The enthalpy and entropy changes associated with protein reduction (deltaHdegrees,(rc), deltaSdegrees,(rc)) were determined for a number of low-potential iron-sulfur proteins through variable temperature direct electrochemical experiments. These data add to previous estimates making available, overall, the reduction thermodynamics for twenty species from various sources containing all the different types of metal centers. These parameters are discussed with reference to structural data and calculated electrostatic metal-environment interaction energies, and redox properties of model complexes. This work, which is the first systematic investigation on the reduction thermodynamics of Fe-S proteins, contributes to the comprehension of the determinants of the differences in reduction potential among different protein families within a novel perspective. Moreover, comparison with analogous data obtained previously for electron transport (ET) metalloproteins with positive reduction potentials, i.e., cytochromes c, blue copper proteins, and HiPIPs, helps our understanding of the factors controlling the reduction potential in ET species containing different metal cofactors. The main results of this work can be summarized as follows.

Nitrous-oxide reductases (N2OR) catalyze the two-electron reduction of N(2)O to N(2). The crystal structure of N2ORs from Pseudomonas nautica (Pn) and Paracoccus denitrificans (Pd) were solved at resolutions of 2.4 and 1.6 A, respectively. The Pn N2OR structure revealed that the catalytic CuZ center belongs to a new type of metal cluster in which four copper ions are liganded by seven histidine residues. A bridging oxygen moiety and two other hydroxide ligands were proposed to complete the ligation scheme (Brown, K., Tegoni, M., Prudencio, M., Pereira, A. S., Besson, S., Moura, J. J. G., Moura, I., and Cambillau, C. (2000) Nat. Struct. Biol. 7, 191-195). However, in the CuZ cluster, inorganic sulfur chemical determination and the high resolution structure of Pd N2OR identified a bridging inorganic sulfur instead of an oxygen. This result reconciles the novel CuZ cluster with the hitherto puzzling spectroscopic data.

Nitrite reductase from the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 is a multihaem (type c) membrane-bound enzyme that catalyzes the dissimilatory conversion of nitrite to ammonia. Crystals of the oxidized form of this enzyme were obtained using PEG and CaCl(2) as precipitants in the presence of 3--(decylmethylammonium)propane-1-sulfonate and belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 78.94, b = 104.59, c = 143.18 A. A complete data set to 2.30 A resolution was collected using synchrotron radiation at the ESRF. However, the crystals may diffract to beyond 1.7 A and high-resolution data will be collected in the near future.

Aldehyde oxidoreductase (AOR) activity has been found in different sulfate reducing organisms (Moura, J. J. G., and Barata, B. A. S. (1994) in Methods in Enzymology (Peck, H. D., Jr., and LeGall, J., Eds.), Vol. 243, Chap. 4. Academic Press; Romao, M. J., Knablein, J., Huber, R., and Moura, J. J. G. (1997) Prog. Biophys. Mol. Biol. 68, 121-144). The enzyme was purified to homogeneity from extracts of Desulfovibrio desulfuricans (Dd) ATCC 27774, a sulfate reducer that can use sulfate or nitrate as terminal respiratory substrates. The protein (AORDd) is described as a homodimer (monomer, circa 100 kDa), contains a Mo-MCD pterin, 2 x [2Fe-2S] clusters, and lacks a flavin group. Visible and EPR spectroscopies indicate a close similarity with the AOR purified from Desulfovibrio gigas (Dg) (Barata, B. A. S., LeGall, J., and Moura, J. J. G. (1993) Biochemistry 32, 11559-11568). Activity and substrate specificity for different aldehydes were determined. EPR studies were performed in native and reduced states of the enzyme and after treatment with ethylene glycol and dithiothreitol. The AORDd was crystallized using ammonium sulfate as precipitant and the crystals belong to the space group P6(1)22, with unit cell dimensions a = b = 156.4 and c = 177.1 A. These crystals diffract to beyond 2.5 A resolution and a full data set was measured on a rotating anode generator. The data were used to solve the structure by Patterson Search methods, using the model of AORDg.

