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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.

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

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

Pessanha, M, Brennan L, Xavier AV, Cuthbertson PM, Reid GA, Chapman SK, Turner DL, Salgueiro CA.  2001.  NMR structure of the haem core of a novel tetrahaem cytochrome isolated from Shewanella frigidimarina: identification of the haem-specific axial ligands and order of oxidation. FEBS Letters. 489(1):8-13. AbstractWebsite

The tetrahaem cytochrome isolated during anaerobic growth of Shewanella frigidimarina NCIMB400 is a small protein (86 residues) involved in electron transfer to Fe(III), which can be used as a terminal respiratory oxidant by this bacterium. A 3D solution structure model of the reduced form of the cytochrome has been determined using NMR data in order to determine the relative orientation of the haems. The haem core architecture of S. frigidimarina tetrahaem cytochrome differs from that found in all small tetrahaem cytochromes c3 so far isolated from strict anaerobes, but has some similarity to the N-terminal cytochrome domain of flavocytochrome c3 isolated from the same bacterium. NMR signals obtained for the four haems of S. frigidimarina tetrahaem cytochrome at all stages of oxidation were cross-assigned to the solution structure using the complete network of chemical exchange connectivities. Thus, the order in which each haem in the structure becomes oxidised was determined.

Pessanha, M, Rothery EL, Miles CS, Reid GA, Chapman SK, Louro RO, Turner DL, Salgueiro CA, Xavier AV.  2009.  Tuning of functional heme reduction potentials in Shewanella fumarate reductases. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1787(2):113-120. AbstractWebsite

The fumarate reductases from S. frigidimarina NCIMB400 and S. oneidensis MR-1 are soluble and monomeric enzymes located in the periplasm of these bacteria. These proteins display two redox active domains, one containing four c-type hemes and another containing FAD at the catalytic site. This arrangement of single-electron redox co-factors leading to multiple-electron active sites is widespread in respiratory enzymes. To investigate the properties that allow a chain of single-electron co-factors to sustain the activity of a multi-electron catalytic site, redox titrations followed by NMR and visible spectroscopies were applied to determine the microscopic thermodynamic parameters of the hemes. The results show that the redox behaviour of these fumarate reductases is similar and dominated by a strong interaction between hemes II and III. This interaction facilitates a sequential transfer of two electrons from the heme domain to FAD via heme IV.

Pessanha, M, Rothery EL, Louro RO, Turner DL, Miles CS, Reid GA, Chapman SK, Xavier AV, Salgueiro CA.  2005.  Elucidation of the Functional Redox Behavior of Fumarate Reductase from Shewanella frigidimarina by NMR. Annals Magnetic Resonance. 4(1/2):24-28. AbstractWebsite

NMR spectroscopy has been applied with great success to study electron transfer proteins
with multiple redox centers. This study aimed to elucidate the redox behavior the enzyme fumarate
reductase from Shewanella frigidimarina and particularly to reveal the electron transfer mechanism
from the N-terminal domain to the active center. We developed a new strategy encompassing the
acquisition of 1H-EXSY bidimensional spectra for observation of chemical exchange connectivities in
partially oxidized samples of fcc3, estimation of the paramagnetic chemical shifts expected for the
heme substituents and their comparison with NMR spectra obtained in the fully oxidized protein. This
study allowed obtaining the order of oxidation of the different groups (II-I-III, IV) and gave insights of
the functional mechanisms that allow fcc3 to efficiently transfer electrons from the N-terminal domain
to the active center.

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.

Pessanha, M, Turner DL, Rothery EL, Pankhurst KL, Reid GA, Chapman SK, Xavier AV, Salgueiro CA.  2003.  NMR redox studies of flavocytochrome c3 from Shewanella frigidimarina. Inorganica Chimica Acta. 356:379-381. AbstractWebsite

Flavocytochrome c3 is a periplasmic fumarate reductase with Mr 63.8 kDa, isolated from Shewanella frigidimarina NCIMB400. NMR spectroscopy was tested for its potential to elucidate the oxidation profile of each of the four haem groups in the enzyme, using the strategy developed previously to perform the thermodynamic characterization of small tetrahaem cytochromes (FEBS Lett. 314 (1992) 155). This work shows that, despite the large size of the protein, 2D-NMR NOESY experiments can be used to obtain the network of chemical exchange connectivities, between the signals of specific haem groups in sequential oxidation stages.

