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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.

Saraiva, LM, Salgueiro CA, Legall J, van Dongen WMAM, Xavier AV.  1996.  Site-directed mutagenesis of a phenylalanine residue strictly conserved in cytochromes c3. Journal of Biological Inorganic Chemistry. 1(6):542-550. AbstractWebsite

Reduction of the haems in tetrahaem cytochromes c3 is a cooperative process, i.e., reduction of each of the haems depends on the redox states of the other haems. Furthermore, electron transfer is coupled to proton transfer (redox-Bohr effect). Two of its haems and a strictly conserved nearby phenylalanine residue, F20, in Desulfovibrio vulgaris (Hildenborough) cytochrome c3 form a structural motif that is present in all cytochromes c3 and also in cytochrome c oxidase. A putative role for this phenylalanine residue in the cooperativity of haem reduction was investigated. Therefore, this phenylalanine was replaced, with genetic techniques, by isoleucine and tyrosine in D. vulgaris (Hildenborough) cytochrome c3. Cyclic voltammetry studies revealed a small increase (30 mV) in one of the macroscopic redox potentials in the mutated cytochromes. EPR showed that the main alterations occurred in the vicinity of haem I, the haem closest to residue 20 and one of the haems responsible for positive cooperativities in electron transfer of D. vulgaris cytochrome c3. NMR studies of F20I cytochrome c3 demonstrated that the haem core architecture is maintained and that the more affected haem proton groups are those near the mutation site. NMR redox titrations of this mutated protein gave evidence for only small changes in the relative redox potentials of the haems. However, electron/electron and proton/electron cooperativity are maintained, indicating that this aromatic residue has no essential role in these processes. Furthermore, chemical modification of the N-terminal amino group of cytochrome c3 backbone, which is also very close to haem I, had no effect on the network of cooperativities.

Dantas, JM, Silva MA, Pantoja-Uceda D, Turner DL, Bruix M, Salgueiro CA.  2017.  Solution structure and dynamics of the outer membrane cytochrome OmcF from Geobacter sulfurreducens. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1858(9):733-741. AbstractWebsite

ABSTRACTGene knock-out studies on Geobacter sulfurreducens cells showed that the outer membrane-associated monoheme cytochrome OmcF is involved in respiratory pathways leading to the extracellular reduction of Fe(III) and U(VI). In addition, microarray analysis of an OmcF-deficient mutant revealed that many of the genes with decreased transcript level were those whose expression is up-regulated in cells grown with a graphite electrode as electron acceptor, suggesting that OmcF also regulates the electron transfer to electrode surfaces and the concomitant electricity production by G. sulfurreducens in microbial fuel cells. 15N,13C–labeled OmcF was produced and NMR spectroscopy was used to determine the solution structure of the protein in the fully reduced state and the pH-dependent conformational changes. In addition, 15N relaxation NMR experiments were used to characterize the overall and internal backbone dynamics of OmcF. The structure obtained is well defined, with an average pairwise root mean square deviation of 0.37 Å for the backbone atoms and 0.98 Å for all heavy atoms. For the first time a solution structure and the protein motions were determined for an outer membrane cytochrome from G. sulfurreducens, which constitutes an important step to understand the extracellular electron transfer mechanism in Geobacter cells.

Dantas, JM, Morgado L, Pokkuluri PR, Turner DL, Salgueiro CA.  2013.  Solution structure of a mutant of the triheme cytochrome PpcA from Geobacter sulfurreducens sheds light on the role of the conserved aromatic residue F15. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1827(4):484-492. AbstractWebsite

