Publications

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Book Chapter
Novel structures in iron-sulfur proteinsBonding Problems, Xavier, António, Moura José, and Moura Isabel , Volume 43, p.187-213, (1981) Abstract
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Journal Article
Amino acid sequence of a 3Fe:3S ferredoxin from the "archaebacterium" Methanosarcina barkeri (DSM 800), Hausinger, R. P., Moura I., Moura J. J., Xavier A. V., Santos M. H., Legall J., and Howard J. B. , J Biol Chem, Dec 10, Volume 257, Number 23, p.14192-7, (1982) AbstractWebsite

The complete amino acid sequence for a 3Fe:3S ferredoxin from the "archaebacterium" Methanosarcina barkeri (DSM 800) was determined by repetitive Edman degradation on the whole protein and peptides derived from trypsin, thermolysin, and Staphylococcus aureus protease digestion. The protein has 59 residues of which 8 are cysteines. The latter have the same spacing and distribution as found for the clostridial-type 2 x 4Fe:4S ferredoxins. Also, the sequence had evidence of internal homology which is indicative of gene duplication prior to the divergence of the archaebacteria and the eubacteria. This is the first sequence to be reported for a methanogen ferredoxin and only the fourth for a 3Fe:3S ferredoxin from any source.

The amino acid sequence of desulforedoxin, a new type of non heme iron protein from Desulfovibrio gigas, Bruschi, M., Moura I., Legall J., Xavier A. V., and Sieker L. C. , Biochemical and Biophysical Research Communications, Volume 90, Number 2, p.596-605, (1979) AbstractWebsite
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Characterization Of Electron-Transfer Proteins From The Nitrogen-Fixing Sulfate-Reducing Bacterium Desulfovibrio-Desulfuricans Berre-Eau, Fauque, G., Moura I., Xavier A. V., Galliano N., Moura J. J. G., and Legall J. , Biochemical Society Transactions, Dec, Volume 15, Number 6, p.1049-1050, (1987) AbstractWebsite
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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.

Characterization of two dissimilatory sulfite reductases (desulforubidin and desulfoviridin) from the sulfate-reducing bacteria. Moessbauer and EPR studies, Moura, I., Legall J., Lino A. R., Peck H. D., Fauque G., Xavier A. V., Dervartanian D. V., Moura J. J. G., and Huynh B. H. , Journal of the American Chemical Society, 1988/02/17, Volume 110, Number 4, p.1075-1082, (1988) AbstractWebsite
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Cobalt containing B12 cofactors from methanogenic bacteria - spectroscopic characterization, Lino, A. R., Xavier A. V., Moura I., Legall J., and Ljungdahl P. O. , Rev Portuguesa de Química, Volume 27, p.175-177, (1985) Abstract
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A cobalt containing protein isolated from Desulfovibrio gigas, a sulfate reducer, Moura, J. J., Moura I., Bruschi M., Legall J., and Xavier A. V. , Biochem Biophys Res Commun, Feb 12, Volume 92, Number 3, p.962-70, (1980) AbstractWebsite
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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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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
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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.

Cytochrome components of nitrate- and sulfate-respiring Desulfovibrio desulfuricans ATCC 27774, Liu, M. C., Costa C., Coutinho I. B., Moura J. J., Moura I., Xavier A. V., and Legall J. , J Bacteriol, Dec, Volume 170, Number 12, p.5545-51, (1988) AbstractWebsite

Three multiheme c-type cytochromes--the tetraheme cytochrome c3 (molecular weight [MW] 13,500), a dodecaheme cytochrome c (MW 40,800), and a "split-Soret" cytochrome c (MW 51,540), which is a dimer with 2 hemes per subunit (MW 26,300)--were isolated from the soluble fraction of Desulfovibrio desulfuricans (ATCC 27774) grown under nitrate- or sulfate-respiring conditions. Two of them, the dodecaheme and the split-Soret cytochromes, showed no similarities to any of the c-type cytochromes isolated from other sulfate-reducing bacteria, while the tetraheme cytochrome c3 appeared to be analogous to the cytochrome c3 found in other sulfate-reducing bacteria. For all three multiheme c-type cytochromes isolated, the homologous proteins from nitrate- and sulfate-grown cells were indistinguishable in amino acid composition, physical properties, and spectroscopic characteristics. It therefore appears that the same c-type cytochrome components are present when D. desulfuricans ATCC 27774 cells are grown under either condition. This is in contrast to the considerable difference found in Pseudomonas perfectomarina (Liu et al., J. Bacteriol. 154:278-286, 1983), a marine denitrifier, when the cells are grown on nitrate or oxygen as the terminal electron acceptor. In addition, two spectroscopy methods capable of revealing minute structural variations in proteins provided identical information about the tetraheme cytochrome c3 from nitrate-grown and sulfate-grown cells.

