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

Multiheme cytochromes have been implicated in Geobacter sulfurreducens extracellular electron transfer (EET). These proteins are potential targets to improve EET and enhance bioremediation and electrical current production by G. sulfurreducens. 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 G. sulfurreducens 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 G. sulfurreducens 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 G. sulfurreducens. For the first time G. sulfurreducens strains have been manipulated by the introduction of mutant forms of essential proteins with the aim to develop and improve bioelectrochemical technologies.

Bacteria play many important roles in animal digestive systems, including the provision of enzymes critical to digestion. Typically, complex communities of bacteria reside in the gut lumen in direct contact with the ingested materials they help to digest. Here, we demonstrate a previously undescribed digestive strategy in the wood-eating marine bivalve Bankia setacea, wherein digestive bacteria are housed in a location remote from the gut. These bivalves, commonly known as shipworms, lack a resident microbiota in the gut compartment where wood is digested but harbor endosymbiotic bacteria within specialized cells in their gills. We show that this comparatively simple bacterial community produces wood-degrading enzymes that are selectively translocated from gill to gut. These enzymes, which include just a small subset of the predicted wood-degrading enzymes encoded in the endosymbiont genomes, accumulate in the gut to the near exclusion of other endosymbiont-made proteins. This strategy of remote enzyme production provides the shipworm with a mechanism to capture liberated sugars from wood without competition from an endogenous gut microbiota. Because only those proteins required for wood digestion are translocated to the gut, this newly described system reveals which of many possible enzymes and enzyme combinations are minimally required for wood degradation. Thus, although it has historically had negative impacts on human welfare, the shipworm digestive process now has the potential to have a positive impact on industries that convert wood and other plant biomass to renewable fuels, fine chemicals, food, feeds, textiles, and paper products.

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.

PpcA is a small protein with 71 residues that contains three covalently bound hemes. The structures of single mutants at residue 58 have shown larger deviations in another part of the protein molecule than at the site of the mutation. Closer examination of the crystal packing has revealed the origin of this unexpected structural change. The site of mutation is within Van der Waals distance from another protein molecule related by a crystallographic twofold axis within the crystal. The structural changes occurred at or near the mutation site have led to a slight adjustment of the surface residues in contact. The observed deviations between the native and the mutant molecular structures are derived from the new crystal packing even though the two crystals are essentially isomorphous. Without careful consideration of the crystal lattice a non-expert looking at only the coordinates deposited in the Protein Data Bank could draw erroneous conclusion that mutation in one part of the molecule affected the structure of the protein in a distant part of the molecule.

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 Met(58), 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.

Cytochromes c(7) are periplasmic triheme proteins that have been reported exclusively in δ-proteobacteria. The structures of five triheme cytochromes identified in Geobacter sulfurreducens and one in Desulfuromonas acetoxidans have been determined. In addition to the hemes and axial histidines, a single aromatic residue is conserved in all these proteins-phenylalanine 15 (F15). PpcA is a member of the G. sulfurreducens cytochrome c(7) family that performs electron/proton energy transduction in addition to electron transfer that leads to the reduction of extracellular electron acceptors. For the first time we probed the role of the F15 residue in the PpcA functional mechanism, by replacing this residue with the aliphatic leucine by site-directed mutagenesis. The analysis of NMR spectra of both oxidized and reduced forms showed that the heme core and the overall fold of the mutated protein were not affected. However, the analysis of (1)H-(15)N heteronuclear single quantum coherence NMR spectra evidenced local rearrangements in the α-helix placed between hemes I and III that lead to structural readjustments in the orientation of heme axial ligands. The detailed thermodynamic characterization of F15L mutant revealed that the reduction potentials are more negative and the redox-Bohr effect is decreased. The redox potential of heme III is most affected. It is of interest that the mutation in F15, located between hemes I and III in PpcA, changes the characteristics of the two hemes differently. Altogether, these modifications disrupt the balance of the global network of cooperativities, preventing the F15L mutant protein from performing a concerted electron/proton transfer., (© SBIC 2011)

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.