Extracellular electron transfer is one of the physiological hallmarks of Geobacteraceae. Most of the Geobacter species encode for more than 100 c-type cytochromes which are, in general, poorly conserved between individual species. An exception to this is the PpcA family of periplasmic triheme c-type cytochromes, which are the most abundant proteins in these bacteria. The functional characterization of PpcA showed that it has the necessary properties to couple electron/proton transfer, a fundamental step for ATP synthesis. The detailed thermodynamic characterization of a PpcA mutant, in which the strictly conserved residue phenylalanine 15 was replaced by leucine, showed that the global redox network of cooperativities among heme groups is altered, preventing the mutant from performing a concerted electron/proton transfer. In this work, we determined the solution structure of PpcA F15L mutant in the fully reduced state using NMR spectroscopy by producing 15N-labeled protein. In addition, pH-dependent conformational changes were mapped onto the structure. The mutant structure obtained is well defined, with an average pairwise root-mean-square deviation of 0.36 Å for the backbone atoms and 1.14 Å for all heavy atoms. Comparison between the mutant and wild-type structures elucidated the contribution of phenylalanine 15 in the modulation of the functional properties of PpcA.

Paixão, VB, Salgueiro CA, Brennan L, Reid GA, Chapman SK, Turner DL.  2008.  The Solution Structure of a Tetraheme Cytochrome from Shewanella frigidimarina Reveals a Novel Family Structural Motif. Biochemistry. 47(46):11973-11980. AbstractWebsite

The bacteria belonging to the genus Shewanella are facultative anaerobes that utilize a variety of terminal electron acceptors which includes soluble and insoluble metal oxides. The tetraheme c-type cytochrome isolated during anaerobic growth of Shewanella frigidimarina NCIMB400 (Sfc) contains 86 residues and is involved in the Fe(III) reduction pathways. Although the functional properties of Sfc redox centers are quite well described, no structures are available for this protein. In this work, we report the solution structure of the reduced form of Sfc. The overall fold is completely different from those of the tetraheme cytochromes c3 and instead has similarities with the tetraheme cytochrome recently isolated from Shewanella oneidensis (Soc). Comparison of the tetraheme cytochromes from Shewanella shows a considerable diversity in their primary structure and heme reduction potentials, yet they have highly conserved heme geometry, as is the case for the family of tetraheme cytochromes isolated from Desulfovibrio spp.

Messias, AC, Aguiar AP, Brennan L, Salgueiro CA, Saraiva LM, Xavier AV, Turner DL.  2006.  Solution structures of tetrahaem ferricytochrome c3 from Desulfovibrio vulgaris (Hildenborough) and its K45Q mutant: The molecular basis of cooperativity. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1757(2):143-153. AbstractWebsite

The NMR structure of the oxidised wild-type cytochrome c3 from Desulfovibrio vulgaris Hildenborough was determined in solution. Using a newly developed methodology, NMR data from the K45Q mutant was then grafted onto data from the wild-type protein to determine the structure in the region of the mutation. The structural origins of the redox-Bohr effect and haem–haem cooperativities are discussed with respect to the redox-related conformational changes observed in solution.

Todorovic, S, Leal SS, Salgueiro CA, Zebger I, Hildebrandt P, Murgida DH, Gomes CM.  2007.  A Spectroscopic Study of the Temperature Induced Modifications on Ferredoxin Folding and Iron−Sulfur Moieties. Biochemistry. 46(37):10733-10738. AbstractWebsite

Thermal perturbation of the dicluster ferredoxin from Acidianus ambivalens was investigated employing a toolbox of spectroscopic methods. FTIR and visible CD were used for assessing changes of the secondary structure and coarse alterations of the [3Fe4S] and [4Fe4S] cluster moieties, respectively. Fine details of the disassembly of the metal centers were revealed by paramagnetic NMR and resonance Raman spectroscopy. Overall, thermally induced unfolding of AaFd is initiated with the loss of α-helical content at relatively low temperatures (Tapp (m) ~ 44 °C), followed by the disruption of both iron−sulfur clusters (Tapp (m) ~ 53−60 °C). The degradation of the metal centers triggers major structural changes on the protein matrix, including the loss of tertiary contacts (Tapp (m) ~ 58 °C) and a change, rather than a significant net loss, of secondary structure (Tapp (m) ~ 60 °C). This latter process triggers a secondary structure reorganization that is consistent with the formation of a molten globule state. The combined spectroscopic approach here reported illustrates how changes in the metalloprotein organization are intertwined with disassembly of the iron−sulfur centers, denoting the conformational interplay of the protein backbone with cofactors.