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, Morgado L, Louro RO, Londer YY, Pokkuluri PR, Schiffer M, Salgueiro CA.  2006.  Thermodynamic Characterization of Triheme Cytochrome PpcA from Geobacter sulfurreducens:  Evidence for a Role Played in e-/H+ Energy Transduction. Biochemistry. 45(46):13910-13917. AbstractWebsite

The facultative aerobic bacterium Geobacter sulfurreducens produces a small periplasmic c-type triheme cytochrome with 71 residues (PpcA) under anaerobic growth conditions, which is involved in the iron respiration. The thermodynamic properties of the PpcA redox centers and of a protonatable center were determined using NMR and visible spectroscopy techniques. The redox centers have negative and different reduction potentials (−162, −143, and −133 mV for heme I, III, and IV, respectively, for the fully reduced and protonated protein), which are modulated by redox interactions among the hemes (covering a range from 10 to 36 mV) and by redox−Bohr interactions (up to −62 mV) between the hemes and a protonatable center located in the proximity of heme IV. All the interactions between the four centers are dominated by electrostatic effects. The microscopic reduction potential of heme III is the one most affected by the oxidation of the other hemes, whereas heme IV is the most affected by the protonation state of the molecule. The thermodynamic properties of PpcA showed that pH strongly modulates the redox behavior of the individual heme groups. A preferred electron transfer pathway at physiologic pH is defined, showing that PpcA has the necessary thermodynamic properties to perform e-/H+ energy transduction, contributing to a H+ electrochemical potential gradient across the periplasmic membrane that drives ATP synthesis. PpcA is 46% identical in sequence to and shares a high degree of structural similarity with a periplasmic triheme cytochrome c7 isolated from Desulfuromonas acetoxidans, a bacterium closely related to the Geobacteracea family. However, the results obtained for PpcA are quite different from those published for D. acetoxidans c7, and the physiological consequences of these differences are discussed.

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.

Pimenta, AI, Paquete CM, Morgado L, Edwards MJ, Clarke TA, Salgueiro CA, Pereira IAC, Duarte AG.  2023.  Characterization of the inner membrane cytochrome ImcH from Geobacter reveals its importance for extracellular electron transfer and energy conservation. Protein Science. 32:e4796., Number 11 AbstractWebsite

Abstract Electroactive bacteria combine the oxidation of carbon substrates with an extracellular electron transfer (EET) process that discharges electrons to an electron acceptor outside the cell. This process involves electron transfer through consecutive redox proteins that efficiently connect the inner membrane to the cell exterior. In this study, we isolated and characterized the quinone-interacting membrane cytochrome c ImcH from Geobacter sulfurreducens, which is involved in the EET process to high redox potential acceptors. Spectroscopic and electrochemical studies show that ImcH hemes have low midpoint redox potentials, ranging from −150 to −358 mV, and connect the oxidation of the quinol-pool to EET, transferring electrons to the highly abundant periplasmic cytochrome PpcA with higher affinity than to its homologues. Despite the larger number of hemes and transmembrane helices, the ImcH structural model has similarities with the NapC/NirT/NrfH superfamily, namely the presence of a quinone-binding site on the P-side of the membrane. In addition, the first heme, likely involved on the quinol oxidation, has apparently an unusual His/Gln coordination. Our work suggests that ImcH is electroneutral and transfers electrons and protons to the same side of the membrane, contributing to the maintenance of a proton motive force and playing a central role in recycling the menaquinone pool.

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.

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.

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.

Portela, PC, Morgado L, Silva MA, Denkhaus L, Einsle O, Salgueiro CA.  2023.  Exploring oxidative stress pathways in Geobacter sulfurreducens: the redox network between MacA peroxidase and triheme periplasmic cytochromes. Frontiers in Microbiology. 14 AbstractWebsite

The recent reclassification of the strict anaerobe Geobacter sulfurreducens bacterium as aerotolerant brought attention for oxidative stress protection pathways. Although the electron transfer pathways for oxygen detoxification are not well established, evidence was obtained for the formation of a redox complex between the periplasmic triheme cytochrome PpcA and the diheme cytochrome peroxidase MacA. In the latter, the reduction of the high-potential heme triggers a conformational change that displaces the axial histidine of the low-potential heme with peroxidase activity. More recently, a possible involvement of the triheme periplasmic cytochrome family (PpcA-E) in the protection from oxidative stress in G. sulfurreducens was suggested. To evaluate this hypothesis, we investigated the electron transfer reaction and the biomolecular interaction between each PpcA-E cytochrome and MacA. Using a newly developed method that relies on the different NMR spectral signatures of the heme proteins, we directly monitored the electron transfer reaction from reduced PpcA-E cytochromes to oxidized MacA. The results obtained showed a complete electron transfer from the cytochromes to the high-potential heme of MacA. This highlights PpcA-E cytochromes’ efficient role in providing the necessary reducing power to mitigate oxidative stress situations, hence contributing to a better knowledge of oxidative stress protection pathways in G. sulfurreducens.

