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Sarcoplasmic reticulum calcium ATPase is inhibited by organic vanadium coordination compounds: pyridine-2,6-dicarboxylatodioxovanadium(V), BMOV, and an amavadine analogue, Aureliano, M., Henao F., Tiago T., Duarte R. O., Moura J. J., Baruah B., and Crans D. C. , Inorg Chem, Jul 7, Volume 47, Number 13, p.5677-84, (2008) AbstractWebsite

The general affinity of the sarcoplasmic reticulum (SR) Ca (2+)-ATPase was examined for three different classes of vanadium coordination complexes including a vanadium(V) compound, pyridine-2,6-dicarboxylatodioxovanadium(V) (PDC-V(V)), and two vanadium(IV) compounds, bis(maltolato)oxovanadium(IV) (BMOV), and an analogue of amavadine, bis( N-hydroxylamidoiminodiacetato)vanadium(IV) (HAIDA-V(IV)). The ability of vanadate to act either as a phosphate analogue or as a transition-state analogue with enzymes' catalysis phosphoryl group transfer suggests that vanadium coordination compounds may reveal mechanistic preferences in these classes of enzymes. Two of these compounds investigated, PDC-V(V) and BMOV, were hydrolytically and oxidatively reactive at neutral pH, and one, HAIDA-V(IV), does not hydrolyze, oxidize, or otherwise decompose to a measurable extent during the enzyme assay. The SR Ca (2+)-ATPase was inhibited by all three of these complexes. The relative order of inhibition was PDC-V(V) > BMOV > vanadate > HAIDA-V(IV), and the IC 50 values were 25, 40, 80, and 325 microM, respectively. Because the observed inhibition is more potent for PDC-V(V) and BMOV than that of oxovanadates, the inhibition cannot be explained by oxovanadate formation during enzyme assays. Furthermore, the hydrolytically and redox stable amavadine analogue HAIDA-V(IV) inhibited the Ca (2+)-ATPase less than oxovanadates. To gauge the importance of the lipid environment, studies of oxidized BMOV in microemulsions were performed and showed that this system remained in the aqueous pool even though PDC-V(V) is able to penetrate lipid interfaces. These findings suggest that the hydrolytic properties of these complexes may be important in the inhibition of the calcium pump. Our results show that two simple coordination complexes with known insulin enhancing effects can invoke a response in calcium homeostasis and the regulation of muscle contraction through the SR Ca (2+)-ATPase.

Purification, crystallization and preliminary X-ray diffraction analysis of adenosine triphosphate sulfurylase (ATPS) from the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774, Gavel, O. Y., Kladova A. V., Bursakov S. A., Dias J. M., Texeira S., Shnyrov V. L., Moura J. J., Moura I., Romao M. J., and Trincao J. , Acta Crystallogr Sect F Struct Biol Cryst Commun, Jul 1, Volume 64, Number Pt 7, p.593-5, (2008) AbstractWebsite

Native zinc/cobalt-containing ATP sulfurylase (ATPS; EC; MgATP:sulfate adenylyltransferase) from Desulfovibrio desulfuricans ATCC 27774 was purified to homogeneity and crystallized. The orthorhombic crystals diffracted to beyond 2.5 A resolution and the X-ray data collected should allow the determination of the structure of the zinc-bound form of this ATPS. Although previous biochemical studies of this protein indicated the presence of a homotrimer in solution, a dimer was found in the asymmetric unit. Elucidation of this structure will permit a better understanding of the role of the metal in the activity and stability of this family of enzymes.