The combination of docking algorithms with NMR data has been developed extensively for the studies of protein-ligand interactions. However, to extend this development for the studies of protein-protein interactions, the intermolecular NOE constraints, which are needed, are more difficult to access. In the present work, we describe a new approach that combines an ab initio docking calculation and the mapping of an interaction site using chemical shift variation analysis. The cytochrome c553-ferredoxin complex is used as a model of numerous electron-transfer complexes. The 15N-labeling of both molecules has been obtained, and the mapping of the interacting site on each partner, respectively, has been done using HSQC experiments. 1H and 15N chemical shift analysis defines the area of both molecules involved in the recognition interface. Models of the complex were generated by an ab initio docking software, the BiGGER program (bimolecular complex generation with global evaluation and ranking). This program generates a population of protein-protein docked geometries ranked by a scoring function, combining relevant stabilization parameters such as geometric complementarity surfaces, electrostatic interactions, desolvation energy, and pairwise affinities of amino acid side chains. We have implemented a new module that includes experimental input (here, NMR mapping of the interacting site) as a filter to select the accurate models. Final structures were energy minimized using the X-PLOR software and then analyzed. The best solution has an interface area (1037.4 A2) falling close to the range of generally observed recognition interfaces, with a distance of 10.0 A between the redox centers.

The aldehyde oxidoreductase (MOD) isolated from the sulfate reducer Desulfovibrio desulfuricans (ATCC 27774) is a member of the xanthine oxidase family of molybdenum-containing enzymes. It has substrate specificity similar to that of the homologous enzyme from Desulfovibrio gigas (MOP) and the primary sequences from both enzymes show 68 % identity. The enzyme was crystallized in space group P6(1)22, with unit cell dimensions of a=b=156.4 A and c=177.1 A, and diffraction data were obtained to beyond 2.8 A. The crystal structure was solved by Patterson search techniques using the coordinates of the D. gigas enzyme. The overall fold of the D. desulfuricans enzyme is very similar to MOP and the few differences are mapped to exposed regions of the molecule. This is reflected in the electrostatic potential surfaces of both homologous enzymes, one exception being the surface potential in a region identifiable as the putative docking site of the physiological electron acceptor. Other essential features of the MOP structure, such as residues of the active-site cavity, are basically conserved in MOD. Two mutations are located in the pocket bearing a chain of catalytically relevant water molecules. As deduced from this work, both these enzymes are very closely related in terms of their sequences as well as 3D structures. The comparison allowed confirmation and establishment of features that are essential for their function; namely, conserved residues in the active-site, catalytically relevant water molecules and recognition of the physiological electron acceptor docking site.

The tungsten-containing formate dehydrogenase (W-FDH) isolated from Desulfovibrio gigas has been crystallized in space group P2(1), with cell parameters a = 73.8 A, b = 111.3 A, c = 156.6 A and beta = 93.7 degrees. These crystals diffract to beyond 2.0 A on a synchrotron radiation source. W-FDH is a heterodimer (92 kDa and 29 kDa subunits) and two W-FDH molecules are present in the asymmetric unit. Although a molecular replacement solution was found using the periplasmic nitrate reductase as a search model, additional phasing information was needed. A multiple-wavelength anomalous dispersion (MAD) dataset was collected at the W- and Fe-edges, at four different wavelengths. Anomalous and dispersive difference data allowed us to unambiguously identify the metal atoms bound to W-FDH as one W atom with a Se-cysteine ligand as well as one [4Fe-4S] cluster in the 92 kDa subunit, and three additional [4Fe-4S] centers in the smaller 29 kDa subunit. The D. gigas W-FDH was previously characterized based on metal analysis and spectroscopic data. One W atom was predicted to be bound to two molybdopterin guanine dinucleotide (MGD) pterin cofactors and two [4Fe-4S] centers were proposed to be present. The crystallographic data now reported reveal a selenium atom (as a Se-cysteine) coordinating to the W site, as well as two extra [4Fe-4S] clusters not anticipated before. The EPR data were re-evaluated in the light of these new results.

