Tuning of functional heme reduction potentials in Shewanella fumarate reductases.

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.

Energy metabolism in Desulfovibrio vulgaris Hildenborough: insights from transcriptome analysis.

Sulphate-reducing bacteria are important players in the global sulphur and carbon cycles, with considerable economical and ecological impact. However, the process of sulphate respiration is still incompletely understood. Several mechanisms of energy conservation have been proposed, but it is unclear how the different strategies contribute to the overall process. In order to obtain a deeper insight into the energy metabolism of sulphate-reducers whole-genome microarrays were used to compare the transcriptional response of Desulfovibrio vulgaris Hildenborough grown with hydrogen/sulphate, pyruvate/sulphate, pyruvate with limiting sulphate, and lactate/thiosulphate, relative to growth in lactate/sulphate. Growth with hydrogen/sulphate showed the largest number of differentially expressed genes and the largest changes in transcript levels. In this condition the most up-regulated energy metabolism genes were those coding for the periplasmic [NiFeSe] hydrogenase, followed by the Ech hydrogenase. The results also provide evidence for the involvement of formate cycling and the recently proposed ethanol pathway during growth in hydrogen. The pathway involving CO cycling is relevant during growth on lactate and pyruvate, but not during growth in hydrogen as the most down-regulated genes were those coding for the CO-induced hydrogenase. Growth on lactate/thiosulphate reveals a down-regulation of several energy metabolism genes similar to what was observed in the presence of nitrite. This study identifies the role of several proteins involved in the energy metabolism of D. vulgaris and highlights several novel genes related to this process, revealing a more complex bioenergetic metabolism than previously considered.

Transcriptional response of Desulfovibrio vulgaris Hildenborough to oxidative stress mimicking environmental conditions.

Sulfate-reducing bacteria (SRB) are anaerobes readily found in oxic-anoxic interfaces. Multiple defense pathways against oxidative conditions were identified in these organisms and proposed to be differentially expressed under different concentrations of oxygen, contributing to their ability to survive oxic conditions. In this study, Desulfovibrio vulgaris Hildenborough cells were exposed to the highest concentration of oxygen that SRB are likely to encounter in natural habitats, and the global transcriptomic response was determined. Three hundred and seven genes were responsive, with cellular roles in energy metabolism, protein fate, cell envelope and regulatory functions, including multiple genes encoding heat shock proteins, peptidases and proteins with heat shock promoters. Of the oxygen reducing mechanisms of D. vulgaris only the periplasmic hydrogen-dependent mechanism was up-regulated, involving the [NiFeSe] hydrogenase, formate dehydrogenase(s) and the Hmc membrane complex. The oxidative defense response concentrated on damage repair by metal-free enzymes. These data, together with the down-regulation of the ferric uptake regulator operon, which restricts the availability of iron, and the lack of response of the peroxide-sensing regulator operon, suggest that a major effect of this oxygen stress is the inactivation and/or degradation of multiple metalloproteins present in D. vulgaris as a consequence of oxidative damage to their metal clusters.

Thermodynamic and kinetic characterisation of individual haems in multicentre cytochromes c3.

The characterisation of individual centres in multihaem proteins is difficult due to the similarities in the redox and spectroscopic properties of the centres. NMR has been used successfully to distinguish redox centres and allow the determination of the microscopic thermodynamic parameters in several multihaem cytochromes c(3) isolated from different sulphate-reducing bacteria. In this article we show that it is also possible to discriminate the kinetic properties of individual centres in multihaem proteins, if the complete microscopic thermodynamic characterisation is available and the system displays fast intramolecular equilibration in the time scale of the kinetic experiment. The deconvolution of the kinetic traces using a model of thermodynamic control provides a reference rate constant for each haem that does not depend on driving force and can be related to structural factors. The thermodynamic characterisation of three tetrahaem cytochromes and their kinetics of reduction by sodium dithionite are reported in this paper. Thermodynamic and kinetic data were fitted simultaneously to a model to obtain microscopic reduction potentials, haem-haem and haem-proton interacting potentials, and reference rate constants for the haems. The kinetic information obtained for these cytochromes and recently published data for other multihaem cytochromes is discussed with respect to the structural factors that determine the reference rates. The accessibility for the reducing agent seems to play an important role in controlling the kinetic rates, although is clearly not the only factor.

Functional properties of type I and type II cytochromes c3 from Desulfovibrio africanus.

