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Marques, AC, Santos L, Costa MN, Dantas JM, Duarte P, Gonçalves A, Martins R, Salgueiro CA, Fortunato E.  2015.  Office Paper Platform for Bioelectrochromic Detection of Electrochemically Active Bacteria using Tungsten Trioxide Nanoprobes. Sci. Rep. 5(9910) AbstractWebsite

Electrochemically active bacteria (EAB) have the capability to transfer electrons to cell exterior, a feature that is currently explored for important applications in bioremediation and biotechnology fields. However, the number of isolated and characterized EAB species is still very limited regarding their abundance in nature. Colorimetric detection has emerged recently as an attractive mean for fast identification and characterization of analytes based on the use of electrochromic materials. In this work, WO3 nanoparticles were synthesized by microwave assisted hydrothermal synthesis and used to impregnate non-treated regular office paper substrates. This allowed the production of a paper-based colorimetric sensor able to detect EAB in a simple, rapid, reliable, inexpensive and eco-friendly method. The developed platform was then tested with Geobacter sulfurreducens, as a proof of concept. G. sulfurreducens cells were detected at latent phase with an RGB ratio of 1.10 ± 0.04, and a response time of two hours.

Morgado, L, Fernandes AP, Dantas JM, Silva MA, Salgueiro CA.  2012.  On the road to improve the bioremediation and electricity-harvesting skills of Geobacter sulfurreducens: functional and structural characterization of multihaem cytochromes. Biochemical Society transactions. 40(6):1295-1301. AbstractWebsite

Extracellular electron transfer is one of the physiological hallmarks of Geobacter sulfurreducens, allowing these bacteria to reduce toxic and/or radioactive metals and grow on electrode surfaces. Aiming to functionally optimize the respiratory electron-transfer chains, such properties can be explored through genetically engineered strains. Geobacter species comprise a large number of different multihaem c-type cytochromes involved in the extracellular electron-transfer pathways. The functional characterization of multihaem proteins is particularly complex because of the coexistence of several microstates in solution, connecting the fully reduced and oxidized states. NMR spectroscopy has been used to monitor the stepwise oxidation of each individual haem and thus to obtain information on each microstate. For the structural study of these proteins, a cost-effective isotopic labelling of the protein polypeptide chains was combined with the comparative analysis of 1H-13C HSQC (heteronuclear single-quantum correlation) NMR spectra obtained for labelled and unlabelled samples. These new methodological approaches allowed us to study G. sulfurreducens haem proteins functionally and structurally, revealing functional mechanisms and key residues involved in their electron-transfer capabilities. Such advances can now be applied to the design of engineered haem proteins to improve the bioremediation and electricity-harvesting skills of G. sulfurreducens.

Morgado, L, Fernandes AP, Londer YY, Bruix M, Salgueiro CA.  2010.  One simple step in the identification of the cofactors signals, one giant leap for the solution structure determination of multiheme proteins. Biochemical and Biophysical Research Communications. 393(3):466-470. AbstractWebsite

Multiheme proteins play major roles in various biological systems. Structural information on these systems in solution is crucial to understand their functional mechanisms. However, the presence of numerous proton-containing groups in the heme cofactors and the magnetic properties of the heme iron, in particular in the oxidised state, complicates significantly the assignment of the NMR signals. Consequently, the multiheme proteins superfamily is extremely under-represented in structural databases, which constitutes a severe bottleneck in the elucidation of their structural–functional relationships. In this work, we present a strategy that simplifies the assignment of the NMR signals in multiheme proteins and, concomitantly, their solution structure determination, using the triheme cytochrome PpcA from the bacterium Geobacter sulfurreducens as a model. Cost-effective isotopic labeling was used to double label (13C/15N) the protein in its polypeptide chain, with the correct folding and heme post-translational modifications. The combined analysis of 1H–13C HSQC NMR spectra obtained for labeled and unlabeled samples of PpcA allowed a straight discrimination between the heme cofactors and the polypeptide chain signals and their confident assignment. The results presented here will be the foundations to assist solution structure determination of multiheme proteins, which are still very scarce in the literature.