The sulfate-reducing bacterium aldehyde oxidoreductase from Desulfovibrio gigas (MOP) is a member of the xanthine oxidase family of enzymes. It has 907 residues on a single polypeptide chain, a molybdopterin cytosine dinucleotide (MCD) cofactor and two [2Fe-2S] iron-sulfur clusters. Synchrotron data to almost atomic resolution were collected for improved cryo-cooled crystals of this enzyme in the oxidized form. The cell constants of a=b=141.78 A and c=160.87 A are about 2% shorter than those of room temperature data, yielding 233,755 unique reflections in space group P6(1)22, at 1.28 A resolution. Throughout the entire refinement the full gradient least-squares method was used, leading to a final R factor of 14.5 and Rfree factor of 19.3 (4sigma cut-off) with "riding" H-atoms at their calculated positions. The model contains 8146 non-hydrogen atoms described by anisotropic displacement parameters with an observations/parameters ratio of 4.4. It includes alternate conformations for 17 amino acid residues. At 1.28 A resolution, three Cl- and two Mg2+ ions from the crystallization solution were clearly identified. With the exception of one Cl- which is buried and 8 A distant from the Mo atom, the other ions are close to the molecular surface and may contribute to crystal packing. The overall structure has not changed in comparison to the lower resolution model apart from local corrections that included some loop adjustments and alternate side-chain conformations. Based on the estimated errors of bond distances obtained by blocked least-squares matrix inversion, a more detailed analysis of the three redox centres was possible. For the MCD cofactor, the resulting geometric parameters confirmed its reduction state as a tetrahydropterin. At the Mo centre, estimated corrections calculated for the Fourier ripples artefact are very small when compared to the experimental associated errors, supporting the suggestion that the fifth ligand is a water molecule rather than a hydroxide. Concerning the two iron-sulfur centres, asymmetry in the Fe-S distances as well as differences in the pattern of NH.S hydrogen-bonding interactions was observed, which influences the electron distribution upon reduction and causes non-equivalence of the individual Fe atoms in each cluster.

Spectroscopy coupled with density functional calculations has been used to define the spin state, oxidation states, spin distribution, and ground state wave function of the mu4-sulfide bridged tetranuclear CuZ cluster of nitrous oxide reductase. Initial insight into the electronic contribution to N2O reduction is developed, which involves a sigma superexchange pathway through the bridging sulfide.

Using a myosin preparation containing endogenous myosin light-chain (LC2) kinase and phosphatase and calmodulin, i.e. near physiological ones, the interaction of vanadate oligomers with phosphorylated myosin was evaluated. Decavanadate or metavanadate solutions (2-15 mM total vanadate) did not prevent the phosphorylation state of the regulatory myosin lightchain, as observed by urea-polyacrylamide gel electrophoresis. The relative order of line broadening upon protein addition, reflecting the interaction of the vanadate oligomers with phosphorylated myosin, was V10 > V-4 > V-1 = 1 whereas, no changes were observed for monomeric vanadate. In the presence of ATP, V-1 signal was shifted upfield 2 ppm and became broadened, while V4 signal became narrowed. Moreover, a significant increase in myosin ATPase inhibition (60%) was observed when decameric vanadate species were present (1.4 mM). It is concluded that, under conditions near physiological ones, decameric vanadate differs from vanadate oligomers present in metavanadate solutions due to its strong interaction with the phosphorylated enzyme and myosin ATPase inhibition. Besides, ATP decreases the affinity of myosin for tetravanadate, induces the interaction with monomeric vanadate, whereas it does not affect decameric vanadate interaction. (C) 2002 Elsevier Science B.V. All rights reserved.

Desulfovibrio gigas formate dehydrogenase is the first representative of a tungsten-containing enzyme from a mesophile that has been structurally characterized. It is a heterodimer of 110 and 24 kDa subunits. The large subunit, homologous to E. coli FDH-H and to D. desulfuricans nitrate reductase, harbors the W site and one [4Fe-4S] center. No small subunit ortholog containing three [4Fe-4S] clusters has been reported. The structural homology with E. coli FDH-H shows that the essential residues (SeCys158, His159, and Arg407) at the active site are conserved. The active site is accessible via a positively charged tunnel, while product release may be facilitated, for H(+) by buried waters and protonable amino acids and for CO(2) through a hydrophobic channel.

