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.

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.

The cytochrome PccH from Geobacter sulfurreducens (Gs) plays a crucial role in current-consuming fumarate-reducing biofilms. Deletion of pccH gene inhibited completely electron transfer from electrodes toward Gs cells. The pccH gene was cloned and the protein heterologously expressed in Escherichia coli. Complementary biophysical techniques including CD, UV-visible and NMR spectroscopy were used to characterize PccH. This cytochrome contains one low-spin c-type heme with His-Met axial coordination and unusual low-reduction potential. This reduction potential is pH-dependent, within the Gs physiological pH range, and is discussed within the context of the electron transfer mechanisms from electrodes to Gs cells.

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.

PpcA is the most abundant member of a family of five triheme cytochromes c7 in the bacterium Geobacter sulfurreducens (Gs) and is the most likely carrier of electrons destined for outer surface during respiration on solid metal oxides, a process that requires extracellular electron transfer. This cytochrome has the highest content of lysine residues (24%) among the family, and it was suggested to be involved in e-/H(+) energy transduction processes. In the present work, we investigated the functional role of lysine residues strategically located in the vicinity of each heme group. Each lysine was replaced by glutamine or glutamic acid to evaluate the effects of a neutral or negatively charged residue in each position. The results showed that replacing Lys9 (located near heme IV), Lys18 (near heme I) or Lys22 (between hemes I and III) has essentially no effect on the redox properties of the heme groups and are probably involved in redox partner recognition. On the other hand, Lys43 (near heme IV), Lys52 (between hemes III and IV) and Lys60 (near heme III) are crucial in the regulation of the functional mechanism of PpcA, namely in the selection of microstates that allow the protein to establish preferential e-/H(+) transfer pathways. The results showed that the preferred e-/H(+) transfer pathways are only established when heme III is the last heme to oxidize, a feature reinforced by a higher difference between its reduction potential and that of its predecessor in the order of oxidation. We also showed that K43 and K52 mutants keep the mechanistic features of PpcA by establishing preferential e-/H+ transfer pathways at lower reduction potential values than the wild-type protein, a property that can enable rational design of Gs strains with optimized extracellular electron transfer capabilities.

The bacterium Geobacter sulfurreducens displays an extraordinary respiratory versatility underpinning the diversity of electron donors and acceptors that can be used to sustain anaerobic growth. Remarkably, G. sulfurreducens can also use as electron donors the reduced forms of some acceptors, such as the humic substance analog anthraquinone-2,6-disulfonate (AQDS), a feature that confers environmentally competitive advantages to the organism. Using UV-visible and stopped-flow kinetic measurements we demonstrate that there is electron exchange between the triheme cytochrome PpcA from Gs and AQDS. 2D-(1)H-(15)N HSQC NMR spectra were recorded for (15)N-enriched PpcA samples, in the absence and presence of AQDS. Chemical shift perturbation measurements, at increasing concentration of AQDS, were used to probe the interaction region and to measure the binding affinity of the PpcA-AQDS complex. The perturbations on the NMR signals corresponding to the PpcA backbone NH and heme substituents showed that the region around heme IV interacts with AQDS through the formation of a complex with a definite life time in the NMR time scale. The comparison of the NMR data obtained for PpcA in the presence and absence of AQDS showed that the interaction is reversible. Overall, this study provides for the first time a clear illustration of the formation of an electron transfer complex between AQDS and a G. sulfurreducens triheme cytochrome, shedding light on the electron transfer pathways underlying the microbial oxidation of humics.