Reduction of ascorbate free radical by the plasma membrane of synaptic terminals from rat brain, Samhan-Arias, A. K., Duarte R. O., Martin-Romero F. J., Moura J. J., and Gutierrez-Merino C. , Arch Biochem Biophys, Jan 15, Volume 469, Number 2, p.243-54, (2008) AbstractWebsite

Synaptic plasma membranes (SPMV) decrease the steady state ascorbate free radical (AFR) concentration of 1mM ascorbate in phosphate/EDTA buffer (pH 7), due to AFR recycling by redox coupling between ascorbate and the ubiquinone content of these membranes. In the presence of NADH, but not NADPH, SPMV catalyse a rapid recycling of AFR which further lower the AFR concentration below 0.05 microM. These results correlate with the nearly 10-fold higher NADH oxidase over NADPH oxidase activity of SPMV. SPMV has NADH-dependent coenzyme Q reductase activity. In the presence of ascorbate the stimulation of the NADH oxidase activity of SPMV by coenzyme Q(1) and cytochrome c can be accounted for by the increase of the AFR concentration generated by the redox pairs ascorbate/coenzyme Q(1) and ascorbate/cytochrome c. The NADH:AFR reductase activity makes a major contribution to the NADH oxidase activity of SPMV and decreases the steady-state AFR concentration well below the micromolar concentration range.

Sub-cellular partitioning of Zn, Cu, Cd and Pb in the digestive gland of native Octopus vulgaris exposed to different metal concentrations (Portugal), Raimundo, J., Vale C., Duarte R., and Moura I. , Science of the Total Environment, Feb 15, Volume 390, Number 2-3, p.410-416, (2008) AbstractWebsite

Cd and Pb and their sub-cellular distributions were determined in Cu Concentrations of Zn,, composite samples of digestive glands of the common octopus, Octopus vulgaris caught from two areas of the Portuguese coast characterised by contrasting metal contamination. Minor contents of Zn (1%), Cu (2%), Cd (6%) and Pb (7%) were found in the insoluble fraction, consisting of nuclei, mitochondria, lysosomes and microsome operationally separated from the whole digestive gland through a sequential centrifugation. A tendency for linear relationships between metal concentrations in nuclei, mitochondria, lysosomes and whole digestive gland was observed. These relationships suggest that despite low metal content organelles responded to the increasing accumulated metals, which means that detoxifying mechanism in cytosol was incomplete. Poorer correlations between microsome and whole digestive gland did not point to metal toxicity in the analysed compartments. However, the high accumulated Cd indicated that O. vulgaris is an important vehicle of this element to its predators in the coastal environment. (c) 2007 Elsevier B.V. All rights reserved.

An improved clean sonoreactor-based method for protein identification by mass spectrometry-based techniques, Santos, H. M., Mota Cristiano, Lodeiro C., Moura Isabel, Isaac Issa, and Capelo J. L. , Talanta, Dec 15, Volume 77, Number 2, p.870-875, (2008) AbstractWebsite

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.

Ultrasonic assisted protein enzymatic digestion for fast protein identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry Sonoreactor versus ultrasonic probe, Rial-Otero, R., Carreira R. J., Cordeiro F. M., Moro A. J., Santos H. M., Vale G., Moura I., and Capelo J. L. , Journal of Chromatography A, Sep 28, Volume 1166, Number 1-2, p.101-107, (2007) AbstractWebsite

Two different ultrasonic energy sources, the sonoreactor and the ultrasonic probe, are compared for enzymatic digestion of proteins for protein identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDl-TOF-MS) using the peptide mass fingerprint (PMF) procedure. Variables such as (i) trypsin/protein ratio; (ii) sonication time; (iii) ultrasound amplitude; and (iv) protein concentration are studied and compared. As a general rule, the trypsin/protein ratio and the minimum protein concentration successfully digested are similar with both ultrasonic energy sources. Results showed that the time needed to digest proteins was shorter with the ultrasonic probe, 60 s versus 120 s, for the same amplitude of sonication, 50%. However, lower standard deviations and cleaner MALDI-TOF-MS spectra were obtained with the sonoreactor. In addition, the sonoreactor device provided higher sample throughput (6 samples for the sonoreactor versus 1 sample for the ultrasonic probe) and easier sample handling for lower sample volumes (25 mu l). Finally, a comparison of both methodologies for the specific identification of the adenylylsulphate reductase alfa subunit from a complex protein mixture from Desulfovibrio desulfuricans ATCC 27774 was done as a proof of the procedure. (c) 2007 Elsevier B.V. All rights reserved.

