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.

Microbial electrosynthesis is an emerging green technology that explores the capability of a particular group of microorganisms to drive their metabolism toward the production of hydrogen or value-added chemicals from electrons supplied by electrode surfaces. The cytochrome PccH showed the largest increase in transcription when electrons are supplied to Geobacter sulfurreducens biofilms. Gene knock-out experiments have shown that the electron transfer toward G. sulfurreducens cells was completely inhibited by the deletion of the gene encoding for cytochrome PccH. This identifies a crucial role for this protein in G. sulfurreducens microbial electrosynthesis mechanisms, which are currently unknown. In this work, we present the backbone (1H, 13C and 15N) and heme assignment for PccH in the oxidized state. The data obtained paves the way to identify and structurally map the molecular interaction regions between the cytochrome PccH and its physiological redox partners.

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.

Gene knock-out studies on Geobacter sulfurreducens cells showed that the periplasmic triheme cytochrome PpcA is involved in respiratory pathways leading to the extracellular reduction of Fe(III) and U(VI) oxides. The crucial role of this protein in bridging the electron transfer between the cytoplasm and cell exterior was further supported by proteomics studies. In comparison with non-heme proteins, the presence of numerous proton-containing groups in the heme groups causes additional challenges to the full protein assignment and structure calculation. Here, we report the complete assignment of the heme proton signals together with the 1H and 15N backbone and side chain assignments of the reduced form of PpcA.

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.

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

The unambiguous assignment of the nuclear magnetic resonance (NMR) signals of the α-substituents of the haems in the tetrahaem cytochrome isolated from Shewanella frigidimarina NCIMB400, was made using a combination of homonuclear and heteronuclear experiments. The paramagnetic 13C shifts of the nuclei directly bound to the porphyrin of each haem group were analysed in the framework of a model for the haem electronic structure. The analysis yields g-tensors for each haem, which allowed the assignment of some electron paramagnetic resonance (EPR) signals to specific haems, and the orientation of the magnetic axes relative to each haem to be established. The orientation of the axial ligands of the haems was determined semi-empirically from the NMR data, and the structural results were compared with those of the homologous tetrahaem cytochrome from Shewanella oneidensis MR-1 showing significant similarities between the two proteins.

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.

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

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

Iron is the most versatile of all biochemically active metals, with variability encompassing its electronic configuration, number of unpaired electrons, type of ligands and iron-complexes stability. The versatility of iron properties is transposed to the proteins it can be associated to, especially relevant in the case of heme proteins. In this Review, the structural and functional properties of heme proteins are revisited, with particular focus on c-type cytochromes. The genome of Geobacter bacteria encodes for an unusually high number of assorted c-type cytochromes and, for this reason, they are used in this Review as a showcase of the cytochrome diversity. In the last decades, a vast portfolio of cytochromes has been revealed in these bacteria, with most of them defining new classes, ranging from monoheme to the recently identified polymeric assembly of multiheme cytochromes that forms micrometer-long electrically conductive filaments. These discoveries were on pace with the development of modern NMR equipment and advances in protein isotopic labeling methods, which are also revisited in this Review. Finally, following the description of the current state of the art of Geobacter cytochromes, examples on how the available structural and functional information was explored to structurally map protein–protein and protein–ligand interacting regions in redox complexes, and hence elucidate Geobacter’s respiratory pathways, are presented.

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.

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 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.

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.

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.

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.

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.

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.

No abstract included.

