The isolation and characterization of a new metalloprotein containing Cu and Fe atoms is reported. The as-isolated Cu-Fe protein shows an UV-visible spectrum with absorption bands at 320 nm, 409 nm and 615 nm. Molecular mass of the native protein along with denaturating electrophoresis and mass spectrometry data show that this protein is a multimer consisting of 14+/-1 subunits of 15254.3+/-7.6 Da. Mossbauer spectroscopy data of the as-isolated Cu-Fe protein is consistent with the presence of [2Fe-2S](2+) centers. Data interpretation of the dithionite reduced protein suggest that the metallic cluster could be constituted by two ferromagnetically coupled [2Fe-2S](+) spin delocalized pairs. The biochemical properties of the Cu-Fe protein are similar to the recently reported molybdenum resistance associated protein from Desulfovibrio, D. alaskensis. Furthermore, a BLAST search from the DNA deduced amino acid sequence shows that the Cu-Fe protein has homology with proteins annotated as zinc resistance associated proteins from Desulfovibrio, D. alaskensis, D. vulgaris Hildenborough, D. piger ATCC 29098. These facts suggest a possible role of the Cu-Fe protein in metal tolerance.

The cytochrome c nitrite reductase is isolated from the membranes of the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 as a heterooligomeric complex composed by two subunits (61 kDa and 19 kDa) containing c-type hemes, encoded by the genes nrfA and nrfH, respectively. The extracted complex has in average a 2NrfA:1NrfH composition. The separation of ccNiR subunits from one another is accomplished by gel filtration chromatography in the presence of SDS. The amino-acid sequence and biochemical subunits characterization show that NrfA contains five hemes and NrfH four hemes. These considerations enabled the revision of a vast amount of existing spectroscopic data on the NrfHA complex that was not originally well interpreted due to the lack of knowledge on the heme content and the oligomeric enzyme status. Based on EPR and Mossbauer parameters and their correlation to structural information recently obtained from X-ray crystallography on the NrfA structure [Cunha, C.A., Macieira, S., Dias, J.M., Almeida, M.G., Goncalves, L.M.L., Costa, C., Lampreia, J., Huber, R., Moura, J.J.G., Moura, I. & Romao, M. (2003) J. Biol. Chem. 278, 17455-17465], we propose the full assignment of midpoint reduction potentials values to the individual hemes. NrfA contains the high-spin catalytic site (-80 mV) as well as a quite unusual high reduction potential (+150 mV)/low-spin bis-His coordinated heme, considered to be the site where electrons enter. In addition, the reassessment of the spectroscopic data allowed the first partial spectroscopic characterization of the NrfH subunit. The four NrfH hemes are all in a low-spin state (S = 1/2). One of them has a gmax at 3.55, characteristic of bis-histidinyl iron ligands in a noncoplanar arrangement, and has a positive reduction potential.

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.

Metal-dependent formate dehydrogenases (Fdh) from prokaryotic organisms are members of the dimethyl sulfoxide reductase family of mononuclear molybdenum-containing and tungsten-containing enzymes. Fdhs catalyze the oxidation of the formate anion to carbon dioxide in a redox reaction that involves the transfer of two electrons from the substrate to the active site. The active site in the oxidized state comprises a hexacoordinated molybdenum or tungsten ion in a distorted trigonal prismatic geometry. Using this structural model, we calculated the catalytic mechanism of Fdh through density functional theory tools. The simulated mechanism was correlated with the experimental kinetic properties of three different Fdhs isolated from three different Desulfovibrio species. Our studies indicate that the C-H bond break is an event involved in the rate-limiting step of the catalytic cycle. The role in catalysis of conserved amino acid residues involved in metal coordination and near the metal active site is discussed on the basis of experimental and theoretical results.

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.

Desulforedoxin and the N-terminus of desulfoferrodoxin share a 36 amino acid domain containing a (Cys-S)(4) metal binding site. Recombinant forms of desulforedoxin, an N-terminal fragment of desulfoferrodoxin, and two desulforedoxin mutant proteins were reconstituted with Fe3+ Cd2+, and Zn2+ and relative metal ion affinities assessed by proton titrations. Protons compete with metal for protein ligands, a process that can be followed by monitoring the optical spectrum of the metal-protein complex as a function of pH. For all polypeptides, Fe3+ bound with the highest affinity, whereas the affinity of Zn2+ was greater than Cd2+ in desulforedoxin and the N-terminal fragment of desulfoferrodoxin, but this order was reversed in desulforedoxin mutant proteins. Metal binding in both mutants was significantly impaired. Furthermore, the Fe3+ complex of both mutants underwent a time-dependent bleaching process which coincided with increased reactivity of cysteine residues to Ellman's reagent and concomitant metal dissociation. It is hypothesized that this results from an autoredox reaction in which Fe3+ is reduced to Fe2+ with attendant oxidation of ligand thiols.

