provided structural evidence for the complex interface established between PpcA and the electron acceptor Fe(III)-citrate. However, genetic studies suggested that this acceptor is mainly reduced by outer membrane cytochomes. In the present work, we used UV-visible measurements to demonstrate that PpcA is able to directly reduce the electron acceptor ferric nitrilotriacetate (Fe-NTA), a more outer membrane permeable iron chelated form. In addition, the molecular interactions between PpcA and Fe-NTA were probed by Nuclear Magnetic Resonance (NMR) spectroscopy. The NMR spectra obtained for PpcA samples in the absence and presence of Fe-NTA showed that the interaction is reversible and encompasses a positively charged surface region located in the vicinity of the heme IV. Overall, the study elucidates the formation of an electron transfer complex between PpcA and a readily outer-membrane permeable iron chelated form. The structural and functional relationships obtained explain how a single cytochrome is designed to effectively interact with a wide range of G. sulfurreducens electron acceptors, a feature that can be explored for optimal bioelectrochemical applications

the redox network between MacA peroxidase and triheme periplasmic cytochromes

This work was supported by Fundação para a Ciência e Tecnologia (FCT – Portugal) through the following grants: PTDC/BIA-BQM/4967/2020 (to CS), 2020.04717.BD (to PP), EXPL/BIA-BQM/0770/2021 (to LM), UIDP/04378/2020 and UIDB/04378/2020 (UCIBIO), LA/P/0140/2020 (Associate Laboratory i4HB), and Deutsche Forschungsgemeinschaft (CRC 1381, project ID 403222702; to OE). The NMR spectrometers are part of the National NMR Network (PTNMR) and are supported by FCT-MCTES (ROTEIRO/0031/2013 – PINFRA/22161/2016) co-funded by FEDER through COMPETE 2020, POCI, and PORL and FCT through PIDDAC. Publisher Copyright: Copyright © 2023 Portela, Morgado, Silva, Denkhaus, Einsle and Salgueiro.The recent reclassification of the strict anaerobe Geobacter sulfurreducens bacterium as aerotolerant brought attention for oxidative stress protection pathways. Although the electron transfer pathways for oxygen detoxification are not well established, evidence was obtained for the formation of a redox complex between the periplasmic triheme cytochrome PpcA and the diheme cytochrome peroxidase MacA. In the latter, the reduction of the high-potential heme triggers a conformational change that displaces the axial histidine of the low-potential heme with peroxidase activity. More recently, a possible involvement of the triheme periplasmic cytochrome family (PpcA-E) in the protection from oxidative stress in G. sulfurreducens was suggested. To evaluate this hypothesis, we investigated the electron transfer reaction and the biomolecular interaction between each PpcA-E cytochrome and MacA. Using a newly developed method that relies on the different NMR spectral signatures of the heme proteins, we directly monitored the electron transfer reaction from reduced PpcA-E cytochromes to oxidized MacA. The results obtained showed a complete electron transfer from the cytochromes to the high-potential heme of MacA. This highlights PpcA-E cytochromes’ efficient role in providing the necessary reducing power to mitigate oxidative stress situations, hence contributing to a better knowledge of oxidative stress protection pathways in G. sulfurreducens.publishersversionpublishe

Alluvial xenotime and heavy minerals assemblage from the northern edge of Nisa-Albuquerque Batholith, eastern Portugal : provenance and geochemical implications

