Thermal perturbation of the dicluster ferredoxin from Acidianus ambivalens was investigated employing a toolbox of spectroscopic methods. FTIR and visible CD were used for assessing changes of the secondary structure and coarse alterations of the [3Fe4S] and [4Fe4S] cluster moieties, respectively. Fine details of the disassembly of the metal centers were revealed by paramagnetic NMR and resonance Raman spectroscopy. Overall, thermally induced unfolding of AaFd is initiated with the loss of α-helical content at relatively low temperatures (Tapp (m) ~ 44 °C), followed by the disruption of both iron−sulfur clusters (Tapp (m) ~ 53−60 °C). The degradation of the metal centers triggers major structural changes on the protein matrix, including the loss of tertiary contacts (Tapp (m) ~ 58 °C) and a change, rather than a significant net loss, of secondary structure (Tapp (m) ~ 60 °C). This latter process triggers a secondary structure reorganization that is consistent with the formation of a molten globule state. The combined spectroscopic approach here reported illustrates how changes in the metalloprotein organization are intertwined with disassembly of the iron−sulfur centers, denoting the conformational interplay of the protein backbone with cofactors.

The NMR structure of the oxidised wild-type cytochrome c3 from Desulfovibrio vulgaris Hildenborough was determined in solution. Using a newly developed methodology, NMR data from the K45Q mutant was then grafted onto data from the wild-type protein to determine the structure in the region of the mutation. The structural origins of the redox-Bohr effect and haem–haem cooperativities are discussed with respect to the redox-related conformational changes observed in solution.

The bacteria belonging to the genus Shewanella are facultative anaerobes that utilize a variety of terminal electron acceptors which includes soluble and insoluble metal oxides. The tetraheme c-type cytochrome isolated during anaerobic growth of Shewanella frigidimarina NCIMB400 (Sfc) contains 86 residues and is involved in the Fe(III) reduction pathways. Although the functional properties of Sfc redox centers are quite well described, no structures are available for this protein. In this work, we report the solution structure of the reduced form of Sfc. The overall fold is completely different from those of the tetraheme cytochromes c3 and instead has similarities with the tetraheme cytochrome recently isolated from Shewanella oneidensis (Soc). Comparison of the tetraheme cytochromes from Shewanella shows a considerable diversity in their primary structure and heme reduction potentials, yet they have highly conserved heme geometry, as is the case for the family of tetraheme cytochromes isolated from Desulfovibrio spp.

Extracellular electron transfer is one of the physiological hallmarks of Geobacteraceae. Most of the Geobacter species encode for more than 100 c-type cytochromes which are, in general, poorly conserved between individual species. An exception to this is the PpcA family of periplasmic triheme c-type cytochromes, which are the most abundant proteins in these bacteria. The functional characterization of PpcA showed that it has the necessary properties to couple electron/proton transfer, a fundamental step for ATP synthesis. The detailed thermodynamic characterization of a PpcA mutant, in which the strictly conserved residue phenylalanine 15 was replaced by leucine, showed that the global redox network of cooperativities among heme groups is altered, preventing the mutant from performing a concerted electron/proton transfer. In this work, we determined the solution structure of PpcA F15L mutant in the fully reduced state using NMR spectroscopy by producing 15N-labeled protein. In addition, pH-dependent conformational changes were mapped onto the structure. The mutant structure obtained is well defined, with an average pairwise root-mean-square deviation of 0.36 Å for the backbone atoms and 1.14 Å for all heavy atoms. Comparison between the mutant and wild-type structures elucidated the contribution of phenylalanine 15 in the modulation of the functional properties of PpcA.

ABSTRACTGene 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 0.37 Å for the backbone atoms and 0.98 Å 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.