Type I cytochrome c(3) is a key protein in the bioenergetic metabolism of Desulfovibrio spp., mediating electron transfer between periplasmic hydrogenase and multihaem cytochromes associated with membrane bound complexes, such as type II cytochrome c(3). This work presents the NMR assignment of the haem substituents in type I cytochrome c(3) isolated from Desulfovibrio africanus and the thermodynamic and kinetic characterisation of type I and type II cytochromes c(3) belonging to the same organism. It is shown that the redox properties of the two proteins allow electrons to be transferred between them in the physiologically relevant direction with the release of energised protons close to the membrane where they can be used by the ATP synthase.

The Tmc complex from Desulfovibrio vulgaris hildenborough is involved in transmembrane electron transfer from periplasmic hydrogen oxidation.

Three membrane-bound redox complexes have been reported in Desulfovibrio spp., whose genes are not found in the genomes of other sulfate reducers such as Desulfotalea psycrophila and Archaeoglobus fulgidus. These complexes contain a periplasmic cytochrome c subunit of the cytochrome c(3) family, and their presence in these organisms probably correlates with the presence of a pool of periplasmic cytochromes c(3), also absent in the two other sulfate reducers. In this work we report the isolation and characterization of the first of such complexes, Tmc from D. vulgaris Hildenborough, which is associated with the tetraheme type II cytochrome c(3). The isolated Tmc complex contains four subunits, including the TpIIc(3) (TmcA), an integral membrane cytochrome b (TmcC), and two cytoplasmically predicted proteins, an iron-sulfur protein (TmcB) and a tryptophan-rich protein (TmcD). Spectroscopic studies indicate the presence of eight hemes c and two hemes b in the complex pointing to an alpha(2)betagammadelta composition (TmcA(2)BCD). EPR analysis reveals the presence of a [4Fe4S](3+) center and up to three other iron-sulfur centers in the cytoplasmic subunit. Nearly full reduction of the redox centers in the Tmc complex could be obtained upon incubation with hydrogenase/TpIc(3), supporting the role of this complex in transmembrane transfer of electrons resulting from periplasmic oxidation of hydrogen.

Solution structures of tetrahaem ferricytochrome c3 from Desulfovibrio vulgaris (Hildenborough) and its K45Q mutant: the molecular basis of cooperativity.

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.

Characterization of the Desulfovibrio desulfuricans ATCC 27774 DsrMKJOP complex--a membrane-bound redox complex involved in the sulfate respiratory pathway.

Sulfate-reducing organisms use sulfate as an electron acceptor in an anaerobic respiratory process. Despite their ubiquitous occurrence, sulfate respiration is still poorly characterized. Genome analysis of sulfate-reducing organisms sequenced to date permitted the identification of only two strictly conserved membrane complexes. We report here the purification and characterization of one of these complexes, DsrMKJOP, from Desulfovibrio desulfuricans ATCC 27774. The complex has hemes of the c and b types and several iron-sulfur centers. The corresponding genes in the genome of Desulfovibrio vulgaris were analyzed. dsrM encodes an integral membrane cytochrome b; dsrK encodes a protein homologous to the HdrD subunit of heterodisulfide reductase; dsrJ encodes a triheme periplasmic cytochrome c; dsrO encodes a periplasmic FeS protein; and dsrM encodes another integral membrane protein. Sequence analysis and EPR studies indicate that DsrJ belongs to a novel family of multiheme cytochromes c and that its three hemes have different types of coordination, one bis-His, one His/Met, and the third a very unusual His/Cys coordination. The His/Cys-coordinated heme is only partially reduced by dithionite. About 40% of the hemes are reduced by menadiol, but no reduction is observed upon treatment with H2 and hydrogenase, irrespective of the presence of cytochrome c3. The aerobically isolated Dsr complex displays an EPR signal with similar characteristics to the catalytic [4Fe-4S]3+ species observed in heterodisulfide reductases. Further five different [4Fe-4S](2+/1+) centers are observed during a redox titration followed by EPR. The role of the DsrMKJOP complex in the sulfate respiratory chain of Desulfovibrio spp. is discussed.

Hydrogenases in Desulfovibrio vulgaris Hildenborough: structural and physiologic characterisation of the membrane-bound [NiFeSe] hydrogenase.