Desulforedoxin: Preliminary X-ray diffraction study of a new iron-containing protein, Sieker, L. C., Jensen L. H., Bruschi M., Legall J., Moura I., and Xavier A. V. , Journal of Molecular Biology, Volume 144, Number 4, p.593-594, (1980) AbstractWebsite
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Desulforedoxin: proposed configuration and preliminary X-ray diffraction study of a two-iron two chain protein, Sieker, L. C., Bruschi M., Legall J., Moura I., and Xavier A. V. , Ciênc. Biol. (Portugal), Volume 5, p.145-147, (1980) Abstract
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Desulfovibrio Gigas hydrogenase: redox properties of the nickel and iron-sulfur centers, Teixeira, M., Moura I., Xavier A. V., Dervartanian D. V., Legall J., Peck, H. D. Jr., Huynh B. H., and Moura J. J. , Eur J Biochem, Feb 15, Volume 130, Number 3, p.481-4, (1983) AbstractWebsite

Below 30 K, oxidized Desulfovibrio gigas hydrogenase presents an intense electron paramagnetic resonance (EPR) signal centered at g = 2.02, typical of an iron-sulfur center. In addition a rhombic EPR signal, attributed to Ni(III) species, is also observed [LeGall, J., Ljungdahl, P., Moura, I., Peck, H.D., Jr, Xavier, A.V., Moura, J.J.G., Teixeira, M., Huynh, B.H., and DerVartanian, D.V. (1982) Biochem. Biophys. Res. Commun. 106, 610-616; and Cammack, R., Patil, D., Aguirre, R., and Hatchikian, E.C., (1982) FEBS Lett. 142, 289-292]. At higher temperatures (77 K) the iron-sulfur EPR signal is broader and all the EPR features of the rhombic nickel signal can easily be observed. We have now obtained additional information concerning the redox properties of these EPR active centers, using an EPR redox titration method in the presence of dye mediators at pH = 8.5. The mid-point potential was determined to be -70 mV for the Fe,S cluster and -220 mV for the Ni center. Intermediate oxidation states were obtained upon partial reduction with either dithionite or hydrogen. Although upon dithionite reduction the centers are reduced in the order of decreasing mid-point reduction potentials, under a hydrogen atmosphere the nickel center reduces preferentially. This suggests a catalytic involvement of the nickel redox center in the binding of hydrogen. Preliminary Mossbauer studies on Desulfovibrio gigas hydrogenase reveal the presence of a paramagnetic 3 Fe center and two 4 Fe centers. The 3 Fe center is responsible for the g = 2.02 EPR signal but the two 4 Fe centers have been so far undetectable by EPR.

Electron paramagnetic resonance studies on the mechanism of activation and the catalytic cycle of the nickel-containing hydrogenase from Desulfovibrio gigas, Teixeira, M., Moura I., Xavier A. V., Huynh B. H., Dervartanian D. V., Peck, H. D. Jr., Legall J., and Moura J. J. , J Biol Chem, Jul 25, Volume 260, Number 15, p.8942-50, (1985) AbstractWebsite

Desulfovibrio gigas hydrogenase (EC 1.12.2.1) is a complex enzyme containing one nickel, one 3Fe, and two [Fe4S4] clusters (Teixeira, M., Moura, I., Xavier, A. V., Der Vartanian, D. V., LeGall, J., Peck, H. D., Jr., Huynh, B. H., and Moura, J. J. G. (1983) Eur. J. Biochem. 130, 481-484). This hydrogenase belongs to a class of enzymes that are inactive "as isolated" (the so-called "oxygen-stable hydrogenases") and must go through an activation process in order to express full activity. The state of characterization of the active centers of the enzyme as isolated prompted us to do a detailed analysis of the redox patterns, activation profile, and catalytic redox cycle of the enzyme in the presence of either the natural substrate (H2) or chemical reductants. The effect of natural cofactors, as cytochrome C3, was also studied. Special focus was given to the intermediate redox species generated during the catalytic cycle of the enzyme and to the midpoint redox potentials associated. The available information is discussed in terms of a "working hypothesis" for the mechanism of the [NiFe] hydrogenases from sulfate reducing organisms in the context of activation process and catalytic cycle.