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.

Dantas, JM, Portela PC, Fernandes AP, Londer YY, Yang X, Duke NEC, Schiffer M, Pokkuluri RP, Salgueiro CA.  2019.  Structural and Functional Relevance of the Conserved Residue V13 in the Triheme Cytochrome PpcA from Geobacter sulfurreducens. The Journal of Physical Chemistry B. 123:3050-3060., Number 14 AbstractWebsite

The triheme cytochrome PpcA from Geobacter sulfurreducens is highly abundant under several growth conditions and is important for extracellular electron transfer. PpcA plays a central role in transferring electrons resulting from the cytoplasmic oxidation of carbon compounds to the cell exterior. This cytochrome is designed to couple electron and proton transfer at physiological pH, a process achieved via the selection of dominant microstates during the redox cycle of the protein, which are ultimately regulated by a well-established order of oxidation of the heme groups. The three hemes are covered only by a polypeptide chain of 71 residues and are located in the small hydrophobic core of the protein. In this work, we used NMR and X-ray crystallography to investigate the structural and functional role of a conserved valine residue (V13) located within van der Waals contact of hemes III and IV. The residue was replaced by alanine (V13A), isoleucine (V13I), serine (V13S), and threonine (V13T) to probe the effects of the side chain volume and polarity. All mutants were found to be as equally thermally stable as the native protein. The V13A and V13T mutants produced crystals and their structures were determined. The side chain of the threonine residue introduced in V13T showed two conformations, but otherwise the two structures did not show significant changes from the native structure. Analysis of the redox behavior of the four mutants showed that for the hydrophobic replacements (V13A and V13I) the redox properties, and hence the order of oxidation of the hemes, were unaffected in spite of the larger side chain, isoleucine, showing two conformations with minor changes of the protein in the heme core. On the other hand, the polar replacements (V13S and V13T) showed the presence of two more distinctive conformations, and the oxidation order of the hemes was altered. Overall, it is striking that a single residue with proper size and polarity, V13, was naturally selected to ensure a unique conformation of the protein and the order of oxidation of the hemes, endowing the cytochrome PpcA with the optimal functional properties necessary to ensure effectiveness in the extracellular electron transfer respiratory pathways of G. sulfurreducens.

Morgado, L, Bruix M, Orshonsky V, Londer YY, Duke NEC, Yang X, Pokkuluri PR, Schiffer M, Salgueiro CA.  2008.  Structural insights into the modulation of the redox properties of two Geobacter sulfurreducens homologous triheme cytochromes. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1777(9):1157-1165. AbstractWebsite

The redox properties of a periplasmic triheme cytochrome, PpcB from Geobacter sulfurreducens, were studied by NMR and visible spectroscopy. The structure of PpcB was determined by X-ray diffraction. PpcB is homologous to PpcA (77% sequence identity), which mediates cytoplasmic electron transfer to extracellular acceptors and is crucial in the bioenergetic metabolism of Geobacter spp. The heme core structure of PpcB in solution, probed by 2D-NMR, was compared to that of PpcA. The results showed that the heme core structures of PpcB and PpcA in solution are similar, in contrast to their crystal structures where the heme cores of the two proteins differ from each other. NMR redox titrations were carried out for both proteins and the order of oxidation of the heme groups was determined. The microscopic properties of PpcB and PpcA redox centers showed important differences: (i) the order in which hemes become oxidized is III–I–IV for PpcB, as opposed to I–IV–III for PpcA; (ii) the redox-Bohr effect is also different in the two proteins. The different redox features observed between PpcB and PpcA suggest that each protein uniquely modulates the properties of their co-factors to assure effectiveness in their respective metabolic pathways. The origins of the observed differences are discussed.