The genome of Desulfovibrio vulgaris Hildenborough (DvH) encodes for six hydrogenases (Hases), making it an interesting organism to study the role of these proteins in sulphate respiration. In this work we address the role of the [NiFeSe] Hase, found to be the major Hase associated with the cytoplasmic membrane. The purified enzyme displays interesting catalytic properties, such as a very high H(2) production activity, which is dependent on the presence of phospholipids or detergent, and resistance to oxygen inactivation since it is isolated aerobically in a Ni(II) oxidation state. Evidence was obtained that the [NiFeSe] Hase is post-translationally modified to include a hydrophobic group bound to the N-terminal, which is responsible for its membrane association. Cleavage of this group originates a soluble, less active form of the enzyme. Sequence analysis shows that [NiFeSe] Hases from Desulfovibrionacae form a separate family from the [NiFe] enzymes of these organisms, and are more closely related to [NiFe] Hases from more distant bacterial species that have a medial [4Fe4S](2+/1+) cluster, but not a selenocysteine. The interaction of the [NiFeSe] Hase with periplasmic cytochromes was investigated and is similar to the [NiFe](1) Hase, with the Type I cytochrome c (3) as the preferred electron acceptor. A model of the DvH [NiFeSe] Hase was generated based on the structure of the Desulfomicrobium baculatum enzyme. The structures of the two [NiFeSe] Hases are compared with the structures of [NiFe] Hases, to evaluate the consensual structural differences between the two families. Several conserved residues close to the redox centres were identified, which may be relevant to the higher activity displayed by [NiFeSe] Hases.

Redox behaviour of the haem domain of flavocytochrome c3 from Shewanella frigidimarina probed by NMR.

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.

Proton-assisted two-electron transfer in natural variants of tetraheme cytochromes from Desulfomicrobium Sp.

The tetraheme cytochrome c3 isolated from Desulfomicrobium baculatum (DSM 1743)(Dsmb) was cloned, and the sequence analysis showed that this cytochrome differs in just three amino acid residues from the cytochrome c3 isolated from Desulfomicrobium norvegicum (Dsmn): (DsmnXXDsmb) Thr-37 --> Ser, Val-45 --> Ala, and Phe-88 --> Tyr. X-ray crystallography was used to determine the structure of cytochrome c3 from Dsmb, showing that it is very similar to the published structure of cytochrome c3 from Dsmn. A detailed thermodynamic and kinetic characterization of these two tetraheme cytochromes c3 was performed by using NMR and visible spectroscopy. The results obtained show that the network of cooperativities between the redox and protonic centers is consistent with a synergetic process to stimulate the hydrogen uptake activity of hydrogenase. This is achieved by increasing the affinity of the cytochrome for protons through binding electrons and, reciprocally, by favoring a concerted two-electron transfer assisted by the binding of proton(s). The data were analyzed within the framework of the differences in the primary and tertiary structures of the two proteins, showing that residue 88, close to heme I, is the main cause for the differences in the microscopic thermodynamic parameters obtained for these two cytochromes c3. This comparison reveals how replacement of a single amino acid can tune the functional properties of energy-transducing proteins, so that they can be optimized to suit the bioenergetic constraints of specific habitats.

Thermodynamic and choreographic constraints for energy transduction by cytochrome c oxidase.

Cooperative effects are fundamental for electroprotonic energy transduction processes, crucial to sustain much of life chemistry. However, the primary cooperative mechanism by which transmembrane proteins couple the downhill transfer of electrons to the uphill activation (acidification) of protic groups is still a matter of great controversy. To understand cooperative processes fully, it is necessary to obtain the microscopic thermodynamic parameters of the functional centres and relate them to the relevant structural features, a task difficult to achieve for large proteins. The approach discussed here explores how this may be done by extrapolation from mechanisms used by simpler proteins operative in similar processes. The detailed study of small, soluble cytochromes performing electroprotonic activation has shown how they use anti-electrostatic effects to control the synchronous movement of charges. These include negative e(-)/H(+) (redox-Bohr effect) cooperativities. This capacity is the basis to discuss an unorthodox mechanism consistent with the available experimental data on the process of electroprotonic energy transduction performed by cytochrome c oxidase (CcO).

A novel membrane-bound respiratory complex from Desulfovibrio desulfuricans ATCC 27774.