Electron transfer mechanism studies of cytochrome c3: pH dependence of the redox equilibria, Santos, H., Moura J. J. G., Xavier A. V., and Legall J. , Inorganica Chimica Acta, Volume 79, p.167-169, (1983) AbstractWebsite
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Electron transport in sulfate-reducing bacteria. Molecular modeling and NMR studies of the rubredoxin--tetraheme-cytochrome-c3 complex, Stewart, D. E., Legall J., Moura I., Moura J. J., Peck, H. D. Jr., Xavier A. V., Weiner P. K., and Wampler J. E. , Eur J Biochem, Nov 20, Volume 185, Number 3, p.695-700, (1989) AbstractWebsite

A hypothetical model of the complex formed between the iron-sulfur protein rubredoxin and the tetraheme cytochrome c3 from the sulfate-reducing bacteria Desulfovibrio vulgaris (Hildenborough) has been proposed utilizing computer graphic modeling, computational methods and NMR spectroscopy. The proposed complex appears feasible on the basis of complementary electrostatic interaction and steric factors and is consistent with the data from NMR experiments. In this model, the non-heme iron atom of rubredoxin is in close proximity to heme 1 of cytochrome c3. The complex is stabilized by charge-pair interactions and hydrogen bonds. This complex is compared to the flavodoxin-cytochrome c3 complex previously proposed [Stewart, D. E., LeGall, J., Moura, I., Moura, J. J. G., Peck, H. D. Jr, Xavier, A. V., Weiner, P. K. & Wampler, J. E. (1988) Biochemistry 27, 2444-2450] and new NMR data shows that both proteins interact with the same heme group of the cytochrome as postulated.

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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Evidence for a three-iron center in a ferredoxin from Desulfovibrio gigas. Mossbauer and EPR studies, Huynh, B. H., Moura J. J., Moura I., Kent T. A., Legall J., Xavier A. V., and Munck E. , J Biol Chem, Apr 25, Volume 255, Number 8, p.3242-4, (1980) AbstractWebsite

The tetrameric form of a Desulfovibrio gigas ferredoxin, named Fd II, mediates electron transfer between cytochrome c3 and sulfite reductase. We have studied two stable oxidation states of this protein with Mossbauer spectroscopy and electron paramagnetic resonance. We found 3 iron atoms/monomer and a spin concentration of 0.9 spins/monomer for the oxidized protein. Taken together, the EPR and Mossbauer data demonstrate conclusively the presence of a spin-coupled structure containing 3 iron atoms and labile sulfur. The Mossbauer data show also that this metal center is structurally similar, if not identical, with the low potential center of a ferredoxin from Azotobacter vinelandii, a novel cluster described recently (Emptage, M.H., Kent, T.A., Huynh, B.H., Rawlings, J., Orme-Johnson, W.H., and Munck, E. (1980) J. Biol. Chem. 255, 1793-1796).

Evidence for nickel and a three-iron center in the hydrogenase of Desulfovibrio desulfuricans, Kruger, H. J., Huynh B. H., Ljungdahl P. O., Xavier A. V., Dervartanian D. V., Moura I., Peck, H. D. Jr., Teixeira M., Moura J. J., and Legall J. , J Biol Chem, Dec 25, Volume 257, Number 24, p.14620-3, (1982) AbstractWebsite

