Superoxide reductase (SOR) is a metalloprotein containing a non-heme iron centre, responsible for the scavenging of superoxide radicals in the cell. The crystal structure of Treponema pallidum (Tp) SOR was determined using soft X-rays and synchrotron radiation. Crystals of the oxidized form were obtained using poly(ethylene glycol) and MgCl2 and diffracted beyond 1.55 A resolution. The overall architecture is very similar to that of other known SORs but TpSOR contains an N-terminal domain in which the desulforedoxin-type Fe centre, found in other SORs, is absent. This domain conserves the beta-barrel topology with an overall arrangement very similar to that of other SOR proteins where the centre is present. The absence of the iron ion and its ligands, however, causes a decrease in the cohesion of the domain and some disorder is observed, particularly in the region where the metal would be harboured. The C-terminal domain exhibits the characteristic immunoglobulin-like fold and harbours the Fe(His)4(Cys) active site. The five ligands of the iron centre are well conserved despite some disorder observed for one of the four molecules in the asymmetric unit. The participation of a glutamate as the sixth ligand of some of the iron centres in Pyrococcus furiosus SOR was not observed in TpSOR. A possible explanation is that either X-ray photoreduction occurred or there was a mixture of redox states at the start of data collection. In agreement with earlier proposals, details in the TpSOR structure also suggest that Lys49 might be involved in attraction of superoxide to the active site.

A biosensor for amperometric determination of nitrite was developed using cytochrome c nitrite reductase (ccNiR) from Desulfovibrio desulfuricans immobilized and electrically connected on a glassy carbon electrode by entrapment into redox active [ZnCr-AQS] layered double hydroxide containing anthraquinone-2-sulfonate (AQS). The transduction step corresponded to the electro-enzymatic reduction of nitrite by immobilized AQS molecules at -0.6 V. The biosensor showed a fast response to nitrite (5 s) with a linear range between 0.015 and 2.35 mu M, a sensitivity of 1.8 A M-1 cm(-2) and a detection limit of 4 nM. The apparent Michaelis-Menten constant (K-M(app)) M was 7.5 mu M. (c) 2007 Elsevier B.V. All rights reserved.

The nickel tetrahedral sulfur-coordinated core formed upon metal replacement of the native iron in Desulfovibrio sp. rubredoxins is shown to mimic the reactivity pattern of nickel-containing hydrogenases with respect to hydrogen production, deuterium-proton exchange, and inhibition by carbon monoxide.

A hypothetical model of the flavodoxin-tetraheme cytochrome c3 electron-transfer complex from the sulfate-reducing bacterium Desulfovibrio vulgaris has been constructed by using interactive computer graphics based on electrostatic potential field calculations and previous NMR experiments. Features of the proposed complex are (1) van der Waals contact between the flavin mononucleotide prosthetic group of flavodoxin and one heme of the cytochrome, (2) unique complementarity of electrostatic fields between the region surrounding this heme and the region surrounding the exposed portion of the flavin mononucleotide group of flavodoxin, and (3) no steric interferences between the two polypeptide chains in the complex. This complex is consistent with all structural and spectroscopic data available.

A new clean fast (8 min) method for in-solution protein digestion Without detergent or urea for protein identification by peptide mass fingerprint and mass spectrometry-based techniques is Proposed. The new method avoids the use of time consuming desalting procedures entailing the following four steps done under the effect of an ultrasonic field provided by a sonoreactor: denaturation (1 min) in a mixed Solution of water:acetonitrile 1/1 (v/v): protein reduction (1 min); protein alkylation (1 min); and protein digestion (5 min). Five Proteins with masses comprised between 14.4 kDa and 97 kDa and the protein splitsoret cytochrome c from D. desulfuricans ATCC27774, Were Successfully identified with this procedure. No differences were found in the sequence coverage or in the number of peptides matched when the new clean method was compared to another one using urea. Twofold better signal-to-noise ratios were obtained in the MALDI spectra from protein samples prepared with the new method when comparing it with a method using urea. The new digestion method avoids the need to remove salt content and increases throughput (six samples at once) while reducing sample loss and contamination from sample handling. (C) 2008 Elsevier B.V. All rights reserved.