Portela, PC, Dantas JM, Salgueiro CA.  2020.  Backbone, side chain and heme resonance assignment of the triheme cytochrome PpcA from Geobacter metallireducens in the oxidized state, 2020. Biomol NMR Assign. 14(1):31-36. AbstractWebsite

The bacterium Geobacter metallireducens is capable of transferring electrons to the cell exterior, a process designated extracellular electron transfer. This mechanism allows the microorganism to reduce extracellular acceptors such as Fe(III) (hydr)oxides and water toxic and/or radioactive contaminants including Cr(VI) and U(VI). It is also capable of oxidizing waste water aromatic organic compounds being an important microorganism for bioremediation of polluted waters. Extracellular electron transfer also allows electricity harvesting from microbial fuel cells, a promising sustainable form of energy production. However, extracellular electron transfer processes in this microorganism are still poorly characterized. The triheme c-type cytochrome PpcA from G. metallireducens is abundant in the periplasm and is crucial for electron transfer between the cytoplasm and the cell’s exterior. In this work, we report near complete assignment of backbone, side chain and heme resonances for PpcA in the oxidized state that will permit its structure determination and identification of interactions with physiological redox partners.

Portela, PC, Silva MA, Teixeira LR, Salgueiro CA.  2021.  A unique aromatic residue modulates the redox range of a periplasmic multiheme cytochrome from Geobacter metallireducens. Journal of Biological Chemistry. 296:100711. AbstractWebsite

Geobacter bacteria are able to transfer electrons to the exterior of the cell and reduce extracellular electron acceptors including toxic/radioactive metals and electrode surfaces, with potential applications in bioremediation or electricity harvesting. The triheme c-type cytochrome PpcA from Geobacter metallireducens plays a crucial role in bridging the electron transfer from the inner to the outer membrane, ensuring an effective extracellular electron transfer. This cytochrome shares 80% identity with PpcA from Geobacter sulfurreducens, but their redox properties are markedly different, thus determining the distinctive working redox potential ranges in the two bacteria. PpcA from G. metallireducens possesses two extra aromatic amino acids (Phe-6 and Trp-45) in its hydrophobic heme core, whereas PpcA from G. sulfurreducens has a leucine and a methionine in the equivalent positions. Given the different nature of these residues in the two cytochromes, we have hypothesized that the extra aromatic amino acids could be partially responsible for the observed functional differences. In this work, we have replaced Phe-6 and Trp-45 residues by their nonaromatic counterparts in PpcA from G. sulfurreducens. Using redox titrations followed by UV–visible and NMR spectroscopy we observed that residue Trp-45 shifted the redox potential range 33% toward that of PpcA from G. sulfurreducens, whereas Phe-6 produced a negligible effect. For the first time, it is shown that the inclusion of an aromatic residue at the heme core can modulate the working redox range in abundant periplasmic proteins, paving the way to engineer bacterial strains for optimal microbial bioelectrochemical applications.

Portela, PC, Fernandes TM, Dantas JM, Ferreira MR, Salgueiro CA.  2018.  Biochemical and functional insights on the triheme cytochrome PpcA from Geobacter metallireducens. Archives of Biochemistry and Biophysics. 644:8-16. AbstractWebsite

G. metallireducens bacterium has highly versatile respiratory pathways that provide the microorganism an enormous potential for many biotechnological applications. However, little is known about the structural and functional properties of its electron transfer components. In this work, the periplasmic cytochrome PpcA from G. metallireducens was studied in detail for the first time using complementary biophysical techniques, including UV–visible, CD and NMR spectroscopy. The results obtained showed that PpcA contains three low-spin c-type heme groups with His-His axial coordination, a feature also observed for its homologue in G. sulfurreducens. However, despite the high sequence homology between the two cytochromes, important structural and functional differences were observed. The comparative analysis of the backbone, side chain and heme substituents NMR signals revealed differences in the relative orientation of the hemes I and III. In addition, redox titrations followed by visible spectroscopy showed that the redox potential values for PpcA from G. metallireducens (−78 and −93 mV at pH 7 and 8, respectively) are considerably less negative. Overall, this study provides biochemical and biophysical data of a key cytochrome from G. metallireducens, paving the way to understand the extracellular electron transfer mechanisms in these bacteria.