Highly sensitive nitrite biosensor based on the electrical wiring of nitrite reductase by ZnCr-AQS LDH, Chen, H., Mousty C., Cosnier S., Silveira C., Moura J. J. G., and Almeida M. G. , Electrochemistry Communications, Sep, Volume 9, Number 9, p.2240-2245, (2007) AbstractWebsite

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.

Improving sample treatment for in-solution protein identification by peptide mass fingerprint using matrix-assisted laser desorption/ionization time-of-flight mass Spectrometry, Santos, H. M., Rial-Otero R., Fernandes L., Vale G., Rivas M. G., Moura I., and Capelo J. L. , Journal of Proteome Research, Sep, Volume 6, Number 9, p.3393-3399, (2007) AbstractWebsite

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.

EPR characterization of the molybdenum(V) forms of formate dehydrogenase from Desulfovibrio desulfuricans ATCC 27774 upon formate reduction, Rivas, M. G., Gonzalez P. J., Brondino C. D., Moura J. J., and Moura I. , J Inorg Biochem, Nov, Volume 101, Number 11-12, p.1617-22, (2007) AbstractWebsite

The EPR characterization of the molybdenum(V) forms obtained on formate reduction of both as-prepared and inhibited formate dehydrogenase from Desulfovibrio desulfuricans ATCC 27774, an enzyme that catalyzes the oxidation of formate to CO(2), is reported. The Mo(V) EPR signal of the as-prepared formate-reduced enzyme is rhombic (g(max)=2.012, g(mid)=1.996, g(min)=1.985) and shows hyperfine coupling with two nuclear species with I=1/2. One of them gives an anisotropic splitting and is not solvent exchangeable (A(max)=11.7, A(mid)=A(min)=non-detectable, A-values in cm(-1)x10(-4)). The second species is exchangeable with solvent and produces a splitting at the three principal g-values (A(max)=7.7, A(mid)=10.0, A(min)=9.3). The hyperfine couplings of the non-solvent and solvent exchangeable nuclei are assigned to the hydrogen atoms of the beta-methylene carbon of a selenocysteine and to a Mo ligand whose nature, sulfydryl or hydroxyl, is still in debate. The Mo(V) species obtained in the presence of inhibitors (azide or cyanide) yields a nearly axial EPR signal showing only one detectable splitting given by nuclear species with I=1/2 (g(max)=2.092, g(mid)=2.000, g(min)=1.989, A(max)=non-detectable, A(mid)=A(min)=7.0), which is originated from the alpha-proton donated by the formate to a proximal ligand of the molybdenum. The possible structures of both paramagnetic molybdenum species (observed upon formate reduction in presence and absence of inhibitors) are discussed in comparison with the available structural information of this enzyme and the structural and EPR properties of the closely related formate dehydrogenase-H from Escherichia coli.

Biosensing nitrite using the system nitrite redutase/Nafion/methyl viologen--a voltammetric study, Almeida, M. G., Silveira C. M., and Moura J. J. , Biosens Bioelectron, May 15, Volume 22, Number 11, p.2485-92, (2007) AbstractWebsite

