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Journal Article
Construção de um Bio-Eléctrodo Específico para Determinação de Nitritos, Almeida, M. G., Tavares P., and Moura J. J. G. , Bol. Soc. Port. Química, Volume 84, p.68-71, (2002) Abstract
Construction of effective disposable biosensors for point-of-care testing of nitrite, Monteiro, T., Rodrigues P. R., Gonçalves A. L., Moura J. J. G., Anorga L., Jubete E., Piknova B., Schechter A. N., Silveira C. M., and Almeida M. G. , Talanta, Volume 142, p.246-251, (2015)
Continuous-wave EPR at 275GHz: application to high-spin Fe(3+) systems, Mathies, G., Blok H., Disselhorst J. A., Gast P., van der Meer H., Miedema D. M., Almeida R. M., Moura J. J., Hagen W. R., and Groenen E. J. , J Magn Reson, May, Volume 210, Number 1, p.126-32, (2011) AbstractWebsite

The 275GHz electron-paramagnetic-resonance spectrometer we reported on in 2004 has been equipped with a new probe head, which contains a cavity especially designed for operation in continuous-wave mode. The sensitivity and signal stability that is achieved with this new probe head is illustrated with 275GHz continuous-wave spectra of a 1mM frozen solution of the complex Fe(III)-ethylenediamine tetra-acetic acid and of 10mM frozen solutions of the protein rubredoxin, which contains Fe(3+) in its active site, from three different organisms. The high quality of the spectra of the rubredoxins allows the determination of the zero-field-splitting parameters with an accuracy of 0.5GHz. The success of our approach results partially from the enhanced absolute sensitivity, which can be reached using a single-mode cavity. At least as important is the signal stability that we were able to achieve with the new probe head.

Control of the spin state of the peroxidatic haem by calcium ions in cytochrome c peroxidase from Paracoccus denitrificans: A 1H NMR study, Prazeres, Susana, Moura Isabel, Moura José J. G., Gilmour Raymond, Goodhew Celia F., and Pettigrew Graham W. , Magnetic Resonance in Chemistry, Volume 31, Number 13, p.S68-S72, (1993) AbstractWebsite

Cytochrome c peroxidase from Paracoccus denitrificans LMD 52.44 was recently identified. The enzyme contains two c-type haems: one is reducible physiologically by cytochrome c550 from the same organism or non-physiologically by ascorbate (high-potential haem) and the other by dithionite (low-potential haem). The enzymatically active form of the peroxidase is the half-reduced enzyme state, in which the high-potential haem is in the iron(II) state and the low-potential haem is in the iron(III) state. It was found that the two haems interact and that the enzyme binds calcium ions near the haem sites which are necessary to promote its activation. In the oxidized form, the high-potential haem is in a high-spin and the low-potential haem is in a low-spin state. The half-reduction of the enzyme with ascorbate-diaminodurol changes the high-potential haem (high-spin) into a low-spin state and the low-potential haem converts from a low- into a high-spin state. This high-spin conversion of the low-potential haem is induced by the presence of calcium ions. These processes of reduction and spin state change can be easily resolved in time by removing the calcium from the enzyme using EDTA, facilitating the observation of the intermediate form by NMR.

Conversion of adrenaline to indolic derivatives by the human erythrocyte plasma membrane, Marques, F., Duarte R. O., Moura J. J., and Bicho M. P. , Biol Signals, Sep-Oct, Volume 5, Number 5, p.275-82, (1996) AbstractWebsite

The conversion of adrenaline to aminochromes by the human erythrocyte plasma membranes at pH 9.5 was shown to be a complex reaction that proceeded at least by two distinct phases. The first one, corresponding to the formation of adrenochrome, is catalyzed in the presence of the membranes, suggesting the involvement of an enzyme-mediated process. Active oxygen species were identified as intermediates during this phase. Oxygen radical scavengers (catalase and superoxide dismutase) suggested H2O2 and O2- involvement. Adrenochrome formation was stimulated by NADH indicating the participation of another enzyme (NADH dehydrogenase) which is known to be present in the human erythrocyte plasma membrane. The second phase, corresponding to the disappearance of adrenochrome, is also stimulated by NADH and inhibited in the presence of the membranes. In this reaction, adrenochrome is converted to aminochromes via adrenochrome semiquinone. The formation of radical species is demonstrated by EPR spectroscopy. The results led to the proposal of a mechanism for the formation of adrenochrome and other oxidation products from adrenaline.

