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Fernandes, TM, Morgado L, Turner DL, Salgueiro CA.  2021.  Protein Engineering of Electron Transfer Components from Electroactive Geobacter Bacteria. Antioxidants. 10, Number 6 AbstractWebsite

Electrogenic 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.

Fernandes, AP, Nunes TC, Paquete CM, Salgueiro CA.  2017.  Interaction studies between periplasmic cytochromes provide insights into extracellular electron transfer pathways of Geobacter sulfurreducens. Biochemical Journal. 474:797–808., Number 5: Portland Press Limited AbstractWebsite

Accepted Manuscript online January 16, 2017.Geobacter bacteria usually prevail among other microorganisms in soils and sediments where Fe(III) reduction has a central role. This reduction is achieved by extracellular electron transfer (EET), where the electrons are exported from the interior of the cell to the surrounding environment. Periplasmic cytochromes play an important role in establishing an interface between inner and outer membrane electron transfer components. In addition, periplasmic cytochromes, in particular nanowire cytochromes that contain at least 12 haem groups, have been proposed to play a role in electron storage in conditions of an environmental lack of electron acceptors. Up to date, no redox partners have been identified in Geobacter sulfurreducens, and concomitantly, the EET and electron storage mechanisms remain unclear. In this work, NMR chemical shift perturbation measurements were used to probe for an interaction between the most abundant periplasmic cytochrome PpcA and the dodecahaem cytochrome GSU1996, one of the proposed nanowire cytochromes in G. sulfurreducens. The perturbations on the haem methyl signals of GSU1996 and PpcA showed that the proteins form a transient redox complex in an interface that involves haem groups from two different domains located at the C-terminal of GSU1996. Overall, the present study provides for the first time a clear evidence for an interaction between periplasmic cytochromes that might be relevant for the EET and electron storage pathways in G. sulfurreducens.1D, one-dimensional; CbcL, c- and b-type cytochrome for low potential; EET, extracellular electron transfer; HP, His-patch; ImcH, inner membrane c-type cytochrome; MacA, metal-reduction-associated cytochrome; NaPi, sodium phosphate; NBAF, acetate-fumarate medium; NMR, nuclear magnetic resonance; PpcA, periplasmic c-type cytochrome; SDS–PAGE, sodium dodecyl sulphate–polyacrylamide gel electrophoresis; STC, small tetrahaem cytochrome.

Fernandes, TM, Silva MA, Morgado L, Salgueiro CA.  2023.  Hemes on a string: insights on the functional mechanisms of PgcA from Geobacter sulfurreducens. Journal of Biological Chemistry. :105167. AbstractWebsite

Microbial extracellular reduction of insoluble compounds requires soluble electron shuttles that diffuse in the extracellular 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 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.

Fernandes, TM, Morgado L, Salgueiro CA, Turner DL.  2019.  Determination of the magnetic properties and orientation of the heme axial ligands of PpcA from G. metallireducens by paramagnetic NMR. Journal of Inorganic Biochemistry. 198:110718. AbstractWebsite

The rising interest in the use of Geobacter bacteria for biotechnological applications demands a deep understanding of how these bacteria are able to thrive in a variety of environments and perform extracellular electron transfer. The Geobacter metallireducens bacterium can couple the oxidation of a wide range of compounds to the reduction of several extracellular acceptors, including heavy metals, toxic organic compounds or electrode surfaces. The periplasmic c-type cytochrome PpcA from this bacterium is a member of a family composed of five periplasmic triheme cytochromes, which are important to bridge the electron transfer between the cytoplasm and the extracellular environment. To better understand the functional mechanism of PpcA it is essential to obtain structural data for this cytochrome. In this work, the geometry of the heme axial ligands, as well as the magnetic properties of the hemes were determined for the oxidized form of the cytochrome, using the 13C NMR chemical shifts of the heme α-substituents. The results were further compared with those previously obtained for the homologous cytochrome from Geobacter sulfurreducens. The orientations of the axial histidine planes and the magnetic properties of the hemes are conserved in both proteins. Overall, the results obtained allowed the definition of the orientation of the magnetic axes of PpcA from G. metallireducens, which will be used as constraints to assist the solution structure determination of the cytochrome in the oxidized form.