Reduction of the haems in tetrahaem cytochromes c3 is a cooperative process, i.e., reduction of each of the haems depends on the redox states of the other haems. Furthermore, electron transfer is coupled to proton transfer (redox-Bohr effect). Two of its haems and a strictly conserved nearby phenylalanine residue, F20, in Desulfovibrio vulgaris (Hildenborough) cytochrome c3 form a structural motif that is present in all cytochromes c3 and also in cytochrome c oxidase. A putative role for this phenylalanine residue in the cooperativity of haem reduction was investigated. Therefore, this phenylalanine was replaced, with genetic techniques, by isoleucine and tyrosine in D. vulgaris (Hildenborough) cytochrome c3. Cyclic voltammetry studies revealed a small increase (30 mV) in one of the macroscopic redox potentials in the mutated cytochromes. EPR showed that the main alterations occurred in the vicinity of haem I, the haem closest to residue 20 and one of the haems responsible for positive cooperativities in electron transfer of D. vulgaris cytochrome c3. NMR studies of F20I cytochrome c3 demonstrated that the haem core architecture is maintained and that the more affected haem proton groups are those near the mutation site. NMR redox titrations of this mutated protein gave evidence for only small changes in the relative redox potentials of the haems. However, electron/electron and proton/electron cooperativity are maintained, indicating that this aromatic residue has no essential role in these processes. Furthermore, chemical modification of the N-terminal amino group of cytochrome c3 backbone, which is also very close to haem I, had no effect on the network of cooperativities.

The bacterium Gs (Geobacter sulfurreducens) is capable of oxidizing a large variety of compounds relaying electrons out of the cytoplasm and across the membranes in a process designated as extracellular electron transfer. The trihaem cytochrome PpcA is highly abundant in Gs and is most probably the reservoir of electrons destined for the outer surface. In addition to its role in electron transfer pathways, we have previously shown that this protein could perform e-/H+ energy transduction. This mechanism is achieved by selecting the specific redox states that the protein can access during the redox cycle and might be related to the formation of proton electrochemical potential gradient across the periplasmic membrane. The regulatory role of haem III in the functional mechanism of PpcA was probed by replacing Met58, a residue that controls the solvent accessibility of haem III, with serine, aspartic acid, asparagine or lysine. The data obtained from the mutants showed that the preferred e-/H+ transfer pathway observed for PpcA is strongly dependent on the reduction potential of haem III. It is striking to note that one residue can fine tune the redox states that can be accessed by the trihaem cytochrome enough to alter the functional pathways.

Detailed thermodynamic and structural data measured in soluble monomeric multiheme cytochromes c provided the basis to investigate the functional significance of interactions between redox co-factors. The steep decay of intramolecular interactions with distance means that close proximity of the redox centers is necessary to modulate the intrinsic reduction potentials in a significant way. This ensures selection of specific populations during redox activity in addition to maintaining fast intramolecular electron transfer. Therefore, intramolecular interactions between redox co-factors play an important role in establishing the biological function of the protein by controlling how electrons flow through and are distributed among the co-factors.

Gs (Geobacter sulfurreducens) can transfer electrons to the exterior of its cells, a property that makes it a preferential candidate for the development of biotechnological applications. Its genome encodes over 100 cytochromes and, despite their abundance and key functional roles, to date there is no structural information for these proteins in solution. The trihaem cytochrome PpcA might have a crucial role in the conversion of electronic energy into protonmotive force, a fundamental step for ATP synthesis in the presence of extracellular electron acceptors. In the present study, 15N-labelled PpcA was produced and NMR spectroscopy was used to determine its solution structure in the fully reduced state, its backbone dynamics and the pH-dependent conformational changes. The structure obtained is well defined, with an average pairwise rmsd (root mean square deviation) of 0.25 Å (1 Å=0.1 nm) for the backbone atoms and 0.99 Å for all heavy atoms, and constitutes the first solution structure of a Gs cytochrome. The redox-Bohr centre responsible for controlling the electron/proton transfer was identified, as well as the putative interacting regions between PpcA and its redox partners. The solution structure of PpcA will constitute the foundation for studies aimed at mapping out in detail these interacting regions.