Conversion of desulforedoxin into a rubredoxin center, Yu, L., Kennedy M., Czaja C., Tavares P., Moura J. J., Moura I., and Rusnak F. , Biochem Biophys Res Commun, Feb 24, Volume 231, Number 3, p.679-82, (1997) AbstractWebsite

Rubredoxin and desulforedoxin both contain an Fe(S-Cys)4 center. However, the spectroscopic properties of the center in desulforedoxin differ from rubredoxin. These differences arise from a distortion of the metal site hypothesized to result from adjacent cysteine residues in the primary sequence of desulforedoxin. Two desulforedoxin mutants were generated in which either a G or P-V were inserted between adjacent cysteines. Both mutants exhibited optical spectra with maxima at 278, 345, 380, 480, and 560 nm while the low temperature X-band EPR spectra indicated highspin Fe3+ ions with large rhombic distortions (E/D = 0.21-0.23). These spectroscopic properties are distinct from wild type desulforedoxin and virtually identical to rubredoxin.

Conversion of [3 Fe-3 S] into [4 Fe-4 S] clusters in a Desulfovibrio gigas ferredoxin and isotopic labeling of iron—sulfur cluster subsites, Kent, T. A., Moura I., Moura J. J. G., Lipscomb J. D., Huynh B. H., Legall J., Xavier A. V., and Münck E. , Febs Letters, Volume 138, Number 1, p.55-58, (1982) AbstractWebsite
Cooperative use of cytochrome cd1 nitrite reductase and its redox partner cytochrome c552 to improve the selectivity of nitrite biosensing, Serra, A. S., Jorge S. R., Silveira C. M., Moura J. J. G., Jubete E., Ochoteco E., Cabañero G., Grande H., and Almeida M. G. , Analytica Chimica Acta, Volume 693, Number 1–2, p.41-46, (2011) AbstractWebsite
Cooperative use of cytochrome cd1 nitrite reductase and its redox partner cytochrome c552 to Improve the selectivity of nitrite biosensing, A.S., Serra, S. Jorge, C. Silveira, J.J.G. Moura, E. Jubete, E. Ochoteco, and G. Almeida M. , Anal Chim Acta, Volume 693, p.41-46, (2011)
A copper protein and a cytochrome bind at the same site on bacterial cytochrome c peroxidase, Pauleta, S. R., Cooper A., Nutley M., Errington N., Harding S., Guerlesquin F., Goodhew C. F., Moura I., Moura J. J., and Pettigrew G. W. , Biochemistry, Nov 23, Volume 43, Number 46, p.14566-76, (2004) AbstractWebsite

Pseudoazurin binds at a single site on cytochrome c peroxidase from Paracoccus pantotrophus with a K(d) of 16.4 microM at 25 degrees C, pH 6.0, in an endothermic reaction that is driven by a large entropy change. Sedimentation velocity experiments confirmed the presence of a single site, although results at higher pseudoazurin concentrations are complicated by the dimerization of the protein. Microcalorimetry, ultracentrifugation, and (1)H NMR spectroscopy studies in which cytochrome c550, pseudoazurin, and cytochrome c peroxidase were all present could be modeled using a competitive binding algorithm. Molecular docking simulation of the binding of pseudoazurin to the peroxidase in combination with the chemical shift perturbation pattern for pseudoazurin in the presence of the peroxidase revealed a group of solutions that were situated close to the electron-transferring heme with Cu-Fe distances of about 14 A. This is consistent with the results of (1)H NMR spectroscopy, which showed that pseudoazurin binds closely enough to the electron-transferring heme of the peroxidase to perturb its set of heme methyl resonances. We conclude that cytochrome c550 and pseudoazurin bind at the same site on the cytochrome c peroxidase and that the pair of electrons required to restore the enzyme to its active state after turnover are delivered one-by-one to the electron-transferring heme.