Dantas, JM, Saraiva IH, Morgado L, Silva MA, Schiffer M, Salgueiro CA, Louro RO.  2011.  Orientation of the axial ligands and magnetic properties of the hemes in the cytochromec7 family from Geobacter sulfurreducens determined by paramagnetic NMR. Dalton Transactions. 40(47):12713-12718. AbstractWebsite

Geobacter sulfurreducens is a sediment bacterium that contains a large number of multiheme cytochromes. The family of five c7 triheme periplasmic cytochromes from Geobacter sulfurreducens shows structural diversity of the heme core. Structural characterization of the relative orientation of the axial ligands of these proteins by 13C-paramagnetic NMR was carried out. The structures in solution were compared with those obtained by X-ray crystallography. For some hemes significant differences exist between the two methods such that orientation of the magnetic axes obtained from NMR data and the orientation taken from the X-ray coordinates differ. The results allowed the orientation of the magnetic axes to be defined confidently with respect to the heme frame in solution, a necessary step for the use of paramagnetic constraints to improve the complete solution structure of these proteins.

Morgado, L, Saraiva IH, Louro RO, Salgueiro CA.  2010.  Orientation of the axial ligands and magnetic properties of the hemes in the triheme ferricytochrome PpcA from G. sulfurreducens determined by paramagnetic NMR. FEBS Letters. 584(15):3442-3445. AbstractWebsite

The geometry of the axial ligands of the hemes in the triheme cytochrome PpcA from Geobacter sulfurreducens was determined in solution for the ferric form using the unambiguous assignment of the NMR signals of the α-substituents of the hemes. The paramagnetic 13C shifts of the hemes can be used to define the heme electronic structure, the geometry of the axial ligands, and the magnetic susceptibility tensor. The latter establishes the magnitude and geometrical dependence of the pseudocontact shifts, which are crucial to warrant reliable structural constraints for a detailed structural characterization of this paramagnetic protein in solution.

Pokkuluri, PR, Londer YY, Wood SJ, Duke NEC, Morgado L, Salgueiro CA, Schiffer M.  2009.  Outer membrane cytochrome c, OmcF, from Geobacter sulfurreducens: High structural similarity to an algal cytochrome c6. Proteins: Structure, Function, and Bioinformatics. 74(1):266-270. AbstractWebsite

No abstract included.

Dantas, JM, Morgado L, Londer YY, Fernandes AP, Louro RO, Pokkuluri PR, Schiffer M, Salgueiro CA.  2012.  Pivotal role of the strictly conserved aromatic residue F15 in the cytochrome c7 family. Journal of Biological Inorganic Chemistry. 17(1):11-24. AbstractWebsite

Cytochromes c7 are periplasmic triheme proteins that have been reported exclusively in δ-proteobacteria. The structures of five triheme cytochromes identified in Geobacter sulfurreducens and one in Desulfuromonas acetoxidans have been determined. In addition to the hemes and axial histidines, a single aromatic residue is conserved in all these proteins - phenylalanine 15 (F15). PpcA is a member of the G. sulfurreducens cytochrome c7 family that performs electron/proton energy transduction in addition to electron transfer that leads to the reduction of extracellular electron acceptors. For the first time we probed the role of the F15 residue in the PpcA functional mechanism, by replacing this residue with the aliphatic leucine by site-directed mutagenesis. The analysis of NMR spectra of both oxidized and reduced forms showed that the heme core and the overall fold of the mutated protein were not affected. However, the analysis of 1H-15N heteronuclear single quantum coherence NMR spectra evidenced local rearrangements in the α-helix placed between hemes I and III that lead to structural readjustments in the orientation of heme axial ligands. The detailed thermodynamic characterization of F15L mutant revealed that the reduction potentials are more negative and the redox-Bohr effect is decreased. The redox potential of heme III is most affected. It is of interest that the mutation in F15, located between hemes I and III in PpcA, changes the characteristics of the two hemes differently. Altogether, these modifications disrupt the balance of the global network of cooperativities, preventing the F15L mutant protein from performing a concerted electron/proton transfer.