Fernandes, TM, Morgado L, Salgueiro CA.  2018.  Thermodynamic and functional characterization of the periplasmic triheme cytochrome PpcA from Geobacter metallireducens. Biochemical Journal. : Portland Press Limited AbstractWebsite

The Geobacter metallireducens bacterium can couple the oxidation of a wide range of compounds to the reduction of several extracellular electron acceptors, including pollutants or electrode surfaces for current production in microbial fuel cells. For these reasons, G. metallireducens are of interest for practical biotechnological applications. The use of such electron acceptors relies on a mechanism that permits electrons to be transferred to the cell exterior. The cytochrome PpcA from G. metallireducens is a member of a family composed by five periplasmic triheme cytochromes, which are important to bridge the electron transfer from the cytoplasmic donors to the extracellular acceptors. Using NMR and visible spectroscopic techniques, a detailed thermodynamic characterization of PpcA was obtained, including the determination of the heme reduction potentials and their redox and redox-Bohr interactions. These parameters revealed unique features for PpcA from G. metallireducens compared to other triheme cytochromes from different microorganisms, namely the less negative heme reduction potentials and concomitant functional working potential ranges. It was also shown that the order of oxidation of the hemes is pH independent, but the protein is designed to couple e-/H+ transfer exclusively at physiological pH.

Fernandes, TM, Folgosa F, Teixeira M, Salgueiro CA, Morgado L.  2021.  Structural and functional insights of GSU0105, a unique multiheme cytochrome from G. sulfurreducens. Biophysical Journal. AbstractWebsite

Geobacter sulfurreducens possesses over 100 cytochromes that assure an effective electron transfer to the cell exterior. The most abundant group of cytochromes in this microorganism is the PpcA family, composed of five periplasmic triheme cytochromes with high structural homology and identical heme coordination (His-His). GSU0105 is a periplasmic triheme cytochrome synthetized by G. sulfurreducens in Fe(III)-reducing conditions but is not present in cultures grown on fumarate. This cytochrome has a low sequence identity with the PpcA family cytochromes and a different heme coordination, based on the analysis of its amino acid sequence. In this work, amino acid sequence analysis, site-directed mutagenesis, and complementary biophysical techniques, including ultraviolet-visible, circular dichroism, electron paramagnetic resonance, and nuclear magnetic resonance spectroscopies, were used to characterize GSU0105. The cytochrome has a low percentage of secondary structural elements, with features of α-helices and β-sheets. Nuclear magnetic resonance shows that the protein contains three low-spin hemes (Fe(II), S = 0) in the reduced state. Electron paramagnetic resonance shows that, in the oxidized state, one of the hemes becomes high-spin (Fe(III), S = 5/2), whereas the two others remain low-spin (Fe(III), S = 1/2). The data obtained also indicate that the heme groups have distinct axial coordination. The apparent midpoint reduction potential of GSU0105 (−154 mV) is pH independent in the physiological range. However, the pH modulates the reduction potential of the heme that undergoes the low- to high-spin interconversion. The reduction potential values of cytochrome GSU0105 are more distinct compared to those of the PpcA family members, providing the protein with a larger functional working redox potential range. Overall, the results obtained, together with an amino acid sequence analysis of different multiheme cytochrome families, indicate that GSU0105 is a member of a new group of triheme cytochromes.

Fernandes, AP, Couto I, Morgado L, Londer YY, Salgueiro CA.  2008.  Isotopic labeling of c-type multiheme cytochromes overexpressed in E. coli. Protein Expression and Purification. 59(1):182-188. AbstractWebsite

Progresses made in bacterial genome sequencing show a remarkable profusion of multiheme c-type cytochromes in many bacteria, highlighting the importance of these proteins in different cellular events. However, the characterization of multiheme cytochromes has been significantly retarded by the numerous experimental challenges encountered by researchers who attempt to overexpress these proteins, especially if isotopic labeling is required. Here we describe a methodology for isotopic labeling of multiheme cytochromes c overexpressed in Escherichia coli, using the triheme cytochrome PpcA from Geobacter sulfurreducens as a model protein. By combining different strategies previously described and using E. coli cells containing the gene coding for PpcA and the cytochrome c maturation gene cluster, an experimental labeling methodology was developed that is based on two major aspects: (i) use of a two-step culture growth procedure, where cell growth in rich media was followed by transfer to minimal media containing 15N-labeled ammonium chloride, and (ii) incorporation of the heme precursor delta-aminolevulinic acid in minimal culture media. The yields of labeled protein obtained were comparable to those obtained for expression of PpcA in rich media. Proper protein folding and labeling were confirmed by UV–visible and NMR spectroscopy. To our knowledge, this is the first report of a recombinant multiheme cytochrome labeling and it represents a major breakthrough for functional and structural studies of multiheme cytochromes.

Ferreira, MR, Salgueiro CA.  2018.  Biomolecular Interaction Studies Between Cytochrome PpcA From Geobacter sulfurreducens and the Electron Acceptor Ferric Nitrilotriacetate (Fe-NTA). Frontiers in Microbiology. 9:2741. AbstractWebsite

Geobacter sulfurreducens is a dissimilatory metal-reducing bacterium that 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. In a previous work 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 nitrilotriacetic acid (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 natural abundance and 15N-enriched 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 provides for the first time a clear illustration of 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.