Copper-containing nitrite reductase from Pseudomonas chlororaphis DSM 50135 - Evidence for modulation of the rate of intramolecular electron transfer through nitrite binding to the type 2 copper center, Pinho, D., Besson S., Brondino C. D., de Castro B., and Moura I. , European Journal of Biochemistry, Jun, Volume 271, Number 12, p.2361-2369, (2004) AbstractWebsite

The nitrite reductase (Nir) isolated from Pseudomonas chlororaphis DSM 50135 is a blue enzyme, with type 1 and type 2 copper centers, as in all copper-containing Nirs described so far. For the first time, a direct determination of the reduction potentials of both copper centers in a Cu-Nir was performed: type 2 copper (T2Cu), 172 mV and type 1 copper (T1Cu), 298 mV at pH 7.6. Although the obtained values seem to be inconsistent with the established electron-transfer mechanism, EPR data indicate that the binding of nitrite to the T2Cu center increases its potential, favoring the electron-transfer process. Analysis of the EPR spectrum of the turnover form of the enzyme also suggests that the electron-transfer process between T1Cu and T2Cu is the fastest of the three redox processes involved in the catalysis: (a) reduction of T1Cu; (b) oxidation of T1Cu by T2Cu; and (c) reoxidation of T2Cu by NO2-. Electrochemical experiments show that azurin from the same organism can donate electrons to this enzyme.

Copper-substituted forms of the wild type and C42A variant of rubredoxin, Thapper, A., Rizzi A. C., Brondino C. D., Wedd A. G., Pais R. J., Maiti B. K., Moura I., Pauleta S. R., and Moura J. J. G. , J Inorg Biochem, Volume 127, p.232-237, (2013)
Core dimensions in the 3Fe cluster of Desulfovibrio gigas ferredoxin II by extended X-ray absorption fine structure spectroscopy, Antonio, M. R., Averill B. A., Moura I., Moura J. J., Orme-Johnson W. H., Teo B. K., and Xavier A. V. , J Biol Chem, Jun 25, Volume 257, Number 12, p.6646-9, (1982) AbstractWebsite

We have obtained the iron K-edge extended X-ray adsorption fine structure spectra of the 3Fe ferredoxin II of Desulfovibrio gigas in the oxidized and reduced states. For both states, interpretation of the EXAFS data suggests that the Fe-S first shell coordination distance is near 2.25 A, in agreement with crystallographic studies of model compounds and proteins containing 2Fe-2S and 4Fe-4S centers, as well as with a recent crystallographic study of Azotobacter vinelandii ferredoxin I (Ghosh, D., Furey, W., Jr., O'Donnell, S., and Stout, C. D. (1981) J. Biol. Chem. 256, 4185-4192). The apparent Fe-Fe distance we obtain for the desulfovibrio protein (2.7 A) also agrees with similar distances seen in other Fe-S centers, except with the 3Fe cluster in the Azotobacter vinelandii ferredoxin I structure, for which an Fe-Fe distance of 4.2 A was reported. We conclude that either the two 3Fe ferredoxins have substantially different core dimensions, a possibility apparently unique to 3Fe centers among known Fe-S systems in proteins, or that one (or more) of the structural studies is in substantial error.

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.

Cross-linking between cytochrome c3 and flavodoxin from Desulfovibrio gigas, Correia, C., Monzani E., Moura I., Lampreia J., and Moura J. J. , Biochem Biophys Res Commun, Mar 16, Volume 256, Number 2, p.367-71, (1999) AbstractWebsite

Tetraheme cytochrome c3 (13 kDa) and flavodoxin (16 kDa), are small electron transfer proteins that have been used to mimic, in vitro, part of the electron-transfer chain that operates between substract electron donors and respiratory electron acceptors partners in Desulfovibrio species (Palma, N., Moura, I., LeGall, J., Van Beeumen, J., Wampler, J., Moura, J. J. G. (1994) Biochemistry 33, 6394-6407). The electron transfer between these two proteins is believed to occur through the formation of a specific complex where electrostatic interaction is the main driving force (Stewart, D., LeGall, J., Moura, I., Moura, J.J.G., Peck, H.D., Xavier, A.V., Weiner, P.K. and Wampler, J.E. (1988) Biochemistry 27, 2444-2450, Stewart, D., LeGall, J., Moura, I., Moura, J.J.G., Peck, H.D., Xavier, A.V., Weiner, P., Wampler, J. (1989) Eur. J. Biochem. 185, 695-700). In order to obtain structural information of the pre-complex, a covalent complex between the two proteins was prepared. A water-soluble carbodiimide [EDC (1-ethyl-3(3 dimethylaminopropyl) carbodiimide hydrochloride] was used for the cross linking reaction. The reaction was optimized varying a wide number of experimental parameters such as ionic strength, protein and cross linker concentration, and utilization of different cross linkers and reaction time between the crosslinker and proteins.

