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2002
Raaijmakers, H, Macieira S, Dias JM, Teixeira S, Bursakov S, Huber R, Moura JJ, Moura I, Romao MJ.  2002.  Gene sequence and the 1.8 A crystal structure of the tungsten-containing formate dehydrogenase from Desulfovibrio gigas, Sep. Structure. 10:1261-72., Number 9 AbstractWebsite

Desulfovibrio gigas formate dehydrogenase is the first representative of a tungsten-containing enzyme from a mesophile that has been structurally characterized. It is a heterodimer of 110 and 24 kDa subunits. The large subunit, homologous to E. coli FDH-H and to D. desulfuricans nitrate reductase, harbors the W site and one [4Fe-4S] center. No small subunit ortholog containing three [4Fe-4S] clusters has been reported. The structural homology with E. coli FDH-H shows that the essential residues (SeCys158, His159, and Arg407) at the active site are conserved. The active site is accessible via a positively charged tunnel, while product release may be facilitated, for H(+) by buried waters and protonable amino acids and for CO(2) through a hydrophobic channel.

Sola, S, Brito MA, Brites D, Moura JJG, Rodrigues CMP.  2002.  Membrane structural changes support the involvement of mitochondria in the bile salt-induced apoptosis of rat hepatocytes, Nov. Clinical Science. 103:475-485., Number 5 AbstractWebsite

The accumulation of toxic bile salts within the hepatocyte plays a key role in organ injury during liver disease. Deoxycholate (DC) and glycochenodeoxycholate (GCDC) induce apoptosis in vitro and in vivo, perhaps through direct perturbation of mitochondrial membrane structure and function. In contrast, ursodeoxycholate (UDC) and its taurine-conjugated form (TUDC) appear to be protective. We show here that hydrophobic bile salts induced apoptosis in cultured rat hepatocytes, without modulating the expression of pro-apoptotic Bax protein, and caused cytochrome c release in isolated mitochondria. Co-incubation with UDC and TUDC prevented cell death and efflux of mitochondrial factors. Using spin-labelling techniques and EPR spectroscopy analysis of isolated rat liver mitochondria, we found significant structural changes at the membrane-water surface in mitochondria exposed to hydrophobic bile salts, including modified lipid polarity and fluidity, altered protein order and increased oxidative injury. UDC, TUDC and cyclosporin A almost completely abrogated DC- and GCDC-induced membrane perturbations. We conclude that the toxicity of hydrophobic bile salts to hepatocytes is mediated by cytochrome c release, through a mechanism associated with marked direct effects on mitochondrial membrane lipid polarity and fluidity, protein order and redox status, without modulation of pro-apoptotic Bax expression. UDC and TUDC can directly suppress disruption of mitochondrial membrane structure, which may represent an important mechanism of hepatoprotection by these bile salts.

Rodrigues, CM, Sola S, Brito MA, Brites D, Moura JJ.  2002.  Bilirubin directly disrupts membrane lipid polarity and fluidity, protein order, and redox status in rat mitochondria, Mar. J Hepatol. 36:335-41., Number 3 AbstractWebsite

BACKGROUND/AIMS: Unconjugated bilirubin (UCB) impairs crucial aspects of cell function and induces apoptosis in primary cultured neurones. While mechanisms of cytotoxicity begin to unfold, mitochondria appear as potential primary targets. METHODS: We used electron paramagnetic resonance spectroscopy analysis of isolated rat mitochondria to test the hypothesis that UCB physically interacts with mitochondria to induce structural membrane perturbation, leading to increased permeability, and subsequent release of apoptotic factors. RESULTS: Our data demonstrate profound changes on mitochondrial membrane properties during incubation with UCB, including modified membrane lipid polarity and fluidity (P<0.01), as well as disrupted protein mobility (P<0.001). Consistent with increased permeability, cytochrome c was released from the intermembrane space (P<0.01), perhaps uncoupling the respiratory chain and further increasing oxidative stress (P<0.01). Both ursodeoxycholate, a mitochondrial-membrane stabilising agent, and cyclosporine A, an inhibitor of the permeability transition, almost completely abrogated UCB-induced perturbation. CONCLUSIONS: UCB directly interacts with mitochondria influencing membrane lipid and protein properties, redox status, and cytochrome c content. Thus, apoptosis induced by UCB may be mediated, at least in part, by physical perturbation of the mitochondrial membrane. These novel findings should ultimately prove useful to our evolving understanding of UCB cytotoxicity.