The membranes of the thermoacidophilic archaeon Sulfolobus metallicus exhibit an oxygen consumption activity of 0.5 nmol O2 min−1 mg−1, which is insensitive to rotenone, suggesting the presence of a type-II NADH dehydrogenase. Following this observation, the enzyme was purified from solubilised membranes and characterised. The pure protein is a monomer with an apparent molecular mass of 49 kDa, having a high N-terminal amino acid sequence similarity towards other prokaryotic enzymes of the same type. It contains a covalently attached flavin, which was identified as being FMN by 31P-NMR spectroscopy, a novelty among type-II NADH dehydrogenases. Metal analysis showed the absence of iron, indicating that no FeS clusters are present in the protein. The average reduction potential of the FMN group was determined to be +160 mV, at 25 °C and pH 6.5, by redox titrations monitored by visible spectroscopy. Catalytically, the enzyme is a NADH:quinone oxidoreductase, as it is capable of transferring electrons from NADH to several quinones, including ubiquinone-1, ubiquinone-2 and caldariella quinone. Maximal turnover rates of 195 μmol NADH oxidized min−1 mg−1 at 60 °C were obtained using ubiquinone-2 as electron acceptor, after enzyme dilution and incubation with phospholipids.

The structural basis for the pH dependence of the redox potential in the tetrahemic Desulfovibrio vulgaris (Hildenborough) cytochrome c3 was investigated by site-directed mutagenesis of charged residues in the vicinity of heme I. Mutation of lysine 45, located in the neighborhood of the propionates of heme I, by uncharged residues, namely threonine, glutamine and leucine, was performed. The replacement of a conserved charged residue, aspartate 7, present in the N-terminal region and near heme I was also attempted. The analysis of the redox interactions as well as the redox-Bohr behavior of the mutated cytochromes c3 allowed the conclusion that residue 45 has a functional role in the control of the pKa of the propionate groups of heme I and confirms the involvement of this residue in the redox-Bohr effect.

The thermodynamic model of five interacting charge centres (four haems and an ionisable centre), which was used in the characterisation of the thermodynamic properties of Desulfovibrio vulgaris (Hildenborough) cytochrome c3 (c3DvH), is now used to reevaluate the thermodynamic properties in Desulfovibrio vulgaris (Miyazaki F) cytochrome c3 (c3DvM) on the basis of published data (Park, J.-S., Ohmura, T., Kano, K., Sagara, T., Niki, K., Kyogoku, Y. and Akutsu, H. (1996) Biochim. Biophys. Acta 1293, 45–54). Contrary to the assertion of Park et al. (1996), the pH dependence of the proton chemical shifts of haem methyls in c3DvM in several stages of oxidation is well described by the model, which involves both homotropic (e–/e–) and heterotropic (e–/H+) cooperativity. This shows that the pH dependence observed for c3DvM is not significantly more complicated than that observed for c3DvH. Since the parameters which we now obtain for c3DvM are generated with the same model as those from c3DvH, albeit using less precise data, it is possible to make a preliminary comparison of the thermodynamic properties of these two proteins and of their role in energy transduction.
The extrinsic dipolar shifts generated for each methyl group by each of the four haems in c3DvM are also determined. A novel method for approximating the magnetic susceptibility tensors is used: the orientations of the principal axes of the tensors have been shown to be closely related to the geometry of the axial ligands, which is available from the X-ray structure of c3DvM, and the components of the tensors are extrapolated from EPR g values. The inclusion of the calculated haem extrinsic contributions clearly describes the pH dependence of the haem methyls in the core of the protein, close to other haems. This description is most remarkable in the case of the haem methyl 21CH3 II I, for which the "unusual pH dependence" commented on by Park et al. (1996) is easily explained using the thermodynamic parameters determined by our model together with the calculated extrinsic dipolar shifts, thus providing a test of the analysis.

Geobacter sulfurreducens is a widely applied microorganism for the reduction of toxic metal salts, as an electron source for bioelectrochemical devices, and as a reagent for the synthesis of nanoparticles. In order to understand the influence of metal salts, and of electron transporting, multiheme c-cytochromes on the electron flux during respiration of G. sulfurreducens, the reduction kinetic of Fe³⁺, Co³⁺, V⁵⁺, Cr⁶⁺, and Mn⁷⁺ containing complexes were measured. Starting from the resting phase, each G. sulfurreducens cell produced an electron flux of 3.7 × 10⁵ electrons per second during the respiration process. Reduction rates were within ± 30% the same for the 6 different metal salts, and reaction kinetics were of zero order. Decrease of c-cytochrome concentrations by downregulation and mutation demonstrated that c-cytochromes stabilized respiration rates by variation of their redox states. Increasing Fe²⁺/heme levels increased electron flux rates, and induced respiration flexibility. The kinetic effects parallel electrochemical results of G. sulfurreducens biofilms on electrodes, and might help to optimize bioelectrochemical devices.