Three ultrasonic energy sources were studied to speed up the sample treatment for in-solution protein identification by peptide mass fingerprint using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Protein reduction, alkylation, and enzymatic digestion steps were done in 15 min. Nine proteins, including zinc resistance-associated protein precursor from Desulfovibrio desulfuricans strain G20 and split-soret cytochrome c from D. desulfuricans ATCC27774 were successfully identified with the new protocol.

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.

In humans, the lymphomyeloid system has a fundamental role on iron metabolism promoting its recycling due to a continuous removal of effete red blood cells. Additionally, one of the most intriguing aspects of metalloporphyrins in biology is their effect on the immune system. However, the process of erythrocyte catabolism is still poorly understood and needs further research. In the present study, we attempt to investigate the nature and the possible physiologic role of Fe compounds released after erythrophagocytosis during the removal of red blood cells. Monocyte erythrophagocytosis in vitro experiments were done to characterize chemically the Fe compounds present inside the cells and in the culture supernatants. We tested the probable immunomodulatory functions of erythrophagocytosis products over lymphocyte cultures activated in vitro with T mitogens (alpha-CD3). Data obtained from atomic absorption spectroscopy confirmed the presence of Fe in the culture supernatants of monocyte cultures after erythrophagocytosis. Also, high-spin haem complexes derived from erythrocyte catabolism were detected by electron paramagnetic electronic resonance. Finally, in vitro activated lymphocyte proliferation experiments indicate the co-mitogenic properties of monocyte culture supernatants after red blood cells phagocytosis. Thus, the results of the present work provide evidence that culture monocyte supernatants after in vitro erythrophagocytosis contain Fe (III) high-spin haem complexes and show lymphocyte proliferation co-stimulatory properties.

A novel sulphate-reducing bacterium (Ind 1) was isolated from a biofilm removed from a severely corroded carbon steel structure in a marine environment. Light microscopy observations revealed that cells were Gram-negative, rod shaped and very motile. Partial 16S rRNA gene sequencing and analysis of the fatty acid profile demonstrated a strong similarity between the new species and members from the Desulfovibrio genus. This was confirmed by the results obtained following purification and characterisation of the key proteins involved in the sulphate-reduction pathway. Several metal-containing proteins, such as two periplasmic proteins: hydrogenase and cytochrome c3, and two cytoplasmic proteins: ferredoxin and sulphite reductase, were isolated and purified. The latter proved to be of the desulfoviridin type which is typical of the Desulfovibrio genus. The study of the remaining proteins revealed a high degree of similarity with the homologous proteins isolated from Desulfovibrio gigas. However, the position of the strain within the phylogenetic tree clearly indicates that the bacterium is closely related to Desulfovibrio gabonensis, and these three strains form a separate cluster in the delta subdivision of the Proteobacteria. On the basis of the results obtained, it is suggested that Ind 1 belongs to a new species of the genus Desulfovibrio, and the name Desulfovibrio indonensis is proposed.

Metallothioneins (MT) were obtained after purification from metal-exposed clams (Ruditapes decussatus) using gel-permeation and ion-exchange chromatography. Four cadmium-metallothioneins (CdMTs) were resolved by ion-exchange chromatography and they all had similar molecular weights, high cadmium content and an absorption spectra indicative of the presence of characteristic Cd-S aggregates. The NH(2)-terminal sequence suggests the presence of at least two class I clam MT isoforms. For the other two putative clam CdMTs isolated, the results of the amino acid determination were inconclusive. One was slightly contaminated and the other one had a blocked NH(2)-terminal. These clam metalothioneins contain glycine, which seems to be a common feature of molluscan MT family and exhibited more similarity to oysters than to mussels. Further investigation on the inducibility of these isoforms will be necessary if clams are to be used as biomarkers of metal exposure.