The purple bacterium Rhodopseudomonas palustris TIE-1 expresses multiple small high-potential redox proteins during photoautotrophic growth, including two high-potential iron-sulfur proteins (HiPIPs) (PioC and Rpal_4085) and a cytochrome c2. We evaluated the role of these proteins in TIE-1 through genetic, physiological, and biochemical analyses. Deleting the gene encoding cytochrome c2 resulted in a loss of photosynthetic ability by TIE-1, indicating that this protein cannot be replaced by either HiPIP in cyclic electron flow. PioC was previously implicated in photoferrotrophy, an unusual form of photosynthesis in which reducing power is provided through ferrous iron oxidation. Using cyclic voltammetry (CV), electron paramagnetic resonance (EPR) spectroscopy, and flash-induced spectrometry, we show that PioC has a midpoint potential of 450 mV, contains all the typical features of a HiPIP, and can reduce the reaction centers of membrane suspensions in a light-dependent manner at a much lower rate than cytochrome c2. These data support the hypothesis that PioC linearly transfers electrons from iron, while cytochrome c2 is required for cyclic electron flow. Rpal_4085, despite having spectroscopic characteristics and a reduction potential similar to those of PioC, is unable to reduce the reaction center. Rpal_4085 is upregulated by the divalent metals Fe(II), Ni(II), and Co(II), suggesting that it might play a role in sensing or oxidizing metals in the periplasm. Taken together, our results suggest that these three small electron transfer proteins perform different functions in the cell.

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.

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.

Abstract Humic substances (HS) constitute a significant fraction of natural organic matter in terrestrial and aquatic environments and can act as terminal electron acceptors in anaerobic microbial respiration. Geobacter sulfurreducens has a remarkable respiratory versatility and can utilize the \{HS\} analog anthraquinone-2,6-disulfonate (AQDS) as a terminal electron acceptor or its reduced form (AH2QDS) as an electron donor. Previous studies set the triheme cytochrome PpcA as a key component for \{HS\} respiration in G. sulfurreducens, but the process is far from fully understood. In this work, \{NMR\} chemical shift perturbation measurements were used to map the interaction region between PpcA and AH2QDS, and to measure their binding affinity. The results showed that the \{AH2QDS\} binds reversibly to the more solvent exposed edge of PpcA heme IV. The \{NMR\} and visible spectroscopies coupled to redox measurements were used to determine the thermodynamic parameters of the PpcA:quinol complex. The higher reduction potential of heme İV\} (− 127 mV) compared to that of \{AH2QDS\} (− 184 mV) explains why the electron transfer is more favorable in the case of reduction of the cytochrome by the quinol. The clear evidence obtained for the formation of an electron transfer complex between \{AH2QDS\} and PpcA, combined with the fact that the protein also formed a redox complex with AQDS, revealed for the first time the bifunctional behavior of PpcA toward an analog of the HS. Such behavior might confer selective advantage to G. sulfurreducens, which can utilize the \{HS\} in any redox state available in the environment for its metabolic needs.

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.

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.

Gene knockout studies on Geobacter sulfurreducens (Gs) cells showed that the outer membrane cytochrome OmcF is involved in respiratory pathways leading to the extracellular reduction of Fe(III) citrate and U(VI) oxide. In addition, microarray analysis of OmcF-deficient mutant versus the wild-type strain revealed that many of the genes with decreased transcript level were those whose expression is upregulated in cells grown with a graphite electrode as electron acceptor. This suggests that OmcF also regulates the electron transfer to electrode surfaces and the concomitant electrical current production by Gs in microbial fuel cells. Extracellular electron transfer processes (EET) constitute nowadays the foundations to develop biotechnological applications in biofuel production, bioremediation and bioenergy. Therefore, the structural characterization of OmcF is a fundamental step to understand the mechanisms underlying EET. Here, we report the complete assignment of the heme proton signals together with (1)H, (13)C and (15)N backbone and side chain assignments of the OmcF, excluding the hydrophobic residues of the N-terminal predicted lipid anchor.

Gene knock-out studies on Geobacter sulfurreducens (Gs) cells showed that the periplasmic triheme cytochrome PpcD is involved in respiratory pathways leading to the extracellular reduction of Fe(III) and U(VI) oxides. More recently, it was also shown that the gene encoding for PpcD has higher transcript abundance when Gs cells utilize graphite electrodes as sole electron donors to reduce fumarate. This sets PpcD as the first multiheme cytochrome to be involved in Gs respiratory pathways that bridge the electron transfer between the cytoplasm and cell exterior in both directions. Nowadays, extracellular electron transfer (EET) processes are explored for several biotechnological applications, which include bioremediation, bioenergy and biofuel production. Therefore, the structural characterization of PpcD is a fundamental step to understand the mechanisms underlying EET. However, compared to non-heme proteins, the presence of numerous proton-containing groups in the redox centers presents additional challenges for protein signal assignment and structure calculation. Here, we report the complete assignment of the heme proton signals together with 1H, 13C and 15N backbone and side chain assignments of the reduced form of PpcD.