Ferreira, MR, Fernandes TM, Salgueiro CA.  2020.  Thermodynamic properties of triheme cytochrome PpcF from Geobacter metallireducens reveal unprecedented functional mechanism. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1861:148271., Number 11 AbstractWebsite

The bacterium Geobacter metallireducens is highly efficient in long-range extracellular electron transfer, a process that relies on an efficient bridging between the cytoplasmic electron donors and the extracellular acceptors. The periplasmic triheme cytochromes are crucial players in these processes and thus the understanding of their functional mechanism is crucial to elucidate the extracellular electron transfer processes in this microorganism. The triheme cytochrome PpcF from G. metallireducens has the lowest amino acid sequence identity with the remaining cytochromes from the PpcA-family of G. sulfurreducens and G. metallireducens, making it an interesting target for structural and functional studies. In this work, we performed a detailed functional and thermodynamic characterization of cytochrome PpcF by the complementary usage of NMR and visible spectroscopic techniques. The results obtained show that the heme reduction potentials are negative, different from each other and are also modulated by the redox and redox-Bohr interactions that assure unprecedented mechanistic features to the protein. The results showed that the order of oxidation of the hemes in cytochrome PpcF is maintained in the entire physiological pH range. The considerable separation of the hemes' redox potential values facilitates a sequential transfer within the chain of redox centers in PpcF, thus assuring electron transfer directionality to the electron acceptors.

Ferreira, MR, Dantas JM, Salgueiro CA.  2018.  The triheme cytochrome PpcF from Geobacter metallireducens exhibits distinct redox properties. FEBS Open Bio. , Number ja AbstractWebsite

Abstract Electrogenic bacteria, such as Geobacter, can couple the oxidation of carbon sources to the reduction of extracellular electron acceptors; such acceptors include toxic and radioactive metals, as well as electrode surfaces, making Geobacter a suitable candidate for applied use in bioremediation and bioenergy generation. Geobacter metallireducens is more promising in this regard than the better studied Geobacter sulfurreducens, as it has more efficient Fe (III) reduction rates and can respire nitrate to ammonia. The operon responsible for nitrate reductase activity in G. metallireducens includes the gene encoding the cytochrome PpcF, which was proposed to exchange electrons with nitrate reductase. In the present work, we perform a biochemical and biophysical characterization of PpcF. Spectroscopic techniques, including circular dichroism (CD), UV-visible, and nuclear magnetic resonance (NMR) revealed that the cytochrome is very stable (Tm > 85 °C), contains three low-spin hemes, and is diamagnetic (S=0) and paramagnetic (S=1/2) in the reduced and oxidized states, respectively. The NMR chemical shifts of the heme substituents were assigned and used to determine the heme core architecture of PpcF. Compared to the PpcA-family from G. sulfurreducens, the spatial disposition of the hemes is conserved, but the functional properties are clearly distinct. In fact, potentiometric titrations monitored by UV-visible absorption reveal that the reduction potential values of PpcF are significantly less negative (-56 and -64 mV, versus the normal hydrogen electrode at pH 7.0 and 8.0, respectively). NMR redox titrations showed that the order of oxidation of the hemes is IV-I-III a feature not observed for G. sulfurreducens. The different redox properties displayed by PpcF, including the small redox-Bohr effect and low reduction potential value of heme IV, were structurally rationalized and attributed to the lower number of positively charged residues located in the vicinity of heme IV. Overall, the redox features of PpcF suggest that biotechnological applications of G. metallireducens may require less negative working functional redox windows than those using by G. sulfurreducens.

Ferreira, MR, Dantas JM, Salgueiro CA.  2017.  Molecular interactions between Geobacter sulfurreducens triheme cytochromes and the electron acceptor Fe(iii) citrate studied by NMR. Dalton Trans.. 46:2350-2359.: The Royal Society of Chemistry AbstractWebsite

