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.

Nuclear Magnetic Resonance (NMR) spectroscopy is extremely powerful to study distinct biological systems ranging from biomolecules to specific metabolites. This chapter presents the basic concepts of the technique and illustrates its potential to study such systems. Similarly, to other spectroscopic techniques, the theoretical background of NMR is sustained by detailed mathematics and physical chemistry concepts, which were kept to the minimum. The intent is to introduce the fundamentals of the technique to science students from different backgrounds. The basic concepts of NMR spectroscopy are briefly presented in the first section, and the following sections describe applications in the biosciences field, using electron transfer proteins as model, particularly cytochromes. The heme groups endow cytochromes with particular features making them excellent examples to illustrate the high versatility of NMR spectroscopy. The main methodologies underlying protein solution structure determination are discussed in the second section. This is followed by a description of the main experiments explored to structurally map protein-protein or protein-ligand interface regions in molecular complexes. Finally, it is shown how NMR spectroscopy can assist in the functional characterization of multiheme cytochromes.

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.

No abstract included.

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.

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.

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.

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.

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 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.

Geobacter sulfurreducens is a dissimilatory metal reducing bacterium with notable properties and significance in biotechnological applications. Biochemical studies suggest that the inner membrane-associated diheme cytochrome MacA and the periplasmic triheme cytochrome PpcA from G. sulfurreducens can exchange electrons. In this work, NMR chemical shift perturbation measurements were used to map the interface region and to measure the binding affinity between PpcA and MacA. The results show that MacA binds to PpcA in a cleft defined by hemes I and IV, favoring the contact between PpcA heme IV and the MacA high potential heme. The dissociation constant values indicate the formation of a low affinity complex between the proteins, which is consistent with the transient interaction observed in electron transfer complexes.This article is protected by copyright. All rights reserved.

The thermodynamic properties of the Desulfovibrio vulgaris (Hildenborough) tetrahaem cytochrome c3 (Dvc3) are rationalised by a model which involves both homotropic (e−/e−) and heterotropic (e−/H+) cooperativity. The paramagnetic shifts of a methyl group from each haem of the DVc3 have been determined in each stage of oxidation at several pH values by means of two-dimensional exchange NMR. The thermodynamic parameters are obtained by fitting the model to the NMR data and to redox titrations followed by visible spectroscopy. They show significant positive cooperativity between two of the haems whereas the remaining interactions appear to be largely electrostatic in origin. These parameters imply that the protein undergoes a proton-assisted two-electron transfer which can be used for energy transduction. Comparison with the crystal structure together with measurement of the kinetics of proton exchange suggest that the pH dependence is mediated by a charged residue(s) readily acessible to the solvent and close to haem I.

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.

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.

Environmental changes trigger the continuous adaptation of bacteria to ensure their survival. This is possible through a variety of signal transduction pathways involving chemoreceptors known as methyl-accepting chemotaxis proteins (MCP) that allow the microorganisms to redirect their mobility towards favorable environments. MCP are two-component regulatory (or signal transduction) systems (TCS) formed by a sensor and a response regulator domain. These domains synchronize transient protein phosphorylation and dephosphorylation events to convert the stimuli into an appropriate cellular response. In this review, the variability of TCS domains and the most common signaling mechanisms are highlighted. This is followed by the description of the overall cellular topology, classification and mechanisms of MCP. Finally, the structural and functional properties of a new family of MCP found in Geobacter sulfurreducens are revisited. This bacterium has a diverse repertoire of chemosensory systems, which represents a striking example of a survival mechanism in challenging environments. Two G. sulfurreducens MCP—GSU0582 and GSU0935—are members of a new family of chemotaxis sensor proteins containing a periplasmic PAS-like sensor domain with a c-type heme. Interestingly, the cellular location of this domain opens new routes to the understanding of the redox potential sensing signaling transduction pathways.

n/a

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.

The bacterium Geobacter sulfurreducens can transfer electrons to quinone moieties of humic substances or to anthraquinone-2,6-disulfonate (AQDS), a model for the humic acids. The reduced form of AQDS (AH2QDS) can also be used as energy source by G. sulfurreducens. Such bidirectional utilization of humic substances confers competitive advantages to these bacteria in Fe(III) enriched environments. Previous studies have shown that the triheme cytochrome PpcA from G. sulfurreducens has a bifunctional behavior toward the humic substance analogue. It can reduce AQDS but the protein can also be reduced by AH2QDS. Using stopped-flow kinetic measurements we were able to demonstrate that other periplasmic members of the PpcA-family in G. sulfurreducens (PpcB, PpcD and PpcE) also showed the same behavior. The extent of the electron transfer is thermodynamically controlled favoring the reduction of the cytochromes. NMR spectra recorded for 13C,15N-enriched samples in the presence increasing amounts of AQDS showed perturbations in the chemical shift signals of the cytochromes. The chemical shift perturbations on cytochromes backbone NH and 1H heme methyl signals were used to map their interaction regions with AQDS, showing that each protein forms a low-affinity binding complex through well-defined positive surface regions in the vicinity of heme IV (PpcB, PpcD and PpcE) and I (PpcE). Docking calculations performed using NMR chemical shift perturbations allowed modeling the interactions between AQDS and each cytochrome at a molecular level. Overall, the results obtained provided important structural-functional relationships to rationalize the microbial respiration of humic substances in G. sulfurreducens.

