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.

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.

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.

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.

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.

No abstract included.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.