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Storage of solar energy as hydrogen provides a platform towards decarbonizing our economy. One emerging strategy for the production of solar fuels is to use photocatalytic biohybrid systems that combine the high catalytic activity of non-photosynthetic microorganisms with the high light-harvesting efficiency of metal semiconductor nanoparticles. However, few such systems have been tested for H 2 production. We investigated light-driven H 2 production by three novel organisms, Desulfovibrio desulfuricans, Citrobacter freundii, and Shewanella oneidensis, self-photosensitized with cadmium sulfide nanoparticles, and compared their performance to Escherichia coli. All biohybrid systems produced H 2 from light, with D. desulfuricans-CdS demonstrating the best activity overall and outperforming the other microbial systems even in the absence of a mediator. With this system, H 2 was continuously produced for more than 10 days with a specific rate of 36 μmol g dcw -1  h -1 . High apparent quantum yields of 23 % and 4 % were obtained, with and without methyl viologen, respectively, exceeding values previously reported., (© 2021 Wiley-VCH GmbH.)

The incorporation of highly active but also highly sensitive catalysts (e.g. the [FeFe] hydrogenase from Desulfovibrio desulfuricans) in biofuel cells is still one of the major challenges in sustainable energy conversion. We report the fabrication of a dual-gas diffusion electrode H 2 /O 2 biofuel cell equipped with a [FeFe] hydrogenase/redox polymer-based high-current-density H 2 -oxidation bioanode. The bioanodes show benchmark current densities of around 14 mA cm -2 and the corresponding fuel cell tests exhibit a benchmark for a hydrogenase/redox polymer-based biofuel cell with outstanding power densities of 5.4 mW cm -2 at 0.7 V cell voltage. Furthermore, the highly sensitive [FeFe] hydrogenase is protected against oxygen damage by the redox polymer and can function under 5 % O 2 ., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

We present a sensitive, selective and robust method for the determination of 14 thiol compounds in aqueous samples. Thiols were derivatized with ω-bromoacetonylquinolinium bromide (BQB) and its deuterium labeled equivalent D7-ω-bromoacetonylquinolinium bromide (D7). Derivatized thiols were preconcentrated by online solid-phase extraction (SPE) followed by liquid chromatography separation and electrospray ionization tandem mass spectrometry determination (SPE/LC-ESI-MS/MS). The robustness of the method was validated for wide ranges in pH, salinity, and concentrations of sulfide and dissolved organic carbon (DOC) to cover contrasting natural water types. The limits of detection (LODs) for the thiols were 3.1-66 pM. Between 6 and 14 of the thiols were detected in different natural sample types at variable concentrations: boreal wetland porewater (0.7-51 nM), estuarine sediment porewater (50 pM-11 nM), coastal sea water (60 pM-16 nM), and sulfate reducing bacterium cultures (80 pM-4 nM). MS/MS fragmentation of the compounds produces two pairs of common product ions, m/z 130.2/137.1 and 218.1/225.1, which enables scanning for unknown thiols in precursor ion scan mode. Using this approach, we identified cysteine, mercaptoacetic acid, N-acetyl-L-cysteine and sulfurothioic S-acid in boreal wetland porewater. The performance of the developed method sets a new state of the art for the determination of thiol compounds in environmental and biological samples., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Gaussian network model (GNM), regarded as the simplest and most representative coarse-grained model, has been widely adopted to analyze and reveal protein dynamics and functions. Designing a variation of the classical GNM, by defining a new Kirchhoff matrix, is the way to improve the residue flexibility modeling. We combined information arising from local relative solvent accessibility (RSA) between two residues into the Kirchhoff matrix of the parameter-free GNM. The undetermined parameters in the new Kirchhoff matrix were estimated by using particle swarm optimization. The usage of RSA was motivated by the fact that our previous work using RSA based linear regression model resulted out higher prediction quality of the residue flexibility when compared with the classical GNM and the parameter free GNM. Computational experiments, conducted based on one training dataset, two independent datasets and one additional small set derived by molecular dynamics simulations, demonstrated that the average correlation coefficients of the proposed RSA based parameter-free GNM, called RpfGNM, were significantly increased when compared with the parameter-free GNM. Our empirical results indicated that a variation of the classical GNMs by combining other protein structural properties is an attractive way to improve the quality of flexibility modeling.