Turner, DL, Salgueiro CA, Legall J, Xavier AV.  1992.  Structural studies of Desulfovibrio vulgaris ferrocytochrome c3 by two-dimensional NMR. European Journal of Biochemistry. 210(3):931-936. AbstractWebsite

Two-dimensional NMR has been used to make specific assignments for the four haems in Desulfovibrio vulgaris (Hildenborough) ferrocytochrome c3 and to determine their haem core architecture. The NMR signals from the haem protons were assigned according to type using two-dimensional NMR experiments which led to four sets of signals, one for each of the haems. Specific assignments were obtained by calculating the ring current shifts which arise from other haems and aromatic residues. Observation of interhaem NOEs confirmed the assignments and established that the relative orientation of the haems is identical to that found in the crystal structure of D. vulgaris (Miyazaki F.) ferricytochrome c3. Assignments were also made for all the aromatic residues except for the haem ligands and F20, which is shifted under the main envelope of signals. The NOEs observed between these aromatic protons and haem protons confirm the similarity between the structures in solution and in the crystal. The assignments reported here are the basis for the cross-assignments of the four microscopic haem redox potentials to specific haems in the protein structure.

Pokkuluri, PR, Londer YY, Duke NEC, Erickson J, Pessanha M, Salgueiro CA, Schiffer M.  2004.  Structure of a novel c7-type three-heme cytochrome domain from a multidomain cytochrome c polymer. Protein Science. 13(6):1684-1692. AbstractWebsite

The structure of a novel c7-type cytochrome domain that has two bishistidine coordinated hemes and one heme with histidine, methionine coordination (where the sixth ligand is a methionine residue) was determined at 1.7 Å resolution. This domain is a representative of domains that form three polymers encoded by the Geobacter sulfurreducens genome. Two of these polymers consist of four and one protein of nine c7-type domains with a total of 12 and 27 hemes, respectively. Four individual domains (termed A, B, C, and D) from one such multiheme cytochrome c (ORF03300) were cloned and expressed in Escherichia coli. The domain C produced diffraction quality crystals from 2.4 M sodium malonate (pH 7). The structure was solved by MAD method and refined to an R-factor of 19.5% and R-free of 21.8%. Unlike the two c7 molecules with known structures, one from G. sulfurreducens (PpcA) and one from Desulfuromonas acetoxidans where all three hemes are bishistidine coordinated, this domain contains a heme which is coordinated by a methionine and a histidine residue. As a result, the corresponding heme could have a higher potential than the other two hemes. The apparent midpoint reduction potential, Eapp, of domain C is −105 mV, 50 mV higher than that of PpcA.

Pokkuluri, PR, Londer YY, Duke NEC, Pessanha M, Yang X, Orshonsky V, Orshonsky L, Erickson J, Zagyanskiy Y, Salgueiro CA, Schiffer M.  2011.  Structure of a novel dodecaheme cytochrome c from Geobacter sulfurreducens reveals an extended 12 nm protein with interacting hemes. Journal of Structural Biology. 174(1):223-233. AbstractWebsite

Multiheme cytochromes c are important in electron transfer pathways in reduction of both soluble and insoluble Fe(III) by Geobacter sulfurreducens. We determined the crystal structure at 3.2 Å resolution of the first dodecaheme cytochrome c (GSU1996) along with its N-terminal and C-terminal hexaheme fragments at 2.6 and 2.15 Å resolution, respectively. The macroscopic reduction potentials of the full-length protein and its fragments were measured. The sequence of GSU1996 can be divided into four c7-type domains (A, B, C and D) with homology to triheme cytochromes c7. In cytochromes c7 all three hemes are bis–His coordinated, whereas in c7-type domains the last heme is His–Met coordinated. The full-length GSU1996 has a 12 nm long crescent shaped structure with the 12 hemes arranged along a polypeptide to form a “nanowire” of hemes; it has a modular structure. Surprisingly, while the C-terminal half of the protein consists of two separate c7-type domains (C and D) connected by a small linker, the N-terminal half of the protein has two c7-type domains (A and B) that form one structural unit. This is also observed in the AB fragment. There is an unexpected interaction between the hemes at the interface of domains A and B, which form a heme-pair with nearly parallel stacking of their porphyrin rings. The hemes adjacent to each other throughout the protein are within van der Waals distance which enables efficient electron exchange between them. For the first time, the structural details of c7-type domains from one multiheme protein were compared.

