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M
Silva, MA, Salgueiro CA.  2021.  Multistep Signaling in Nature: A Close-Up of Geobacter Chemotaxis Sensing. International Journal of Molecular Sciences. 22, Number 16 AbstractWebsite

Environmental changes trigger the continuous adaptation of bacteria to ensure their survival. This is possible through a variety of signal transduction pathways involving chemoreceptors known as methyl-accepting chemotaxis proteins (MCP) that allow the microorganisms to redirect their mobility towards favorable environments. MCP are two-component regulatory (or signal transduction) systems (TCS) formed by a sensor and a response regulator domain. These domains synchronize transient protein phosphorylation and dephosphorylation events to convert the stimuli into an appropriate cellular response. In this review, the variability of TCS domains and the most common signaling mechanisms are highlighted. This is followed by the description of the overall cellular topology, classification and mechanisms of MCP. Finally, the structural and functional properties of a new family of MCP found in Geobacter sulfurreducens are revisited. This bacterium has a diverse repertoire of chemosensory systems, which represents a striking example of a survival mechanism in challenging environments. Two G. sulfurreducens MCP—GSU0582 and GSU0935—are members of a new family of chemotaxis sensor proteins containing a periplasmic PAS-like sensor domain with a c-type heme. Interestingly, the cellular location of this domain opens new routes to the understanding of the redox potential sensing signaling transduction pathways.

Salgueiro, CA, Dantas JM.  2016.  Multiheme Cytochromes. Multiheme Cytochromes. :1–39., Berlin, Heidelberg: Springer Berlin Heidelberg Abstract

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Paquete, CM, Morgado L, Salgueiro CA, Louro RO.  2022.  Molecular Mechanisms of Microbial Extracellular Electron Transfer: The Importance of Multiheme Cytochromes, 2022-06-27. FBL. 27(6) AbstractWebsite

Extracellular electron transfer is a key metabolic process of many organismsthat enables them to exchange electrons with extracellular electrondonors/acceptors. The discovery of organisms with these abilities and theunderstanding of their electron transfer processes has become a priority for thescientific and industrial community, given the growing interest on the use ofthese organisms in sustainable biotechnological processes. For example, inbioelectrochemical systems electrochemical active organisms can exchangeelectrons with an electrode, allowing the production of energy and added-valuecompounds, among other processes. In these systems, electrochemical activeorganisms exchange electrons with an electrode through direct or indirectmechanisms, using, in most cases, multiheme cytochromes. In numerouselectroactive organisms, these proteins form a conductive pathway that allowselectrons produced from cellular metabolism to be transferred across the cellsurface for the reduction of an electrode, or vice-versa. Here, the mechanisms bywhich the most promising electroactive bacteria perform extracellular electrontransfer will be reviewed, emphasizing the proteins involved in these pathways.The ability of some of the organisms to perform bidirectional electron transferand the pathways used will also be highlighted.

Dantas, JM, Ferreira MR, Catarino T, Kokhan O, Pokkuluri RP, Salgueiro CA.  2018.  Molecular interactions between Geobacter sulfurreducens triheme cytochromes and the redox active analogue for humic substances. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1859:619-630., Number 8 AbstractWebsite

The bacterium Geobacter sulfurreducens can transfer electrons to quinone moieties of humic substances or to anthraquinone-2,6-disulfonate (AQDS), a model for the humic acids. The reduced form of AQDS (AH2QDS) can also be used as energy source by G. sulfurreducens. Such bidirectional utilization of humic substances confers competitive advantages to these bacteria in Fe(III) enriched environments. Previous studies have shown that the triheme cytochrome PpcA from G. sulfurreducens has a bifunctional behavior toward the humic substance analogue. It can reduce AQDS but the protein can also be reduced by AH2QDS. Using stopped-flow kinetic measurements we were able to demonstrate that other periplasmic members of the PpcA-family in G. sulfurreducens (PpcB, PpcD and PpcE) also showed the same behavior. The extent of the electron transfer is thermodynamically controlled favoring the reduction of the cytochromes. NMR spectra recorded for 13C,15N-enriched samples in the presence increasing amounts of AQDS showed perturbations in the chemical shift signals of the cytochromes. The chemical shift perturbations on cytochromes backbone NH and 1H heme methyl signals were used to map their interaction regions with AQDS, showing that each protein forms a low-affinity binding complex through well-defined positive surface regions in the vicinity of heme IV (PpcB, PpcD and PpcE) and I (PpcE). Docking calculations performed using NMR chemical shift perturbations allowed modeling the interactions between AQDS and each cytochrome at a molecular level. Overall, the results obtained provided important structural-functional relationships to rationalize the microbial respiration of humic substances in 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.

Dantas, JM, Kokhan O, Pokkuluri RP, Salgueiro CA.  2015.  Molecular interaction studies revealed the bifunctional behavior of triheme cytochrome PpcA from Geobacter sulfurreducens toward the redox active analog of humic substances. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1847:1129-1138., Number 10 AbstractWebsite

Abstract Humic substances (HS) constitute a significant fraction of natural organic matter in terrestrial and aquatic environments and can act as terminal electron acceptors in anaerobic microbial respiration. Geobacter sulfurreducens has a remarkable respiratory versatility and can utilize the \{HS\} analog anthraquinone-2,6-disulfonate (AQDS) as a terminal electron acceptor or its reduced form (AH2QDS) as an electron donor. Previous studies set the triheme cytochrome PpcA as a key component for \{HS\} respiration in G. sulfurreducens, but the process is far from fully understood. In this work, \{NMR\} chemical shift perturbation measurements were used to map the interaction region between PpcA and AH2QDS, and to measure their binding affinity. The results showed that the \{AH2QDS\} binds reversibly to the more solvent exposed edge of PpcA heme IV. The \{NMR\} and visible spectroscopies coupled to redox measurements were used to determine the thermodynamic parameters of the PpcA:quinol complex. The higher reduction potential of heme İV\} (− 127 mV) compared to that of \{AH2QDS\} (− 184 mV) explains why the electron transfer is more favorable in the case of reduction of the cytochrome by the quinol. The clear evidence obtained for the formation of an electron transfer complex between \{AH2QDS\} and PpcA, combined with the fact that the protein also formed a redox complex with AQDS, revealed for the first time the bifunctional behavior of PpcA toward an analog of the HS. Such behavior might confer selective advantage to G. sulfurreducens, which can utilize the \{HS\} in any redox state available in the environment for its metabolic needs.

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.

Louro, RO, Catarino T, Salgueiro CA, Legall J, Turner DL, Xavier AV.  1998.  Molecular Basis for Energy Transduction: Mechanisms of Cooperativity in Multihaem Cytochromes. Biological Electron Transfer Chains: Genetics, Composition and Mode of Operation NATO ASI Series Volume 512. (Canters, G.W., Vijgenboom, E., Eds.).:209-223.: Springer Netherlands Abstract

Energy transduction through electron/proton cooperativity is at the heart of the metabolism of every living organism Nonetheless, the search for the structural bases sustaining these phenomena has been hindered by the fact that they are usually associated with complex transmembrane proteins of high molecular weight.

Teixeira, LR, Cordas CM, Fonseca MP, Duke NEC, Pokkuluri PR, Salgueiro CA.  2020.  Modulation of the Redox Potential and Electron/Proton Transfer Mechanisms in the Outer Membrane Cytochrome OmcF From Geobacter sulfurreducens. Frontiers in Microbiology. 10:2941. AbstractWebsite

The 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.