Metallothionein-like proteins (MT) and V, Cr, Co, Ni, Zn, Cu, As and Cd were determined in digestive gland, gills, kidney and gonads of Octopus vulgaris, from the Portuguese coast. To our knowledge these are the first data on MT in octopus. High concentrations (mu g g(-1), dry mass) of Zn (48050) and Cd (555) were found in digestive gland, and MT reached levels one order of magnitude above the ones registered in wild bivalves. Significantly higher levels of MT in digestive gland and gills of specimens from A and B were in line with elevated Cd concentrations. Principal component analyses (PCA) point to MT-Cd and MT-Cr associations in digestive gland and gills. Despite the high levels of Zn in specimens from B, association with Zn was not obtained. Due to the affinity of MT to various elements, it should not be excluded the possibility of Cd replacing Zn in Zn-MT. Kidney presented higher levels of Cd, Co, Ni and As than gills and gonads, and in the case of As surpassing the levels in digestive gland, but PCA showed no relation with MT. Likewise the MT levels in gonads had no correspondence to the metal concentration variation. (C) 2010 Elsevier Inc. All rights reserved.

Molybdenum and tungsten are second- and third-row transition elements, respectively, which are found in a mononuclear form in the active site of a diverse group of enzymes that generally catalyze oxygen atom transfer reactions. Mononuclear Mo-containing enzymes have been classified into three families: xanthine oxidase, DMSO reductase, and sulfite oxidase. The proteins of the DMSO reductase family present the widest diversity of properties among its members and our knowledge about this family was greatly broadened by the study of the enzymes nitrate reductase and formate dehydrogenase, obtained from different sources. We discuss in this review the information of the better characterized examples of these two types of Mo enzymes and W enzymes closely related to the members of the DMSO reductase family. We briefly summarize, also, the few cases reported so far for enzymes that can function either with Mo or W at their active site.

Metallothionein (MT) in the liver of gilthead seabreams (Sparus aurata L., 1758) exposed to Sado estuary (Portugal) sediments was quantified to assess the MT induction potential as a biomarker of sediment-based contamination by copper (Cu), cadmium (U), lead (Pb) and arsenic (As). Sediments were collected from two control sites and four sites with different levels of contamination. Sediment Cu, Cd, Pb, As, total organic matter (TOM) and fine fraction (FF) levels were determined. Generalized linear models (GLM) allowed integration of sediment parameters with liver Cu, Cd, Pb, As and MT concentrations. Although sediment metal levels were lower than expected, we relate NIT with liver Cd and also with interactions between liver and sediment Cu and between liver Cu and TOM. We suggest integrating biomarkers and environmental parameters using statistical models such as GLM as a more sensitive and reliable technique for sediment risk assessment than traditional isolated biomarker approaches. (C) 2007 Elsevier Inc. All rights reserved.

In this report, we describe the isolation of a 4020-bp genomic PstI fragment of Desulfovibrio gigas harboring the aldehyde oxido-reductase gene. The aldehyde oxido-reductase gene spans 2718 bp of genomic DNA and codes for a protein with 906 residues. The protein sequence shows an average 52% (+/- 1.5%) similarity to xanthine dehydrogenase from different organisms. The codon usage of the aldehyde oxidoreductase is almost identical to a calculated codon usage of the Desulfovibrio bacteria.

Mononuclear molybdenum and tungsten are found in the active site of a diverse group of enzymes that, in general, catalyze oxygen atom transfer reactions. Enzymes of the xanthine oxidase family are the best-characterized mononuclear Mo-containing enzymes. Several 3D structures of diverse members of this family are known. Recently, the structures of substrate-bound and arsenite-inhibited forms of two members of this family have also been reported. In addition, spectroscopic studies have been utilized to elucidate fine details that complement the structural information. Altogether, these studies have provided an important amount of information on the characteristics of the active site and the electron transfer pathways.