Proteomic and genetic studies have identified a family of five triheme cytochromes (PpcA-E) that are essential in the iron respiratory pathways of Geobacter sulfurreducens. These include the reduction of Fe(iii) soluble chelated forms or Fe(iii) oxides{,} which can be used as terminal acceptors by G. sulfurreducens. The relevance of these cytochromes in the respiratory pathways of soluble or insoluble forms of iron is quite distinct. In fact{,} while PpcD had a higher abundance in the Fe(iii) oxides supplanted G. sulfurreducens cultures{,} PpcA{,} PpcB and PpcE were important in Fe(iii) citrate supplanted cultures. Based on these observations we probed the molecular interactions between these cytochromes and Fe(iii) citrate by NMR spectroscopy. NMR spectra were recorded for natural abundance and 15N-enriched PpcA{,} PpcB or PpcE samples at increasing amounts of Fe(iii) citrate. The addition of this molecule caused pronounced perturbations on the line width of the protein{'}s NMR signals{,} which were used to map the interaction region between each cytochrome and the Fe(iii) citrate molecule. The perturbations on the NMR signals corresponding to the backbone NH and heme methyl substituents showed that complex interfaces consist of a well-defined patch{,} which surrounds the more solvent-exposed heme IV methyl groups in each cytochrome. Overall{,} this study provides for the first time a clear illustration of the formation of an electron transfer complex between Fe(iii) citrate and G. sulfurreducens triheme cytochromes{,} shown to be crucial in this respiratory pathway.

Ferreira, MR, Fernandes TM, Turner DL, Salgueiro CA.  2022.  Molecular geometries of the heme axial ligands from the triheme cytochrome PpcF from Geobacter metallireducens reveal a conserved heme core architecture. Archives of Biochemistry and Biophysics. 723:109220. AbstractWebsite

Electroactive Geobacter bacteria can perform extracellular electron transfer and present a wide metabolic versatility. These bacteria reduce organic, toxic and radioactive compounds, and produce electric current while interacting with electrodes, making them interesting targets for numerous biotechnological applications. Their global electrochemical responses rely on an efficient interface between the inside and the cell's exterior, which is driven by the highly abundant periplasmic triheme PpcA-family cytochromes. The functional features of these cytochromes have been studied in G. sulfurreducens and G. metallireducens, and although they share a high degree of structural homology and sequence identity, their properties are quite distinct. In this work, the heme axial ligand geometries and the magnetic properties of PpcF from G. metallireducens were determined. The data obtained constitute important constraints for the determination of its solution structure in the oxidized state and indicate that the (i) heme core architecture; (ii) axial ligands geometries and (iii) magnetic properties of the cytochrome are conserved compared to the other members of the PpcA-families. Furthermore, the results also indicate that the heme arrangement is crucial to maintain an intrinsic regulation of the protein's redox properties and hence its electron transfer efficiency and functionality.

Fonseca, BM, Saraiva IH, Paquete CM, Soares CM, Pacheco I, Salgueiro CA, Louro RO.  2009.  The tetraheme cytochrome from Shewanella oneidensis MR-1 shows thermodynamic bias for functional specificity of the hemes. Journal of Biological Inorganic Chemistry. 14(3):375-385. AbstractWebsite

Bacteria of the genus Shewanella contain an abundant small tetraheme cytochrome in their periplasm when growing anaerobically. Data collected for the protein isolated from S. oneidensis MR-1 and S. frigidimarina indicate differences in the order of oxidation of the hemes. A detailed thermodynamic characterization of the cytochrome from S. oneidensis MR-1 in the physiological pH range was performed, with data collected in the pH range 5.5-9.0 from NMR experiments using partially oxidized samples and from redox titrations followed by visible spectroscopy. These data allow the parsing of the redox and redox-protonation interactions that occur during the titration of hemes. The results show that electrostatic effects dominate the heme-heme interactions, in agreement with modest redox-linked structural modifications, and protonation has a considerable influence on the redox properties of the hemes in the physiological pH range. Theoretical calculations using the oxidized and reduced structures of this protein reveal that the bulk redox-Bohr effect arises from the aggregate fractional titration of several of the heme propionates. This detailed characterization of the thermodynamic properties of the cytochrome shows that only a few of the multiple microscopic redox states that the protein can access are significantly populated at physiological pH. On this basis a functional pathway for the redox activity of the small tetraheme cytochrome from S. oneidensis MR-1 is proposed, where reduction and protonation are thermodynamically coupled in the physiological range. The differences between the small tetraheme cytochromes from the two organisms are discussed in the context of their biological role.

Fonseca, BM, Paquete CM, Salgueiro CA, Louro RO.  2012.  The role of intramolecular interactions in the functional control of multiheme cytochromes c. FEBS Lett. 586(5):504-509. AbstractWebsite

Detailed thermodynamic and structural data measured in soluble monomeric multiheme cytochromes c provided the basis to investigate the functional significance of interactions between redox co-factors. The steep decay of intramolecular interactions with distance means that close proximity of the redox centers is necessary to modulate the intrinsic reduction potentials in a significant way. This ensures selection of specific populations during redox activity in addition to maintaining fast intramolecular electron transfer. Therefore, intramolecular interactions between redox co-factors play an important role in establishing the biological function of the protein by controlling how electrons flow through and are distributed among the co-factors.