The production of cytochrome c peroxidase (CCP) from Pseudomonas (Ps.) stutzeri (ATCC 11607) was optimized by adjusting the composition of the growth medium and aeration of the culture. The protein was isolated and characterized biochemically and spectroscopically in the oxidized and mixed valence forms. The activity of Ps. stutzeri CCP was studied using two different ferrocytochromes as electron donors: Ps. stutzeri cytochrome C-551 (the physiological electron donor) and horse heart cytochrome c. These electron donors interact differently with Ps. stutzeri CCP, exhibiting different ionic strength dependence. The CCP from Paracoccus (Pa.) denitrificans was proposed to have two different Ca2+ binding sites: one usually occupied (site I) and the other either empty or partially occupied in the oxidized enzyme (site II). The Ps. stutzeri enzyme was purified in a form with tightly bound Ca2+. The affinity for Ca2+ in the mixed valence enzyme is so high that Ca2+ returns to it from the EGTA which was added to empty the site in the oxidized enzyme. Molecular mass determination by ultracentrifugation and behavior on gel filtration chromatography have revealed that this CCP is isolated as an active dimer, in contrast to the Pa. denitrificans CCP which requires added Ca2+ for formation of the dimer and also for activation of the enzyme. This is consistent with the proposal that Ca2+ in the bacterial peroxidases influences the monomer/dimer equilibrium and the transition to the active form of the enzyme. Additional Ca2+ does affect both the kinetics of oxidation of horse heart cytochrome c (but not cytochrome C-551) and higher aggregation states of the enzyme. This suggests the presence of a superficial Ca2+ binding site of low affinity.

The gene encoding cytochrome c nitrite reductase (NrfA) from Desulfovibrio desulfuricans ATCC 27774 was sequenced and the crystal structure of the enzyme was determined to 2.3-A resolution. In comparison with homologous structures, it presents structural differences mainly located at the regions surrounding the putative substrate inlet and product outlet, and includes a well defined second calcium site with octahedral geometry, coordinated to propionates of hemes 3 and 4, and caged by a loop non-existent in the previous structures. The highly negative electrostatic potential in the environment around hemes 3 and 4 suggests that the main role of this calcium ion may not be electrostatic but structural, namely in the stabilization of the conformation of the additional loop that cages it and influences the solvent accessibility of heme 4. The NrfA active site is similar to that of peroxidases with a nearby calcium site at the heme distal side nearly in the same location as occurs in the class II and class III peroxidases. This fact suggests that the calcium ion at the distal side of the active site in the NrfA enzymes may have a similar physiological role to that reported for the peroxidases.

A biosensor for nitrite analytical determination was developed using a cytochrome c nitrite reductase (ccNiR) from Desulfovibrio desufuricans ATCC 27774 immobilized and electrically connected on a glassy carbon electrode by entrapment in an electrogencrated poly(pyrrole-viologen) matrix. The modified bioelectrode was studied by cyclic voltammetry and a catalytic current was observed in presence of nitrite. The linear range of the electrode response was 5.4-43.4 muM. The detection limit and the sensitivity were 5.4 muM and 1721 mA M-1 cm(-2), respectively. The K-M(app) value determined from the Lineweaver-Burk plot was 86 muM. The biosensor fully maintained its electroenzymatic activity towards nitrite after four days.. No catalytic response was observed in the presence of nitrate ions while interference from sulfites was considered negligible. Finally, the biosensor composition was optimized in term of monomer-enzyme ratio. (C) 2004 Elsevier B.V. All rights reserved.