Catarino, T, Pessanha M, Candia ADG, Gouveia Z, Fernandes AP, Pokkuluri PR, Murgida D, Marti MA, Todorovic S, Salgueiro CA.  2010.  Probing the Chemotaxis Periplasmic Sensor Domains from Geobacter sulfurreducens by Combined Resonance Raman and Molecular Dynamic Approaches: NO and CO Sensing. The Journal of Physical Chemistry B. 114 (34):11251-11260. AbstractWebsite

The periplasmic sensor domains encoded by genes gsu0582 and gsu0935 are part of methyl accepting chemotaxis proteins in the bacterium Geobacter sulfurreducens (Gs). The sensor domains of these proteins contain a heme-c prosthetic group and a PAS-like fold as revealed by their crystal structures. Biophysical studies of the two domains showed that nitric oxide (NO) binds to the heme in both the ferric and ferrous forms, whereas carbon monoxide (CO) binds only to the reduced form. In order to address these exogenous molecules as possible physiological ligands, binding studies and resonance Raman (RR) spectroscopic characterization of the respective CO and NO adducts were performed in this work. In the absence of exogenous ligands, typical RR frequencies of five-coordinated (5c) high-spin and six-coordinated (6c) low-spin species were observed in the oxidized form. In the reduced state, only frequencies corresponding to the latter were detected. In both sensors, CO binding yields 6c low-spin adducts by replacing the endogenous distal ligand. The binding of NO by the two proteins causes partial disruption of the proximal Fe-His bond, as revealed by the RR fingerprint features of 5cFe-NO and 6cNO-Fe-His species. The measured CO and NO dissociation constants of ferrous GSU0582 and GSU0935 sensors reveal that both proteins have high and similar affinity toward these molecules (Kd ≈ 0.04−0.08 μM). On the contrary, in the ferric form, sensor GSU0582 showed a much higher affinity for NO (Kd ≈ 0.3 μM for GSU0582 versus 17 μM for GSU0935). Molecular dynamics calculations revealed a more open heme pocket in GSU0935, which could account for the different affinities for NO. Taken together, spectroscopic data and MD calculations revealed subtle differences in the binding properties and structural features of formed CO and NO adducts, but also indicated a possibility that a (5c) high-spin/(6c) low-spin redox-linked equilibrium could drive the physiological sensing of Gs cells.

Dantas, JM, Morgado L, Marques AC, Salgueiro CA.  2014.  Probing the effect of ionic strength on the functional robustness of the triheme cytochrome PpcA from Geobacter sulfurreducens: a contribution for optimizing biofuel cell's power density. J Phys Chem B. 118(43):12416-12425. AbstractWebsite

The increase of conductivity of electrolytes favors the current production in microbial fuel cells (MFCs). Adaptation of cell cultures to higher ionic strength is a promising strategy to increase electricity production. The bacterium Geobacter sulfurreducens is considered a leading candidate for MFCs. Therefore, it is important to evaluate the impact of the ionic strength on the functional properties of key periplasmic proteins that warrants electron transfer to cell exterior. The effect of the ionic strength on the functional properties of triheme cytochrome PpcA, the most abundant periplasmic cytochrome in G. sulfurreducens, was investigated by NMR and potentiometric methods. The redox properties of heme IV are the most affected ones. Chemical shift perturbation measurements on the backbone NMR signals, at increasing ionic strength, also showed that the region close to heme IV is the most affected due to the large number of positively charged residues, which confer a highly positive electrostatic surface around this heme. The shielding of these positive charges at high ionic strength explain the observed decrease in the reduction potential of heme IV and shows that PpcA was designed to maintain its functional mechanistic features even at high ionic strength.

Boscolo, B, Leal SS, Salgueiro CA, Ghibaudi EM, Gomes CM.  2009.  The prominent conformational plasticity of lactoperoxidase: A chemical and pH stability analysis. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 1794(7):1041-1048. AbstractWebsite

Lactoperoxidase (LPO) is a structurally complex and stable mammalian redox enzyme. Here we aim at evaluating the influence of ionic interactions and how these intertwine with the structural dynamics, stability and activity of LPO. In this respect, we have compared LPO guanidinium hydrochloride (GdmCl) and urea denaturation pathways and performed a detailed investigation on the effects of pH on the LPO conformational dynamics and stability. Our experimental findings using far-UV CD, Trp fluorescence emission and ESR spectroscopies clearly indicate that LPO charged-denaturation with GdmCl induced a sharp two-step process versus a three-step unfolding mechanism induced by urea. This differential effect between GdmCl and urea suggests that ionic interactions must play a rather prominent role in the stabilization of LPO. With both denaturants, the protein core was shown to retain activity up to near the respective Cm values. Moreover, a pH titration of LPO evidenced no significant conformational alterations or perturbation of heme activity within the 4 to 11 pH interval. In contrast, alterations of ionic interactions by poising LPO at pH 3, 2 and 12 resulted in a loss of secondary structure, loosening of tertiary contacts and loss of activity, which appear to be associated with the perturbation of the hydrophobic core, as evidenced by ANS binding, as well as disruption of the heme pocket demonstrated by optical and EPR spectroscopies. Overall, LPO is characterised by a high degree of peripheral structural plasticity without perturbation of the core heme moiety. The possible physiological meaning of such features is discussed.