Multiheme c-type cytochromes from members of the Desulfovibrionacea and Geobactereacea families play crucial roles in the bioenergetics of these microorganisms. Thermodynamic studies using NMR and visible spectroscopic techniques on tetraheme cytochromes c3 isolated from Desulfovibrio spp. and more recently on a triheme cytochrome from Geobacter sulfurreducens showed that the properties of each redox centre are modulated by the neighbouring redox centres enabling these proteins to perform energy transduction and thus contributing to cellular energy conservation. Electron/proton transfer coupling relies on redox-linked conformational changes that were addressed for some multiheme cytochromes from the comparison of protein structure of fully reduced and fully oxidised forms. In this work, we identify for the first time in a multiheme cytochrome the simultaneous presence of two different conformations in solution. This was achieved by probing the different oxidation stages of a triheme cytochrome isolated from G. sulfurreducens using 2D-NMR techniques. The results presented here will be the foundations to evaluate the modulation of the redox centres properties by conformational changes that occur during the reoxidation of a multiheme protein.

Using potentiometric titrations, two protons were found to participate in the redox-Bohr effect observed for cytochrome c3 from Desulfovibrio vulgaris (Hildenborough). Within the framework of the thermodynamic model previously presented, this finding supports the occurrence of a concerted proton-assisted 2e– step, ideally suited for the coupling role of cytochrome c3 to hydrogenase. Furthermore, at physiological pH, it is shown that when sulfate-reducing bacteria use H2 as energy source, cytochrome c3 can be used as a charge separation device, achieving energy transduction by energising protons which can be left in the acidic periplasmic side and transferring deenergised electrons to sulfate respiration. This mechanism for energy transduction, using a full thermodynamic data set, is compared to that put forward to explain the proton-pumping function of cytochrome c oxidase.

Accepted Manuscript online November 14, 2016.The periplasmic triheme cytochrome PpcA from Geobacter sulfurreducens is highly abundant; it is the likely reservoir of electrons to the outer surface to assist the reduction of extracellular terminal acceptors; these include insoluble metal oxides in natural habitats and electrode surfaces from which electricity can be harvested. A detailed thermodynamic characterization of PpcA showed that it has an important redox-Bohr effect that might implicate the protein in e-/H+ coupling mechanisms to sustain cellular growth. This functional mechanism requires control of both the redox state and the protonation state. In the present study, isotope-labeled PpcA was produced and the three-dimensional structure of PpcA in the oxidized form was determined by NMR. This is the first solution structure of a G. sulfurreducens cytochrome in the oxidized state. The comparison of oxidized and reduced structures revealed that the heme I axial ligand geometry changed and there were other significant changes in the segments near heme I. The pH-linked conformational rearrangements observed in the vicinity of the redox-Bohr center, both in the oxidized and reduced structures, constitute the structural basis for the differences observed in the pKa values of the redox-Bohr center, providing insights into the e-/H+ coupling molecular mechanisms driven by PpcA in G. sulfurreducens.EET, extracellular electron transfer; IM, inner membrane; IPTG, isopropyl β-d-thiogalactoside; MFCs, microbial fuel cells; NOE, Nuclear Overhauser effect; OM, outer membrane; rmsd, root mean square deviation.