Ligand K-edge XAS of an [Fe3S4]0 model complex is reported. The pre-edge can be resolved into contributions from the mu(2)S(sulfide), mu(3)S(sulfide), and S(thiolate) ligands. The average ligand-metal bond covalencies obtained from these pre-edges are further distributed between Fe(3+) and Fe(2.5+) components using DFT calculations. The bridging ligand covalency in the [Fe2S2]+ subsite of the [Fe3S4]0 cluster is found to be significantly lower than its value in a reduced [Fe2S2] cluster (38% vs 61%, respectively). This lowered bridging ligand covalency reduces the superexchange coupling parameter J relative to its value in a reduced [Fe2S2]+ site (-146 cm(-1) vs -360 cm(-1), respectively). This decrease in J, along with estimates of the double exchange parameter B and vibronic coupling parameter lambda2/k(-), leads to an S = 2 delocalized ground state in the [Fe3S4]0 cluster. The S K-edge XAS of the protein ferredoxin II (Fd II) from the D. gigas active site shows a decrease in covalency compared to the model complex, in the same oxidation state, which correlates with the number of H-bonding interactions to specific sulfur ligands present in the active site. The changes in ligand-metal bond covalencies upon redox compared with DFT calculations indicate that the redox reaction involves a two-electron change (one-electron ionization plus a spin change of a second electron) with significant electronic relaxation. The presence of the redox inactive Fe(3+) center is found to decrease the barrier of the redox process in the [Fe3S4] cluster due to its strong antiferromagnetic coupling with the redox active Fe2S2 subsite.

Cytochrome c peroxidase (CCP) catalyses the reduction of H2O2 to H2O, an important step in the cellular detoxification process. The crystal structure of the di-heme CCP from Pseudomonas nautica 617 was obtained in two different conformations in a redox state with the electron transfer heme reduced. Form IN, obtained at pH 4.0, does not contain Ca2+ and was refined at 2.2 Angstrom resolution. This inactive form presents a closed conformation where the peroxidatic heme adopts a six-ligand coordination, hindering the peroxidatic reaction from taking place. Form OUT is Ca2+ dependent and was crystallized at pH 5.3 and refined at 2.4 Angstrom resolution. This active form shows an open conformation, with release of the distal histidine (His71) ligand, providing peroxide access to the active site. This is the first time that the active and inactive states are reported for a di-heme peroxidase.

An orange-coloured protein (ORP) isolated from Desulfovibrio gigas, a sulphate reducer, has been previously shown by extended X-ray absorption fine structure (EXAFS) to contain a novel mixed-metal sulphide cluster of the type [S(2)MoS(2)CuS(2)MoS(2)] [J. Am. Chem. Soc. 122 (2000) 8321]. We report here the purification and the biochemical/spectroscopic characterisation of this novel protein. ORP is a soluble monomeric protein (11.8 kDa). The cluster is non-covalently bound to the polypeptide chain. The presence of a MoS(4)(2-) moiety in the structure of the cofactor contributes with a quite characteristic UV-Vis spectra, exhibiting an orange colour, with intense absorption peaks at 480 and 338 nm. Pure ORP reveals an Abs(480)/Abs(338) ratio of 0.535. The gene sequence coding for ORP as well as the amino acid sequence was determined. The putative biological function of ORP is discussed.

High-molecular-weight cytochromes (Hmcs) belong to a large family of multihaem cytochromes in sulfate-reducing bacteria. HmcA is the first cytochrome reported to have 16 c-type haems arranged in its polypeptide chain. The function of this cytochrome is still unknown, although it is clear that it belongs to a membrane-bound complex involved in electron transfer from the periplasm to the membrane. HmcA from Desulfovibrio gigas has been purified and successfully crystallized using the hanging-drop vapour-diffusion method. The crystals grew using PEG and zinc acetate as precipitants to maximum dimensions of 0.2 x 0.2 x 0.2 mm in an orthorhombic space group, with unit-cell parameters a = 88.9, b = 90.9, c = 83.7 Angstrom. The crystals diffracted to beyond 2.07 Angstrom and a MAD data set was collected.

