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.

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 redox properties of a periplasmic triheme cytochrome, PpcB from Geobacter sulfurreducens, were studied by NMR and visible spectroscopy. The structure of PpcB was determined by X-ray diffraction. PpcB is homologous to PpcA (77% sequence identity), which mediates cytoplasmic electron transfer to extracellular acceptors and is crucial in the bioenergetic metabolism of Geobacter spp. The heme core structure of PpcB in solution, probed by 2D-NMR, was compared to that of PpcA. The results showed that the heme core structures of PpcB and PpcA in solution are similar, in contrast to their crystal structures where the heme cores of the two proteins differ from each other. NMR redox titrations were carried out for both proteins and the order of oxidation of the heme groups was determined. The microscopic properties of PpcB and PpcA redox centers showed important differences: (i) the order in which hemes become oxidized is III–I–IV for PpcB, as opposed to I–IV–III for PpcA; (ii) the redox-Bohr effect is also different in the two proteins. The different redox features observed between PpcB and PpcA suggest that each protein uniquely modulates the properties of their co-factors to assure effectiveness in their respective metabolic pathways. The origins of the observed differences are discussed.

Multihaem cytochromes that could form protein “nanowires” were identified in the Geobacter sulfurreducens genome, and represent a new type of multihaem cytochrome. The sequences of these proteins, two with 12 haems (GSU1996, GSU0592) and one with 27 haems (GSU2210), suggest that they are formed with domains homologous to the trihaem cytochrome c7. Although all three haems have bis-His co-ordination in cytochromes c7, in each domain of the above polymers, the haem equivalent to haem IV has His-Met co-ordination. We previously determined the structure and measured the macroscopic redox potential of one representative domain (domain C) of a dodecahaem cytochrome (GSU1996). In the present study, the microscopic redox properties of the individual haem groups of domain C were determined using NMR and UV–visible spectroscopies. The reduction potentials of the haems for the fully reduced and protonated protein are different from each other (haem I, −106 mV; haem III, −136 mV; and haem IV, −125 mV) and are strongly modulated by redox interactions. This result is rather surprising since the His-Met co-ordinated haem IV does not have the highest potential as was expected. The polypeptide environment of each haem group and the strong haem pairwise redox interactions must play a dominant role in controlling the individual haem potentials. The strong redox interactions between the haems extend the range of their operating potentials at physiological pH (haem I, −71 mV, haem III, −146 mV and haem IV, −110 mV). Such a modulation in haem potentials is likely to have a functional significance in the metabolism of G. sulfurreducens.

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.

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.

Multiheme proteins play major roles in various biological systems. Structural information on these systems in solution is crucial to understand their functional mechanisms. However, the presence of numerous proton-containing groups in the heme cofactors and the magnetic properties of the heme iron, in particular in the oxidised state, complicates significantly the assignment of the NMR signals. Consequently, the multiheme proteins superfamily is extremely under-represented in structural databases, which constitutes a severe bottleneck in the elucidation of their structural–functional relationships. In this work, we present a strategy that simplifies the assignment of the NMR signals in multiheme proteins and, concomitantly, their solution structure determination, using the triheme cytochrome PpcA from the bacterium Geobacter sulfurreducens as a model. Cost-effective isotopic labeling was used to double label (13C/15N) the protein in its polypeptide chain, with the correct folding and heme post-translational modifications. The combined analysis of 1H–13C HSQC NMR spectra obtained for labeled and unlabeled samples of PpcA allowed a straight discrimination between the heme cofactors and the polypeptide chain signals and their confident assignment. The results presented here will be the foundations to assist solution structure determination of multiheme proteins, which are still very scarce in the literature.

