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Silva, MA, Portela PC, Salgueiro CA.  2021.  Rational design of electron/proton transfer mechanisms in the exoelectrogenic bacteria Geobacter sulfurreducens, 07. Biochemical Journal. 478:2871-2887., Number 14 AbstractWebsite

{The redox potential values of cytochromes can be modulated by the protonation/deprotonation of neighbor groups (redox-Bohr effect), a mechanism that permits the proteins to couple electron/proton transfer. In the respiratory chains, this effect is particularly relevant if observed in the physiological pH range, as it may contribute to the electrochemical gradient for ATP synthesis. A constitutively produced family of five triheme cytochromes (PpcA−E) from the bacterium Geobacter sulfurreducens plays a crucial role in extracellular electron transfer, a hallmark that permits this bacterium to be explored for several biotechnological applications. Two members of this family (PpcA and PpcD) couple electron/proton transfer in the physiological pH range, a feature not shared with PpcB and PpcE. That ability is crucial for G. sulfurreducens’ growth in Fe(III)-reducing habitats since extra contributors to the electrochemical gradient are needed. It was postulated that the redox-Bohr effect is determined by the nature of residue 6, a leucine in PpcA/PpcD and a phenylalanine in PpcB/PpcE. To confirm this hypothesis, Phe6 was replaced by leucine in PpcB and PpcE. The functional properties of these mutants were investigated by NMR and UV–visible spectroscopy to assess their capability to couple electron/proton transfer in the physiological pH range. The results obtained showed that the mutants have an increased redox-Bohr effect and are now capable of coupling electron/proton transfer. This confirms the determinant role of the nature of residue 6 in the modulation of the redox-Bohr effect in this family of cytochromes, opening routes to engineer Geobacter cells with improved biomass production.}

Dantas, J, Morgado L, Aklujkar M, Bruix M, Londer Y, Schiffer M, Pokkuluri RP, Salgueiro C.  2015.  Rational engineering of Geobacter sulfurreducens electron transfer components: a foundation for building improved Geobacter-based bioelectrochemical technologies. Frontiers in Microbiology. 6:752. AbstractWebsite

Multiheme cytochromes have been implicated in Geobacter sulfurreducens (Gs) extracellular electron transfer (EET). These proteins are potential targets to improve EET and enhance bioremediation and electrical current production by Gs. However, the functional characterization of multiheme cytochromes is particularly complex due to the co-existence of several microstates in solution, connecting the fully reduced and fully oxidized states. Over the last decade, new strategies have been developed to characterize multiheme redox proteins functionally and structurally. These strategies were used to reveal the functional mechanism of Gs multiheme cytochromes and also to identify key residues in these proteins for EET. In previous studies, we set the foundations for enhancement of the EET abilities of Gs by characterizing a family of five triheme cytochromes (PpcA-E). These periplasmic cytochromes are implicated in electron transfer between the oxidative reactions of metabolism in the cytoplasm and the reduction of extracellular terminal electron acceptors at the cell’s outer surface. The results obtained suggested that PpcA can couple e-/H+ transfer, a property that might contribute to the proton electrochemical gradient across the cytoplasmic membrane for metabolic energy production. The structural and functional properties of PpcA were characterized in detail and used for rational design of a family of 23 single site PpcA mutants. In this review, we summarize the functional characterization of the native and mutant proteins. Mutants that retain the mechanistic features of PpcA and adopt preferential e-/H+ transfer pathways at lower reduction potential values compared to the wild-type protein were selected for in vivo studies as the best candidates to increase the electron transfer rate of Gs. For the first time Gs strains have been manipulated by the introduction of mutant forms of essential proteins with the aim to develop and improve bioelectrochemical technologies.