This work describes the construction and voltammetric characterization of a nitrite biosensor based on a cytochrome c-type nitrite reductase (ccNiR) and the Nafion ionomeric matrix loaded with methyl viologen as redox mediator. Despite the potential electrostatic repulsions between the anionic substrate and the Nafion sulfonate groups, the resulting bioelectrode exhibited electrocatalytic activity toward nitrite. This phenomenon must be due to the nonuniformity of the enzyme/Nafion membrane, which allows the direct interaction between the substrate and numerous enzyme molecules. Nevertheless, the anionic nature of Nafion exerted a certain diffusion barrier to nitrite, as revealed by the unusually elevated limits of the linear dynamic range and k(m)(app). The irregularity of the composite membrane also contributed to slow down the rate of charge transfer throughout the Nafion polymer. The level of viologens incorporated within the Nafion membrane had a strong influence in the analytical parameters: as much mediator was present, lower was the sensitivity and wider was the linear range. For an optimized ratio enzyme/mediator the sensitivity was 445+/-8 mA M(-1)cm(-2), within the linear range 75-800 microM; the lowest detected nitrite concentration was 60 microM. The operational stability of the biosensor and the influence of some possible interferences were evaluated.

Gas chromatography mass spectrometry determination of acaricides from honey after a new fast ultrasonic-based solid phase micro-extraction sample treatment, Rial-Otero, R., Gaspar E. M., Moura I., and Capelo J. L. , Talanta, Mar 30, Volume 71, Number 5, p.1906-1914, (2007) AbstractWebsite

A method is reported for the determination of acaricides (amitraz, bromopropylate, coumaphos and fluvalinate) from honey by gas chromatography mass spectrometry after a new fast solid phase micro-extraction, SPME, procedure. Six different fibers were assessed for micro-extraction purpose studying the following variables: (i) SPME coating, (ii) extraction temperature, (iii) extraction time, (iv) desorption conditions and (v) agitation conditions. The new ultrasonic bath technology providing different sonication frequencies (35 and 130 kHz) and different working modes (Sweep, Standard and Degas) was studied and optimized for speeding up the acaricide micro-extraction. The best extraction results were achieved with the polyacrylate fiber. The extraction process was done in 30 min using the ultrasonic bath at 130 kHz in the Standard mode. Quality parameters of the proposed method show a good precision (<11%) and detection and quantitation limits lower than 6 and 15 ng/g, respectively, except for fluvalinate. Eleven Portuguese commercial honey samples were analyzed with the developed method in order to assess the performance of the method with real samples and to determine whether the concentration of acaricides in honey exceed their maximum residue levels (MRLs). Acaricide residues detected were lower than those established by the legislation. (c) 2006 Elsevier B.V. All rights reserved.

Application of lactate amperometric sol-gel biosensor to sequential injection determination of L-lactate, Gomes, S. P., Odlozilikova M., Almeida M. G., Araujo A. N., Couto C. M., and Montenegro M. C. , J Pharm Biomed Anal, Mar 12, Volume 43, Number 4, p.1376-81, (2007) AbstractWebsite

This work describes the construction and evaluation of lactate sol-gel biosensors to accomplish the determination of lactate in pharmaceutical products. Lactate oxidase was incorporated in a porous sol-gel film placed onto a platinum-based electrode. Acid and basic catalysis were assessed. When coupled to a sequential injection system (SIA) the biosensor, based on (3-aminopropyl)trimethoxysilane, 2-(3,4-epoxycyclohexyl)ethyl-trimethoxysilane, deionised water, polyethylene glycol 6000 and acid catalyst, presented a range of linearity of 5x10(-5) to 5x10(-3)M. The analytical usefulness of the developed biosensor was evaluated through analysis of commercial pharmaceutical products containing lactate with a sampling rate of 40 samples h(-1). The enzyme remained active for at least 30 days, enabling about 700 determinations without sensitivity decrease.