In the anaerobic respiration of sulfate, performed by sulfate-reducing prokaryotes, reduction of the terminal electron acceptor takes place in the cytoplasm. The membrane-associated electron transport chain that feeds electrons to the cytoplasmic reductases is still very poorly characterized. In this study we report the isolation and characterization of a novel membrane-bound redox complex from Desulfovibrio desulfuricans ATCC 27774. This complex is formed by three subunits, and contains two hemes b, two FAD groups and several iron-sulfur centers. The two hemes b are low-spin, with macroscopic redox potentials of +75 and -20 mV at pH 7.6. Both hemes are reduced by menadiol, a menaquinone analogue, indicating a function for this complex in the respiratory electron-transport chain. EPR studies of the as-isolated and dithionite-reduced complex support the presence of a [3Fe-4S](1+/0) center and at least four [4Fe-4S](2+/1+) centers. Cloning of the genes coding for the complex subunits revealed that they form a putative transcription unit and have homology to subunits of heterodisulfide reductases (Hdr). The first and second genes code for soluble proteins that have homology to HdrA, whereas the third gene codes for a novel type of membrane-associated protein that contains both a hydrophobic domain with homology to the heme b protein HdrE and a hydrophilic domain with homology to the iron-sulfur protein HdrC. Homologous operons are found in the genomes of other sulfate-reducing organisms and in the genome of the green-sulfur bacterium Chlorobium tepidum TLS. The isolated complex is the first example of a new family of respiratory complexes present in anaerobic prokaryotes.

Response of a strict anaerobe to oxygen: survival strategies in Desulfovibrio gigas.

The biochemical response to oxygen of the strictly anaerobic sulfate-reducing bacterium Desulfovibrio gigas was studied with the goal of elucidating survival strategies in oxic environments. Cultures of D. gigas on medium containing lactate and sulfate were exposed to oxygen (concentration 5-120 micro M). Growth was fully inhibited by oxygen, but the cultures resumed growth as soon as they were shifted back to anoxic conditions. Following 24 h exposure to oxygen the growth rate was as high as 70 % of the growth rates observed before oxygenation. Catalase levels and activity were enhanced by exposure to oxygen whereas superoxide-scavenging and glutathione reductase activities were not affected. The general pattern of cellular proteins as analysed by two-dimensional electrophoresis was altered in the presence of oxygen, the levels of approximately 12 % of the detected proteins being markedly increased. Among the induced proteins, a homologue of a 60 kDa eukaryotic heat-shock protein (Hsp60) was identified by immunoassay analysis. In the absence of external substrates, the steady-state levels of nucleoside triphosphates detected by in vivo (31)P-NMR under saturating concentrations of oxygen were 20 % higher than under anoxic conditions. The higher energy levels developed under oxygen correlated with a lower rate of substrate (glycogen) mobilization, but no experimental evidence for a contribution from oxidative phosphorylation was found. The hypothesis that oxygen interferes with ATP dissipation processes is discussed.

Reduced hybrid cluster proteins (HCP) from Desulfovibrio desulfuricans ATCC 27774 and Desulfovibrio vulgaris (Hildenborough): X-ray structures at high resolution using synchrotron radiation.

The hybrid cluster proteins from the sulfate reducing bacteria Desulfovibrio desulfuricans ATCC 27774 ( Dd) and Desulfovibrio vulgaris strain Hildenborough ( Dv) have been isolated and crystallized anaerobically. In each case, the protein has been reduced with dithionite and the crystal structure of the reduced form elucidated using X-ray synchrotron radiation techniques at 1.25 A and 1.55 A resolution for Dd and Dv, respectively. Although the overall structures of the proteins are unchanged upon reduction, there are significant changes at the hybrid cluster centres. These include significant movements in the position of the iron atom linked to the persulfide moiety in the oxidized as-isolated proteins and the sulfur atom of the persulfide itself. The nature of these changes is described and the implications with respect to the function of hybrid cluster proteins are discussed.

The nature of the di-iron site in the bacterioferritin from Desulfovibrio desulfuricans.

The first crystal structure of a native di-iron center in an iron-storage protein (bacterio)ferritin is reported. The protein, isolated from the anaerobic bacterium Desulfovibrio desulfuricans, has the unique property of having Fe-coproporphyrin III as its heme cofactor. The three-dimensional structure of this bacterioferritin was determined in three distinct catalytic/redox states by X-ray crystallography (at 1.95, 2.05 and 2.35 A resolution), corresponding to different intermediates of the di-iron ferroxidase site. Conformational changes associated with these intermediates support the idea of a route for iron entry into the protein shell through a pore that passes through the di-iron center. Molecular surface and electrostatic potential calculations also suggest the presence of another ion channel, distant from the channels at the three- and four-fold axes proposed as points of entry for the iron atoms.

A novel iron centre in the split-Soret cytochrome c from Desulfovibrio desulfuricans ATCC 27774.