A nitrate reductase was solubilized with Triton X-100 from the membranes of Pseudomonas chlororaphis DSM 50135 grown microaerobically in the presence of nitrate. Like other membrane-bound nitrate reductases, it contains three subunits, of 129, 66 (64) and 24 kDa, referred to in the literature as alpha, beta and gamma, respectively. Electrocatalytic studies revealed that only the membrane-bound, not the solubilized form of the enzyme, can accept electrons from a menaquinone analog, menadione, whereas both forms can accept electrons from methylviologen. The isolated enzyme possesses several iron-sulfur clusters and a molybdopterin guanine dinucleotide active center. The iron-sulfur clusters can be grouped in two classes according to their redox properties, the high-potential and low-potential clusters. In the as-isolated enzyme, two forms of the molybdenum center, high- and low-pH, are detectable by electron paramagnetic resonance spectroscopy. The low-pH form shows a hyperfine splitting due to a proton, suggesting the presence of an -OHx ligand. Dithionite reduces the Mo(V) center to Mo(W) and subsequent reoxidization with nitrate originates a new Mo(V) signal, identical to the oxidized low-pH form but lacking its characteristic hyperfine splitting. The isolated preparation also contains heme c (in a sub-stoichiometric amount) with the ability to relay electrons to the molybdenum center, suggesting that this nitrate reductase may contain heme c instead of the heme b usually found in this class of enzymes. (c) 2005 Elsevier B.V. All rights reserved.

Aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is a member of the xanthine oxidase (XO) family of mononuclear Mo-enzymes that catalyzes the oxidation of aldehydes to carboxylic acids. The molybdenum site in the enzymes of the XO family shows a distorted square pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. We report here steady-state kinetic studies of DgAOR with the inhibitors cyanide, ethylene glycol, glycerol, and arsenite, together with crystallographic and EPR studies of the enzyme after reaction with the two alcohols. In contrast to what has been observed in other members of the XO family, cyanide, ethylene glycol, and glycerol are reversible inhibitors of DgAOR. Kinetic data with both cyanide and samples prepared from single crystals confirm that DgAOR does not need a sulfido ligand for catalysis and confirm the absence of this ligand in the coordination sphere of the molybdenum atom in the active enzyme. Addition of ethylene glycol and glycerol to dithionite-reduced DgAOR yields rhombic Mo(V) EPR signals, suggesting that the nearly square pyramidal coordination of the active enzyme is distorted upon alcohol inhibition. This is in agreement with the X-ray structure of the ethylene glycol and glycerol-inhibited enzyme, where the catalytically labile OH/OH(2) ligand is lost and both alcohols coordinate the Mo site in a eta(2) fashion. The two adducts present a direct interaction between the molybdenum and one of the carbon atoms of the alcohol moiety, which constitutes the first structural evidence for such a bond in a biological system.

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.

The monohemic cytochrome c552from Pseudomonas nautica (c552-Pn) is thought to be the electron donor to cytochrome cd1, the so-called nitrite reductase (NiR). It shows as high levels of activity and affinity for the P. nautica NiR (NiR-Pn), as the Pseudomonas aeruginosa enzyme (NiR-Pa). Since cytochrome c552is by far the most abundant electron carrier in the periplasm, it is probably involved in numerous other reactions. Its sequence is related to that of the c type cytochromes, but resembles that of the dihemic c4cytochromes even more closely. The three-dimensional structure of P. nautica cytochrome c552has been solved to 2.2 A resolution using the multiple wavelength anomalous dispersion (MAD) technique, taking advantage of the presence of the eight Fe heme ions in the asymmetric unit. Density modification procedures involving 4-fold non-crystallographic averaging yielded a model with an R -factor value of 17.8 % (Rfree=20.8 %). Cytochrome c552forms a tight dimer in the crystal, and the dimer interface area amounts to 19% of the total cytochrome surface area. Four tighly packed dimers form the eight molecules of the asymmetric unit. The c552dimer is superimposable on each domain of the monomeric cytochrome c4from Pseudomomas stutzeri (c4-Ps), a dihemic cytochrome, and on the dihemic c domain of flavocytochrome c of Chromatium vinosum (Fcd-Cv). The interacting residues which form the dimer are both similar in character and position, which is also true for the propionates. The dimer observed in the crystal also exists in solution. It has been hypothesised that the dihemic c4-Ps may have evolved via monohemic cytochrome c gene duplication followed by evolutionary divergence and the adjunction of a connecting linker. In this process, our dimeric c552structure might be said to constitute a "living fossile" occurring in the course of evolution between the formation of the dimer and the gene duplication and fusion. The availability of the structure of the cytochrome c552-Pn and that of NiR from P. aeruginosa made it possible to identify putative surface patches at which the docking of c552to NiR-Pn may occur.