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.

Silva, MA, Lucas TG, Salgueiro CA, Gomes CM.  2012.  Protein Folding Modulates the Swapped Dimerization Mechanism of Methyl-Accepting Chemotaxis Heme Sensors. PLoS ONE. 7(9):e46328. AbstractWebsite

The periplasmic sensor domains GSU0582 and GSU0935 are part of methyl accepting chemotaxis proteins in the bacterium Geobacter sulfurreducens. Both contain one c-type heme group and their crystal structures revealed that these domains form swapped dimers with a PAS fold formed from the two protein chains. The swapped dimerization of these sensors is related to the mechanism of signal transduction and the formation of the swapped dimer involves significant folding changes and conformational rearrangements within each monomeric component. However, the structural changes occurring during this process are poorly understood and lack a mechanistic framework. To address this issue, we have studied the folding and stability properties of two distinct heme-sensor PAS domains, using biophysical spectroscopies. We observed substantial differences in the thermodynamic stability (ΔG = 14.6 kJ.mol−1 for GSU0935 and ΔG = 26.3 kJ.mol−1 for GSU0582), and demonstrated that the heme moiety undergoes conformational changes that match those occurring at the global protein structure. This indicates that sensing by the heme cofactor induces conformational changes that rapidly propagate to the protein structure, an effect which is directly linked to the signal transduction mechanism. Interestingly, the two analyzed proteins have distinct levels of intrinsic disorder (25% for GSU0935 and 13% for GSU0582), which correlate with conformational stability differences. This provides evidence that the sensing threshold and intensity of the propagated allosteric effect is linked to the stability of the PAS-fold, as this property modulates domain swapping and dimerization. Analysis of the PAS-domain shows that disorder segments are found either at the hinge region that controls helix motions or in connecting segments of the β-sheet interface. The latter is known to be widely involved in both intra- and intermolecular interactions, supporting the view that it's folding and stability are at the basis of the specificity and regulation of many types of PAS-containing signaling proteins.

Inoue, K, Qian X, Morgado L, Kim B-C, Mester T, Izallalen M, Salgueiro CA, Lovley DR.  2010.  Purification and Characterization of OmcZ, an Outer-Surface, Octaheme c-Type Cytochrome Essential for Optimal Current Production by Geobacter sulfurreducens. Applied and Environmental Microbiology. 76(12):3999-4007. AbstractWebsite

Previous studies have demonstrated that Geobacter sulfurreducens requires the c-type cytochrome OmcZ, which is present in large (OmcZL; 50-kDa) and small (OmcZS; 30-kDa) forms, for optimal current production in microbial fuel cells. This protein was further characterized to aid in understanding its role in current production. Subcellular-localization studies suggested that OmcZS was the predominant extracellular form of OmcZ. N- and C-terminal amino acid sequence analysis of purified OmcZS and molecular weight measurements indicated that OmcZS is a cleaved product of OmcZL retaining all 8 hemes, including 1 heme with the unusual c-type heme-binding motif CX14CH. The purified OmcZS was remarkably thermally stable (thermal-denaturing temperature, 94.2°C). Redox titration analysis revealed that the midpoint reduction potential of OmcZS is approximately −220 mV (versus the standard hydrogen electrode [SHE]) with nonequivalent heme groups that cover a large reduction potential range (−420 to −60 mV). OmcZS transferred electrons in vitro to a diversity of potential extracellular electron acceptors, such as Fe(III) citrate, U(VI), Cr(VI), Au(III), Mn(IV) oxide, and the humic substance analogue anthraquinone-2,6-disulfonate, but not Fe(III) oxide. The biochemical properties and extracellular localization of OmcZ suggest that it is well suited for promoting electron transfer in current-producing biofilms of G. sulfurreducens.

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.