The levels of sulfate-reducing bacteria (SRB), including Desulfovibrionaceae, in the gut increase following a fat-enriched diet. Endotoxins from gut microbiota contribute to the inflammation process, leading to metabolic diseases. Thus, we sought to characterize the lipid A structures of Desulfovibrionaceae lipopolysaccharides (LPS) that are associated with the microbiota inflammatory properties. LPS variants were obtained from two SRB isolates from the gut of a single individual. These LPS variants shared similar lipid A moieties with Enterobacterial LPS, but differed from one another with regard to fatty-acid numbers and endotoxic activity. This first complete structural characterization of Desulfovibrio lipid A gives new insights into previously published data on Desulfovibrio lipid A biosynthesis. LPS microdiversity within SRBs illustrates how adaptation can influence pro-inflammatory potential., (Copyright © 2014. Published by Elsevier B.V.)

The two cyanide ligands in the assembled cluster of [FeFe] hydrogenase originate from exogenous l-tyrosine. Using selectively labeled tyrosine substrates, the cyanides were isotopically labeled via a recently developed in vitro maturation procedure allowing advanced electron paramagnetic resonance techniques to probe the electronic structure of the catalytic core of the enzyme. The ratio of the isotropic (13)C hyperfine interactions for the two CN(-) ligands-a reporter of spin density on their respective coordinating iron ions-collapses from ≈5.8 for the Hox form of hydrogenase to <2 for the CO-inhibited form. Additionally, when the maturation was carried out using [(15)N]-tyrosine, no features previously ascribed to the nitrogen of the bridging dithiolate ligand were observed suggesting that this bridge is not sourced from tyrosine.

Glutathione is the metal stress protector and changes of its level in the sulfate-reducing bacteria cells under the influence of heavy metal salts have not been studied yet. CdCl2, Pb(NO3)2, CuCl2, and ZnCl2 influence on the total glutathione level in cell-free extracts of sulfate-reducing bacteria Desulfovibrio desulfuricans IMV K-6 was studied. The research has been carried out using Ellman, Lowry methods, statistical processing of the results. It was shown that the glutathione level depends on the heavy metal salts concentration in the medium. The total glutathione level was the highest under the influence of Pb(NO3)2. Other salts were also toxic to bacteria because glutathione level increased in bacterial cells after addition of these salts to the medium. On the basis of the results of our work the range of heavy metal salts influence on D. desulfuricans IMV K-6 cells glutathione level has been formed for the first time: Pb(NO3)2 > CuCl2 > CdCl2 > ZnCl2.

Lipopolysaccharides also called endotoxins are an integral component of the outer membrane of Gram-negative bacteria. When released from the bacterial surface, they interact with a host immune system, triggering excessive inflammatory response. Lipid A is the biologically most active part of endotoxin, and its activity is modulated by the quantity, quality and arrangement of its fatty acids. Desulfovibrio desulfuricans is sulfate-reducing, Gram-negative bacterium that is supposed to be opportunistic pathogens of humans and animals. In the present study, chemical composition of lipid A from various strains of D. desulfuricans was analyzed by gas chromatography/mass spectrometry. It was found that the fatty acid component of the lipid A contains dodecanoic, tetradecanoic, 3-hydroxytetradecanoic and hexadecanoic acids, and its carbohydrate core is composed of glucosamine. The analysis of 3-acyloxyacyl residue of the lipid A revealed the presence of amide-bound 3-(dodecanoyloxy)tetradecanoic and 3-(hexadecanoyloxy)tetradecanoic acids and ester-bound 3-(tetradecanoyloxy)tetradecanoic acid. It was concluded that both fatty acid and 3-acyloxyacyl residue profiles of the lipid A from the studied bacteria were similar to those of E. coli and S.enterica.

Despite extensive investigations of the active site of the [FeFe] hydrogenases, many details concerning the properties of the "hydrogen converting cluster" are not yet fully understood. The complexity of the so-called H-cluster is one of the main difficulties in studying the properties of its components. The present study is aimed at the mixed-valence EPR active [Fe2(μ-CO)(CO)3(CN)2{MeSCH2C(Me)(CH2S)2}](1-) that is structurally closely related to the redox active binuclear part of the H-cluster in its CO-inhibited oxidized state. In this work, we present a characterization of this compound by advanced pulse EPR methods. The accurate determination of the (57)Fe, (1)H, (2)H, (14)N, and (15)N electron nuclear hyperfine interactions provided a very detailed picture of the electronic structure of this complex. A theoretical study using density functional theory (DFT) calculations identified possible isomers of the compound and further refined the knowledge about its properties. It was found that upon one electron oxidation of the parent Fe(I)-Fe(I) complex, the dominant mixed-valence Fe(I)-Fe(II) species is the one in which the CN ligand of the iron center that is distal to the thioether moves from the basal to the apical position. The unpaired spin distribution of the model complex is found to be clearly different from that of the native H-cluster. These differences are discussed and provide new insight into the functional features of the [FeFe] hydrogenase active site.