The characterization of a novel Mo-Fe protein (MorP) associated with a system that responds to Mo in Desulfovibrio alaskensis is reported. Biochemical characterization shows that MorP is a periplasmic homomultimer of high molecular weight (260 +/- 13 kDa) consisting of 16-18 monomers of 15321.1 +/- 0.5 Da. The UV/visible absorption spectrum of the as-isolated protein shows absorption peaks around 280, 320, and 570 nm with extinction coefficients of 18700, 12800, and 5000 M(-1) cm(-1), respectively. Metal content, EXAFS data and DFT calculations support the presence of a Mo-2S-[2Fe-2S]-2S-Mo cluster never reported before. Analysis of the available genomes from Desulfovibrio species shows that the MorP encoding gene is located downstream of a sensor and a regulator gene. This type of gene arrangement, called two component system, is used by the cell to regulate diverse physiological processes in response to changes in environmental conditions. Increase of both gene expression and protein production was observed when cells were cultured in the presence of 45 microM molybdenum. Involvement of this system in Mo tolerance of sulfate reducing bacteria is proposed.

Mossbauer and electron paramagnetic resonance (EPR) spectroscopies were used to characterize the diheme cytochrome c peroxidase from Paracoccus denitrificans (L.M.D. 52.44). The spectra of the oxidized enzyme show two distinct spectral components characteristic of low spin ferric hemes (S = 1/2), revealing different heme environments for the two heme groups. The Paracoccus peroxidase can be non-physiologically reduced by ascorbate. Mossbauer investigation of the ascorbate-reduced peroxidase shows that only one heme (the high potential heme) is reduced and that the reduced heme is diamagnetic (S = 0). The other heme (the low potential heme) remains oxidized, indicating that the enzyme is in a mixed valence, half-reduced state. The EPR spectrum of the half-reduced peroxidase, however, shows two low spin ferric species with gmax = 2.89 (species I) and gmax = 2.78 (species II). This EPR observation, together with the Mossbauer result, suggests that both species are arising from the low potential heme. More interestingly, the spectroscopic properties of these two species are distinct from that of the low potential heme in the oxidized enzyme, providing evidence for heme-heme interaction induced by the reduction of the high potential heme. Addition of calcium ions to the half-reduced enzyme converts species II to species I. Since calcium has been found to promote peroxidase activity, species I may represent the active form of the peroxidatic heme.

Mossbauer spectroscopy was used to study the tetraheme cytochrome c3 from Desulfovibrio baculatus (DSM 1743). Samples with different degrees of reduction were prepared using a redoxtitration technique. In the reduced cytochrome c3, all four hemes are reduced and exhibit diamagnetic Mossbauer spectra typical for low-spin ferrous hemes (S = 0). In the oxidized protein, the hemes are low-spin ferric (S = 1/2) and exhibit overlapping magnetic Mossbauer spectra. A method of differential spectroscopy was applied to deconvolute the four overlapping heme spectra and a crystal-field model was used for data analysis. Characteristic Mossbauer spectral components for each heme group are obtained. Hyperfine and crystal-field parameters for all four hemes are determined from these deconvoluted spectra.

Treponema pallidum, the causative agent of venereal syphilis, is a microaerophilic obligate pathogen of humans. As it disseminates hematogenously and invades a wide range of tissues, T. pallidum presumably must tolerate substantial oxidative stress. Analysis of the T. pallidum genome indicates that the syphilis spirochete lacks most of the iron-binding proteins present in many other bacterial pathogens, including the oxidative defense enzymes superoxide dismutase, catalase, and peroxidase, but does possess an orthologue (TP0823) for neelaredoxin, an enzyme of hyperthermophilic and sulfate-reducing anaerobes shown to possess superoxide reductase activity. To analyze the potential role of neelaredoxin in treponemal oxidative defense, we examined the biochemical, spectroscopic, and antioxidant properties of recombinant T. pallidum neelaredoxin. Neelaredoxin was shown to be expressed in T. pallidum by reverse transcriptase-polymerase chain reaction and Western blot analysis. Recombinant neelaredoxin is a 26-kDa alpha(2) homodimer containing, on average, 0.7 iron atoms/subunit. Mossbauer and EPR analysis of the purified protein indicates that the iron atom exists as a mononuclear center in a mixture of high spin ferrous and ferric oxidation states. The fully oxidized form, obtained by the addition of K(3)(Fe(CN)(6)), exhibits an optical spectrum with absorbances at 280, 320, and 656 nm; the last feature is responsible for the protein's blue color, which disappears upon ascorbate reduction. The fully oxidized protein has a A(280)/A(656) ratio of 10.3. Enzymatic studies revealed that T. pallidum neelaredoxin is able to catalyze a redox equilibrium between superoxide and hydrogen peroxide, a result consistent with it being a superoxide reductase. This finding, the first description of a T. pallidum iron-binding protein, indicates that the syphilis spirochete copes with oxidative stress via a primitive mechanism, which, thus far, has not been described in pathogenic bacteria.