Cytochromes c7 are periplasmic triheme proteins that have been reported exclusively in δ-proteobacteria. The structures of five triheme cytochromes identified in Geobacter sulfurreducens and one in Desulfuromonas acetoxidans have been determined. In addition to the hemes and axial histidines, a single aromatic residue is conserved in all these proteins - phenylalanine 15 (F15). PpcA is a member of the G. sulfurreducens cytochrome c7 family that performs electron/proton energy transduction in addition to electron transfer that leads to the reduction of extracellular electron acceptors. For the first time we probed the role of the F15 residue in the PpcA functional mechanism, by replacing this residue with the aliphatic leucine by site-directed mutagenesis. The analysis of NMR spectra of both oxidized and reduced forms showed that the heme core and the overall fold of the mutated protein were not affected. However, the analysis of 1H-15N heteronuclear single quantum coherence NMR spectra evidenced local rearrangements in the α-helix placed between hemes I and III that lead to structural readjustments in the orientation of heme axial ligands. The detailed thermodynamic characterization of F15L mutant revealed that the reduction potentials are more negative and the redox-Bohr effect is decreased. The redox potential of heme III is most affected. It is of interest that the mutation in F15, located between hemes I and III in PpcA, changes the characteristics of the two hemes differently. Altogether, these modifications disrupt the balance of the global network of cooperativities, preventing the F15L mutant protein from performing a concerted electron/proton transfer.

The periplasmic sensor domains encoded by genes gsu0582 and gsu0935 are part of methyl accepting chemotaxis proteins in the bacterium Geobacter sulfurreducens (Gs). The sensor domains of these proteins contain a heme-c prosthetic group and a PAS-like fold as revealed by their crystal structures. Biophysical studies of the two domains showed that nitric oxide (NO) binds to the heme in both the ferric and ferrous forms, whereas carbon monoxide (CO) binds only to the reduced form. In order to address these exogenous molecules as possible physiological ligands, binding studies and resonance Raman (RR) spectroscopic characterization of the respective CO and NO adducts were performed in this work. In the absence of exogenous ligands, typical RR frequencies of five-coordinated (5c) high-spin and six-coordinated (6c) low-spin species were observed in the oxidized form. In the reduced state, only frequencies corresponding to the latter were detected. In both sensors, CO binding yields 6c low-spin adducts by replacing the endogenous distal ligand. The binding of NO by the two proteins causes partial disruption of the proximal Fe-His bond, as revealed by the RR fingerprint features of 5cFe-NO and 6cNO-Fe-His species. The measured CO and NO dissociation constants of ferrous GSU0582 and GSU0935 sensors reveal that both proteins have high and similar affinity toward these molecules (Kd ≈ 0.04−0.08 μM). On the contrary, in the ferric form, sensor GSU0582 showed a much higher affinity for NO (Kd ≈ 0.3 μM for GSU0582 versus 17 μM for GSU0935). Molecular dynamics calculations revealed a more open heme pocket in GSU0935, which could account for the different affinities for NO. Taken together, spectroscopic data and MD calculations revealed subtle differences in the binding properties and structural features of formed CO and NO adducts, but also indicated a possibility that a (5c) high-spin/(6c) low-spin redox-linked equilibrium could drive the physiological sensing of Gs cells.

{The redox potential values of cytochromes can be modulated by the protonation/deprotonation of neighbor groups (redox-Bohr effect), a mechanism that permits the proteins to couple electron/proton transfer. In the respiratory chains, this effect is particularly relevant if observed in the physiological pH range, as it may contribute to the electrochemical gradient for ATP synthesis. A constitutively produced family of five triheme cytochromes (PpcA−E) from the bacterium Geobacter sulfurreducens plays a crucial role in extracellular electron transfer, a hallmark that permits this bacterium to be explored for several biotechnological applications. Two members of this family (PpcA and PpcD) couple electron/proton transfer in the physiological pH range, a feature not shared with PpcB and PpcE. That ability is crucial for G. sulfurreducens’ growth in Fe(III)-reducing habitats since extra contributors to the electrochemical gradient are needed. It was postulated that the redox-Bohr effect is determined by the nature of residue 6, a leucine in PpcA/PpcD and a phenylalanine in PpcB/PpcE. To confirm this hypothesis, Phe6 was replaced by leucine in PpcB and PpcE. The functional properties of these mutants were investigated by NMR and UV–visible spectroscopy to assess their capability to couple electron/proton transfer in the physiological pH range. The results obtained showed that the mutants have an increased redox-Bohr effect and are now capable of coupling electron/proton transfer. This confirms the determinant role of the nature of residue 6 in the modulation of the redox-Bohr effect in this family of cytochromes, opening routes to engineer Geobacter cells with improved biomass production.}

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.