Pessanha, M, Rothery EL, Louro RO, Turner DL, Miles CS, Reid GA, Chapman SK, Xavier AV, Salgueiro CA.  2004.  Redox behaviour of the haem domain of flavocytochrome c3 from Shewanella frigidimarina probed by NMR. FEBS Letters. 578(1/2):185-190. AbstractWebsite

Flavocytochrome c3 from Shewanella frigidimarina (fcc3) is a tetrahaem periplasmic protein of 64 kDa with fumarate reductase activity. This work reports the first example of NMR techniques applied to the assignment of the thermodynamic order of oxidation of the four individual haems for such large protein, expanding its applicability to a wide range of proteins. NMR data from partially and fully oxidised samples of fcc3 and a mutated protein with an axial ligand of haem IV replaced by alanine were compared with calculated chemical shifts, allowing the structural assignment of the signals and the unequivocal determination of the order of oxidation of the haems. As oxidation progresses the fcc3 haem domain is polarised, with haems I and II much more oxidised than haems III and IV, haem IV being the most reduced. Thus, during catalysis as an electron is taken by the flavin adenosine dinucleotide from haem IV, haem III is eager to re-reduce haem IV, allowing the transfer of two electrons to the active site.

Pessanha, M, Londer YY, Long WC, Erickson J, Pokkuluri PR, Schiffer M, Salgueiro CA.  2004.  Redox Characterization of Geobacter sulfurreducens Cytochrome c7:  Physiological Relevance of the Conserved Residue F15 Probed by Site-Specific Mutagenesis. Biochemistry. 43(30):9909-9917. AbstractWebsite

The complete genome sequence of the δ-proteobacterium Geobacter sulfurreducens reveals a large abundance of multiheme cytochromes. Cytochrome c7, isolated from this metal ion-reducing bacterium, is a triheme periplasmic electron-transfer protein with Mr 9.6 kDa. This protein is involved in metal ion-reducing pathways and shares 56% sequence identity with a triheme cytochrome isolated from the closely related δ-proteobacterium Desulfuromonas acetoxidans (Dac7). In this work, two-dimensional NMR was used to monitor the heme core and the general folding in solution of the G. sulfurreducens triheme cytochrome c7 (PpcA). NMR signals obtained for the three hemes of PpcA at different stages of oxidation were cross-assigned to the crystal structure [Pokkuluri, P. R., Londer, Y. Y., Duke, N. E. C., Long, W. C., and Schiffer, M. (2004) Biochemistry 43, 849−859] using the complete network of chemical exchange connectivities, and the order in which each heme becomes oxidized was determined at pH 6.0 and 8.2. Redox titrations followed by visible spectroscopy were also performed in order to monitor the macroscopic redox behavior of PpcA. The results obtained showed that PpcA and Dac7 have different redox properties:  (i) the order in which each heme becomes oxidized is different; (ii) the reduction potentials of the heme groups and the global redox behavior of PpcA are pH dependent (redox−Bohr effect) in the physiological pH range, which is not observed with Dac7. The differences observed in the redox behavior of PpcA and Dac7 may account for the different functions of these proteins and constitute an excellent example of how homologous proteins can perform different physiological functions. The redox titrations followed by visible spectroscopy of PpcA and two mutants of the conserved residue F15 (PpcAF15Y and PpcAF15W) lead to the conclusion that F15 modulates the redox behavior of PpcA, thus having an important physiological role.

Morgado, L, Bruix M, Pokkuluri RP, Salgueiro CA, Turner DL.  2017.  Redox- and pH-linked conformational changes in triheme cytochrome PpcA from Geobacter sulfurreducens. Biochemical Journal. 474:231–246., Number 2: Portland Press Limited AbstractWebsite