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 3.2Å resolution of the first dodecaheme cytochrome c (GSU1996) along with its N-terminal and C-terminal hexaheme fragments at 2.6 and 2.15Å 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 c(7)-type domains (A, B, C and D) with homology to triheme cytochromes c(7). In cytochromes c(7) all three hemes are bis-His coordinated, whereas in c(7)-type domains the last heme is His-Met coordinated. The full-length GSU1996 has a 12nm 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 c(7)-type domains (C and D) connected by a small linker, the N-terminal half of the protein has two c(7)-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 c(7)-type domains from one multiheme protein were compared., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Answering questions about proteins' structures and functions in the new era of systems biology and genomics requires the development of new methods for heterologous production of numerous proteins from newly sequenced genomes. Cytochromes c - electron transfer proteins carrying one or more hemes covalently bound to the polypeptide chain - are one of the most recalcitrant classes of proteins with respect to heterologous expression because post-translational incorporation of hemes is required for proper folding and stability. However, significant advances in expression of recombinant cytochromes c have been made during the last decade. It has been shown that a single gene cluster, ccmA-H, is responsible for cytochrome c maturation in Escherichia coli under anaerobic conditions and that constitutive co-expression of this cluster under aerobic conditions is sufficient to provide heme incorporation in many different types of cytochromes c, regardless of their origin, as long as the nascent polypeptide is translocated to the periplasm. Using conditions that result in sub-maximal protein induction can dramatically increase the yield of mature protein. The intrinsic peroxidase activity of hemes can be used as a highly selective and sensitive detection method of mature cytochromes in samples resolved by gel electrophoresis.

Engineering efficient, directional electronic communication between living and nonliving systems has the potential to combine the unique characteristics of both materials for advanced biotechnological applications. However, the cell membrane is designed by nature to be an insulator, restricting the flow of charged species; therefore, introducing a biocompatible pathway for transferring electrons across the membrane without disrupting the cell is a significant challenge. Here we describe a genetic strategy to move intracellular electrons to an inorganic extracellular acceptor along a molecularly defined route. To do so, we reconstitute a portion of the extracellular electron transfer chain of Shewanella oneidensis MR-1 into the model microbe Escherichia coli. This engineered E. coli can reduce metal ions and solid metal oxides ∼8× and ∼4× faster than its parental strain. We also find that metal oxide reduction is more efficient when the extracellular electron acceptor has nanoscale dimensions. This work demonstrates that a genetic cassette can create a conduit for electronic communication from living cells to inorganic materials, and it highlights the importance of matching the size scale of the protein donors to inorganic acceptors.

A family of five periplasmic triheme cytochromes (PpcA-E) was identified in Geobacter sulfurreducens, where they play a crucial role by driving electron transfer from the cytoplasm to the cell exterior and assisting the reduction of extracellular acceptors. The thermodynamic characterization of PpcA using NMR and visible spectroscopies was previously achieved under experimental conditions identical to those used for the triheme cytochrome c(7) from Desulfuromonas acetoxidans. Under such conditions, attempts to obtain NMR data were complicated by the relatively fast intermolecular electron exchange. This work reports the detailed thermodynamic characterization of PpcB, PpcD, and PpcE under optimal experimental conditions. The thermodynamic characterization of PpcA was redone under these new conditions to allow a proper comparison of the redox properties with those of other members of this family. The heme reduction potentials of the four proteins are negative, differ from each other, and cover different functional ranges. These reduction potentials are strongly modulated by heme-heme interactions and by interactions with protonated groups (the redox-Bohr effect) establishing different cooperative networks for each protein, which indicates that they are designed to perform different functions in the cell. PpcA and PpcD appear to be optimized to interact with specific redox partners involving e(-)/H(+) transfer via different mechanisms. Although no evidence of preferential electron transfer pathway or e(-)/H(+) coupling was found for PpcB and PpcE, the difference in their working potential ranges suggests that they may also have different physiological redox partners. This is the first study, to our knowledge, to characterize homologous cytochromes from the same microorganism and provide evidence of their different mechanistic and functional properties. These findings provide an explanation for the coexistence of five periplasmic triheme cytochromes in G. sulfurreducens., (Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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., (2010 Elsevier Inc. All rights reserved.)