Alluvial xenotime and heavy minerals assemblage from the northern edge of Nisa-Albuquerque Batholith, eastern Portugal : provenance and geochemical implications / Rute Salgueiro... [et al.]. - Amsterdam : Elsevier, 2014. - [18] p. : il., 13 figuras e 8 tabelas ; 30 cm The xenotime-bearing heavy mineral assemblages present in the alluvial samples from Vila Velha de Ródão, Nisa, Póvoa e Meadas and Sto. António das Areias, localized in the northern edge of Nisa-Albuquerque Batholith, Eastern Portugal, were studied in detail. The insights for mineral provenance came from the analysis of the drainage network and from the composition of the regional alluvial samples and outcropping lithologies. Since xenotime is a mineral with economic interest, the motivation of the current work is its contribution for exploration studies, considering the potential information contained in the alluvial samples. In the study region, the alluvial heavy minerals and their morphology reflect clearly the mineralogy and relative distance to their source, respectively. In alluvial samples with direct provenance from the Nisa granite (late Carboniferous), the increase in xenotime concentration and decrease in monazite, apatite, zircon, ilmenite and iron oxide concentration, from W (Nisa) to E (Sto. António das Areias), matches the increase in P2O5 and decrease in REE, CaO, Zr, TiO2, and Fe2O3 contents in granite rocks. The geochemical signature of xenotime studied reveals an igneous source, characterized by the characteristic strong Eu negative anomaly in REE patterns. Their YPO4 values (72–78 mol%) are similar to xenotime from Erzgebirge (Germany) granites. The high values of Y/Ho (50–71) and, in some cases, downward kinks at Ho in REE plots, suggest generation in late stages of crystallization and association with a siliceous evolved magmatic system. The slight decrease in HREE contents in xenotime, from W to E, and Ho anomalies in the normalized REE pattern are similar to those identified in the Nisa granite, which supports the sourcing of xenotime from these granitic rocks. The genetic development of the batholith chemical zonation (more evolved to E) seems to have favoured the generation of xenotime instead of apatite, monazite and zircon, in the eastern end area (Sto. António das Areias). Supporting the geochemical signature, the alluvial zircons also display morphological typologies that are compatible with provenance from peraluminous granites formed at temperatures of 600–700 °C, possibly from the Nisa granite. Since all xenotime grains show the same magmatic affinity, this leads to the hypothesis that the xenotime from the Vila Velha de Ródão sample, with provenance from Cenozoic sedimentary rocks, has been transported to this northern area, after been disaggregated from Nisa batholith granites, most probably by braided anastomosing and erratically sandy channels, that explains its actual position in the opposite bank of the Tagus river. This phosphate has been deposited with other sediments in tectonic depressions and subsequently included in the formation of sedimentary rocks. The greater development of Ca, Th and U phosphate inclusions/substitutions in these xenotime grains can be explained by the chemical mobility provided during all the geological processes

Modulation of the Redox Potential and Electron/Proton Transfer Mechanisms in the Outer Membrane Cytochrome OmcF From Geobacter sulfurreducens

PD/00193/2012 UID/FIS/00068/2019 PTDC/BBBBQB/3554/2014 PTDC/BIA-BQM/31981/2017 PD/BD/114445/2016 UID/Multi/04378/2019 ROTEIRO/0031/2013 -PINFRA/22161/2016The monoheme outer membrane cytochrome F (OmcF) from Geobacter sulfurreducens plays an important role in Fe(III) reduction and electric current production. The electrochemical characterization of this cytochrome has shown that its redox potential is modulated by the solution pH (redox-Bohr effect) endowing the protein with the necessary properties to couple electron and proton transfer in the physiological range. The analysis of the OmcF structures in the reduced and oxidized states showed that with the exception of the side chain of histidine 47 (His47), all other residues with protonatable side chains are distant from the heme iron and, therefore, are unlikely to affect the redox potential of the protein. The protonatable site at the imidazole ring of His47 is in the close proximity to the heme and, therefore, this residue was suggested as the redox-Bohr center. In the present work, we tested this hypothesis by replacing the His47 with non-protonatable residues (isoleucine – OmcFH47I and phenylalanine – OmcFH47F). The structure of the mutant OmcFH47I was determined by X-ray crystallography to 1.13 Å resolution and showed only minimal changes at the site of the mutation. Both mutants were 15N-labeled and their overall folding was confirmed to be the same as the wild-type by NMR spectroscopy. The pH dependence of the redox potential of the mutants was measured by cyclic voltammetry. Compared to the wild-type protein, the magnitude of the redox-Bohr effect in the mutants was smaller, but not fully abolished, confirming the role of His47 on the pH modulation of OmcF’s redox potential. However, the pH effect on the heme substituents’ NMR chemical shifts suggested that the heme propionate P13 also contributes to the overall redox-Bohr effect in OmcF. In physiological terms, the contribution of two independent acid–base centers to the observed redox-Bohr effect confers OmcF a higher versatility to environmental changes by coupling electron/proton transfer within a wider pH range.publishersversionpublishe