Correlating EPR and X-ray structural analysis of arsenite-inhibited forms of aldehyde oxidoreductase, Thapper, A., Boer D. R., Brondino C. D., Moura J. J., and Romao M. J. , J Biol Inorg Chem, Mar, Volume 12, Number 3, p.353-66, (2007) AbstractWebsite

Two arsenite-inhibited forms of each of the aldehyde oxidoreductases from Desulfovibrio gigas and Desulfovibrio desulfuricans have been studied by X-ray crystallography and electron paramagnetic resonance (EPR) spectroscopy. The molybdenum site of these enzymes 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. Arsenite addition to active as-prepared enzyme or to a reduced desulfo form yields two different species called A and B, respectively, which show different Mo(V) EPR signals. Both EPR signals show strong hyperfine and quadrupolar couplings with an arsenic nucleus, which suggests that arsenic interacts with molybdenum through an equatorial ligand. X-ray data of single crystals prepared from EPR-active samples show in both inhibited forms that the arsenic atom interacts with the molybdenum ion through an oxygen atom at the catalytic labile site and that the sulfido ligand is no longer present. EPR and X-ray data indicate that the main difference between both species is an equatorial ligand to molybdenum which was determined to be an oxo ligand in species A and a hydroxyl/water ligand in species B. The conclusion that the sulfido ligand is not essential to determine the EPR properties in both Mo-As complexes is achieved through EPR measurements on a substantial number of randomly oriented chemically reduced crystals immediately followed by X-ray studies on one of those crystals. EPR saturation studies show that the electron transfer pathway, which is essential for catalysis, is not modified upon inhibition.

New findings for in-gel digestion accelerated by high-intensity focused ultrasound for protein identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, Carreira, R. J., Cordeiro F. M., Moro A. J., Rivas M. G., Rial-Otero R., Gaspar E. M., Moura I., and Capelo J. L. , Journal of Chromatography A, Jun 15, Volume 1153, Number 1-2, p.291-299, (2007) AbstractWebsite

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.

Heterodimeric nitrate reductase (NapAB) from Cupriavidus necator H16: purification, crystallization and preliminary X-ray analysis, Coelho, C., Gonzalez P. J., Trincao J., Carvalho A. L., Najmudin S., Hettman T., Dieckman S., Moura J. J., Moura I., and Romao M. J. , Acta Crystallogr Sect F Struct Biol Cryst Commun, Jun 1, Volume 63, Number Pt 6, p.516-9, (2007) AbstractWebsite

The periplasmic nitrate reductase from Cupriavidus necator (also known as Ralstonia eutropha) is a heterodimer that is able to reduce nitrate to nitrite. It comprises a 91 kDa catalytic subunit (NapA) and a 17 kDa subunit (NapB) that is involved in electron transfer. The larger subunit contains a molybdenum active site with a bis-molybdopterin guanine dinucleotide cofactor as well as one [4Fe-4S] cluster, while the small subunit is a di-haem c-type cytochrome. Crystals of the oxidized form of this enzyme were obtained using polyethylene glycol 3350 as precipitant. A single crystal grown at the High Throughput Crystallization Laboratory of the EMBL in Grenoble diffracted to beyond 1.5 A at the ESRF (ID14-1), which is the highest resolution reported to date for a nitrate reductase. The unit-cell parameters are a = 142.2, b = 82.4, c = 96.8 A, beta = 100.7 degrees, space group C2, and one heterodimer is present per asymmetric unit.

Mediated catalysis of Paracoccus pantotrophus cytochrome c peroxidase by P. pantotrophus pseudoazurin: kinetics of intermolecular electron transfer, de Sousa, P. M., Pauleta S. R., Goncalves M. L., Pettigrew G. W., Moura I., Dos Santos M. M., and Moura J. J. , J Biol Inorg Chem, Jun, Volume 12, Number 5, p.691-8, (2007) AbstractWebsite

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.