Periplasmic cytochromes c(7) are important in electron transfer pathway(s) in Fe(III) respiration by Geobacter sulfurreducens. The genome of G. sulfurreducens encodes a family of five 10-kDa, three-heme cytochromes c(7). The sequence identity between the five proteins (designated PpcA, PpcB, PpcC, PpcD, and PpcE) varies between 45% and 77%. Here, we report the high-resolution structures of PpcC, PpcD, and PpcE determined by X-ray diffraction. This new information made it possible to compare the sequences and structures of the entire family. The triheme cores are largely conserved but are not identical. We observed changes, due to different crystal packing, in the relative positions of the hemes between two molecules in the crystal. The overall protein fold of the cytochromes is similar. The structure of PpcD differs most from that of the other homologs, which is not obvious from the sequence comparisons of the family. Interestingly, PpcD is the only cytochrome c(7) within the family that has higher abundance when G. sulfurreducens is grown on insoluble Fe(III) oxide compared to ferric citrate. The structures have the highest degree of conservation around "heme IV"; the protein surface around this heme is positively charged in all of the proteins, and therefore all cytochromes c(7) could interact with similar molecules involving this region. The structures and surface characteristics of the proteins near the other two hemes, "heme I" and "heme III", differ within the family. The above observations suggest that each of the five cytochromes c(7) could interact with its own redox partner via an interface involving the regions of heme I and/or heme III; this provides a possible rationalization for the existence of five similar proteins in G. sulfurreducens., (2009 Elsevier B.V. All rights reserved.)

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.

Integrated studies that address proteins structure and function in the new era of systems biology and genomics often require the application of high-throughput approaches for parallel production of many different purified proteins from the same organism. Cytochromes c-electron transfer proteins carrying one or more hemes covalently bound to the polypeptide chain-are essential in most organisms. However, they are one of the most recalcitrant classes of proteins with respect to heterologous expression because post-translational incorporation of hemes is required for proper folding and stability. We have addressed this challenge by designing two families of vectors (total of 6 vectors) suitable for ligation-independent cloning and developing a pipeline for expression and solubility analysis of cytochromes c. This system has been validated by expression analysis of thirty genes from Shewanella oneidensis coding for cytochromes c or cytochromes c-type domains predicted to have 1-4 hemes. Out of 30 targets, 26 (87%) were obtained in soluble form in one or more vectors. This work establishes a methodology for high-throughput expression of this class of proteins and provides a clone resource for the microbiological and functional genomics research communities.

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.

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

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.

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 c(3) 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 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.

Geobacter sulfurreducens encodes one of the largest numbers of proteins annotated as parts of the two-component signal transduction and/or chemotaxis pathways. Ten of these signal transducers have homologous periplasmic sensor domains that contain the sequence signature for c-type hemes. One such sensor domain encoded by gene GSU0303 was isolated and characterized. The protein was expressed in Escherichia coli and was isolated as two colored species (green and red). The green species is a monomer of the sensor domain with a five-coordinated high-spin heme and the red species is probably a noncovalent dimer of the sensor domain which might have an uncharacterized ligand bound to the dimer. The UV-VIS spectrum of the green species indicates that it has a c'-type heme, but its structure is predicted to be homologous to CitA, a periplasmic PAS domain that does not contain heme. The GSU0303 sensor domain represents a previously unreported family of PAS-type periplasmic sensor domains that contain c-type hemes; these proteins could be part of an important mechanism for sensing redox potential or small ligands in the periplasm. Homologs to the sensor domains we identified in G. sulfurreducens are observed in various bacteria although they occur in larger numbers in the Geobacteraceae.