Accepted Manuscript online November 14, 2016.The periplasmic triheme cytochrome PpcA from Geobacter sulfurreducens is highly abundant; it is the likely reservoir of electrons to the outer surface to assist the reduction of extracellular terminal acceptors; these include insoluble metal oxides in natural habitats and electrode surfaces from which electricity can be harvested. A detailed thermodynamic characterization of PpcA showed that it has an important redox-Bohr effect that might implicate the protein in e-/H+ coupling mechanisms to sustain cellular growth. This functional mechanism requires control of both the redox state and the protonation state. In the present study, isotope-labeled PpcA was produced and the three-dimensional structure of PpcA in the oxidized form was determined by NMR. This is the first solution structure of a G. sulfurreducens cytochrome in the oxidized state. The comparison of oxidized and reduced structures revealed that the heme I axial ligand geometry changed and there were other significant changes in the segments near heme I. The pH-linked conformational rearrangements observed in the vicinity of the redox-Bohr center, both in the oxidized and reduced structures, constitute the structural basis for the differences observed in the pKa values of the redox-Bohr center, providing insights into the e-/H+ coupling molecular mechanisms driven by PpcA in G. sulfurreducens.EET, extracellular electron transfer; IM, inner membrane; IPTG, isopropyl β-d-thiogalactoside; MFCs, microbial fuel cells; NOE, Nuclear Overhauser effect; OM, outer membrane; rmsd, root mean square deviation.

Louro, RO, Catarino T, Salgueiro CA, Legall J, Xavier AV.  1996.  Redox-Bohr effect in the tetrahaem cytochrome c3 from Desulfovibrio vulgaris: a model for energy transduction mechanisms. Journal of Biological Inorganic Chemistry. 1(1):34-38. AbstractWebsite

Using potentiometric titrations, two protons were found to participate in the redox-Bohr effect observed for cytochrome c3 from Desulfovibrio vulgaris (Hildenborough). Within the framework of the thermodynamic model previously presented, this finding supports the occurrence of a concerted proton-assisted 2e– step, ideally suited for the coupling role of cytochrome c3 to hydrogenase. Furthermore, at physiological pH, it is shown that when sulfate-reducing bacteria use H2 as energy source, cytochrome c3 can be used as a charge separation device, achieving energy transduction by energising protons which can be left in the acidic periplasmic side and transferring deenergised electrons to sulfate respiration. This mechanism for energy transduction, using a full thermodynamic data set, is compared to that put forward to explain the proton-pumping function of cytochrome c oxidase.

Morgado, L, Bruix M, Londer YY, Pokkuluri PR, Schiffer M, Salgueiro CA.  2007.  Redox-linked conformational changes of a multiheme cytochrome from Geobacter sulfurreducens. Biochemical and Biophysical Research Communications. 360(1):194-198. AbstractWebsite

Multiheme c-type cytochromes from members of the Desulfovibrionacea and Geobactereacea families play crucial roles in the bioenergetics of these microorganisms. Thermodynamic studies using NMR and visible spectroscopic techniques on tetraheme cytochromes c3 isolated from Desulfovibrio spp. and more recently on a triheme cytochrome from Geobacter sulfurreducens showed that the properties of each redox centre are modulated by the neighbouring redox centres enabling these proteins to perform energy transduction and thus contributing to cellular energy conservation. Electron/proton transfer coupling relies on redox-linked conformational changes that were addressed for some multiheme cytochromes from the comparison of protein structure of fully reduced and fully oxidised forms. In this work, we identify for the first time in a multiheme cytochrome the simultaneous presence of two different conformations in solution. This was achieved by probing the different oxidation stages of a triheme cytochrome isolated from G. sulfurreducens using 2D-NMR techniques. The results presented here will be the foundations to evaluate the modulation of the redox centres properties by conformational changes that occur during the reoxidation of a multiheme protein.

Karamash, M, Stumpe M, Dengjel J, Salgueiro CA, Giese B, Fromm KM.  2022.  Reduction Kinetic of Water Soluble Metal Salts by Geobacter sulfurreducens: Fe2+/Hemes Stabilize and Regulate Electron Flux Rates. Frontiers in Microbiology. 13 AbstractWebsite

Geobacter sulfurreducens is a widely applied microorganism for the reduction of toxic metal salts, as an electron source for bioelectrochemical devices, and as a reagent for the synthesis of nanoparticles. In order to understand the influence of metal salts, and of electron transporting, multiheme c-cytochromes on the electron flux during respiration of G. sulfurreducens, the reduction kinetic of Fe3+, Co3+, V5+, Cr6+, and Mn7+ containing complexes were measured. Starting from the resting phase, each G. sulfurreducens cell produced an electron flux of 3.7 × 105 electrons per second during the respiration process. Reduction rates were within ± 30% the same for the 6 different metal salts, and reaction kinetics were of zero order. Decrease of c-cytochrome concentrations by downregulation and mutation demonstrated that c-cytochromes stabilized respiration rates by variation of their redox states. Increasing Fe2+/heme levels increased electron flux rates, and induced respiration flexibility. The kinetic effects parallel electrochemical results of G. sulfurreducens biofilms on electrodes, and might help to optimize bioelectrochemical devices.