The complete genome sequence of the δ-proteobacterium Geobacter sulfurreducens reveals a large abundance of multiheme cytochromes. Cytochrome c7, isolated from this metal ion-reducing bacterium, is a triheme periplasmic electron-transfer protein with Mr 9.6 kDa. This protein is involved in metal ion-reducing pathways and shares 56% sequence identity with a triheme cytochrome isolated from the closely related δ-proteobacterium Desulfuromonas acetoxidans (Dac7). In this work, two-dimensional NMR was used to monitor the heme core and the general folding in solution of the G. sulfurreducens triheme cytochrome c7 (PpcA). NMR signals obtained for the three hemes of PpcA at different stages of oxidation were cross-assigned to the crystal structure [Pokkuluri, P. R., Londer, Y. Y., Duke, N. E. C., Long, W. C., and Schiffer, M. (2004) Biochemistry 43, 849−859] using the complete network of chemical exchange connectivities, and the order in which each heme becomes oxidized was determined at pH 6.0 and 8.2. Redox titrations followed by visible spectroscopy were also performed in order to monitor the macroscopic redox behavior of PpcA. The results obtained showed that PpcA and Dac7 have different redox properties:  (i) the order in which each heme becomes oxidized is different; (ii) the reduction potentials of the heme groups and the global redox behavior of PpcA are pH dependent (redox−Bohr effect) in the physiological pH range, which is not observed with Dac7. The differences observed in the redox behavior of PpcA and Dac7 may account for the different functions of these proteins and constitute an excellent example of how homologous proteins can perform different physiological functions. The redox titrations followed by visible spectroscopy of PpcA and two mutants of the conserved residue F15 (PpcAF15Y and PpcAF15W) lead to the conclusion that F15 modulates the redox behavior of PpcA, thus having an important physiological role.

Flavocytochrome c3 from Shewanella frigidimarina (fcc3) is a tetrahaem periplasmic protein of 64 kDa with fumarate reductase activity. This work reports the first example of NMR techniques applied to the assignment of the thermodynamic order of oxidation of the four individual haems for such large protein, expanding its applicability to a wide range of proteins. NMR data from partially and fully oxidised samples of fcc3 and a mutated protein with an axial ligand of haem IV replaced by alanine were compared with calculated chemical shifts, allowing the structural assignment of the signals and the unequivocal determination of the order of oxidation of the haems. As oxidation progresses the fcc3 haem domain is polarised, with haems I and II much more oxidised than haems III and IV, haem IV being the most reduced. Thus, during catalysis as an electron is taken by the flavin adenosine dinucleotide from haem IV, haem III is eager to re-reduce haem IV, allowing the transfer of two electrons to the active site.

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.

{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.}

Previous studies have demonstrated that Geobacter sulfurreducens requires the c-type cytochrome OmcZ, which is present in large (OmcZL; 50-kDa) and small (OmcZS; 30-kDa) forms, for optimal current production in microbial fuel cells. This protein was further characterized to aid in understanding its role in current production. Subcellular-localization studies suggested that OmcZS was the predominant extracellular form of OmcZ. N- and C-terminal amino acid sequence analysis of purified OmcZS and molecular weight measurements indicated that OmcZS is a cleaved product of OmcZL retaining all 8 hemes, including 1 heme with the unusual c-type heme-binding motif CX14CH. The purified OmcZS was remarkably thermally stable (thermal-denaturing temperature, 94.2°C). Redox titration analysis revealed that the midpoint reduction potential of OmcZS is approximately −220 mV (versus the standard hydrogen electrode [SHE]) with nonequivalent heme groups that cover a large reduction potential range (−420 to −60 mV). OmcZS transferred electrons in vitro to a diversity of potential extracellular electron acceptors, such as Fe(III) citrate, U(VI), Cr(VI), Au(III), Mn(IV) oxide, and the humic substance analogue anthraquinone-2,6-disulfonate, but not Fe(III) oxide. The biochemical properties and extracellular localization of OmcZ suggest that it is well suited for promoting electron transfer in current-producing biofilms of G. sulfurreducens.

The periplasmic sensor domains GSU0582 and GSU0935 are part of methyl accepting chemotaxis proteins in the bacterium Geobacter sulfurreducens. Both contain one c-type heme group and their crystal structures revealed that these domains form swapped dimers with a PAS fold formed from the two protein chains. The swapped dimerization of these sensors is related to the mechanism of signal transduction and the formation of the swapped dimer involves significant folding changes and conformational rearrangements within each monomeric component. However, the structural changes occurring during this process are poorly understood and lack a mechanistic framework. To address this issue, we have studied the folding and stability properties of two distinct heme-sensor PAS domains, using biophysical spectroscopies. We observed substantial differences in the thermodynamic stability (ΔG = 14.6 kJ.mol−1 for GSU0935 and ΔG = 26.3 kJ.mol−1 for GSU0582), and demonstrated that the heme moiety undergoes conformational changes that match those occurring at the global protein structure. This indicates that sensing by the heme cofactor induces conformational changes that rapidly propagate to the protein structure, an effect which is directly linked to the signal transduction mechanism. Interestingly, the two analyzed proteins have distinct levels of intrinsic disorder (25% for GSU0935 and 13% for GSU0582), which correlate with conformational stability differences. This provides evidence that the sensing threshold and intensity of the propagated allosteric effect is linked to the stability of the PAS-fold, as this property modulates domain swapping and dimerization. Analysis of the PAS-domain shows that disorder segments are found either at the hinge region that controls helix motions or in connecting segments of the β-sheet interface. The latter is known to be widely involved in both intra- and intermolecular interactions, supporting the view that it's folding and stability are at the basis of the specificity and regulation of many types of PAS-containing signaling proteins.