Dantas, JM, Campelo LM, Duke NEC, Salgueiro CA, Pokkuluri PR.  2015.  The structure of PccH from Geobacter sulfurreducens: a novel low reduction potential monoheme cytochrome essential for accepting electrons from an electrode. FEBS J. 282(11):2215-2231. AbstractWebsite

The structure of cytochrome c (GSU3274) designated as PccH from Geobacter sulfurreducens was determined at a resolution of 2.0 Å. PccH is a small (15 kDa) cytochrome containing one c-type heme, found to be essential for the growth of G. sulfurreducens with respect to accepting electrons from graphite electrodes poised at -300 mV versus standard hydrogen electrode. with fumarate as the terminal electron acceptor. The structure of PccH is unique among the monoheme cytochromes described to date. The structural fold of PccH can be described as forming two lobes with the heme sandwiched in a cleft between the two lobes. In addition, PccH has a low reduction potential of -24 mV at pH 7, which is unusual for monoheme cytochromes. Based on difference in structure, together with sequence phylogenetic analysis, we propose that PccH can be regarded as a first characterized example of a new subclass of class I monoheme cytochromes. The low reduction potential of PccH may enable the protein to be redox active at the typically negative potential ranges encountered by G. sulfurreducens. Because PccH is predicted to be located in the periplasm of this bacterium, it could not be involved in the first step of accepting electrons from the electrode but is very likely involved in the downstream electron transport events in the periplasm.

Silveira, CM, Castro MA, Dantas JM, Salgueiro C, Murgida DH, Todorovic S.  2017.  Structure, electrocatalysis and dynamics of immobilized cytochrome PccH and its microperoxidase, 2017. Physical Chemistry Chemical Physics. 19(13):8908-8918.: The Royal Society of Chemistry AbstractWebsite

Geobacter sulfurreducens cells have the ability to exchange electrons with conductive materials, and the periplasmic cytochrome PccH plays an essential role in the direct electrode-to-cell electron transfer in this bacterium. It has atypically low redox potential and unique structural features that differ from those observed in other c-type cytochromes. We report surface enhanced resonance Raman spectroscopic and electrochemical characterization of the immobilized PccH, together with molecular dynamics simulations that allow for the rationalization of experimental observations. Upon attachment to electrodes functionalized with partially or fully hydrophobic self-assembled monolayers, PccH displays a distribution of native and non-native heme spin configurations, similar to those observed in horse heart cytochrome c. The native structural and thermodynamic features of PccH are preserved upon attachment mixed hydrophobic (-CH3/-NH2) surfaces, while pure -OH, -NH2 and -COOH surfaces do not provide suitable platforms for its adsorption, indicating that its still unknown physiological redox partner might be membrane integrated. Neither of the employed immobilization strategies results in electrocatalytically active PccH capable of the reduction of hydrogen peroxide. Pseudoperoxidase activity is observed in immobilized microperoxidase, which is enzymatically produced from PccH and spectroscopically characterized. Further improvement of PccH microperoxidase stability is required for its application in electrochemical biosensing of hydrogen peroxide.