Cytochrome c3 from Desulfovibrio desulfuricans ATCC 27774 appears to be capable of receiving two protons and two electrons from hydrogenase for transport to the membrane, and converting electronic energy into proton motive force. Detailed studies of the mechanism require control both of the redox state and of the protonation state of the protein; hence, structure determination of the protein in solution by NMR is the preferred method. This work compares the structures of the protonated protein in the fully oxidized and fully reduced states as a first step toward elucidating the pH-dependent and redox-state-dependent conformational changes that drive the energy transduction. These high-resolution structures revealed significant localized differences upon change of redox state, even though the global folds of the two families of structures are similar. There are concerted redox-linked motions within the protein that bring E61 and K75 closer to heme II in the oxidized form. This is consistent with an electrostatically driven movement that may provide an important contribution to the previously measured positive cooperativity between hemes I and II. No significant conformational changes were observed that might be related to redox−Bohr effects; the families of structures represent mainly protonated forms, and therefore, pH dependence should not play a major role in the observed structural rearrangements.

Cells and metabolic products of Desulfovibrio desulfuricans were successfully used to separate quartz from hematite through environmentally benign microbially induced flotation. Bacterial metabolic products such as extracellular proteins and polysaccharides were isolated from both unadapted and mineral-adapted bacterial metabolite and their basic characteristics were studied in order to get insight into the changes brought about on bioreagents during adaptation. Interaction between bacterial cells and metabolites with minerals like hematite and quartz brought about significant surface-chemical changes on both the minerals. Quartz was rendered more hydrophobic, while hematite became more hydrophilic after biotreatment. The predominance of bacterial polysaccharides on interacted hematite and of proteins on quartz was responsible for the above surface-chemical changes, as attested through adsorption studies. Surface-chemical changes were also observed on bacterial cells after adaptation to the above minerals. Selective separation of quartz from hematite was achieved through interaction with quartz-adapted bacterial cells and metabolite. Mineral-specific proteins secreted by quartz-adapted cells were responsible for conferment of hydrophobicity on quartz resulting in enhanced separation from hematite through flotation., (2010 Elsevier B.V. All rights reserved.)

Flavodoxin adopts the common repeat beta/alpha topology and folds in a complex kinetic reaction with intermediates. To better understand this reaction, we analyzed a set of Desulfovibrio desulfuricans apoflavodoxin variants with point mutations in most secondary structure elements by in vitro and in silico methods. By equilibrium unfolding experiments, we first revealed how different secondary structure elements contribute to overall protein resistance to heat and urea. Next, using stopped-flow mixing coupled with far-UV circular dichroism, we probed how individual residues affect the amount of structure formed in the experimentally detected burst-phase intermediate. Together with in silico folding route analysis of the same point-mutated variants and computation of growth in nucleation size during early folding, computer simulations suggested the presence of two competing folding nuclei at opposite sides of the central beta-strand 3 (i.e., at beta-strands 1 and 4), which cause early topological frustration (i.e., misfolding) in the folding landscape. Particularly, the extent of heterogeneity in folding nuclei growth correlates with the in vitro burst-phase circular dichroism amplitude. In addition, phi-value analysis (in vitro and in silico) of the overall folding barrier to apoflavodoxin's native state revealed that native-like interactions in most of the beta-strands must form in transition state. Our study reveals that an imbalanced competition between the two sides of apoflavodoxin's central beta-sheet directs initial misfolding, while proper alignment on both sides of beta-strand 3 is necessary for productive folding., (Copyright 2009 Elsevier Ltd. All rights reserved.)