Multiheme cytochromes c are difficult to produce in heterologous systems. The genome of delta-proteobacterium Geobacter sulfurreducens contains more than a hundred genes coding for c-type cytochromes. Among those are two dodecaheme cytochromes c representing a new class of multiheme cytochromes, whose putative structure is a one-dimensional array of small highly homologous domains that contain three hemes and are covalently bound by short linkers. They are likely to form "nanowires" that are part of the electron transfer chain. We cloned the genes coding for the two cytochromes into a vector we developed for ligation-independent cloning of proteins targeted to the Escherichia coli periplasmic space. We expressed the proteins in E. coli co-transformed with a plasmid harboring the cytochrome c maturation genes. Expression levels were optimized by varying IPTG concentrations used for induction. Although both proteins appeared insoluble or strongly associated with cell membranes, they were solubilized using 0.5 M sodium chloride which was more selective than conventional solubilizing agents, such as HEGA-10 or beta-octylglucoside. The solubilized proteins were dialyzed and purified by cation exchange chromatography followed by gel filtration. Mass-spectrometry analysis confirmed that both purified proteins contained the complete set of covalently attached hemes, 12 per molecule. Their visible spectra were typical of c-type cytochromes. Both proteins were successfully crystallized.

Multiheme cytochromes c are of great interest for researchers for a variety of reasons but difficult to obtain in quantities sufficient for the majority of studies. The genome of delta-proteobacterium Geobacter sulfurreducens contains more than a hundred genes coding for c-type cytochromes. Three of them represent a new class of multiheme cytochromes characterized by a mixed type of heme coordination and multidomain organization. We cloned and expressed in Escherichia coli three hexaheme fragments corresponding to two-domain fragments of one such protein containing 12 heme binding motifs and believed to consist of four triheme domains. Despite high sequence similarity among the fragments, expression levels varied significantly. Expression was optimized either by host strain variation or by reducing the rate of apoprotein synthesis. All three fragments were purified by cation exchange followed by gel filtration and were shown to contain six covalently attached hemes as confirmed by mass spectrometry. Their visible spectra are typical of c-type cytochromes. One of the fragments was crystallized and its preliminary X-ray structure shows two separate domains.

The complete genome sequence of the delta-proteobacterium Geobacter sulfurreducens reveals a large abundance of multiheme cytochromes. Cytochrome c(7), isolated from this metal ion-reducing bacterium, is a triheme periplasmic electron-transfer protein with M(r) 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 delta-proteobacterium Desulfuromonas acetoxidans (Dac(7)). 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 c(7) (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 Dac(7) 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 Dac(7). The differences observed in the redox behavior of PpcA and Dac(7) 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 structure of a novel c(7)-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 A 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 c(7)-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 c(7) 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, E(app), of domain C is -105 mV, 50 mV higher than that of PpcA.

The structure of a cytochrome c(7) (PpcA) from Geobacter sulfurreducens was determined by X-ray diffraction at 1.45 A resolution; the R factor is 18.2%. The protein contains a three-heme core that is surrounded by 71 amino acid residues. An unusual feature of this cytochrome is that it has 17 lysine residues, but only nine hydrophobic residues that are larger than alanine. The details of the structure are described and compared with those of cytochrome c(7) from Desulfuromonas acetoxidans and with cytochromes c(3). The two cytochrome c(7) molecules have sequences that are 46% identical, but the arrangements of the hemes in the two structures differ; the rms deviation of all alpha-carbons is 2.5 A. These cytochromes can reduce various metal ions. The reduction site of the chromate ion in D. acetoxidans is occupied by a sulfate ion in the crystal structure of PpcA. We identified four additional homologues of cytochrome c(7) in the G. sulfurreducens genome and three polymers of c(7)-type domains. Of the polymers, two have four repeats and one has nine repeats. On the basis of sequence alignments, one of the hemes in each of the cytochrome c(7)-type domains does not have the bis-histidine coordination. The packing of the molecules in the crystal structure of PpcA suggests that the polymers have an elongated conformation and might form a "nanowire".

