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ArsC3 from Desulfovibrio alaskensis G20, a cation and sulfate-independent highly efficient arsenate reductase, Nunes, C. I., Brás J. L., Najmudin S., Moura J. J. G., Moura I., and Carepo M. , J Biol Inorg Chem, Volume 19, p.1277-1285, (2014)
Evaluation of Sweat Sampling Procedures for Human Stress Biomarkers Detection, Nunes, M. J., Moura J. J. G., Noronha J. P., Branco L. C., Samhan-Arias A., Sousa J. P., Rouco C., and Cordas C. , Analytica, Volume 3, p.178–194, (2022)
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.

Periplasmic nitrate reductase revisited: a sulfur atom completes the sixth coordination of the catalytic molybdenum, Najmudin, S., Gonzalez P. J., Trincao J., Coelho C., Mukhopadhyay A., Cerqueira N. M., Romao C. C., Moura I., Moura J. J., Brondino C. D., and Romao M. J. , J Biol Inorg Chem, Jun, Volume 13, Number 5, p.737-53, (2008) AbstractWebsite

Nitrate reductase from Desulfovibrio desulfuricans ATCC 27774 (DdNapA) is a monomeric protein of 80 kDa harboring a bis(molybdopterin guanine dinucleotide) active site and a [4Fe-4S] cluster. Previous electron paramagnetic resonance (EPR) studies in both catalytic and inhibiting conditions showed that the molybdenum center has high coordination flexibility when reacted with reducing agents, substrates or inhibitors. As-prepared DdNapA samples, as well as those reacted with substrates and inhibitors, were crystallized and the corresponding structures were solved at resolutions ranging from 1.99 to 2.45 A. The good quality of the diffraction data allowed us to perform a detailed structural study of the active site and, on that basis, the sixth molybdenum ligand, originally proposed to be an OH/OH(2) ligand, was assigned as a sulfur atom after refinement and analysis of the B factors of all the structures. This unexpected result was confirmed by a single-wavelength anomalous diffraction experiment below the iron edge (lambda = 1.77 A) of the as-purified enzyme. Furthermore, for six of the seven datasets, the S-S distance between the sulfur ligand and the Sgamma atom of the molybdenum ligand Cys(A140) was substantially shorter than the van der Waals contact distance and varies between 2.2 and 2.85 A, indicating a partial disulfide bond. Preliminary EPR studies under catalytic conditions showed an EPR signal designated as a turnover signal (g values 1.999, 1.990, 1.982) showing hyperfine structure originating from a nucleus of unknown nature. Spectropotentiometric studies show that reduced methyl viologen, the electron donor used in the catalytic reaction, does not interact directly with the redox cofactors. The turnover signal can be obtained only in the presence of the reaction substrates. With use of the optimized conditions determined by spectropotentiometric titration, the turnover signal was developed with (15)N-labeled nitrate and in D(2)O-exchanged DdNapA samples. These studies indicate that this signal is not associated with a Mo(V)-nitrate adduct and that the hyperfine structure originates from two equivalent solvent-exchangeable protons. The new coordination sphere of molybdenum proposed on the basis of our studies led us to revise the currently accepted reaction mechanism for periplasmic nitrate reductases. Proposals for a new mechanism are discussed taking into account a molybdenum and ligand-based redox chemistry, rather than the currently accepted redox chemistry based solely on the molybdenum atom.

The 1.4 angstrom resolution structure of Paracoccus pantotrophus pseudoazurin, Najmudin, Shabir, Pauleta Sofia R., Moura Isabel, and Romao Maria J. , Acta Crystallographica Section F-Structural Biology and Crystallization Communications, Jun, Volume 66, p.627-635, (2010) AbstractWebsite

Pseudoazurins are small type 1 copper proteins that are involved in the flow of electrons between various electron donors and acceptors in the bacterial periplasm, mostly under denitrifying conditions. The previously determined structure of Paracoccus pantotrophus pseudoazurin in the oxidized form was improved to a nominal resolution of 1.4 angstrom, with R and R(free) values of 0.188 and 0.206, respectively. This high-resolution structure makes it possible to analyze the interactions between the monomers and the solvent structure in detail. Analysis of the high-resolution structure revealed the structural regions that are responsible for monomer-monomer recognition during dimer formation and for protein-protein interaction and that are important for partner recognition. The pseudoazurin structure was compared with other structures of various type 1 copper proteins and these were grouped into families according to similarities in their secondary structure; this may be useful in the annotation of copper proteins in newly sequenced genomes and in the identification of novel copper proteins.