Crystal structure of the 16 heme cytochrome from Desulfovibrio gigas: A glycosylated protein in a sulphate-reducing bacterium, Santos-Silva, Teresa, Dias Joao Miguel, Dolla Alain, Durand Marie-Claire, Goncalves Luisa L., Lampreia Jorge, Moura Isabel, and Romao Maria Joao , Journal of Molecular Biology, Jul 20, Volume 370, Number 4, p.659-673, (2007) AbstractWebsite

Sulphate-reducing bacteria have a wide variety of periplasmic cytochromes involved in electron transfer from the periplasm to the cytoplasm. HmcA is a high molecular mass cytochrome of 550 amino acid residues that harbours 16 c-type heme groups. We report the crystal structure of HmcA isolated from the periplasm of Desulfovibrio gigas. Crystals were grown. using polyethylene glycol 8K and zinc acetate, and diffracted beyond 2.1 angstrom resolution. A multiple-wavelength anomalous dispersion experiment at the iron absorption edge enabled us to obtain good-quality phases for structure solution and model building. DgHmcA has a V-shape architecture, already observed in HmcA isolated from Desulfovibrio vulgaris Hildenborough. The presence of an oligosaccharide molecule covalently bound to an Asn residue was observed in the electron density maps of DgHmcA and confirmed by mass spectrometry. Three modified monosaccharides appear at the highly hydrophobic vertex, possibly acting as an anchor of the protein to the cytoplasmic membrane. (c) 2007 Elsevier Ltd. All rights reserved.

NMR assignment of the apo-form of a Desulfovibrio gigas protein containing a novel Mo-Cu cluster, Pauleta, S. R., Duarte A. G., Carepo M. S., Pereira A. S., Tavares P., Moura I., and Moura J. J. , Biomol NMR Assign, Jul, Volume 1, Number 1, p.81-3, (2007) AbstractWebsite

We report the 98% assignment of the apo-form of an orange protein, containing a novel Mo-Cu cluster isolated from Desulfovibrio gigas. This protein presents a region where backbone amide protons exchange fast with bulk solvent becoming undetectable. These residues were assigned using 13C-detection experiments.

A needle in a haystack: the active site of the membrane-bound complex cytochrome c nitrite reductase, Almeida, M. G., Silveira C. M., Guigliarelli B., Bertrand P., Moura J. J., Moura I., and Leger C. , FEBS Lett, Jan 23, Volume 581, Number 2, p.284-8, (2007) AbstractWebsite

Cytochrome c nitrite reductase is a multicenter enzyme that uses a five-coordinated heme to perform the six-electron reduction of nitrite to ammonium. In the sulfate reducing bacterium Desulfovibrio desulfuricans ATCC 27774, the enzyme is purified as a NrfA2NrfH complex that houses 14 hemes. The number of closely-spaced hemes in this enzyme and the magnetic interactions between them make it very difficult to study the active site by using traditional spectroscopic approaches such as EPR or UV-Vis. Here, we use both catalytic and non-catalytic protein film voltammetry to simply and unambiguously determine the reduction potential of the catalytic heme over a wide range of pH and we demonstrate that proton transfer is coupled to electron transfer at the active site.

Vanadium distribution, lipid peroxidation and oxidative stress markers upon decavanadate in vivo administration, Soares, S. S., Martins H., Duarte R. O., Moura J. J., Coucelo J., Gutierrez-Merino C., and Aureliano M. , J Inorg Biochem, Jan, Volume 101, Number 1, p.80-8, (2007) AbstractWebsite

The contribution of decameric vanadate species to vanadate toxic effects in cardiac muscle was studied following an intravenous administration of a decavanadate solution (1mM total vanadium) in Sparus aurata. Although decameric vanadate is unstable in the assay medium, it decomposes with a half-life time of 16 allowing studying its effects not only in vitro but also in vivo. After 1, 6 and 12h upon decavanadate administration the increase of vanadium in blood plasma, red blood cells and in cardiac mitochondria and cytosol is not affected in comparison to the administration of a metavanadate solution containing labile oxovanadates. Cardiac tissue lipid peroxidation increases up to 20%, 1, 6 and 12h after metavanadate administration, whilst for decavanadate no effects were observed except 1h after treatment (+20%). Metavanadate administration clearly differs from decavanadate by enhancing, 12h after exposure, mitochondrial superoxide dismutase (SOD) activity (+115%) and not affecting catalase (CAT) activity whereas decavanadate increases SOD activity by 20% and decreases (-55%) mitochondrial CAT activity. At early times of exposure, 1 and 6h, the only effect observed upon decavanadate administration was the increase by 20% of SOD activity. In conclusion, decavanadate has a different response pattern of lipid peroxidation and oxidative stress markers, in spite of the same vanadium distribution in cardiac cells observed after decavanadate and metavanadate administration. It is suggested that once formed decameric vanadate species has a different reactivity than vanadate, thus, pointing out that the differential contribution of vanadium oligomers should be taken into account to rationalize in vivo vanadate toxicity.