Electrogenic microorganisms possess unique redox biological features, being capable of transferring electrons to the cell exterior and converting highly toxic compounds into nonhazardous forms. These microorganisms have led to the development of Microbial Electrochemical Technologies (METs), which include applications in the fields of bioremediation and bioenergy production. The optimization of these technologies involves efforts from several different disciplines, ranging from microbiology to materials science. Geobacter bacteria have served as a model for understanding the mechanisms underlying the phenomenon of extracellular electron transfer, which is highly dependent on a multitude of multiheme cytochromes (MCs). MCs are, therefore, logical targets for rational protein engineering to improve the extracellular electron transfer rates of these bacteria. However, the presence of several heme groups complicates the detailed redox characterization of MCs. In this Review, the main characteristics of electroactive Geobacter bacteria, their potential to develop microbial electrochemical technologies and the main features of MCs are initially highlighted. This is followed by a detailed description of the current methodologies that assist the characterization of the functional redox networks in MCs. Finally, it is discussed how this information can be explored to design optimal Geobacter-mutated strains with improved capabilities in METs.

Lactoperoxidase (LPO) is a structurally complex and stable mammalian redox enzyme. Here we aim at evaluating the influence of ionic interactions and how these intertwine with the structural dynamics, stability and activity of LPO. In this respect, we have compared LPO guanidinium hydrochloride (GdmCl) and urea denaturation pathways and performed a detailed investigation on the effects of pH on the LPO conformational dynamics and stability. Our experimental findings using far-UV CD, Trp fluorescence emission and ESR spectroscopies clearly indicate that LPO charged-denaturation with GdmCl induced a sharp two-step process versus a three-step unfolding mechanism induced by urea. This differential effect between GdmCl and urea suggests that ionic interactions must play a rather prominent role in the stabilization of LPO. With both denaturants, the protein core was shown to retain activity up to near the respective Cm values. Moreover, a pH titration of LPO evidenced no significant conformational alterations or perturbation of heme activity within the 4 to 11 pH interval. In contrast, alterations of ionic interactions by poising LPO at pH 3, 2 and 12 resulted in a loss of secondary structure, loosening of tertiary contacts and loss of activity, which appear to be associated with the perturbation of the hydrophobic core, as evidenced by ANS binding, as well as disruption of the heme pocket demonstrated by optical and EPR spectroscopies. Overall, LPO is characterised by a high degree of peripheral structural plasticity without perturbation of the core heme moiety. The possible physiological meaning of such features is discussed.

The increase of conductivity of electrolytes favors the current production in microbial fuel cells (MFCs). Adaptation of cell cultures to higher ionic strength is a promising strategy to increase electricity production. The bacterium Geobacter sulfurreducens is considered a leading candidate for MFCs. Therefore, it is important to evaluate the impact of the ionic strength on the functional properties of key periplasmic proteins that warrants electron transfer to cell exterior. The effect of the ionic strength on the functional properties of triheme cytochrome PpcA, the most abundant periplasmic cytochrome in G. sulfurreducens, was investigated by NMR and potentiometric methods. The redox properties of heme IV are the most affected ones. Chemical shift perturbation measurements on the backbone NMR signals, at increasing ionic strength, also showed that the region close to heme IV is the most affected due to the large number of positively charged residues, which confer a highly positive electrostatic surface around this heme. The shielding of these positive charges at high ionic strength explain the observed decrease in the reduction potential of heme IV and shows that PpcA was designed to maintain its functional mechanistic features even at high ionic strength.