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)
AbstractThe 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.
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)
AbstractThe 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)
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)
AbstractTwo 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.
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)
AbstractBACKGROUND: 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 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)
AbstractAdenylate 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)
AbstractThe 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 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)
AbstractPeriplasmic 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.
Crystallization and preliminary X-ray diffraction analysis of the 16-haem cytochrome of Desulfovibrio gigas,
Santos-Silva, T., Diasa J. M., Bourenkov G., Bartunik H., Moura I., and Romao M. J.
, Acta Crystallographica Section D-Biological Crystallography, May, Volume 60, p.968-970, (2004)
AbstractHigh-molecular-weight cytochromes (Hmcs) belong to a large family of multihaem cytochromes in sulfate-reducing bacteria. HmcA is the first cytochrome reported to have 16 c-type haems arranged in its polypeptide chain. The function of this cytochrome is still unknown, although it is clear that it belongs to a membrane-bound complex involved in electron transfer from the periplasm to the membrane. HmcA from Desulfovibrio gigas has been purified and successfully crystallized using the hanging-drop vapour-diffusion method. The crystals grew using PEG and zinc acetate as precipitants to maximum dimensions of 0.2 x 0.2 x 0.2 mm in an orthorhombic space group, with unit-cell parameters a = 88.9, b = 90.9, c = 83.7 Angstrom. The crystals diffracted to beyond 2.07 Angstrom and a MAD data set was collected.
Crystallization and preliminary X-ray diffraction analysis of the di-haem cytochrome c peroxidase from Pseudomonas stutzeri,
Bonifácio, Cecília, Cunha Carlos A., Müller Axel, Timóteo Cristina G., Dias João M., Moura Isabel, and Romão Maria João
, Acta Crystallographica Section D, Volume 59, Number 2, p.345-347, (2003)
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Crystallization and preliminary X-ray diffraction analysis of two pH-dependent forms of a di-haem cytochrome c peroxidase from Pseudomonas nautica,
Dias, João M., Bonifácio Cecília, Alves Teresa, Moura José J. G., Moura Isabel, and Romão Maria João
, Acta Crystallographica Section D, Volume 58, Number 4, p.697-699, (2002)
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A cytochrome c peroxidase from Pseudomonas nautica 617 active at high ionic strength: expression, purification and characterization,
Alves, T., Besson S., Duarte L. C., Pettigrew G. W., Girio F. M. F., Devreese B., Vandenberghe I., Van Beeumen J., Fauque G., and Moura I.
, Biochimica Et Biophysica Acta-Protein Structure and Molecular Enzymology, Oct 12, Volume 1434, Number 2, p.248-259, (1999)
AbstractCytochrome c peroxidase was expressed in cells of Pseudomonas nautica strain 617 grown under microaerophilic conditions. The 36.5 kDa dihaemic enzyme was purified to electrophoretic homogeneity in three chromatographic steps. N-terminal sequence comparison showed that the Ps. nautica enzyme exhibits a high similarity with the corresponding proteins from Paracoccus denitrificans and Pseudomonas aeruginosa. UV-visible spectra confirm calcium activation of the enzyme through spin state transition of the peroxidatic haem. Monohaemic cytochrome c(552) from Ps. nautica was identified as the physiological electron donor, with a half-saturating concentration of 122 mu M and allowing a maximal catalytic centre activity of 116 000 min(-1). Using this cytochrome the enzyme retained the same activity even at high ionic strength. There are indications that the interactions between the two redox partners are mainly hydrophobic in nature. (C) 1999 Elsevier Science B.V. All rights reserved.
A cytochrome cd1-type nitrite reductase isolated from the marine denitrifier Pseudomonas nautica 617: purification and characterization,
Besson, S., Carneiro C., Moura J. J., Moura I., and Fauque G.
, Anaerobe, Aug, Volume 1, Number 4, p.219-26, (1995)
AbstractNitrite reductase (cytochrome cd1) was purified to electrophoretic homogeneity from the soluble extract of the marine denitrifying bacterium Pseudomonas nautica strain 617. Cells were anaerobically grown with 10 mM nitrate as final electron acceptor. The soluble fraction was purified by four successive chromatographic steps and the purest cytochrome cd1 exhibited an A280 nm(oxidized)/A410nm(oxidized) coefficient of 0.90. In the course of purification, cytochrome cd1 specific activity presented a maximum value of 0.048 units/mg of protein. This periplasmic enzyme is a homodimer and each 60 kDa subunit contains one heme c and one heme d1 as prosthetic moieties, both in a low spin state. Redox potentials of hemes c and d1 were determined at three different pH values (6.6, 7.6 and 8.6) and did not show any pH dependence. The first 20 amino acids of the NH2-terminal region of the protein were identified and the sequence showed 45% identity with the corresponding region of Pseudomonas aeruginosa nitrite reductase but no homology to Pseudomonas stutzeri and Paracoccus denitrificans enzymes. Spectroscopic properties of Pseudomonas nautica 617 cytochrome cd1 in the ultraviolet-visible range and in electron paramagnetic resonance are described. The formation of a heme d1 -nitric-oxide complex as an intermediate of nitrite reduction was demonstrated by electron paramagnetic resonance experiments.