Chromatographic-based methods for pesticide determination in honey: An overview, Rial-Otero, R., Gaspar E. M., Moura I., and Capelo J. L. , Talanta, Feb 15, Volume 71, Number 2, p.503-514, (2007) AbstractWebsite

Nowadays the control of pesticides in honey is an issue of primary health importance as consequence of the increasing content of these chemicals in the aforementioned matrix. This poisoning has led to the worldwide increasing loss of bees since 1995. From Europe to Canada, scientist, beekeepers and chemical companies disagree about the reasons that have led to colony losses higher than 50% in some areas. This problem has become a public health issue due to the high honey worldwide consumption. The presence of pesticides in honey has been directly related to bees' mortality by some researchers through pesticide presence in (1) pollen, (2) honeycomb walls, (3) own bees and (4) honey. In this work we describe the actual state-of-the-art for pesticides determination in honey along with a review in this subject focused on sample treatments and instrumentation. Finally, future trends are also commented. (c) 2006 Elsevier B.V. All rights reserved.

Sonoreactor-based technology for fast high-throughput proteolytic digestion of proteins, Rial-Otero, R., Carreira R. J., Cordeiro F. M., Moro A. J., Fernandes L., Moura I., and Capelo J. L. , Journal of Proteome Research, Feb, Volume 6, Number 2, p.909-912, (2007) AbstractWebsite

Fast (120 s) and high-throughput (more than six samples at once) in-gel trypsin digestion of proteins using sonoreactor technology has been achieved. Successful protein identification was done by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF-MS. Specific identification of the adenylylsulphate reductase alfa subunit from a complex protein mixture from Desulfovibrio desulfuricans ATCC 27774 was done as a proof of the methodology. The new sample treatment is of easy implementation, saves time and money, and can be adapted to online procedures and robotic platforms.

NADH oxidase activity of rat and human liver xanthine oxidoreductase: potential role in superoxide production, Maia, L., Duarte R. O., Ponces-Freire A., Moura J. J., and Mira L. , J Biol Inorg Chem, Aug, Volume 12, Number 6, p.777-87, (2007) AbstractWebsite

To characterise the NADH oxidase activity of both xanthine dehydrogenase (XD) and xanthine oxidase (XO) forms of rat liver xanthine oxidoreductase (XOR) and to evaluate the potential role of this mammalian enzyme as an O2*- source, kinetics and electron paramagnetic resonance (EPR) spectroscopic studies were performed. A steady-state kinetics study of XD showed that it catalyses NADH oxidation, leading to the formation of one O2*- molecule and half a H(2)O(2) molecule per NADH molecule, at rates 3 times those observed for XO (29.2 +/- 1.6 and 9.38 +/- 0.31 min(-1), respectively). EPR spectra of NADH-reduced XD and XO were qualitatively similar, but they were quantitatively quite different. While NADH efficiently reduced XD, only a great excess of NADH reduced XO. In agreement with reductive titration data, the XD specificity constant for NADH (8.73 +/- 1.36 microM(-1) min(-1)) was found to be higher than that of the XO specificity constant (1.07 +/- 0.09 microM(-1) min(-1)). It was confirmed that, for the reducing substrate xanthine, rat liver XD is also a better O2*- source than XO. These data show that the dehydrogenase form of liver XOR is, thus, intrinsically more efficient at generating O2*- than the oxidase form, independently of the reducing substrate. Most importantly, for comparative purposes, human liver XO activity towards NADH oxidation was also studied, and the kinetics parameters obtained were found to be very similar to those of the XO form of rat liver XOR, foreseeing potential applications of rat liver XOR as a model of the human liver enzyme.