Hydrogenase from Desulfovibrio desulfuricans (ATCC No. 27774) grown in unenriched and in enriched 61Ni and 57Fe media has been purified to apparent homogeneity. Two fractions of enzymes with hydrogenase activity were separated and were termed hydrogenase I and hydrogenase II. they were shown to have similar molecular weights (77,600 for hydrogenase I and 75,500 for hydrogenase II), to be composed of two polypeptide chains, and to contain Ni and non-heme iron. Because of its higher specific activity (152 versus 97) hydrogenase II was selected for EPR and Mossbauer studies. As isolated, hydrogenase II exhibits an "isotropic" EPR signal at g = 2.02 and a rhombic EPR signal at g = 2.3, 2.2, and 2.0. Isotopic substitution of 61Ni proves that the rhombic signal is due to Ni. Combining the Mossbauer and EPR data, the isotropic g = 2.02 EPR signal was shown to originate from a 3Fe cluster which may have oxygenous or nitrogenous ligands. In addition, the Mossbauer data also revealed two [4Fe-4S]2+ clusters iun each molecule of hydrogenase II. The EPR and Mossbauer data of hydrogenase I were found to be identical to those of hydrogenase II, indicating that both enzymes have common metallic centers.

Ferredoxin from Methanosarcina barkeri: evidence for the presence of a three-iron center, Moura, I., Moura J. J., Huynh B. H., Santos H., Legall J., and Xavier A. V. , Eur J Biochem, Aug, Volume 126, Number 1, p.95-8, (1982) AbstractWebsite

Methanosarcina barkeri ferredoxin was purified and characterized by electron paramagnetic resonance (EPR) and Mossbauer spectroscopy. The purification procedure included chromatographic steps on DEAE-cellulose and gel filtration. The isolated protein is unstable under aerobic conditions. The ferredoxin exhibits charge transfer bands at 283 nm and 405 nm with an absorption ratio A405/A283 = 0.73. Its molecular weight has been estimated to be 20000-22000 by gel filtration chromatography. The native ferredoxin exhibits an intense EPR signal at g = 2.02 and only a very weak g = 1.94 signal develops upon reduction with dithionite. The Mossbauer spectra of the reduced protein are characteristic of a [3Fe-3S] center. The combined EPR and Mossbauer studies show that M. barkeri ferredoxin contains only [3Fe-3S] clusters, similar to Azotobacter vinelandii Fd[Emptage, M.H., Kent, T.A., Huynh, B.H., Rawlings, J., Orme-Johnson, W.H. & Munck, M. (1980) J. Biol. Chem. 255, 1793-1796], Desulfovibrio gigas FdII [Huynh, B.H., Moura, J.J.G., Moura, I., Kent, T.A., LeGall, J., Xavier, A.V. & Munck, E. (1980) J. Biol. Chem. 255, 3242-3244] and mitochondrial beef heart aconitase [Kent, T.A., Dreyer, J.-L., Kennedy, M.C., Huynh, B.H., Emptage, M.H., Beinert, H. & Munck, E. (1982) Proc. Natl Acad. Sci. USA, 79, 1096-1100].

Hydrogen production and deuterium-proton exchange reactions catalyzed by Desulfovibrio nickel(II)-substituted rubredoxins, Saint-Martin, P., Lespinat P. A., Fauque G., Berlier Y., Legall J., Moura I., Teixeira M., Xavier A. V., and Moura J. J. , Proc Natl Acad Sci U S A, Dec, Volume 85, Number 24, p.9378-80, (1988) AbstractWebsite

The nickel tetrahedral sulfur-coordinated core formed upon metal replacement of the native iron in Desulfovibrio sp. rubredoxins is shown to mimic the reactivity pattern of nickel-containing hydrogenases with respect to hydrogen production, deuterium-proton exchange, and inhibition by carbon monoxide.

A hypothetical model of the flavodoxin-tetraheme cytochrome c3 complex of sulfate-reducing bacteria, Stewart, D. E., Legall J., Moura I., Moura J. J., Peck, H. D. Jr., Xavier A. V., Weiner P. K., and Wampler J. E. , Biochemistry, Apr 5, Volume 27, Number 7, p.2444-50, (1988) AbstractWebsite

A hypothetical model of the flavodoxin-tetraheme cytochrome c3 electron-transfer complex from the sulfate-reducing bacterium Desulfovibrio vulgaris has been constructed by using interactive computer graphics based on electrostatic potential field calculations and previous NMR experiments. Features of the proposed complex are (1) van der Waals contact between the flavin mononucleotide prosthetic group of flavodoxin and one heme of the cytochrome, (2) unique complementarity of electrostatic fields between the region surrounding this heme and the region surrounding the exposed portion of the flavin mononucleotide group of flavodoxin, and (3) no steric interferences between the two polypeptide chains in the complex. This complex is consistent with all structural and spectroscopic data available.