Changes in metabolic pathways of Desulfovibrio alaskensis G20 cells induced by molybdate excess, Nair, R. R., Silveira C. M., Diniz M. S., Almeida M. G., Moura J. J. G., and Rivas M. G. , J Biol Inorg Chem, Volume 20, p.311–322, (2015)
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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.

Influence of storage solution on enamel demineralization submitted to pH cycling, Moura, J. S., Rodrigues L. K., Del Bel Cury A. A., Lima E. M., and Garcia R. M. , J Appl Oral Sci, Sep, Volume 12, Number 3, p.205-8, (2004) AbstractWebsite

Extracted human teeth are frequently used for research or educational purposes. Therefore, it is necessary to store them in disinfectant solutions that do not alter dental structures. Thus, this study evaluated the influence of storage solution on enamel demineralization. For that purpose, sixty samples were divided into the following groups: enamel stored in formaldehyde (F1), stored in thymol (T1), stored in formaldehyde and submitted to pH cycling (F2), stored in thymol and submitted to pH cycling (T2). All samples were evaluated by cross-sectional microhardness analysis and had their percentage of mineral volume versus micrometer (integrated area) determined. Differences between groups were found up to 30-microm depth from the enamel surface (p < 0.05), where samples from group T2 were more demineralized. It was concluded that the storage solution influenced the reaction of a dental substrate to a cariogenic challenge, suggesting that formaldehyde may increase enamel resistance to demineralization, when compared to demineralization occurring in enamel stored in thymol solution.

Structural aspects of denitrifying enzymes, Moura, I., and Moura J. J. , Curr Opin Chem Biol, Apr, Volume 5, Number 2, p.168-75, (2001) AbstractWebsite

The reduction of nitrate to nitrogen gas via nitrite, nitric oxide and nitrous oxide is the metabolic pathway usually known as denitrification, a key step in the nitrogen cycle. As observed for other elemental cycles, a battery of enzymes are utilized, namely the reductases for nitrate, nitrite, nitric oxide and nitrous oxide, as well as multiple electron donors that interact with these enzymes, in order to carry out the stepwise reactions that involve key intermediates. Because of the importance of this pathway (of parallel importance to the nitrogen-fixation pathway), efforts are underway to understand the structures of the participating enzymes and to uncover mechanistic aspects. Three-dimensional structures have been solved for the majority of these enzymes in the past few years, revealing the architecture of the active metal sites as well as global structural aspects, and possible mechanistic aspects. In addition, the recognition of specific electron-transfer partners raises important questions regarding specific electron-transfer pathways, partner recognition and control of metabolism.

A comparative spectroscopic study of two non-haem iron proteins lacking labile sulphide from Desulphovibrio gigas, Moura, I., Xavier A. V., Cammack R., Bruschi M., and Legall J. , Biochimica et Biophysica Acta (BBA) - Protein Structure, Volume 533, Number 1, p.156-162, (1978) AbstractWebsite
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[15] Characterization of three proteins containing multiple iron sites: Rubrerythrin, desulfoferrodoxin, and a protein containing a six-iron cluster, Moura, Isabel, Tavares Pedro, and Ravi Natarajan , Methods in Enzymology, Volume Volume 243, p.216-240, (1994) Abstract
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Structural and functional approach toward a classification of the complex cytochrome c system found in sulfate-reducing bacteria, Moura, J. J., Costa C., Liu M. Y., Moura I., and Legall J. , Biochim Biophys Acta, May 23, Volume 1058, Number 1, p.61-6, (1991) AbstractWebsite

Following the discovery of the tetraheme cytochrome c3 in the strict anaerobic sulfate-reducing bacteria (Postgate, J.R. (1954) Biochem. J. 59, xi; Ishimoto et al. (1954) Bull. Chem. Soc. Japan 27, 564-565), a variety of c-type cytochromes (and others) have been reported, indicating that the array of heme proteins in these bacteria is complex. We are proposing here a tentative classification of sulfate- (and sulfur-) reducing bacteria cytochromes c based on: number of hemes per monomer, heme axial ligation, heme spin state and primary structures (whole or fragmentary). Different and complementary spectroscopic tools have been used to reveal the structural features of the heme sites.