Spectroscopic, computational, and kinetic studies of the mu4-sulfide-bridged tetranuclear CuZ cluster in N2O reductase: pH effect on the edge ligand and its contribution to reactivity, Ghosh, S., Gorelsky S. I., George S. D., Chan J. M., Cabrito I., Dooley D. M., Moura J. J., Moura I., and Solomon E. I. , J Am Chem Soc, Apr 4, Volume 129, Number 13, p.3955-65, (2007) AbstractWebsite

A combination of spectroscopy and density functional theory (DFT) calculations has been used to evaluate the pH effect at the CuZ site in Pseudomonas nautica (Pn) nitrous oxide reductase (N2OR) and Achromobacter cycloclastes (Ac) N2OR and its relevance to catalysis. Absorption, magnetic circular dichroism, and electron paramagnetic resonance with sulfur K-edge X-ray absorption spectra of the enzymes at high and low pH show minor changes. However, resonance Raman (rR) spectroscopy of PnN2OR at high pH shows that the 415 cm-1 Cu-S vibration (observed at low pH) shifts to higher frequency, loses intensity, and obtains a 9 cm-1 18O shift, implying significant Cu-O character, demonstrating the presence of a OH- ligand at the CuICuIV edge. From DFT calculations, protonation of either the OH- to H2O or the mu4-S2- to mu4-SH- would produce large spectral changes which are not observed. Alternatively, DFT calculations including a lysine residue at an H-bonding distance from the CuICuIV edge ligand show that the position of the OH- ligand depends on the protonation state of the lysine. This would change the coupling of the Cu-(OH) stretch with the Cu-S stretch, as observed in the rR spectrum. Thus, the observed pH effect (pKa approximately 9.2) likely reflects protonation equilibrium of the lysine residue, which would both raise E degrees and provide a proton for lowering the barrier for the N-O cleavage and for reduction of the [Cu4S(im)7OH]2+ to the fully reduced 4CuI active form for turnover.

Simplifying sample handling for protein identification by peptide mass fingerprint using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, Cordeiro, Francisco M., Carreira Ricardo J., Rial-Otero Raquel, Rivas Gabriela M., Moura Isabel, and Capelo Jose-Luis , Rapid Communications in Mass Spectrometry, 2007, Volume 21, Number 20, p.3269-3278, (2007) AbstractWebsite

An ultrasonic bath, an ultrasonic probe and a sonoreactor were used to speed up the kinetics of the reactions involved in each step of the sample handling for in-gel protein identification by peptide mass fingerprint, PMF, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The following steps were successfully accelerated using ultrasonic energy: gel washing, protein reduction, and protein alkylation. As a result, a reduction comprising 80% to 90% of the total time involved in the classic approach was achieved. In addition the sample handling was also drastically simplified. The number of peptides identified and the protein sequence coverage obtained for the new procedure were comparable to those obtained with the traditional sample treatment for the following protein standards: glycogen phosphorylase b, BSA, ovalbumin, carbonic anhydrase, trypsin inhibitor and alpha-lactalbumin. Finally, as a proof of the procedure, specific proteins were identified from complex protein mixtures obtained from three different sulphate- reducing bacteria: Desulfovibrio, desulfuricans G20, Desulfuvibrio gigas NCIB 9332, and Desulfuvibrio desulfuricans ATCC 27774. Copyright (c) 2007 John Wiley & Sons, Ltd.