Pokkuluri, PR, Pessanha M, Londer YY, Wood SJ, Duke NEC, Wilton R, Catarino T, Salgueiro CA, Schiffer M.  2008.  Structures and Solution Properties of Two Novel Periplasmic Sensor Domains with c-Type Heme from Chemotaxis Proteins of Geobacter sulfurreducens: Implications for Signal Transduction. Journal of Molecular Biology. 377(5):1498-1517. AbstractWebsite

Periplasmic sensor domains from two methyl-accepting chemotaxis proteins from Geobacter sulfurreducens (encoded by genes GSU0935 and GSU0582) were expressed in Escherichia coli. The sensor domains were isolated, purified, characterized in solution, and their crystal structures were determined. In the crystal, both sensor domains form swapped dimers and show a PAS-type fold. The swapped segment consists of two helices of about 45 residues at the N terminus with the hemes located between the two monomers. In the case of the GSU0582 sensor, the dimer contains a crystallographic 2-fold symmetry and the heme is coordinated by an axial His and a water molecule. In the case of the GSU0935 sensor, the crystals contain a non-crystallographic dimer, and surprisingly, the coordination of the heme in each monomer is different; monomer A heme has His-Met ligation and monomer B heme has His-water ligation as found in the GSU0582 sensor. The structures of these sensor domains are the first structures of PAS domains containing covalently bound heme. Optical absorption, electron paramagnetic resonance and NMR spectroscopy have revealed that the heme groups of both sensor domains are high-spin and low-spin in the oxidized and reduced forms, respectively, and that the spin-state interconversion involves a heme axial ligand replacement. Both sensor domains bind NO in their ferric and ferrous forms but bind CO only in the reduced form. The binding of both NO and CO occurs via an axial ligand exchange process, and is fully reversible. The reduction potentials of the sensor domains differ by 95 mV (− 156 mV and − 251 mV for sensors GSU0582 and GSU0935, respectively). The swapped dimerization of these sensor domains and redox-linked ligand switch might be related to the mechanism of signal transduction by these chemotaxis proteins.

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.

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.

Santos, TC, de Oliveira AR, Dantas JM, Salgueiro CA, Cordas CM.  2015.  Thermodynamic and kinetic characterization of PccH, a key protein in microbial electrosynthesis processes in Geobacter sulfurreducens. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1847:1113-1118., Number 10 AbstractWebsite

Abstract The monoheme c-type cytochrome PccH from Geobacter sulfurreducens, involved in the pathway of current-consumption in biofilms, was electrochemically characterized in detail. Cyclic voltammetry was used to determine the kinetics and thermodynamics properties of PccH redox behavior. Entropy, enthalpy and Gibbs free energy changes associated with the redox center transition between the ferric and the ferrous state were determined, indicating an enhanced solvent exposure. The midpoint redox potential is considerably low for a monoheme c-type cytochrome and the heterogeneous electron transfer constant rate reflects a high efficiency of electron transfer process in PccH. The midpoint redox potential dependence on the pH (redox-Bohr effect) was investigated, over the range of 2.5 to 9.1, and is described by the protonation/deprotonation events of two distinct centers in the vicinity of the heme group with pKa values of 2.7 (pKox1); 4.1 (pKred1) and 5.9 (pKox2); 6.4 (pKred2). Based on the inspection of PccH structure, these centers were assigned to heme propionic acids \{P13\} and P17, respectively. The observed redox-Bohr effect indicates that PccH is able to thermodynamically couple electron and proton transfer in the G. sulfurreducens physiological pH range.

Silva, MA, Valente RC, Pokkuluri PR, Turner DL, Salgueiro CA, Catarino T.  2014.  Thermodynamic and kinetic characterization of two methyl-accepting chemotaxis heme sensors from Geobacter sulfurreducens reveals the structural origin of their functional difference. Biochim Biophys Acta. 1837(6):920-928. AbstractWebsite