The versatility and reaction specificity of a novel bioinorganic catalyst is demonstrated in various reactions. Palladized cells (bioPd) of the sulphate-reducing bacterium Desulfovibrio desulfuricans showed an increased product selectivity and a catalytic activity comparable to a commercial Pd catalyst in several industrially relevant hydrogenations and hydrogenolyses (reductive dehalogenations). The ability of palladized cells to promote the reductive debromination of a polybrominated diphenyl ether (PBDE #47) is demonstrated, although chemically reduced Pd(II) and commercial Pd(0) were more effective debromination agents. Polybrominated diphenyl ethers are being supplanted as flame retardants by other compounds, e.g. tris(chloroisopropyl)phosphate (TCPP), the concentration of which was seen to increase approximately 10-fold in groundwater samples between 2000 and 2004. BioPd dechlorinated TCPP in groundwater samples with >90% recovery of free chloride ion, and was five times more effective than using commercial Pd(0) catalyst. Examination of the spent groundwater using 31P NMR showed a phosphorus species novel to the bioPd-treated solution, which was not evident in a commercial reference sample of TCPP.

The determination of methylmercury (MeHg) in environmental samples by ethylation derivation-gas chromatography-atomic fluorescence spectrometry (ED-GC-AFS) is associated with an intimate problem of water moisture accumulation introduced in the ethylation step, which enters the detection system and cause a spectroscopic interference. With a simple modification on the GC-AFS system, this problem was eliminated and the analytical quality of the measurements was significantly improved. The presence of dissolved sulfide in samples can also cause serious chemical interference in the ethylation step resulting in lower or total loss of the MeHg signal. It was found that a masking system of CuSO(4)-Na(2)C(2)O(4) was able to eliminate this interference. With this system, the accurate determination of trace amount of MeHg in high dissolved sulfide containing samples was achieved. Satisfactory analytical results were obtained with the certified reference sediment IAEA405, sulfate reducing bacteria culture and sulfide containing water samples. The limit of detection and quantitation of this masking system is 0.01 and 0.04ngL(-1) respectively. Other factors affecting ethylation are also discussed.

Bacteria of Desulfovibrio desulfuricans species are Gram-negative, anaerobic rods selectively reducing sulphates and colonizing oxygen-free ecosystems. They are ubiquitous in the natural environment and have been also found to reside in the human digestive tract. They are suggested to be involved in the pathogenesis ofulcerative colitis and Crohn's disease. The D. desulfuricans wild strains were isolated from feces and bioptate of patients suffering from various digestive tract disorders. LPSs were isolated from the wild enteric strains and soil type strain La 2226 of D. desulfuricans and analyzed in terms of their 2-keto-3-deoxyoctulosonic acid (Kdo) component content. The obtained spectrophotometric data indicate that Kdo content is characteristic of each of the investigated strains and it ranges from 0.48% to 2.86% (w/w) of the total LPS mass. Statistically significant interstrain differences of Kdo quantity seem to suggest the differences in the O-antigen content. Comparative analysis of Kdo content in LPSs of D. desulfuricans strains in relation to that of the reference endotoxin from Salmonella spp. allows us to suggest that D. desulfuricans bacteria possess O-antigen polysaccharides composed of diverse number of carbohydrate units.

We have systematically explored the Hofmeister effects of cations and anions (0.3-1.75 M range) for acidic Desulfovibrio desulfuricans apoflavodoxin (net charge -19, pH 7) and basic horse heart cytochrome c (net charge +17, pH 4.5). The Hofmeister effect of the ions on protein thermal stability was assessed by the parameter dT trs/d[ion] (T trs; thermal midpoint). We show that dT trs/d[ion] correlates with ion partition coefficients between surface and bulk water and ion surface tension effects: this suggests direct interactions between ions and proteins. Surprisingly, the stability effects of the different ions on the two model proteins are similar, implying a major role of the peptide backbone, instead of charged groups, in mediation of the interactions. Upon assessing chemical/physical properties of the ions responsible for the Hofmeister effects on protein stability, ion charge density was identified as most important. Taken together, our study suggests key roles for ion hydration and the peptide group in facilitating interactions between Hofmeister ions and proteins.