New findings in sample treatment based on high-intensity focused ultrasound (HIFU) for protein digestion after polyacrylamide gel electrophoresis separation are presented. The following variables were studied: (i) sample volume; (ii) sonotrode diameter; (iii) previous protein denaturation; (iv) cooling; (v) enzyme concentration; and (vi) protein concentration. Results showed that positive protein identification could be done after protein separation by gel electrophoresis through peptide mass fingerprint (PMF) in a volume as low as 25 mu L. The time needed was less than 2 min and no cooling was necessary. The importance of the sonotrode diameter was negligible. On the other hand, protein denaturation before sonication was a trade-off for the success of procedure here described. The protein coverage was raised from 5 to 30%, and the number of peptides matching the proteins was also increased in a percentage ranging 10-100% when the classical overnight treatment is compared with the proposed HIFU procedure. The minimum amount of protein that can be identified using the HIFU sample treatment by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was 0.06 mu g. The lower concentration of trypsin successfully used to obtain an adequate protein digestion was 3.6 mu g/mL. (c) 2006 Elsevier B.V. All rights reserved.

Desulforedoxin (Dx) is a simple homodimeric protein isolated from Desulfovibrio gigas (Dg) containing a distorted rubredoxin-like center with one iron coordinated by four cysteinyl residues (7.9 kDa with 36 amino acids per monomer). In order to probe the geometry and the H-bonding at the active site of Dx, the protein was reconstituted with 113Cd and the solution structure determined using 2D NMR methods. The structure of this derivative was initially compared with the NMR solution structure of the Zn form (Goodfellow BJ et al., 1996, J Biol Inorg Chem 1:341-353). Backbone amide protons for G4, D5, G13, L11 NH, and the Q14 NH side-chain protons, H-bonded in the X-ray structure, were readily exchanged with solvent. Chemical shift differences observed for amide protons near the metal center confirm the H-bonding pattern seen in the X-ray model (Archer M et al., 1995, J Mol Biol 251:690-702) and also suggest that H-bond lengths may vary between the Fe, Zn, and 113Cd forms. The H-bonding pattern was further probed using a heteronuclear spin echo difference (HSED) experiment; the results confirm the presence of NH-S H-bonds inferred from D2O exchange data and observed in the NMR family of structures. The presence of "H-bond mediated" coupling in Dx indicates that the NH-S H-bonds at the metal center have significant covalent character. The HSED experiment also identified an intermonomer "through space" coupling for one of the L26 methyl groups, indicating its proximity to the 113Cd center in the opposing monomer. This is the first example of an intermonomer "through space" coupling. Initial structure calculations produced subsets of NMR families with the S of C28 pointing away from or toward the L26 methyl: only the subset with the C28 sulfur pointing toward the L26 methyl could result in a "through space" coupling. The HSED result was therefore included in the structure calculations. Comparison of the Fe, Zn, and 113Cd forms of Dx suggests that the geometry of the metal center and the global fold of the protein does not vary to any great extent, although the H-bond network varies slightly when Cd is introduced. The similarity between the H-bonding pattern seen at the metal center in Dx, Rd (including H-bonded and through space-mediated coupling), and many zinc-finger proteins suggests that these H-bonds are structurally vital for stabilization of the metal centers in these proteins.

The differences in geometry at the metal centres in the two known [Fe-4S] proteins rubredoxin (Rd) and desulforedoxin (Dx) are postulated to be a result of the different spacing of the C-terminal cysteine pair in the two proteins. In order to address this question, two mutants of Desulfovibrio gigas Dx with modified cysteinyl spacing were prepared and their solution structures have been determined by NMR. Mutant 1 of Dx (DxM1) has a single glycine inserted between the adjacent cysteines (C28 and C29) found in the wild type Dx sequence. Mutant 3 (DxM3) has two amino acid residues, -P-V-, inserted between C28 and C29 in order to mimic the primary sequence found in Rd from Desulfovibrio gigas. The solution structure of DxM1 exists, like wild type Dx, as a dimer in solution although the single glycine inserted between the adjacent cysteines disrupts the stability of the dimer resulting in exchange between a dimer state and a small population of another, probably monomeric, state. For DxM3 the two amino acid residues inserted between the adjacent cysteines results in a monomeric protein that has a global fold near the metal centre very similar to that found in Rd.