Rodrigues, CM, Sola S, Castro RE, Laires PA, Brites D, Moura JJ.  2002.  Perturbation of membrane dynamics in nerve cells as an early event during bilirubin-induced apoptosis, Jun. J Lipid Res. 43:885-94., Number 6 AbstractWebsite

Increased levels of unconjugated bilirubin, the end product of heme catabolism, impair crucial aspects of nerve cell function. In previous studies, we demonstrated that bilirubin toxicity may be due to cell death by apoptosis. To characterize the sequence of events leading to neurotoxicity, we exposed developing rat brain astrocytes and neurons to unconjugated bilirubin and investigated whether changes in membrane dynamic properties can mediate apoptosis. Bilirubin induced a rapid, dose-dependent increase in apoptosis, which was nevertheless preceded by impaired mitochondrial metabolism. Using spin labels and electron paramagnetic resonance spectroscopy analysis of whole cell and isolated mitochondrial membranes exposed to bilirubin, we detected major membrane perturbation. By physically interacting with cell membranes, bilirubin induced an almost immediate increase in lipid polarity sensed at a superficial level. The enhanced membrane permeability coincided with an increase in lipid fluidity and protein mobility and was associated with significant oxidative injury to membrane lipids. In conclusion, apoptosis of nerve cells induced by bilirubin is mediated by its primary effect at physically perturbing the cell membrane. Bilirubin directly interacts with membranes influencing lipid polarity and fluidity, protein order, and redox status. These data suggest that nerve cell membranes are primary targets of bilirubin toxicity.

Cabrita, EJ, Berger S, Brauer P, Karger J.  2002.  High-resolution DOSY NMR with spins in different chemical surroundings: Influence of particle exchange, JUL 2002. Journal of Magnetic Resonance. 157:124-131., Number 1 Abstract

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Carepo, M, Tierney DL, Brondino CD, Yang TC, Pamplona A, Telser J, Moura I, Moura JJ, Hoffman BM.  2002.  17O ENDOR detection of a solvent-derived Ni-(OH(x))-Fe bridge that is lost upon activation of the hydrogenase from Desulfovibrio gigas, Jan 16. J Am Chem Soc. 124:281-6., Number 2 AbstractWebsite

Crystallographic studies of the hydrogenases (Hases) from Desulfovibrio gigas (Dg) and Desulfovibrio vulgaris Miyazaki (DvM) have revealed heterodinuclear nickel-iron active centers in both enzymes. The structures, which represent the as-isolated (unready) Ni-A (S = (1)/(2)) enzyme state, disclose a nonprotein ligand (labeled as X) bridging the two metals. The bridging atom was suggested to be an oxygenic (O(2)(-) or OH(-)) species in Dg Hase and an inorganic sulfide in DvM Hase. To determine the nature and chemical characteristics of the Ni-X-Fe bridging ligand in Dg Hase, we have performed 35 GHz CW (17)O ENDOR measurements on the Ni-A form of the enzyme, exchanged into H(2)(17)O, on the active Ni-C (S = (1)/(2)) form prepared by H(2)-reduction of Ni-A in H(2)(17)O, and also on Ni-A formed by reoxidation of Ni-C in H(2)(17)O. In the native state of the protein (Ni-A), the bridging ligand does not exchange with the H(2)(17)O solvent. However, after a reduction/reoxidation cycle (Ni-A --> Ni-C --> Ni-A), an (17)O label is introduced at the active site, as seen by ENDOR. Detailed analysis of a 2-D field-frequency plot of ENDOR spectra taken across the EPR envelope of Ni-A((17)O) shows that the incorporated (17)O has a roughly axial hyperfine tensor, A((17)O) approximately [5, 7, 20] MHz, discloses its orientation relative to the g tensor, and also yields an estimate of the quadrupole tensor. The substantial isotropic component (a(iso)((17)O) approximately 11 MHz) of the hyperfine interaction indicates that a solvent-derived (17)O is indeed a ligand to Ni and thus that the bridging ligand X in the Ni-A state of Dg Hase is indeed an oxygenic (O(2)(-) or OH(-)) species; comparison with earlier EPR results by others indicates that the same holds for Ni-B. The small (57)Fe hyperfine coupling seen previously for Ni-A (A((57)Fe) approximately 0.9 MHz) is now shown to persist in Ni-C, A((57)Fe) approximately 0.8 MHz. However, the (17)O signal is lost upon reductive activation to the Ni-C state; reoxidation to Ni-A leads to the reappearance of the signal. Consideration of the electronic structure of the EPR-active states of the dinuclear center leads us to suggest that the oxygenic bridge in Ni-A(B) is lost in Ni-C and is re-formed from solvent upon reoxidation to Ni-A. This implies that the reductive activation to Ni-C opens Ni/Fe coordination sites which may play a central role in the enzyme's activity.