In paper I [Janner (2008). Acta Cryst. A64, 494-502], the enclosing forms of the monomers of four octahedral holoenzymes (bacterio and mitochondrial ferritins, small heat-shock protein and sulfur oxygenase reductase) were derived, with vertices at points of a cubic lattice and indexed accordingly. The correspondence between vertices and neighboring residues allows a sequential ordering of the vertices within the polyline defined by the C(alpha) atoms of the primary structure. The alignment of these sequences shows that the form vertices denoted as turning points delimit the elements of the secondary structure (alpha-helices, beta-strands and loops). This relation is analyzed further in a plot of angular changes in orientations of the polyline segments and planes as a function of the residue numbers and of the form vertices, respectively, leading to an alternative characterization of the ternary structure. Finally, two simple connectivity models of monomers, oriented according to the symmetry axes of the octahedral point group 432, suggest possible patterns in the self-assembly process from clusters of monomers to the quaternary structure of the cubic cage.

The architectural elements of four protein cages (bacterio ferritin, human mitochondrial ferritin, sulfur oxygenase reductase and small heat-shock protein) are compared top-to-bottom. The starting points are polyhedra with octahedral symmetry 432 enclosing the cage and delimiting the central cavity, respectively, which have vertices at points of a species-dependent cubic form lattice. The approach is extended from the whole cage to axial-symmetric clusters down to polyhedral forms of single monomers viewed along the fourfold, the threefold and the twofold axes, respectively. The corresponding projected monomeric forms can be approximated by two-dimensional tiles, 13 enantiomorphic pairs in total. The determination of the cubic indices of the monomeric vertices opens the possibility of the way back analysis, bottom-to-top, as discussed in paper II [Janner (2008). Acta Cryst. A64, 503-512].

Apoflavodoxin from the sulfate reducing bacteria Desulfovibrio desulfuricans is a small, acidic protein with a net charge of -19 at neutral pH. Here, we show that monovalent cations in biologically relevant amounts have dramatic effects on apoflavodoxin stability. The effect is largest for Gdm(+) and decreases as a function of increased cation charge density (Gdm(+)>NH(4)(+)K(+) approximately Cs(+) approximately Na(+)>Li(+)). A linear correlation of stabilizing effects with cation hydration properties suggests an important role of dehydration in efficient cation interaction with the protein. The effects on stability are due to preferential binding of one cation to native apoflavodoxin and results in an increase in thermal midpoint of 20 degrees C and the free energy of unfolding (at 20 degrees C) increases fivefold. Tuning of biophysical properties (such as folding and ligand/cofactor binding) of acidic proteins by cation binding may be important in vivo.

To investigate the consequences of macromolecular crowding on the behavior of a globular protein, we performed a combined experimental and computational study on the 148-residue single-domain alpha/beta protein, Desulfovibrio desulfuricans apoflavodoxin. In vitro thermal unfolding experiments, as well as assessment of native and denatured structures, were probed by using far-UV CD in the presence of various amounts of Ficoll 70, an inert spherical crowding agent. Ficoll 70 has a concentration-dependent effect on the thermal stability of apoflavodoxin (DeltaT(m) of 20 degrees C at 400 mg/ml; pH 7). As judged by CD, addition of Ficoll 70 causes an increase in the amount of secondary structure in the native-state ensemble (pH 7, 20 degrees C) but only minor effects on the denatured state. Theoretical calculations, based on an off-lattice model and hard-sphere particles, are in good agreement with the in vitro data. The simulations demonstrate that, in the presence of 25% volume occupancy of spheres, native flavodoxin is thermally stabilized, and the free energy landscape shifts to favor more compact structures in both native and denatured states. The difference contact map reveals that the native-state compaction originates in stronger interactions between the helices and the central beta-sheet, as well as by less fraying in the terminal helices. This study demonstrates that macromolecular crowding has structural effects on the folded ensemble of polypeptides.

In bacteria, the intracellular metal content or metallome reflects the metabolic requirements of the cell. When comparing the composition of metals in phytoplankton and bacteria that make up the macronutrients and the trace elements, we have determined that the content of trace elements in both of these microorganisms is markedly similar. The trace metals consisting of transition metals plus zinc are present in a stoichometric molar formula that we have calculated to be as follows: Fe(1)Mn(0.3)Zn(0.26)Cu(0.03)Co(0.03)Mo(0.03). Under conditions of routine cultivation, trace metal homeostasis may be maintained by a series of transporter systems that are energized by the cell. In specific environments where heavy metals are present at toxic levels, some bacteria have developed a detoxification strategy where the metallic ion is reduced outside of the cell. The result of this extracellular metabolism is that the bacterial metallome specific for trace metals is not disrupted. One of the microorganisms that reduces toxic metals outside of the cell is the sulfate-reducing bacterium Desulfovibrio desulfuricans. While D. desulfuricans reduces metals by enzymatic processes involving polyhemic cytochromes c3 and hydrogenases, which are all present inside the cell; we report the presence of chain B cytochrome c nitrite reductase, NrfA, in the outer membrane fraction of D. desulfuricans ATCC 27774 and discuss its activity as a metal reductase.