Salgueiro, CA, Turner DL, Legall J, Xavier AV, Legall J.  1997.  Reevaluation of the redox and redox-Bohr cooperativity in tetrahaem Desulfovibrio vulgaris (Miyazaki F) cytochrome c3. Journal of Biological Inorganic Chemistry. 2(3):343-349. AbstractWebsite

The thermodynamic model of five interacting charge centres (four haems and an ionisable centre), which was used in the characterisation of the thermodynamic properties of Desulfovibrio vulgaris (Hildenborough) cytochrome c3 (c3DvH), is now used to reevaluate the thermodynamic properties in Desulfovibrio vulgaris (Miyazaki F) cytochrome c3 (c3DvM) on the basis of published data (Park, J.-S., Ohmura, T., Kano, K., Sagara, T., Niki, K., Kyogoku, Y. and Akutsu, H. (1996) Biochim. Biophys. Acta 1293, 45–54). Contrary to the assertion of Park et al. (1996), the pH dependence of the proton chemical shifts of haem methyls in c3DvM in several stages of oxidation is well described by the model, which involves both homotropic (e–/e–) and heterotropic (e–/H+) cooperativity. This shows that the pH dependence observed for c3DvM is not significantly more complicated than that observed for c3DvH. Since the parameters which we now obtain for c3DvM are generated with the same model as those from c3DvH, albeit using less precise data, it is possible to make a preliminary comparison of the thermodynamic properties of these two proteins and of their role in energy transduction.
The extrinsic dipolar shifts generated for each methyl group by each of the four haems in c3DvM are also determined. A novel method for approximating the magnetic susceptibility tensors is used: the orientations of the principal axes of the tensors have been shown to be closely related to the geometry of the axial ligands, which is available from the X-ray structure of c3DvM, and the components of the tensors are extrapolated from EPR g values. The inclusion of the calculated haem extrinsic contributions clearly describes the pH dependence of the haem methyls in the core of the protein, close to other haems. This description is most remarkable in the case of the haem methyl 21CH3 II I, for which the "unusual pH dependence" commented on by Park et al. (1996) is easily explained using the thermodynamic parameters determined by our model together with the calculated extrinsic dipolar shifts, thus providing a test of the analysis.

Saraiva, LM, Salgueiro CA, da Costa PN, Messias AC, Legall J, van Dongen WMAM, Xavier AV.  1998.  Replacement of Lysine 45 by Uncharged Residues Modulates the Redox-Bohr Effect in Tetraheme Cytochrome c3 of Desulfovibrio vulgaris (Hildenborough). Biochemistry. 37(35):12160-12165. AbstractWebsite

The structural basis for the pH dependence of the redox potential in the tetrahemic Desulfovibrio vulgaris (Hildenborough) cytochrome c3 was investigated by site-directed mutagenesis of charged residues in the vicinity of heme I. Mutation of lysine 45, located in the neighborhood of the propionates of heme I, by uncharged residues, namely threonine, glutamine and leucine, was performed. The replacement of a conserved charged residue, aspartate 7, present in the N-terminal region and near heme I was also attempted. The analysis of the redox interactions as well as the redox-Bohr behavior of the mutated cytochromes c3 allowed the conclusion that residue 45 has a functional role in the control of the pKa of the propionate groups of heme I and confirms the involvement of this residue in the redox-Bohr effect.