Biochemical, Biophysical, and Structural Analysis of an Unusual DyP from the Extremophile Deinococcus radiodurans

Funding Information: This research was funded by FCT, Fundação para a Ciência e a Tecnologia, I.P., through MOSTMICRO-ITQB R&D Unit (UIDB/04612/2020, UIDP/04612/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020), with national funds through FCT, Fundação para a Ciência e a Tecnologia (PTDC/BBBEBB/0122/2014, IF/00710/2014, PTDC/BII-BBF/29564/2017 and PTDC/BIA-BFS/31026/2017), post doc fellowship SFRH/BPD/97493/2013 (EM), and PhD fellowship COVID/BD/152598/2022 (BS). Funding is also acknowledged for the TIMB3, European Union’s Horizon 2020 research and innovation program, under grant agreement No. 810856. Publisher Copyright: © 2024 by the authors.Dye-decolorizing peroxidases (DyPs) are heme proteins with distinct structural properties and substrate specificities compared to classical peroxidases. Here, we demonstrate that DyP from the extremely radiation-resistant bacterium Deinococcus radiodurans is, like some other homologues, inactive at physiological pH. Resonance Raman (RR) spectroscopy confirms that the heme is in a six-coordinated-low-spin (6cLS) state at pH 7.5 and is thus unable to bind hydrogen peroxide. At pH 4.0, the RR spectra of the enzyme reveal the co-existence of high-spin and low-spin heme states, which corroborates catalytic activity towards H2O2 detected at lower pH. A sequence alignment with other DyPs reveals that DrDyP possesses a Methionine residue in position five in the highly conserved GXXDG motif. To analyze whether the presence of the Methionine is responsible for the lack of activity at high pH, this residue is substituted with a Glycine. UV-vis and RR spectroscopies reveal that the resulting DrDyPM190G is also in a 6cLS spin state at pH 7.5, and thus the Methionine does not affect the activity of the protein. The crystal structures of DrDyP and DrDyPM190G, determined to 2.20 and 1.53 Å resolution, respectively, nevertheless reveal interesting insights. The high-resolution structure of DrDyPM190G, obtained at pH 8.5, shows that one hydroxyl group and one water molecule are within hydrogen bonding distance to the heme and the catalytic Asparagine and Arginine. This strong ligand most likely prevents the binding of the H2O2 substrate, reinforcing questions about physiological substrates of this and other DyPs, and about the possible events that can trigger the removal of the hydroxyl group conferring catalytic activity to DrDyP.publishersversionpublishe

Biological Assays and Chemical Composition of Volatile Oils of Bupleurum fruticosum L. (Apiaceae)

The composition of supercritical CO 2 extracts and essential oils obtained by hydrodistillation of Bupleurum fruticosum L., growing spontaneously in Italy and Portugal, and its antifungal activity is reported. The collected extracts were analyzed by GC-FID and GC-MS methods. The minimal inhibitory concentration (MIC) and the minimal lethal concentration (MLC) were used to evaluate the antifungal activity of the oils against Candida albicans, C. tropicalis, C. krusei, C. guillermondii, C. parapsilosis, Cryptococcus neoformans, Trichophyton rubrum, T. mentagrophytes, Microsporum canis, M. gypseum, Epidermophyton floccosum, Aspergillus niger, A. fumigatus and A. flavus