Extracted human teeth are frequently used for research or educational purposes. Therefore, it is necessary to store them in disinfectant solutions that do not alter dental structures. Thus, this study evaluated the influence of storage solution on enamel demineralization. For that purpose, sixty samples were divided into the following groups: enamel stored in formaldehyde (F1), stored in thymol (T1), stored in formaldehyde and submitted to pH cycling (F2), stored in thymol and submitted to pH cycling (T2). All samples were evaluated by cross-sectional microhardness analysis and had their percentage of mineral volume versus micrometer (integrated area) determined. Differences between groups were found up to 30-microm depth from the enamel surface (p < 0.05), where samples from group T2 were more demineralized. It was concluded that the storage solution influenced the reaction of a dental substrate to a cariogenic challenge, suggesting that formaldehyde may increase enamel resistance to demineralization, when compared to demineralization occurring in enamel stored in thymol solution.

The gene for pseudoazurin was isolated from Paracoccus pantotrophus LMD 52.44 and expressed in a heterologous system with a yield of 54.3 mg of pure protein per liter of culture. The gene and protein were shown to be identical to those from P. pantotrophus LMD 82.5. The extinction coefficient of the protein was re-evaluated and was found to be 3.00 mM(-1) cm(-1) at 590 nm. It was confirmed that the oxidized protein is in a weak monomer/dimer equilibrium that is ionic- strength-dependent. The pseudoazurin was shown to be a highly active electron donor to cytochrome c peroxidase, and activity showed an ionic strength dependence consistent with an electrostatic interaction. The pseudoazurin has a very large dipole moment, the vector of which is positioned at the putative electron-transfer site, His81, and is conserved in this position across a wide range of blue copper proteins. Binding of the peroxidase to pseudoazurin causes perturbation of a set of NMR resonances associated with residues on the His81 face, including a ring of lysine residues. These lysines are associated with acidic residues just back from the rim, the resonances of which are also affected by binding to the peroxidase. We propose that these acidic residues moderate the electrostatic influence of the lysines and so ensure that specific charge interactions do not form across the interface with the peroxidase.

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Among the biotargets interacting with vanadium is the calcium pump from the sarcoplasmic reticulum (SR). To this end, initial research efforts were launched with two vanadium(V)-citrate complexes, namely (NH(4))(6)[V(2)O(4)(C(6)H(4)O(7))(2)].6H(2)O and (NH(4))(6)[V(2)O(2)(O(2))(2)(C(6)H(4)O(7))(2)].4H(2)O, potentially capable of interacting with the SR calcium pump by combining kinetic studies with (51)V NMR spectroscopy. Upon dissolution in the reaction medium (concentration range: 4-0.5mM), both vanadium(V):citrate (VC) and peroxovanadium(V):citrate (PVC) complexes are partially converted into vanadate oligomers. A 1mM solution of the PVC complex, containing 184microM of the PVC complex, 94microM oxoperoxovanadium(V) (PV) species, 222microM monomeric (V1), 43microM dimeric (V2) and 53microM tetrameric (V4) species, inhibits Ca(2+) accumulation by 75 %, whereas a solution of the VC complex of the same vanadium concentration, containing 98microM of the VC complex, 263microM monomeric (V1), 64microM dimeric (V2) and 92microM tetrameric (V4) species inhibits the calcium pump activity by 33 %. In contrast, a 1 mM metavanadate solution, containing 460microM monomeric (V1), 90.2microM dimeric (V2) and 80microM tetrameric (V4) species, has no effect on Ca(2+) accumulation. The NMR signals from the VC complex (-548.0ppm), PVC complex (-551.5ppm) and PV (-611.1ppm) are broadened upon SR vesicle addition (2.5mg/ml total protein). The relative order for the half width line broadening of the NMR signals, which reflect the interaction with the protein, was found to be V4>PVC>VC>PV>V2=V1=1, with no effect observed for the V1 and V2 signals. Putting it all together the effects of two vanadium(V)-citrate complexes on the modulation of calcium accumulation and ATP hydrolysis by the SR calcium pump reflected the observed variable reactivity into the nature of key species forming upon dissolution of the title complexes in the reaction media.