The crystal structure of Cupriavidus necator nitrate reductase in oxidized and partially reduced states, Coelho, C., Gonzalez P. J., Moura J. G., Moura I., Trincao J., and Joao Romao M. , J Mol Biol, May 20, Volume 408, Number 5, p.932-48, (2011) AbstractWebsite

The periplasmic nitrate reductase (NapAB) from Cupriavidus necator is a heterodimeric protein that belongs to the dimethyl sulfoxide reductase family of mononuclear Mo-containing enzymes and catalyzes the reduction of nitrate to nitrite. The protein comprises a large catalytic subunit (NapA, 91 kDa) containing the molybdenum active site plus one [4Fe-4S] cluster, as well as a small subunit (NapB, 17 kDa), which is a diheme c-type cytochrome involved in electron transfer. Crystals of the oxidized form of the enzyme diffracted beyond 1.5 A at the European Synchrotron Radiation Facility. This is the highest resolution reported to date for a nitrate reductase, providing true atomic details of the protein active center, and this showed further evidence on the molybdenum coordination sphere, corroborating previous data on the related Desulfovibrio desulfuricans NapA. The molybdenum atom is bound to a total of six sulfur atoms, with no oxygen ligands or water molecules in the vicinity. In the present work, we were also able to prepare partially reduced crystals that revealed two alternate conformations of the Mo-coordinating cysteine. This crystal form was obtained by soaking dithionite into crystals grown in the presence of the ionic liquid [C(4)mim]Cl(-). In addition, UV-Vis and EPR spectroscopy studies showed that the periplasmic nitrate reductase from C. necator might work at unexpectedly high redox potentials when compared to all periplasmic nitrate reductases studied to date.

Crystal structure of desulforedoxin from Desulfovibrio gigas determined at 1.8 A resolution: a novel non-heme iron protein structure, Archer, M., Huber R., Tavares P., Moura I., Moura J. J., Carrondo M. A., Sieker L. C., Legall J., and Romao M. J. , J Mol Biol, Sep 1, Volume 251, Number 5, p.690-702, (1995) AbstractWebsite

The crystal structure of desulforedoxin from Desulfovibrio gigas, a new homo-dimeric (2 x 36 amino acids) non-heme iron protein, has been solved by the SIRAS method using the indium-substituted protein as the single derivative. The structure was refined to a crystallographic R-factor of 16.9% at 1.8 A resolution. Native desulforedoxin crystals were grown from either PEG 4K or lithium sulfate, with cell constants a = b = 42.18 A, c = 72.22 A (for crystals grown from PEG 4K), and they belong to space group P3(2)21. The indium-substituted protein crystallized isomorphously under the same conditions. The 2-fold symmetric dimer is firmly hydrogen bonded and folds as an incomplete beta-barrel with the two iron centers placed on opposite poles of the molecule. Each iron atom is coordinated to four cysteinyl residues in a distorted tetrahedral arrangement. Both iron atoms are 16 A apart but connected across the 2-fold axis by 14 covalent bonds along the polypeptide chain plus two hydrogen bonds. Desulforedoxin and rubredoxin share some structural features but show significant differences in terms of metal environment and water structure, which account for the known spectroscopic differences between rubredoxin and desulforedoxin.

