Kinetics and Mechanism of Mineral Respiration: How Iron Hemes Synchronize Electron Transfer Rates.

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.

Molecular interactions between Geobacter sulfurreducens triheme cytochromes and the redox active analogue for humic substances.

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.

Biochemical and functional insights on the triheme cytochrome PpcA from Geobacter metallireducens.

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

Solution structure and dynamics of the outer membrane cytochrome OmcF from Geobacter sulfurreducens.

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

Structure, electrocatalysis and dynamics of immobilized cytochrome PccH and its microperoxidase.

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.

Molecular interaction studies revealed the bifunctional behavior of triheme cytochrome PpcA from Geobacter sulfurreducens toward the redox active analog of humic substances.

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 IV (-127mV) compared to that of AH2QDS (-184mV) 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.

Thermodynamic and kinetic characterization of PccH, a key protein in microbial electrosynthesis processes in Geobacter sulfurreducens.

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.

Evidence for interaction between the triheme cytochrome PpcA from Geobacter sulfurreducens and anthrahydroquinone-2,6-disulfonate, an analog of the redox active components of humic substances.

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.

Solution structure of a mutant of the triheme cytochrome PpcA from Geobacter sulfurreducens sheds light on the role of the conserved aromatic residue F15.

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 (15)N-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 0.36Å for the backbone atoms and 1.14Å 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.

On the road to improve the bioremediation and electricity-harvesting skills of Geobacter sulfurreducens: functional and structural characterization of multihaem cytochromes.

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.

Pivotal role of the strictly conserved aromatic residue F15 in the cytochrome c7 family.

Cytochromes c(7) 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 c(7) 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 (1)H-(15)N 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.

Fine Tuning of Redox Networks on Multiheme Cytochromes from Geobacter sulfurreducens Drives Physiological Electron/Proton Energy Transduction.

The bacterium Geobacter sulfurreducens (Gs) can grow in the presence of extracellular terminal acceptors, a property that is currently explored to harvest electricity from aquatic sediments and waste organic matter into microbial fuel cells. A family composed of five triheme cytochromes (PpcA-E) was identified in Gs. These cytochromes play a crucial role by bridging the electron transfer from oxidation of cytoplasmic donors to the cell exterior and assisting the reduction of extracellular terminal acceptors. The detailed thermodynamic characterization of such proteins showed that PpcA and PpcD have an important redox-Bohr effect that might implicate these proteins in the e(-)/H(+) coupling mechanisms to sustain cellular growth. The physiological relevance of the redox-Bohr effect in these proteins was studied by determining the fractional contribution of each individual redox-microstate at different pH values. For both proteins, oxidation progresses from a particular protonated microstate to a particular deprotonated one, over specific pH ranges. The preferred e(-)/H(+) transfer pathway established by the selected microstates indicates that both proteins are functionally designed to couple e(-)/H(+) transfer at the physiological pH range for cellular growth.

Backbone, side chain and heme resonance assignment of the triheme cytochrome PpcA from Geobacter metallireducens in the oxidized state.

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

Structural and Functional Relevance of the Conserved Residue V13 in the Triheme Cytochrome PpcA from Geobacter sulfurreducens.

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

The triheme cytochrome PpcF from Geobacter metallireducens exhibits distinct redox properties.

Electrogenic bacteria, such as Geobacter, can couple the oxidation of carbon sources to the reduction of extracellular electron acceptors; such acceptors include toxic and radioactive metals, as well as electrode surfaces, making Geobacter a suitable candidate for applied use in bioremediation and bioenergy generation. Geobacter metallireducens is more promising in this regard than the better studied Geobacter sulfurreducens, as it has more efficient Fe (III) reduction rates and can convert nitrate to ammonia. The operon responsible for nitrate reductase activity in G. metallireducens includes the gene encoding the cytochrome PpcF, which was proposed to exchange electrons with nitrate reductase. In the present work, we perform a biochemical and a biophysical characterization of PpcF. Spectroscopic techniques, including circular dichroism (CD), UV-visible, and nuclear magnetic resonance (NMR), revealed that the cytochrome is very stable (T m > 85 °C), contains three low-spin hemes, and is diamagnetic (S = 0) and paramagnetic (S = 1/2) in the reduced and oxidized states, respectively. The NMR chemical shifts of the heme substituents were assigned and used to determine the heme core architecture of PpcF. Compared to the PpcA-family from G. sulfurreducens, the spatial disposition of the hemes is conserved, but the functional properties are clearly distinct. In fact, potentiometric titrations monitored by UV-visible absorption reveal that the reduction potential values of PpcF are significantly less negative (-56 and -64 mV, versus the normal hydrogen electrode at pH 7.0 and 8.0, respectively). NMR redox titrations showed that the order of oxidation of the hemes is IV-I-III, a feature not observed for G. sulfurreducens. The different redox properties displayed by PpcF, including the small redox-Bohr effect and low reduction potential value of heme IV, were structurally rationalized and attributed to the lower number of positively charged residues located in the vicinity of heme IV. Overall, the redox features of PpcF suggest that biotechnological applications of G. metallireducens may require less negative working functional redox windows than those using by G. sulfurreducens.

Thermodynamic and kinetic properties of the outer membrane cytochrome OmcF, a key protein for extracellular electron transfer in Geobacter sulfurreducens.

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

Molecular interactions between Geobacter sulfurreducens triheme cytochromes and the electron acceptor Fe(iii) citrate studied by NMR.

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.

NMR studies of the interaction between inner membrane-associated and periplasmic cytochromes from Geobacter sulfurreducens.

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.

Unveiling the Structural Basis That Regulates the Energy Transduction Properties within a Family of Triheme Cytochromes from Geobacter sulfurreducens.

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.

Backbone, side chain and heme resonance assignments of the triheme cytochrome PpcD from Geobacter sulfurreducens.

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 (1)H, (13)C and (15)N backbone and side chain assignments of the reduced form of PpcD.