The periplasmic sensor domains GSU582 and GSU935 are part of methyl-accepting chemotaxis proteins of the bacterium Geobacter sulfurreducens containing one c-type heme and a PAS-like fold. Their spectroscopic properties were shown previously to share similar spectral features. In both sensors, the heme group is in the high-spin form in the oxidized state and low-spin after reduction and binding of a methionine residue. Therefore, it was proposed that this redox-linked ligand switch might be related to the signal transduction mechanism. We now report the thermodynamic and kinetic characterization of the sensors GSU582 and GSU935 by visible spectroscopy and stopped-flow techniques, at several pH and ionic strength values. Despite their similar spectroscopic features, the midpoint reduction potentials and the rate constants for reduction by dithionite are considerably different in the two sensors. The reduction potentials of both sensors are negative and well framed within the typical anoxic subsurface environments in which Geobacter species predominate. The midpoint reduction potentials of sensor GSU935 are lower than those of GSU582 at all pH and ionic strength values and the same was observed for the reduction rate constants. The origin of the different functional properties of these closely related sensors is rationalized in the terms of the structures. The results suggest that the sensors are designed to function in different working potential ranges, allowing the bacteria to trigger an adequate cellular response in different anoxic subsurface environments. These findings provide an explanation for the co-existence of two similar methyl-accepting chemotaxis proteins in G. sulfurreducens.

Teixeira, LR, Dantas JM, Salgueiro CA, Cordas CM.  2018.  Thermodynamic and kinetic properties of the outer membrane cytochrome OmcF, a key protein for extracellular electron transfer in Geobacter sulfurreducens. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1859(10):1132-1137. AbstractWebsite

Gene knock-out studies on Geobacter sulfurreducens have shown that the monoheme c-type cytochrome OmcF is essential for the extracellular electron transfer pathways involved in the reduction of iron and uranium oxy-hydroxides, as well as, on electricity production in microbial fuel cells. A detailed electrochemical characterization of OmcF was performed for the first time, allowing attaining kinetics and thermodynamic data. The heterogeneous electron transfer rate constant was determined at pH 7 (0.16 ± 0.01 cm s−1) indicating that the protein displays high electron transfer efficiency compared to other monoheme cytochromes. The pH dependence of the redox potential indicates that the protein has an important redox-Bohr effect in the physiological pH range for G. sulfurreducens growth. The analysis of the structures of OmcF allowed us to assign the redox-Bohr centre to the side chain of His47 residue and its pKa values in the reduced and oxidized states were determined (pKox = 6.73; pKred = 7.55). The enthalpy, entropy and Gibbs free energy associated with the redox transaction were calculated, pointing the reduced form of the cytochrome as the most favourable. The data obtained indicate that G. sulfurreducens cells evolved to warrant a down-hill electron transfer from the periplasm to the outer-membrane associated cytochrome OmcF.

Pessanha, M, Louro RO, Correia IJ, Rothery EL, Pankhurst KL, Reid GA, Chapman SK, Turner DL, Salgueiro CA.  2003.  Thermodynamic characterization of a tetrahaem cytochrome isolated from a facultative aerobic bacterium, Shewanella frigidimarina: a putative redox model for flavocytochrome c3. Biochemical Journal. 370(Pt. 2):489-495. AbstractWebsite

The facultative aerobic bacterium Shewanella frigidimarina produces a small c-type tetrahaem cytochrome (86 residues) under anaerobic growth conditions. This protein is involved in the respiration of iron and shares 42% sequence identity with the N-terminal domain of a soluble flavocytochrome, isolated from the periplasm of the same bacterium, which also contains four c-type haem groups. The thermodynamic properties of the redox centres and of an ionizable centre in the tetrahaem cytochrome were determined using NMR and visible spectroscopy techniques. This is the first detailed thermodynamic study performed on a tetrahaem cytochrome isolated from a facultative aerobic bacterium and reveals that this protein presents unique features. The redox centres have negative and different redox potentials, which are modulated by redox interactions between the four haems (covering a range of 8–56mV) and by redox–Bohr interactions between the haems and an ionizable centre (-4 to -36mV) located in close proximity to haem III. All of the interactions between the five centres are clearly dominated by electrostatic effects and the microscopic reduction potential of haem III is the one most affected by the oxidation of the other haems and by the protonation state of the molecule. Altogether, this study indicates that the tetrahaem cytochrome isolated from S. frigidimarina (Sfc) has the thermodynamic properties to work as an electron wire between its redox partners. Considering the high degree of sequence identity between Sfc and the cytochrome domain of flavocytochrome c3, the structural similarities of the haem core, and that the macroscopic potentials are also identical, the results obtained in this work are rationalized in order to put forward a putative redox model for flavocytochrome c3.