Crystal structure of flavodoxin from Desulfovibrio desulfuricans ATCC 27774 in two oxidation states, Romero, A., Caldeira J., Legall J., Moura I., Moura J. J., and Romao M. J. , Eur J Biochem, Jul 1, Volume 239, Number 1, p.190-6, (1996) AbstractWebsite

The crystal structures of the flavodoxin from Desulfovibrio desulfuricans ATCC 27774 have been determined and refined for both oxidized and semi-reduced forms to final crystallographic R-factors of 17.9% (0.8-0.205-nm resolution) and 19.4% (0.8-0.215-nm resolution) respectively. Native flavodoxin crystals were grown from ammonium sulfate with cell constants a = b = 9.59 nm, c=3.37nm (oxidized crystals) and they belong to space group P3(2)21. Semireduced crystals showed some changes in cell dimensions: a = b = 9.51 nm, c=3.35 nm. The three-dimensional structures are similar to other known flavodoxins and deviations are found essentially in the isoalloxazine ring environment. Conformational changes are observed between both redox states and a flip of the Gly61-Met62 peptide bond occurs upon one-electron reduction of the FMN group. These changes influence the redox potential of the oxidized/semiquinone couple. Modulation of the redox potentials is known to be related to the association constant of the FMN group to the protein. The flavodoxin from D. desulfuricans now studied has a large span between E2 (oxidized --> semiquinone) and E1 (semiquinone --> hydroquinone) redox potentials, both these values being substantially more positive within known flavodoxins. A comparison of their FMN environment was made in both oxidation states in order to correlate functional and structural differences.

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.

Crystal structure of the first dissimilatory nitrate reductase at 1.9 A solved by MAD methods, Dias, J. M., Than M. E., Humm A., Huber R., Bourenkov G. P., Bartunik H. D., Bursakov S., Calvete J., Caldeira J., Carneiro C., Moura J. J., Moura I., and Romao M. J. , Structure, Jan 15, Volume 7, Number 1, p.65-79, (1999) AbstractWebsite

BACKGROUND: The periplasmic nitrate reductase (NAP) from the sulphate reducing bacterium Desulfovibrio desulfuricans ATCC 27774 is induced by growth on nitrate and catalyses the reduction of nitrate to nitrite for respiration. NAP is a molybdenum-containing enzyme with one bis-molybdopterin guanine dinucleotide (MGD) cofactor and one [4Fe-4S] cluster in a single polypeptide chain of 723 amino acid residues. To date, there is no crystal structure of a nitrate reductase. RESULTS: The first crystal structure of a dissimilatory (respiratory) nitrate reductase was determined at 1.9 A resolution by multiwavelength anomalous diffraction (MAD) methods. The structure is folded into four domains with an alpha/beta-type topology and all four domains are involved in cofactor binding. The [4Fe-4S] centre is located near the periphery of the molecule, whereas the MGD cofactor extends across the interior of the molecule interacting with residues from all four domains. The molybdenum atom is located at the bottom of a 15 A deep crevice, and is positioned 12 A from the [4Fe-4S] cluster. The structure of NAP reveals the details of the catalytic molybdenum site, which is coordinated to two MGD cofactors, Cys140, and a water/hydroxo ligand. A facile electron-transfer pathway through bonds connects the molybdenum and the [4Fe-4S] cluster. CONCLUSIONS: The polypeptide fold of NAP and the arrangement of the cofactors is related to that of Escherichia coli formate dehydrogenase (FDH) and distantly resembles dimethylsulphoxide reductase. The close structural homology of NAP and FDH shows how small changes in the vicinity of the molybdenum catalytic site are sufficient for the substrate specificity.

Crystal structure of the xanthine oxidase-related aldehyde oxido-reductase from D. gigas, Romao, M. J., Archer M., Moura I., Moura J. J., Legall J., Engh R., Schneider M., Hof P., and Huber R. , Science, Nov 17, Volume 270, Number 5239, p.1170-6, (1995) AbstractWebsite

The crystal structure of the aldehyde oxido-reductase (Mop) from the sulfate reducing anaerobic Gram-negative bacterium Desulfovibrio gigas has been determined at 2.25 A resolution by multiple isomorphous replacement and refined. The protein, a homodimer of 907 amino acid residues subunits, is a member of the xanthine oxidase family. The protein contains a molybdopterin cofactor (Mo-co) and two different [2Fe-2S] centers. It is folded into four domains of which the first two bind the iron sulfur centers and the last two are involved in Mo-co binding. Mo-co is a molybdenum molybdopterin cytosine dinucleotide. Molybdopterin forms a tricyclic system with the pterin bicycle annealed to a pyran ring. The molybdopterin dinucleotide is deeply buried in the protein. The cis-dithiolene group of the pyran ring binds the molybdenum, which is coordinated by three more (oxygen) ligands.