{Cytochromes are electron transfer proteins essential in various biological systems, playing crucial roles in the respiratory chains of bacteria. These proteins are particularly abundant in electrogenic microorganisms and are responsible for the efficient delivery of electrons to the cells’ exterior. The capability of sending electron outside the cells open new avenues to be explored for emerging biotechnological applications in bioremediation, microbial electrosynthesis and bioenergy fields. To develop these applications, it is critical to identify the different redox partners and elucidate the stepwise electron transfer along the respiratory paths. However, investigating direct electron transfer events between proteins with identical features in nearly all spectroscopic techniques is extremely challenging. NMR spectroscopy offers the possibility to overcome this difficulty by analysing the alterations of the spectral signatures of each protein caused by electron exchange events. The uncrowded NMR spectral regions containing the heme resonances of the cytochromes display unique and distinct signatures in the reduced and oxidized states, which can be explored to monitor electron transfer within the redox complex. In this study, we present a strategy for a fast and straightforward monitorization of electron transfer between c-type cytochromes, using as model a triheme periplasmic cytochrome (PpcA) and a membrane associated monoheme cytochrome (OmcF) from the electrogenic bacterium Geobacter sulfurreducens. The comparison between the 1D 1H NMR spectra obtained for samples containing the two cytochromes and for samples containing the individual proteins clearly demonstrated a unidirectional electron transfer within the redox complex. This strategy provides a simple and straightforward means to elucidate complex biologic respiratory electron transfer chains.}

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.

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.

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.

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 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.

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.

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.

The facultative aerobic bacterium Geobacter sulfurreducens produces a small periplasmic c-type triheme cytochrome with 71 residues (PpcA) under anaerobic growth conditions, which is involved in the iron respiration. The thermodynamic properties of the PpcA redox centers and of a protonatable center were determined using NMR and visible spectroscopy techniques. The redox centers have negative and different reduction potentials (−162, −143, and −133 mV for heme I, III, and IV, respectively, for the fully reduced and protonated protein), which are modulated by redox interactions among the hemes (covering a range from 10 to 36 mV) and by redox−Bohr interactions (up to −62 mV) between the hemes and a protonatable center located in the proximity of heme IV. All the interactions between the four centers are dominated by electrostatic effects. The microscopic reduction potential of heme III is the one most affected by the oxidation of the other hemes, whereas heme IV is the most affected by the protonation state of the molecule. The thermodynamic properties of PpcA showed that pH strongly modulates the redox behavior of the individual heme groups. A preferred electron transfer pathway at physiologic pH is defined, showing that PpcA has the necessary thermodynamic properties to perform e-/H+ energy transduction, contributing to a H+ electrochemical potential gradient across the periplasmic membrane that drives ATP synthesis. PpcA is 46% identical in sequence to and shares a high degree of structural similarity with a periplasmic triheme cytochrome c7 isolated from Desulfuromonas acetoxidans, a bacterium closely related to the Geobacteracea family. However, the results obtained for PpcA are quite different from those published for D. acetoxidans c7, and the physiological consequences of these differences are discussed.

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 fumarate reductases from S. frigidimarina NCIMB400 and S. oneidensis MR-1 are soluble and monomeric enzymes located in the periplasm of these bacteria. These proteins display two redox active domains, one containing four c-type hemes and another containing FAD at the catalytic site. This arrangement of single-electron redox co-factors leading to multiple-electron active sites is widespread in respiratory enzymes. To investigate the properties that allow a chain of single-electron co-factors to sustain the activity of a multi-electron catalytic site, redox titrations followed by NMR and visible spectroscopies were applied to determine the microscopic thermodynamic parameters of the hemes. The results show that the redox behaviour of these fumarate reductases is similar and dominated by a strong interaction between hemes II and III. This interaction facilitates a sequential transfer of two electrons from the heme domain to FAD via heme IV.

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.

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 facultative aerobic bacterium Shewanella frigidimarina produces a small c-type tetrahaem cytochrome (86 residues) under anaerobic growth conditions. This protein is involved in the respiration of iron and shares 42% sequence identity with the N-terminal domain of a soluble flavocytochrome, isolated from the periplasm of the same bacterium, which also contains four c-type haem groups. The thermodynamic properties of the redox centres and of an ionizable centre in the tetrahaem cytochrome were determined using NMR and visible spectroscopy techniques. This is the first detailed thermodynamic study performed on a tetrahaem cytochrome isolated from a facultative aerobic bacterium and reveals that this protein presents unique features. The redox centres have negative and different redox potentials, which are modulated by redox interactions between the four haems (covering a range of 8–56mV) and by redox–Bohr interactions between the haems and an ionizable centre (-4 to -36mV) located in close proximity to haem III. All of the interactions between the five centres are clearly dominated by electrostatic effects and the microscopic reduction potential of haem III is the one most affected by the oxidation of the other haems and by the protonation state of the molecule. Altogether, this study indicates that the tetrahaem cytochrome isolated from S. frigidimarina (Sfc) has the thermodynamic properties to work as an electron wire between its redox partners. Considering the high degree of sequence identity between Sfc and the cytochrome domain of flavocytochrome c3, the structural similarities of the haem core, and that the macroscopic potentials are also identical, the results obtained in this work are rationalized in order to put forward a putative redox model for flavocytochrome c3.