Metal corroding sulfate reducing bacteria have been poorly characterized at molecular level due to difficulties pertaining to isolation and handling of anaerobes. We report here for the first time the presence and characterization of thioredoxin 2 in an obligate anaerobic dissimilatory sulfate reducing bacterium Desulfovibrio desulfuricans. In silico analysis of the D. desulfuricans genome revealed the presence of thioredoxin 1 (dstrx1), thioredoxin 2 (dstrx2) and thioredoxin reductase (dstrxR) genes. These genes were found to be actively expressed by the bacteria under the anaerobic growth conditions. We have overexpressed the anaerobic thioredoxin genes in E. coli to produce functionally active recombinant proteins. Recombinant DsTrxR recognized both DsTrx1 and DsTrx2 as its substrate. Mutation studies revealed that the activity of DsTrx2 can be completely abolished with a single amino acid mutation (C69A) in the signature motif 'WCGPC'. Furthermore, the N-terminal domain of DsTrx2 containing two extra CXXC motifs was found to have a negative regulation on its biochemical activity. In conclusion, we have shown the presence of thioredoxin 2 for the first time in an obligate anaerobe which in this anaerobe may be required for its survival under either oxidative stress conditions or metal ion hemostasis.

Folding of cofactor-binding proteins involves ligand binding in addition to polypeptide folding. We here assess the kinetic folding/binding landscape for Desulfovibrio desulfuricans flavodoxin that coordinates an FMN cofactor. The apo-form folds in a two-step process involving a burst-phase intermediate. Studies on Tyr98Ala and Trp60Ala variants reveal that these aromatics-that stack with the FMN in the holo-form-are not participating in the apo-protein folding pathway. However, these residues are essential for FMN interactions with the unfolded protein during refolding of holo-flavodoxin. Unfolding of wild-type holo-flavodoxin is coupled to FMN dissociation whereas for Tyr98Ala and Trp60Ala holo-variants, FMN dissociates before polypeptide unfolding. Both variants refold as apo-proteins before FMN rebinds. In sharp contrast, refolding of unfolded wild-type holo-flavodoxin is over an order of magnitude faster than that of the apo-form, the pathway does not include a burst-phase intermediate, and the speed is independent of FMN excess ratio. These observations demonstrate that FMN binds rapidly to the unfolded polypeptide and guides folding straight to the native state. As this path to functional D. desulfuricans holo-flavodoxin is faster than if the cofactor binds to pre-folded apo-protein, this is one of few examples where molecular recognition via a "fly-casting" mechanism is kinetically favored.

The structure of the type I tetraheme cytochrome c(3) from Desulfovibrio desulfuricans G20 was determined to 1.5 Angstrom by X-ray crystallography. In addition to the oxidized form, the structure of the molybdate-bound form of the protein was determined from oxidized crystals soaked in sodium molybdate. Only small structural shifts were obtained with metal binding, consistent with the remarkable structural stability of this protein. In vitro experiments with pure cytochrome showed that molybdate could oxidize the reduced cytochrome, although not as rapidly as U(VI) present as uranyl acetate. Alterations in the overall conformation and thermostability of the metal-oxidized protein were investigated by circular dichroism studies. Again, only small changes in protein structure were documented. The location of the molybdate ion near heme IV in the crystal structure suggested heme IV as the site of electron exit from the reduced cytochrome and implicated Lys14 and Lys56 in binding. Analysis of structurally conserved water molecules in type I cytochrome c(3) crystal structures identified interactions predicted to be important for protein stability and possibly for intramolecular electron transfer among heme molecules.

The crystallographic structure of an engineered flavodoxin mutant from Desulfovibrio vulgaris has been analysed. Site-directed mutagenesis was used to substitute serine 35 with a cysteine to provide a possible covalent linkage. The crystal structure of the semiquinone form of this mutant is similar to the corresponding oxidation state of the wild-type flavodoxin. Analysis of the structural changes reveals the interaction between N(5)H of the flavin and the carbonyl O atom of Gly61 to be critical for modulation of the electrochemical properties of the protein.