The Role Of Nickel And Iron Sulfur Centers In The Bioproduction Of Hydrogen, Moura, J. J. G., Teixeira M., and Moura I. , Pure and Applied Chemistry, May, Volume 61, Number 5, p.915-921, (1989) AbstractWebsite
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Molybdenum and tungsten enzymes: from biology to chemistry and back, Moura, J. J. G., Bernhardt P. V., Maia L. B., and Gonzalez P. J. , J Biol Inorg Chem, Volume 20, p.181-182, (2015)
Nitrate and nitrite utilization in sulfate-reducing bacteria, Moura, I., Bursakov S., Costa C., and Moura J. J. , Anaerobe, Oct, Volume 3, Number 5, p.279-90, (1997) AbstractWebsite
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Redox studies on rubredoxins from sulphate and sulphur reducing bacteria, Moura, I., Moura J. J., Santos M. H., Xavier A. V., and Legall J. , FEBS Lett, Nov 15, Volume 107, Number 2, p.419-21, (1979) AbstractWebsite
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Interconversion from 3Fe into 4Fe clusters in the presence of Desulfovibrio gigas cell extracts, Moura, J. J., Legall J., and Xavier A. V. , Eur J Biochem, Jun 1, Volume 141, Number 2, p.319-22, (1984) AbstractWebsite

Desulfovibrio gigas ferredoxin II (FdII) contains a single 3Fe cluster [Huynh, B.H., Moura, J.J.G., Moura, I., Kent, T.A., LeGall, J., Xavier, A.V., and Munck, E. (1980) J. Biol. Chem. 255, 3242-3244]. In the oxidized state the protein exhibits an intense electron paramagnetic resonance (EPR) signal at g = 2.02. Upon one-electron reduction the center becomes EPR silent. In the presence of D. gigas crude cell extracts, devoid of acidic electron carriers and supplemented with pyruvate and FdII, an EPR signal typical of reduced [4Fe-4S] centers is obtained. The appearance of this signal correlates with the beginning of stimulation of the phosphoroclastic reaction as judged by the production of H2. These results, supported by the occurrence of easy chemical conversion of the 3Fe cluster of D. gigas ferredoxin into 4Fe structures [Moura, J.J.G., Moura, I., Kent, T.A., Lipscomb, J.D., Huynh, B.H., LeGall, J., Xavier, A.V., and Munch, E. (1982) J. Biol. Chem. 257, 6259-6267], suggest that cluster conversion takes place in conditions close to the situation in vivo. This cluster interconversion is discussed in the context of some of the relevant metabolic pathways of Desulfovibrio spp.

Unambiguous identification of the nickel EPR signal in 61Ni-enriched Desulfovibrio gigas hydrogenase, Moura, J. J., Moura I., Huynh B. H., Kruger H. J., Teixeira M., DuVarney R. C., Dervartanian D. V., Xavier A. V., Peck, H. D. Jr., and Legall J. , Biochem Biophys Res Commun, Oct 29, Volume 108, Number 4, p.1388-93, (1982) AbstractWebsite
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Evidence for the formation of a cobalt-iron-sulfur (CoFe3S4) cluster in Desulfovibrio gigas ferredoxin II, Moura, Isabel, Moura Jose J. G., Munck Eckard, Papaefthymiou Vasilios, and Legall Jean , Journal of the American Chemical Society, 1986/01/01, Volume 108, Number 2, p.349-351, (1986) AbstractWebsite
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Spectroscopic characterization of a high-potential monohaem cytochrome from Wolinella succinogenes, a nitrate-respiring organism. Redox and spin equilibria studies, Moura, I., Liu M. Y., Costa C., Liu M. C., Pai G., Xavier A. V., Legall J., Payne W. J., and Moura J. J. , Eur J Biochem, Nov 15, Volume 177, Number 3, p.673-82, (1988) AbstractWebsite