Geobacter bacteria have a remarkable respiratory versatility that includes the dissimilatory reduction of insoluble metal oxides in natural habitats and electron transfer to electrode surfaces from which electricity can be harvested. In both cases, electrons need to be exported from the cell interior to the exterior via a mechanism designated as extracellular electron transfer (EET). Several c-type cytochromes from G. sulfurreducens (Gs) were identified as key players in this process. Biochemical and biophysical data have been obtained for ten Gs cytochromes, including inner-membrane associated (MacA), periplasmic (PpcA, PpcB, PpcC, PpcD, PpcE and GSU1996) and outer membrane-associated (OmcF, OmcS and OmcZ). The redox properties of these cytochromes have been determined, except for PpcC and GSU1996. In this perspective, the reduction potentials of these two cytochromes were determined by potentiometric redox titrations followed by visible spectroscopy. The data obtained are taken together with those available for other key cytochromes to present a thorough overview of the current knowledge of Gs EET mechanisms and provide a possible rationalization for the existence of several multiheme cytochromes involved in the same respiratory pathways.

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.

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.

A family of triheme cytochromes from Geobacter sulfurreducens plays an important role in extracellular electron transfer. In addition to their role in electron transfer pathways, two members of this family (PpcA and PpcD) were also found to be able to couple e–/H+ transfer through the redox Bohr effect observed in the physiological pH range, a feature not observed for cytochromes PpcB and PpcE. In attempting to understand the molecular control of the redox Bohr effect in this family of cytochromes, which is highly homologous both in amino acid sequence and structures, it was observed that residue 6 is a conserved leucine in PpcA and PpcD, whereas in the other two characterized members (PpcB and PpcE) the equivalent residue is a phenylalanine. To determine the role of this residue located close to the redox Bohr center, we replaced Leu6 in PpcA with Phe and determined the redox properties of the mutant, as well as its solution structure in the fully reduced state. In contrast with the native form, the mutant PpcAL6F is not able to couple the e–/H+ pathway. We carried out the reverse mutation in PpcB and PpcE (i.e., replacing Phe6 in these two proteins by leucine) and the mutated proteins showed an increased redox Bohr effect. The results clearly establish the role of residue 6 in the control of the redox Bohr effect in this family of cytochromes, a feature that could enable the rational design of G. sulfurreducens strains that carry mutant cytochromes with an optimal redox Bohr effect that would be suitable for various biotechnological applications.

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The extracellular electron transfer metabolism of Geobacter sulfurreducens is sustained by several multiheme c-type cytochromes. One of these is the dodecaheme cytochrome GSU1996 that belongs to a new sub-class of c-type cytochromes. GSU1996 is composed by four similar triheme domains (A-D). The C-terminal half of the molecule encompasses the domains C and D, which are connected by a small linker and the N-terminal half of the protein contains two domains (A and B) that form one structural unit. It was proposed that this protein works as an electrically conductive device in Geobacter sulfurreducens, transferring electrons within the periplasm or to outer-membrane cytochromes. In this work, a novel strategy was applied to characterize in detail the thermodynamic and kinetic properties of the hexaheme fragment CD of GSU1996. This characterization revealed the electron transfer process of GSU1996 for the first time, showing that a heme at the edge of the C-terminal of the protein is thermodynamic and kinetically competent to receive electrons from physiological redox partners. This information contributes towards understanding how this new sub-class of cytochromes functions as nanowires, and also increases the current knowledge of the extracellular electron transfer mechanisms in Geobacter sulfurreducens.