The facultative sulfate/nitrate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 harbours a split-Soret cytochrome c. This cytochrome is a homodimeric protein, having two bis-histidinyl c-type haems per monomer. It has an unique architecture at the haem domain: each haem has one of the coordinating histidines provided by the other monomer, and in each monomer the haems are parallel to each other, almost in van der Waals contact. This work reports the cloning and sequencing of the gene encoding for this cytochrome and shows, by transcriptional analysis, that it is more expressed in nitrate-grown cells than in sulfate-grown ones. In addition, the gene-deduced amino acid sequence revealed two new cysteine residues that could be involved in the binding of a non-haem iron centre. Indeed, the presence of a novel type of an iron-sulfur centre (possibly of the [2Fe-2S] type) was demonstrated by EPR spectroscopy, and putative models for its localization and structure in the cytochrome molecule are proposed on the basis of the so-far-known 3D crystallographic structure of the aerobically purified split-Soret cytochrome, which lacks this centre.

A mechano-chemical model for energy transduction in cytochrome c oxidase: the work of a Maxwell's god.

Cytochrome c3 has a central role in the energetics of Desulfovibrio sp., where it performs an electroprotonic energy transduction step. This process uses a network of cooperativities, largely based on anti-Coulomb components, resulting from a mechano-chemical energy coupling mechanism. This mechanism provides a model coherent with the data available for the redox chemistry of haem a of cytochrome c oxidase and its link to the activation of protons. A crucial feature of the model is an anti-Coulomb effect that sets the stage for a molecular ratchet, ensuring vectoriality for the redox-driven localised movement of protons across the membrane, against an electrochemical gradient.

Thermodynamic and kinetic characterization of trihaem cytochrome c3 from Desulfuromonas acetoxidans.

Trihaem cytochrome c3 (also known as cytochrome c551.5 and cytochrome c7) is isolated from the periplasmic space of Desulfuromonas acetoxidans, a sulfur-reducing bacterium. Thermodynamic and kinetic data for the trihaem cytochrome c3 are presented and discussed in the context of the possible physiological implications of its functional properties with respect to the natural habitat of D. acetoxidans, namely as a symbiont with green sulfur bacteria working as a mini-sulfuretum. The thermodynamic properties were determined through the fit of redox titration data, followed by NMR and visible spectroscopy, to a model of four functional centres that describes the network of cooperativities between the three haems and one protolytic centre. The kinetics of trihaem cytochrome c3 reduction by sodium dithionite were studied using the stopped-flow technique and the data were fitted to a kinetic model that makes use of the thermodynamic properties to obtain the rate constants of the individual haems. This analysis indicates that the electrons enter the cytochrome mainly via haem I. The reduction potentials of the haems in this cytochrome show little variation with pH within the physiological range, and the kinetic studies show that the rates of reduction are also independent of pH in the range studied. Thus, although the trihaem cytochrome c3 is readily reduced by hydrogenases from Desulfovibrio sp. and its haem core is similar to that of the homologous tetrahaem cytochromes c3, its physico-chemical properties are quite different, which suggests that these multihaem cytochromes with similar structures perform different functions.

Sulfate respiration in Desulfovibrio vulgaris Hildenborough. Structure of the 16-heme cytochrome c HmcA AT 2.5-A resolution and a view of its role in transmembrane electron transfer.

The crystal structure of the high molecular mass cytochrome c HmcA from Desulfovibrio vulgaris Hildenborough is described. HmcA contains the unprecedented number of sixteen hemes c attached to a single polypeptide chain, is associated with a membrane-bound redox complex, and is involved in electron transfer from the periplasmic oxidation of hydrogen to the cytoplasmic reduction of sulfate. The structure of HmcA is organized into four tetraheme cytochrome c(3)-like domains, of which the first is incomplete and contains only three hemes, and the final two show great similarity to the nine-heme cytochrome c from Desulfovibrio desulfuricans. An isoleucine residue fills the vacant coordination space above the iron atom in the five-coordinated high-spin Heme 15. The characteristics of each of the tetraheme domains of HmcA, as well as its surface charge distribution, indicate this cytochrome has several similarities with the nine-heme cytochrome c and the Type II cytochrome c(3) molecules, in agreement with their similar genetic organization and mode of reactivity and further support an analogous physiological function for the three cytochromes. Based on the present structure, the possible electron transfer sites between HmcA and its redox partners (namely Type I cytochrome c(3) and other proteins of the Hmc complex), as well as its physiological role, are discussed.