The flavin mononucleotide (FMN) cofactor in Desulfovibrio desulfuricans flavodoxin stays associated with the polypeptide upon guanidine hydrochloride (GuHCl) induced unfolding. Using isothermal titration calorimetry (ITC), we determined the affinity of FMN for the flavodoxin polypeptide as a function of both urea and GuHCl concentrations (pH 7, 25 degrees C). The FMN affinity for folded and GuHCl-unfolded flavodoxin differs 10-fold, which is in agreement with the difference in thermodynamic stability between the apo- and holo-forms. In contrast, the urea-unfolded protein does not interact with FMN and equilibrium unfolding of holo-flavodoxin in urea results in FMN dissociation prior to polypeptide unfolding. ANS-binding, near-UV circular dichroism (CD), acrylamide quenching and FMN-emission experiments reveal the presence of native-like intermediates, not detected by far-UV CD and aromatic fluorescence detection methods, in low concentrations of both denaturants. Time-resolved experiments show that FMN binding is fastest at GuHCl concentrations where the native-like intermediate species is populated.

A novel bioinorganic catalyst was obtained via reduction of Pd(II) to Pd0 on to the surface of cells of Desulfovibrio desulfuricans at the expense of H2. Palladised biomass, supplied with formate or H2 as an electron donor, catalysed the dehalogenation of 2-chlorophenol and polychlorinated biphenyls. In the example of 2,3,4,5-tetrachlorobiphenyl, the bioinorganic catalyst promoted a rate of chloride release of 9.33 +/- 0.17 nmol min(-1) mg (-1) and only approximately 5% of this value was obtained using chemically reduced or commercially available Pd0. In the case of 2,2',4,4',6,6'-hexachlorobiphenyl the rate was more than four orders of magnitude faster than the degradation reported using a sulfidogenic culture. Negligible chloride release occurred from any of the chloroaromatic compounds using biomass alone, or from palladised biomass challenged with hexane carrier solvent only. Analysis of the spent solution showed that in addition to catalysis of reductive dehalogenation the new material was able to remove very effectively the organic residua, with neither any PCB nor any breakdown products identifiable by GC/MS.

We here compare thermal unfolding of the apo and holo forms of Desulfovibrio desulfuricans flavodoxin, which noncovalently binds a flavin mononucleotide (FMN) cofactor. In the case of the apo form, fluorescence and far-UV circular dichroism (CD) detected transitions are reversible but do not overlap (T(m) of 50 and 60 degrees C, respectively, pH 7). The thermal transitions for the holo form follow the same pattern but occur at higher temperatures (T(m) of 60 and 67 degrees C for fluorescence and CD transitions, respectively, pH 7). The holoprotein transitions are also reversible and exhibit no protein concentration dependence (above 10 microM), indicating that the FMN remains bound to the polypeptide throughout. Global analysis shows that the thermal reactions for both apo and holo forms proceed via an equilibrium intermediate that has approximately 90% nativelike secondary structure and significant enthalpic stabilization relative to the unfolded states. Incubation of unfolded holoflavodoxin at high temperatures results in FMN dissociation. Rebinding of FMN at these conditions is nominal, and therefore, cooling of holoprotein heated to 95 degrees C follows the refolding pathway of the apo form. However, FMN readily rebinds to the apoprotein at lower temperatures. We conclude that (1) a three-state thermal unfolding behavior appears to be conserved among long- and short-chain, as well as apo and holo forms of, flavodoxins and (2) flavodoxin's thermal stability (in both native and intermediate states) is augmented by the presence of the FMN cofactor.

The tetraheme cytochrome c3 is a small metalloprotein with ca. 13,000 Da found in sulfate-reducing bacteria, which is believed to act as a partner of hydrogenase. The three-dimensional structure of the oxidized and reduced forms of cytochrome c3 from Desulfovibrio desulfuricans ATCC 27774 at pH 7.6 were determined using high-resolution X-ray crystallography and were compared with the previously determined oxidized form at pH 4.0. Theoretical calculations were performed with both structures, using continuum electrostatic calculations and Monte Carlo sampling of protonation and redox states, in order to understand the molecular basis of the redox-Bohr and cooperativity effects related to the coupled transfer of electrons and protons. We were able to identify groups that showed redox-linked conformational changes. In particular, Glu61, His76, and propionate D of heme II showed important contributions to the redox-cooperativity, whereas His76, propionate A of heme I, and propionate D of heme IV were the key residues for the redox-Bohr effect. Upon reduction, an important movement of the backbone region surrounding hemes I and II was also identified, that, together with a few redox-linked conformational changes in side-chain residues, results in a significant decrease in the solvent accessibility of hemes I and II., (Copyright 2003 Wiley-Liss, Inc.)