Accepted Manuscript online January 16, 2017.Geobacter bacteria usually prevail among other microorganisms in soils and sediments where Fe(III) reduction has a central role. This reduction is achieved by extracellular electron transfer (EET), where the electrons are exported from the interior of the cell to the surrounding environment. Periplasmic cytochromes play an important role in establishing an interface between inner and outer membrane electron transfer components. In addition, periplasmic cytochromes, in particular nanowire cytochromes that contain at least 12 haem groups, have been proposed to play a role in electron storage in conditions of an environmental lack of electron acceptors. Up to date, no redox partners have been identified in Geobacter sulfurreducens, and concomitantly, the EET and electron storage mechanisms remain unclear. In this work, NMR chemical shift perturbation measurements were used to probe for an interaction between the most abundant periplasmic cytochrome PpcA and the dodecahaem cytochrome GSU1996, one of the proposed nanowire cytochromes in G. sulfurreducens. The perturbations on the haem methyl signals of GSU1996 and PpcA showed that the proteins form a transient redox complex in an interface that involves haem groups from two different domains located at the C-terminal of GSU1996. Overall, the present study provides for the first time a clear evidence for an interaction between periplasmic cytochromes that might be relevant for the EET and electron storage pathways in G. sulfurreducens.1D, one-dimensional; CbcL, c- and b-type cytochrome for low potential; EET, extracellular electron transfer; HP, His-patch; ImcH, inner membrane c-type cytochrome; MacA, metal-reduction-associated cytochrome; NaPi, sodium phosphate; NBAF, acetate-fumarate medium; NMR, nuclear magnetic resonance; PpcA, periplasmic c-type cytochrome; SDS–PAGE, sodium dodecyl sulphate–polyacrylamide gel electrophoresis; STC, small tetrahaem cytochrome.

Proteomic and genetic studies have identified a family of five triheme cytochromes (PpcA-E) that are essential in the iron respiratory pathways of Geobacter sulfurreducens. These include the reduction of Fe(iii) soluble chelated forms or Fe(iii) oxides{,} which can be used as terminal acceptors by G. sulfurreducens. The relevance of these cytochromes in the respiratory pathways of soluble or insoluble forms of iron is quite distinct. In fact{,} while PpcD had a higher abundance in the Fe(iii) oxides supplanted G. sulfurreducens cultures{,} PpcA{,} PpcB and PpcE were important in Fe(iii) citrate supplanted cultures. Based on these observations we probed the molecular interactions between these cytochromes and Fe(iii) citrate by NMR spectroscopy. NMR spectra were recorded for natural abundance and 15N-enriched PpcA{,} PpcB or PpcE samples at increasing amounts of Fe(iii) citrate. The addition of this molecule caused pronounced perturbations on the line width of the protein{'}s NMR signals{,} which were used to map the interaction region between each cytochrome and the Fe(iii) citrate molecule. The perturbations on the NMR signals corresponding to the backbone NH and heme methyl substituents showed that complex interfaces consist of a well-defined patch{,} which surrounds the more solvent-exposed heme IV methyl groups in each cytochrome. Overall{,} this study provides for the first time a clear illustration of the formation of an electron transfer complex between Fe(iii) citrate and G. sulfurreducens triheme cytochromes{,} shown to be crucial in this respiratory pathway.

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.

The discovery of microorganisms with the ability of Extracellular Electron Transfer (EET), nearly three decades ago, sparked interest due to their ability to be used in diverse applications that can range from bioremediation to electricity production in Microbial Fuel Cells (MFC). Microbial respiration is based on electron transfer from a donor to an electron acceptor, through a series of stepwise electron transfer events that generate the necessary metabolic energy. Some microorganisms, such as Pseudomonas species, Shewanella putrefaciens or Geothrix fermentans are able to produce electrochemical mediators to increase the EET. The mechanical stability of the biofilm is provided by the biofilm matrix, a hydrated extracellular polymeric matrix that encases the biofilm cells. The biofilm matrix could potentially offer a resistance pathway to EET unless bacteria develop strategies to increase its conductivity. MFC devices currently being used and studied do not generate sufficient power to support widespread and cost-effective applications.