Multiheme cytochromes c have been found in a number of sulfate- and metal ion-reducing bacteria. Geobacter sulfurreducens is one of a family of microorganisms that oxidize organic compounds, with Fe(III) oxide as the terminal electron acceptor. A triheme 9.6 kDa cytochrome c(7) from G. sulfurreducens is a part of the metal ion reduction pathway. We cloned the gene for cytochrome c(7) and expressed it in Escherichia coli together with the cytochrome c maturation gene cluster, ccmABCDEFGH, on a separate plasmid. We designed two constructs, with and without an N-terminal His-tag. The untagged version provided a good yield (up to 6 mg/l of aerobic culture) of the fully matured protein, with all three hemes attached, while the N-terminal His-tag appeared to be detrimental for proper heme incorporation. The recombinant protein (untagged) is properly folded, it has the same molecular weight and displays the same absorption spectra, both in reduced and in oxidized forms, as the protein isolated from G. sulfurreducens and it is capable of reducing metal ions in vitro. The shape parameters for the recombinant cytochrome c(7) determined by small angle X-ray scattering are in good agreement with the ones calculated from a homologous cytochrome c(7) of known structure.

Fibritin is a segmented coiled-coil homotrimer of the 486-residue product of phage T4 gene wac. This protein attaches to a phage particle by the N-terminal region and forms fibrous whiskers of 530 A, which perform a chaperone function during virus assembly. The short C-terminal region has a beta-annulus-like structure. We engineered a set of fibritin deletion mutants sequentially truncated from the N-termini, and the mutants were studied by differential scanning calorimetry (DSC) and CD measurements. The analysis of DSC curves indicates that full-length fibritin exhibits three thermal-heat-absorption peaks centred at 321 K (Delta H=1390 kJ x mol trimer(-1)), at 336 K (Delta H=7600 kJ x mol trimer(-1)), and at 345 K (Delta H=515 kJ x mol trimer(-1)). These transitions were assigned to the N-terminal, segmented coiled-coil, and C-terminal functional domains, respectively. The coiled-coil region, containing 13 segments, melts co-operatively as a single domain with a mean enthalpy Delta Hres=21 kJ x mol residue(-1). The ratio of Delta HVH/Delta Hcal for the coiled-coil part of the 120-, 182-, 258- and 281-residue per monomer mutants, truncated from the N-termini, and for full-length fibritin are 0.91, 0.88, 0.42, 0.39, and 0.13, respectively. This gives an indication of the decrease of the 'all-or-none' character of the transition with increasing protein size. The deletion of the 12-residue-long loop in the 120-residue fibritin increases the thermal stability of the coiled-coil region. According to CD data, full-length fibritin and all the mutants truncated from the N-termini refold properly after heat denaturation. In contrast, fibritin XN, which is deleted for the C-terminal domain, forms aggregates inside the cell. The XN protein can be partially refolded by dilution from urea and does not refold after heat denaturation. These results confirm that the C-terminal domain is essential for correct fibritin assembly both in vivo and in vitro and acts as a foldon.

Bacteriophage T4 fibritin is a triple-stranded, parallel, segmented alpha-helical coiled-coil protein. Earlier we showed that the C-terminal globular domain (foldon) of fibritin is essential for correct trimerization and folding of the protein. We constructed the chimerical fusion protein W31 in which the fibritin foldon sequence is followed by the small globular non-alpha-helical protein gp31 of the T4 phage. We showed that the foldon is capable of trimerization in the absence of the coiled-coil part of fibritin. A deletion mutant of fibritin (NB1) with completely deleted foldon is unable to fold and trimerize correctly. An excess of this mutant protein did not influence the refolding of fibritin in vitro, and the chimerical protein inhibited this process efficiently. Our conclusion is that the trimerization of the foldon is the initial step of fibritin refolding and is followed by the formation of the coiled-coil structure.