Morgado, L, Bruix M, Pessanha M, Londer YY, Salgueiro CA.  2010.  Thermodynamic Characterization of a Triheme Cytochrome Family from Geobacter sulfurreducens Reveals Mechanistic and Functional Diversity. Biophysical Journal. 99(1):293-301. AbstractWebsite

A family of five periplasmic triheme cytochromes (PpcA-E) was identified in Geobacter sulfurreducens, where they play a crucial role by driving electron transfer from the cytoplasm to the cell exterior and assisting the reduction of extracellular acceptors. The thermodynamic characterization of PpcA using NMR and visible spectroscopies was previously achieved under experimental conditions identical to those used for the triheme cytochrome c7 from Desulfuromonas acetoxidans. Under such conditions, attempts to obtain NMR data were complicated by the relatively fast intermolecular electron exchange. This work reports the detailed thermodynamic characterization of PpcB, PpcD, and PpcE under optimal experimental conditions. The thermodynamic characterization of PpcA was redone under these new conditions to allow a proper comparison of the redox properties with those of other members of this family. The heme reduction potentials of the four proteins are negative, differ from each other, and cover different functional ranges. These reduction potentials are strongly modulated by heme-heme interactions and by interactions with protonated groups (the redox-Bohr effect) establishing different cooperative networks for each protein, which indicates that they are designed to perform different functions in the cell. PpcA and PpcD appear to be optimized to interact with specific redox partners involving e−/H+ transfer via different mechanisms. Although no evidence of preferential electron transfer pathway or e−/H+ coupling was found for PpcB and PpcE, the difference in their working potential ranges suggests that they may also have different physiological redox partners. This is the first study, to our knowledge, to characterize homologous cytochromes from the same microorganism and provide evidence of their different mechanistic and functional properties. These findings provide an explanation for the coexistence of five periplasmic triheme cytochromes in G. sulfurreducens.

Morgado, L, Fernandes AP, Londer YY, Pokkuluri PR, Schiffer M, Salgueiro CA.  2009.  Thermodynamic characterization of the redox centres in a representative domain of a novel c-type multihaem cytochrome. Biochemical Journal. 420(3):485-492. AbstractWebsite

Multihaem cytochromes that could form protein “nanowires” were identified in the Geobacter sulfurreducens genome, and represent a new type of multihaem cytochrome. The sequences of these proteins, two with 12 haems (GSU1996, GSU0592) and one with 27 haems (GSU2210), suggest that they are formed with domains homologous to the trihaem cytochrome c7. Although all three haems have bis-His co-ordination in cytochromes c7, in each domain of the above polymers, the haem equivalent to haem IV has His-Met co-ordination. We previously determined the structure and measured the macroscopic redox potential of one representative domain (domain C) of a dodecahaem cytochrome (GSU1996). In the present study, the microscopic redox properties of the individual haem groups of domain C were determined using NMR and UV–visible spectroscopies. The reduction potentials of the haems for the fully reduced and protonated protein are different from each other (haem I, −106 mV; haem III, −136 mV; and haem IV, −125 mV) and are strongly modulated by redox interactions. This result is rather surprising since the His-Met co-ordinated haem IV does not have the highest potential as was expected. The polypeptide environment of each haem group and the strong haem pairwise redox interactions must play a dominant role in controlling the individual haem potentials. The strong redox interactions between the haems extend the range of their operating potentials at physiological pH (haem I, −71 mV, haem III, −146 mV and haem IV, −110 mV). Such a modulation in haem potentials is likely to have a functional significance in the metabolism of G. sulfurreducens.