Crystal structure of the zinc-, cobalt-, and iron-containing adenylate kinase from Desulfovibrio gigas: a novel metal-containing adenylate kinase from Gram-negative bacteria, Mukhopadhyay, A., Kladova A. V., Bursakov S. A., Gavel O. Y., Calvete J. J., Shnyrov V. L., Moura I., Moura J. J., Romao M. J., and Trincao J. , J Biol Inorg Chem, Jan, Volume 16, Number 1, p.51-61, (2011) AbstractWebsite

Adenylate kinases (AK) from Gram-negative bacteria are generally devoid of metal ions in their LID domain. However, three metal ions, zinc, cobalt, and iron, have been found in AK from Gram-negative bacteria. Crystal structures of substrate-free AK from Desulfovibrio gigas with three different metal ions (Zn(2+), Zn-AK; Co(2+), Co-AK; and Fe(2+), Fe-AK) bound in its LID domain have been determined by X-ray crystallography to resolutions 1.8, 2.0, and 3.0 A, respectively. The zinc and iron forms of the enzyme were crystallized in space group I222, whereas the cobalt-form crystals were C2. The presence of the metals was confirmed by calculation of anomalous difference maps and by X-ray fluorescence scans. The work presented here is the first report of a structure of a metal-containing AK from a Gram-negative bacterium. The native enzyme was crystallized, and only zinc was detected in the LID domain. Co-AK and Fe-AK were obtained by overexpressing the protein in Escherichia coli. Zn-AK and Fe-AK crystallized as monomers in the asymmetric unit, whereas Co-AK crystallized as a dimer. Nevertheless, all three crystal structures are very similar to each other, with the same LID domain topology, the only change being the presence of the different metal atoms. In the absence of any substrate, the LID domain of all holoforms of AK was present in a fully open conformational state. Normal mode analysis was performed to predict fluctuations of the LID domain along the catalytic pathway.

Crystallization and crystallographic analysis of the apo form of the orange protein (ORP) from Desulfovibrio gigas, Najmudin, S., Bonifacio C., Duarte A. G., Pauleta S. R., Moura I., Moura J. J., and Romao M. J. , Acta Crystallogr Sect F Struct Biol Cryst Commun, Jul 1, Volume 65, Number Pt 7, p.730-2, (2009) AbstractWebsite

The orange-coloured protein (ORP) from Desulfovibrio gigas is a 12 kDa protein that contains a novel mixed-metal sulfide cluster of the type [S(2)MoS(2)CuS(2)MoS(2)]. Diffracting crystals of the apo form of ORP have been obtained. Data have been collected for the apo form of ORP to 2.25 A resolution in-house and to beyond 2.0 A resolution at ESRF, Grenoble. The crystals belonged to a trigonal space group, with unit-cell parameters a = 43, b = 43, c = 106 A.

Crystallization and preliminary X-ray analysis of a membrane-bound nitrite reductase from Desulfovibrio desulfuricans ATCC 27774, Dias, J. M., Cunha C. A., Teixeira S., Almeida G., Costa C., Lampreia J., Moura J. J., Moura I., and Romao M. J. , Acta Crystallogr D Biol Crystallogr, Feb, Volume 56, Number Pt 2, p.215-7, (2000) AbstractWebsite

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.

Crystallization and preliminary x-ray analysis of a nitrate reductase from Desulfovibrio desulfuricans ATCC 27774, Dias, J. M., Bursakov S., Carneiro C., Moura J. J., Moura I., and Romao M. J. , Acta Crystallogr D Biol Crystallogr, Apr, Volume 55, Number Pt 4, p.877-9, (1999) AbstractWebsite

Periplasmic nitrate reductase from the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 contains two molybdopterin guanine dinucleotide cofactors and one [4Fe-4S] cluster as prosthetic groups and catalyzes the conversion of nitrate to nitrite. Crystals of the oxidized form of this enzyme were obtained using PEG as precipitant and belong to space group P3121 or P3221, with unit-cell dimensions a = b = 106.3, c = 135.1 A. There is one monomer of 80 kDa in the asymmetric unit, which corresponds to a Matthews ratio of 2.75 A3 Da-1. Using cryo-cooling procedures and X-rays from a rotating-anode generator, diffraction was observed to beyond 3.0 A resolution.