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.

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.

Electroactive Geobacter bacteria can perform extracellular electron transfer and present a wide metabolic versatility. These bacteria reduce organic, toxic and radioactive compounds, and produce electric current while interacting with electrodes, making them interesting targets for numerous biotechnological applications. Their global electrochemical responses rely on an efficient interface between the inside and the cell's exterior, which is driven by the highly abundant periplasmic triheme PpcA-family cytochromes. The functional features of these cytochromes have been studied in G. sulfurreducens and G. metallireducens, and although they share a high degree of structural homology and sequence identity, their properties are quite distinct. In this work, the heme axial ligand geometries and the magnetic properties of PpcF from G. metallireducens were determined. The data obtained constitute important constraints for the determination of its solution structure in the oxidized state and indicate that the (i) heme core architecture; (ii) axial ligands geometries and (iii) magnetic properties of the cytochrome are conserved compared to the other members of the PpcA-families. Furthermore, the results also indicate that the heme arrangement is crucial to maintain an intrinsic regulation of the protein's redox properties and hence its electron transfer efficiency and functionality.

Energy transduction through electron/proton cooperativity is at the heart of the metabolism of every living organism Nonetheless, the search for the structural bases sustaining these phenomena has been hindered by the fact that they are usually associated with complex transmembrane proteins of high molecular weight.

The monoheme outer membrane cytochrome F (OmcF) from Geobacter sulfurreducens plays an important role in Fe(III) reduction and electric current production. The electrochemical characterization of this cytochrome has shown that its redox potential is modulated by the solution pH (redox-Bohr effect) endowing the protein with the necessary properties to couple electron and proton transfer in the physiological range. The analysis of the OmcF structures in the reduced and oxidized states showed that with the exception of the side chain of histidine 47 (His⁴⁷), all other residues with protonatable side chains are distant from the heme iron and, therefore, are unlikely to affect the redox potential of the protein. The protonatable site at the imidazole ring of His⁴⁷ is in the close proximity to the heme and, therefore, this residue was suggested as the redox-Bohr center. In the present work, we tested this hypothesis by replacing the His⁴⁷ with non-protonatable residues (isoleucine – OmcFH47I and phenylalanine – OmcFH47F). The structure of the mutant OmcFH47I was determined by X-ray crystallography to 1.13 Å resolution and showed only minimal changes at the site of the mutation. Both mutants were ¹⁵N-labeled and their overall folding was confirmed to be the same as the wild-type by NMR spectroscopy. The pH dependence of the redox potential of the mutants was measured by cyclic voltammetry. Compared to the wild-type protein, the magnitude of the redox-Bohr effect in the mutants was smaller, but not fully abolished, confirming the role of His⁴⁷ on the pH modulation of OmcF’s redox potential. However, the pH effect on the heme substituents’ NMR chemical shifts suggested that the heme propionate P₁₃ also contributes to the overall redox-Bohr effect in OmcF. In physiological terms, the contribution of two independent acid–base centers to the observed redox-Bohr effect confers OmcF a higher versatility to environmental changes by coupling electron/proton transfer within a wider pH range.

Abstract Anaerobic microorganisms of the Geobacter genus are effective electron sources for the synthesis of nanoparticles, for bioremediation of polluted water, and for the production of electricity in fuel cells. In multistep reactions, electrons are transferred via iron/heme cofactors of c-type cytochromes from the inner cell membrane to extracellular metal ions, which are bound to outer membrane cytochromes. We measured electron production and electron flux rates to 5×105 e s−1 per G. sulfurreducens. Remarkably, these rates are independent of the oxidants, and follow zero order kinetics. It turned out that the microorganisms regulate electron flux rates by increasing their Fe2+/Fe3+ ratios in the multiheme cytochromes whenever the activity of the extracellular metal oxidants is diminished. By this mechanism the respiration remains constant even when oxidizing conditions are changing. This homeostasis is a vital condition for living systems, and makes G. sulfurreducens a versatile electron source.

Progresses made in bacterial genome sequencing show a remarkable profusion of multiheme c-type cytochromes in many bacteria, highlighting the importance of these proteins in different cellular events. However, the characterization of multiheme cytochromes has been significantly retarded by the numerous experimental challenges encountered by researchers who attempt to overexpress these proteins, especially if isotopic labeling is required. Here we describe a methodology for isotopic labeling of multiheme cytochromes c overexpressed in Escherichia coli, using the triheme cytochrome PpcA from Geobacter sulfurreducens as a model protein. By combining different strategies previously described and using E. coli cells containing the gene coding for PpcA and the cytochrome c maturation gene cluster, an experimental labeling methodology was developed that is based on two major aspects: (i) use of a two-step culture growth procedure, where cell growth in rich media was followed by transfer to minimal media containing 15N-labeled ammonium chloride, and (ii) incorporation of the heme precursor delta-aminolevulinic acid in minimal culture media. The yields of labeled protein obtained were comparable to those obtained for expression of PpcA in rich media. Proper protein folding and labeling were confirmed by UV–visible and NMR spectroscopy. To our knowledge, this is the first report of a recombinant multiheme cytochrome labeling and it represents a major breakthrough for functional and structural studies of multiheme cytochromes.