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.

Multiheme cytochromes c are important in electron transfer pathways in reduction of both soluble and insoluble Fe(III) by Geobacter sulfurreducens. We determined the crystal structure at 3.2 Å resolution of the first dodecaheme cytochrome c (GSU1996) along with its N-terminal and C-terminal hexaheme fragments at 2.6 and 2.15 Å resolution, respectively. The macroscopic reduction potentials of the full-length protein and its fragments were measured. The sequence of GSU1996 can be divided into four c7-type domains (A, B, C and D) with homology to triheme cytochromes c7. In cytochromes c7 all three hemes are bis–His coordinated, whereas in c7-type domains the last heme is His–Met coordinated. The full-length GSU1996 has a 12 nm long crescent shaped structure with the 12 hemes arranged along a polypeptide to form a “nanowire” of hemes; it has a modular structure. Surprisingly, while the C-terminal half of the protein consists of two separate c7-type domains (C and D) connected by a small linker, the N-terminal half of the protein has two c7-type domains (A and B) that form one structural unit. This is also observed in the AB fragment. There is an unexpected interaction between the hemes at the interface of domains A and B, which form a heme-pair with nearly parallel stacking of their porphyrin rings. The hemes adjacent to each other throughout the protein are within van der Waals distance which enables efficient electron exchange between them. For the first time, the structural details of c7-type domains from one multiheme protein were compared.

The structure of a novel c7-type cytochrome domain that has two bishistidine coordinated hemes and one heme with histidine, methionine coordination (where the sixth ligand is a methionine residue) was determined at 1.7 Å resolution. This domain is a representative of domains that form three polymers encoded by the Geobacter sulfurreducens genome. Two of these polymers consist of four and one protein of nine c7-type domains with a total of 12 and 27 hemes, respectively. Four individual domains (termed A, B, C, and D) from one such multiheme cytochrome c (ORF03300) were cloned and expressed in Escherichia coli. The domain C produced diffraction quality crystals from 2.4 M sodium malonate (pH 7). The structure was solved by MAD method and refined to an R-factor of 19.5% and R-free of 21.8%. Unlike the two c7 molecules with known structures, one from G. sulfurreducens (PpcA) and one from Desulfuromonas acetoxidans where all three hemes are bishistidine coordinated, this domain contains a heme which is coordinated by a methionine and a histidine residue. As a result, the corresponding heme could have a higher potential than the other two hemes. The apparent midpoint reduction potential, Eapp, of domain C is −105 mV, 50 mV higher than that of PpcA.

Periplasmic sensor domains from two methyl-accepting chemotaxis proteins from Geobacter sulfurreducens (encoded by genes GSU0935 and GSU0582) were expressed in Escherichia coli. The sensor domains were isolated, purified, characterized in solution, and their crystal structures were determined. In the crystal, both sensor domains form swapped dimers and show a PAS-type fold. The swapped segment consists of two helices of about 45 residues at the N terminus with the hemes located between the two monomers. In the case of the GSU0582 sensor, the dimer contains a crystallographic 2-fold symmetry and the heme is coordinated by an axial His and a water molecule. In the case of the GSU0935 sensor, the crystals contain a non-crystallographic dimer, and surprisingly, the coordination of the heme in each monomer is different; monomer A heme has His-Met ligation and monomer B heme has His-water ligation as found in the GSU0582 sensor. The structures of these sensor domains are the first structures of PAS domains containing covalently bound heme. Optical absorption, electron paramagnetic resonance and NMR spectroscopy have revealed that the heme groups of both sensor domains are high-spin and low-spin in the oxidized and reduced forms, respectively, and that the spin-state interconversion involves a heme axial ligand replacement. Both sensor domains bind NO in their ferric and ferrous forms but bind CO only in the reduced form. The binding of both NO and CO occurs via an axial ligand exchange process, and is fully reversible. The reduction potentials of the sensor domains differ by 95 mV (− 156 mV and − 251 mV for sensors GSU0582 and GSU0935, respectively). The swapped dimerization of these sensor domains and redox-linked ligand switch might be related to the mechanism of signal transduction by these chemotaxis proteins.