When purified, a high-potential c-type monohaem cytochrome from the nitrate-respiring organism, Wollinella succinogenes (VPI 10659), displayed a minimum molecular mass of 8.2 kDa and 0.9 mol iron and 0.95 mol haem groups/mol protein. Visible light spectroscopy suggested the presence of an equilibrium between two ligand arrangements around the haem, i.e. an absorption band at 695 nm characteristic of haem-methionine coordination (low-spin form) coexisting with a high-spin form revealed by a band at 619 nm and a shoulder at 498 nm. The mid-point redox potential measured by visible redox titration of the low-spin form was approximately +100 mV. Binding cyanide (Ka = 5 x 10(5) M-1) resulted in the displacement of the methionyl axial residue, and full conversion to a low-spin, cyanide-bound form. Structural features were studied by 300-MHz 1H-NMR spectroscopy. In the oxidized state, the pH dependence of the haem methyl resonances (pH range 5-10) and the magnetic susceptibility measurements (using an NMR method) were consistent with the visible light spectroscopic data for the presence of a high-spin/low-spin equilibrium with a transition pKa of 7.3. The spin equilibrium was fast on the NMR time scale. The haem methyl resonances presented large downfield chemical shifts. An unusually broad methyl resonance at around 35 ppm (pH = 7.5, 25 degrees C) was extremely temperature-dependent [delta(323 K) - delta(273 K) = 7.2 ppm] and was assigned to the S-CH3 group of the axial methionine. In the ferrous state only a low-spin form is present. The haem meso protons, the methyl group and the methylene protons from the axial methionine were identified in the reduced form. The resonances from the aromatic residues (three tyrosines and one phenylalanine) were also assigned. Detailed monitoring of the NMR-redox pattern of the monohaem cytochrome from the fully reduced up to the fully oxidized state revealed that the rate of the intermolecular electronic exchange process was approximately 6 x 10(6) M-1 s-1 at 303 K and pH = 6.31. A dihaem cytochrome also present in the crude cell extract and purified to a homogeneous state, exhibited a molecular mass of 11 kDa and contained 2.43 mol iron and 1.89 mol haem c moieties/mol cytochrome. The absorption spectrum in the visible region exhibited no band at 695 nm, suggesting that methione is not a ligand for either of the two haems. Recovery of only small amounts of this protein prevented more detailed structural analyzes.

EPR and Mossbauer studies of desulforedoxin from Desulfovibrio gigas, Moura, I., Huynh B., Legall J., Xavier A. V., and Munck E. , Ciênc. Biol. (Portugal), Volume 5, p.199-201, (1980) Abstract
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Analysis, design and engineering of simple iron-sulfur proteins: Tales from rubredoxin and desulforedoxin, Moura, J. J. G., Goodfellow B. J., Romao M. J., Rusnak F., and Moura I. , Comments on Inorganic Chemistry, 1996, Volume 19, Number 1, p.47-+, (1996) AbstractWebsite

The most thoroughly characterized non-heme iron center in biology is Rubredoxin, the simplest member of the iron-sulfur: class of metalloproteins. Rubredoxin contains a high-spin iron atom with tetrahedral coordination by four cysteinyl sulfur atoms. A structural variant of this center is found in Desulforedoxin, the smallest known Rubredoxin type protein. The 3D structure of both Rd and Dr has been determined at high resolution. These two proteins can therefore be used as case studies in which structural control by the polypeptide chain over the metal site can be discussed in detail.

Characterization of the cytochrome system of a nitrogen-fixing strain of a sulfate-reducing bacterium: Desulfovibrio desulfuricans strain Berre-Eau, Moura, I., Fauque G., Legall J., Xavier A. V., and Moura J. J. , Eur J Biochem, Feb 2, Volume 162, Number 3, p.547-54, (1987) AbstractWebsite

Two c-type cytochromes were purified and characterized by electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopic techniques, from the sulfate-reducer nitrogen-fixing organism, Desulfovibrio desulfuricans strain Berre-Eau (NCIB 8387). The purification procedures included several chromatographic steps on alumina, carboxymethylcellulose and gel filtration. A tetrahaem and a monohaem cytochrome were identified. The multihaem cytochrome has visible, EPR and NMR spectra with general properties similar to other low-potential bis-histidinyl axially bound haem proteins, belonging to the class of tetrahaem cytochrome c3 isolated from other Desulfovibrio species. The monohaem cytochrome c553 is ascorbate-reducible and its EPR and NMR data are characteristic of a cytochrome with methionine-histidine ligation. Their properties are compared with other homologous proteins isolated from sulfate-reducing bacteria.

NMR redox studies of Desulfovibrio vulgaris Cytochrome c3. Electron transfer mechanisms, Moura, J. J., Santos H., Moura I., Legall J., Moore G. R., Williams R. J., and Xavier A. V. , Eur J Biochem, Sep, Volume 127, Number 1, p.151-5, (1982) AbstractWebsite

The 300-MHz proton NMR spectra of the tetrahaem cytochrome c3 from Desulfovibrio vulgaris were examined while varying the pH and the redox potential. The analysis of the complete NMR reoxidation pattern was done taking into account all the 16 redox states that can be present in the redox titration of a tetra-redox-center molecule. A network of saturation transfer experiments performed at different oxidation stages, between the fully reduced and the fully oxidized states, allowed the observation of different resonances for some of the haem methyl groups. In the present experimental conditions, some of the haems show a fast intramolecular electron exchange rate, but the intermolecular electron exchange is always slow. In intermediate reoxidation stages, large shifts of the resonances of some haem methyl groups were observed upon changing the pH. These shifts are discussed in terms of a pH dependence of the haem midpoint redox potentials. The physiological relevance of this pH dependence is discussed.