Bandeiras, TM, Salgueiro CA, Huber H, Gomes CM, Teixeira M.  2003.  The respiratory chain of the thermophilic archaeon Sulfolobus metallicus: studies on the type-II NADH dehydrogenase. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1557(1-3):13-19. AbstractWebsite

The membranes of the thermoacidophilic archaeon Sulfolobus metallicus exhibit an oxygen consumption activity of 0.5 nmol O2 min−1 mg−1, which is insensitive to rotenone, suggesting the presence of a type-II NADH dehydrogenase. Following this observation, the enzyme was purified from solubilised membranes and characterised. The pure protein is a monomer with an apparent molecular mass of 49 kDa, having a high N-terminal amino acid sequence similarity towards other prokaryotic enzymes of the same type. It contains a covalently attached flavin, which was identified as being FMN by 31P-NMR spectroscopy, a novelty among type-II NADH dehydrogenases. Metal analysis showed the absence of iron, indicating that no FeS clusters are present in the protein. The average reduction potential of the FMN group was determined to be +160 mV, at 25 °C and pH 6.5, by redox titrations monitored by visible spectroscopy. Catalytically, the enzyme is a NADH:quinone oxidoreductase, as it is capable of transferring electrons from NADH to several quinones, including ubiquinone-1, ubiquinone-2 and caldariella quinone. Maximal turnover rates of 195 μmol NADH oxidized min−1 mg−1 at 60 °C were obtained using ubiquinone-2 as electron acceptor, after enzyme dilution and incubation with phospholipids.

Morgado, L, Paixão VB, Schiffer M, Pokkuluri PR, Bruix M, Salgueiro CA.  2012.  Revealing the structural origin of the redox-Bohr effect: the first solution structure of a cytochrome from Geobacter sulfurreducens. Biochemical Journal. 441(1):179-187. AbstractWebsite

Gs (Geobacter sulfurreducens) can transfer electrons to the exterior of its cells, a property that makes it a preferential candidate for the development of biotechnological applications. Its genome encodes over 100 cytochromes and, despite their abundance and key functional roles, to date there is no structural information for these proteins in solution. The trihaem cytochrome PpcA might have a crucial role in the conversion of electronic energy into protonmotive force, a fundamental step for ATP synthesis in the presence of extracellular electron acceptors. In the present study, 15N-labelled PpcA was produced and NMR spectroscopy was used to determine its solution structure in the fully reduced state, its backbone dynamics and the pH-dependent conformational changes. The structure obtained is well defined, with an average pairwise rmsd (root mean square deviation) of 0.25 Å (1 Å=0.1 nm) for the backbone atoms and 0.99 Å for all heavy atoms, and constitutes the first solution structure of a Gs cytochrome. The redox-Bohr centre responsible for controlling the electron/proton transfer was identified, as well as the putative interacting regions between PpcA and its redox partners. The solution structure of PpcA will constitute the foundation for studies aimed at mapping out in detail these interacting regions.

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.

Morgado, L, Dantas JM, Simões T, Londer YY, Pokkuluri PR, Salgueiro CA.  2013.  Role of Met58 in the regulation of electron/proton transfer in trihaem cytochrome PpcA from Geobacter sulfurreducens. Bioscience Reports. 33(1):11-22. AbstractWebsite

The bacterium Gs (Geobacter sulfurreducens) is capable of oxidizing a large variety of compounds relaying electrons out of the cytoplasm and across the membranes in a process designated as extracellular electron transfer. The trihaem cytochrome PpcA is highly abundant in Gs and is most probably the reservoir of electrons destined for the outer surface. In addition to its role in electron transfer pathways, we have previously shown that this protein could perform e-/H+ energy transduction. This mechanism is achieved by selecting the specific redox states that the protein can access during the redox cycle and might be related to the formation of proton electrochemical potential gradient across the periplasmic membrane. The regulatory role of haem III in the functional mechanism of PpcA was probed by replacing Met58, a residue that controls the solvent accessibility of haem III, with serine, aspartic acid, asparagine or lysine. The data obtained from the mutants showed that the preferred e-/H+ transfer pathway observed for PpcA is strongly dependent on the reduction potential of haem III. It is striking to note that one residue can fine tune the redox states that can be accessed by the trihaem cytochrome enough to alter the functional pathways.