The cytochrome c nitrite reductase (ccNiR) from Desulfovibrio desulfuricans ATCC 27774 is able to reduce nitrite to ammonia in a six-electron transfer reaction. Although extensively characterized from the spectroscopic and structural points-of-view, some of its kinetic aspects are still under explored. In this work the kinetic behaviour of ccNiR has been evaluated in a systematic manner using two different spectrophotometric assays carried out in the presence of different redox mediators and a direct electrochemical approach. Solution assays have proved that the specific activity of ccNiR decreases with the reduction potential of the electronic carriers and ammonium is always the main product of nitrite reduction. The catalytic parameters were discussed on the basis of the mediator reducing power and also taking into account the location of their putative docking sites with ccNiR. Due to the fast kinetics of ccNiR, electron delivering from reduced electron donors is rate-limiting in all spectrophotometric assays, so the estimated kinetic constants are apparent only. Nevertheless, this limitation could be overcome by using a direct electrochemical approach which shows that the binding affinity for nitrite decreases whilst turnover increases with the reductive driving force.

This work reports the direct electrochemistry of Paracoccus pantotrophus pseudoazurin and the mediated catalysis of cytochrome c peroxidase from the same organism. The voltammetric behaviour was examined at a gold membrane electrode, and the studies were performed in the presence of calcium to enable the peroxidase activation. A formal reduction potential, E (0)', of 230 +/- 5 mV was determined for pseudoazurin at pH 7.0. Its voltammetric signal presented a pH dependence, defined by pK values of 6.5 and 10.5 in the oxidised state and 7.2 in the reduced state, and was constant up to 1 M NaCl. This small copper protein was shown to be competent as an electron donor to cytochrome c peroxidase and the kinetics of intermolecular electron transfer was analysed. A second-order rate constant of 1.4 +/- 0.2 x 10(5) M(-1) s(-1) was determined at 0 M NaCl. This parameter has a maximum at 0.3 M NaCl and is pH-independent between pH 5 and 9.

The accumulation of toxic bile salts within the hepatocyte plays a key role in organ injury during liver disease. Deoxycholate (DC) and glycochenodeoxycholate (GCDC) induce apoptosis in vitro and in vivo, perhaps through direct perturbation of mitochondrial membrane structure and function. In contrast, ursodeoxycholate (UDC) and its taurine-conjugated form (TUDC) appear to be protective. We show here that hydrophobic bile salts induced apoptosis in cultured rat hepatocytes, without modulating the expression of pro-apoptotic Bax protein, and caused cytochrome c release in isolated mitochondria. Co-incubation with UDC and TUDC prevented cell death and efflux of mitochondrial factors. Using spin-labelling techniques and EPR spectroscopy analysis of isolated rat liver mitochondria, we found significant structural changes at the membrane-water surface in mitochondria exposed to hydrophobic bile salts, including modified lipid polarity and fluidity, altered protein order and increased oxidative injury. UDC, TUDC and cyclosporin A almost completely abrogated DC- and GCDC-induced membrane perturbations. We conclude that the toxicity of hydrophobic bile salts to hepatocytes is mediated by cytochrome c release, through a mechanism associated with marked direct effects on mitochondrial membrane lipid polarity and fluidity, protein order and redox status, without modulation of pro-apoptotic Bax expression. UDC and TUDC can directly suppress disruption of mitochondrial membrane structure, which may represent an important mechanism of hepatoprotection by these bile salts.