The chemical structure determining properties and biological functions of endotoxins derived from Desulfovibrio desulfuricans species has not been recognized, which considerably hinders the choice of an effective extraction procedure of these lipopolysaccharides (LPS) from the bacterial outer cell membrane. We aimed at selecting the most effective method of LPS isolation from D. desulfuricans in terms of the most efficient extraction solution, the appropriate conditions of isolation and adequate purification technique. For this purpose we tested a few literature-based procedures utilizing various extraction mixtures (phenol-water, phenol-chloroform-petroleum ether and Tri-Reagent, i.e. aqueous solution of guanidinum thiocyanate and phenol). The best yield and purity of the isolated LPS were provided by the application of the extraction with phenol-water according to the modified by Shnyra et.al. (2000) procedure of Westphal et. al. (1952). A satisfactory method of isolation in micro scale appeared to be that based on Tri-Reagent and propagated by Yi and Hackett in 2000. The extraction of LPS from D. desulfuricans with phenol-chloroform-petroleum ether should not be recommended due to its low efficiency.

The hybrid cluster protein (HCP) from the sulfate-reducing bacteria Desulfovibrio desulfuricans ATCC 27774 has been isolated and crystallized anaerobically. The protein sample used in the crystallization studies was several months old, having been stored at 193 K, and initial crystal structure studies were unable to fully resolve details of the hybrid cluster despite the use of high-resolution data to 1.25 A collected at the ESRF, Grenoble, France. Full elucidation of the structure has only become possible with the complete knowledge of the as-isolated and fully reduced crystal structures. The analysis clarifies the significant movements in the position of the Fe atom linked to the persulfide moiety in the oxidized as-isolated protein and the S atom of the persulfide itself as the protein is reduced. The structures of the as-isolated and reduced states are discussed in terms of the putative function of the HCP proteins.

Flavodoxins catalyze redox reactions using the isoalloxazine moiety of the flavin mononucleotide (FMN) cofactor stacked between two aromatic residues located in two peptide loops. At high FMN concentrations that favor stacked FMN dimers in solution, isothermal titration calorimetric studies show that these dimers bind strongly to apo-flavodoxin from Desulfovibrio desulfuricans (30 degrees C, 20 mM Hepes, pH 7, K(D) = 5.8 microM). Upon increasing the temperature so the FMN dimers dissociate (as shown by (1)H NMR), only one-to-one (FMN-to-protein) binding is observed. Calorimetric titrations result in one-to-one binding also in the presence of phosphate or sulfate (30 degrees C, 13 mM anion, pH 7, K(D) = 0.4 microM). FMN remains dimeric in the presence of phosphate and sulfate, suggesting that specific binding of a divalent anion to the phosphate-binding site triggers ordering of the peptide loops so only one isoalloxazine can fit. Although the physiological relevance of FMN and other nucleotides as dimers has not been explored, our study shows that high-affinity binding to proteins of such dimers can occur in vitro. This emphasizes that the cofactor-binding site in flavodoxin is more flexible than previously expected.

The aim of this study was to determine whether Desulfovibrio desulfuricans-derived LPS stimulate the release of IL-6 and IL-8 from ECs and the expression of their adhesion molecules at the transcriptional level. Confluent monolayers of HUVEC were incubated in the absence or presence of 20 microg/ml and 60 microg/ml LPSs derived from the DdT and DdA bacterial strains. Also, the simultaneous stimulation of cells with LPSs and IL-1beta was evaluated. The levels of cytokines released were measured using ELISA. LPS-activated HUVEC increased the secretion of both IL-6 and IL-8, which was not LPS dose dependent. The expression of E-selectin and VCAM-1 was assessed by TR-PCR. The transcripts were detectable at all the concentrations (20, 40, 60 microg/ml) of LPSs used. These results suggest that D. desulfuricans LPS may activate immune functions in endothelial cells and influence the inflammatory response during bacteremia caused by these bacteria.