Cabrita, EJ, Berger S.  2002.  HR-DOSY as a new tool for the study of chemical exchange phenomena, DEC 2002. Magnetic Resonance in Chemistry. 40:S122-S127. Abstract

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Carepo, M, Baptista JF, Pamplona A, Fauque G, Moura JJ, Reis MA.  2002.  Hydrogen metabolism in Desulfovibrio desulfuricans strain New Jersey (NCIMB 8313)--comparative study with D. vulgaris and D. gigas species, Dec. Anaerobe. 8:325-32., Number 6 AbstractWebsite

This article aims to study hydrogen production/consumption in Desulfovibrio (D.) desulfuricans strain New Jersey, a sulfate reducer isolated from a medium undergoing active biocorrosion and to compare its hydrogen metabolism with two other Desulfovibrio species, D. gigas and D. vulgaris Hildenborough. Hydrogen production was followed during the growth of these three bacterial species under different growth conditions: no limitation of sulfate and lactate, sulfate limitation, lactate limitation, pyruvate/sulfate medium and in the presence of molybdate. Hydrogen production/consumption by D. desulfuricans shows a behavior similar to that of D. gigas but a different one from that of D. vulgaris, which produces higher quantities of hydrogen on lactate/sulfate medium. The three species are able to increase the hydrogen production when the sulfate became limiting. Moreover, in a pyruvate/sulfate medium hydrogen production was lower than on lactate/sulfate medium. Hydrogen production by D. desulfuricans in presence of molybdate is extremely high. Hydrogenases are key enzymes on production/consumption of hydrogen in sulfate reducing organisms. The specific activity, number and cellular localization of hydrogenases vary within the three Desulfovibrio species used in this work, which could explain the differences observed on hydrogen utilization.

Schlorer, NE, Cabrita EJ, Berger S.  2002.  Characterization of reactive intermediates by diffusion-ordered NMR spectroscopy: A snapshot of the reaction of (CO2)-C-13 with [Cp2Zr(Cl)H], 2002. Angewandte Chemie-International Edition. 41:107-109., Number 1 Abstract

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Bandeiras, TM, Salgueiro CA, Kletzin A, Gomes CM, Teixeira M.  2002.  Acidianus ambivalens type-II NADH dehydrogenase: genetic characterisation and identification of the flavin moiety as FMN. FEBS Letters. 531(2):273-277. AbstractWebsite

The thermoacidophilic archaeon Acidianus ambivalens contains a monomeric 47 kDa type-II NADH dehydrogenase (NDH), which contains a covalently bound flavin. In this work, by a combination of several methods, namely 31P-nuclear magnetic resonance and fluorescence spectroscopies, it is proven that this enzyme contains covalent FMN, a novelty among this family of enzymes, which were so far thought to mainly have the flavin dinucleotide form. Discrimination between several possible covalent flavin linkages was achieved by spectral and fluorescence experiments, which identified an 8α-N(1)-histidylflavin-type of linkage. Analysis of the gene-deduced amino acid sequence of type-II NDH showed no transmembranar helices and allowed the definition of putative dinucleotide and quinone binding motifs. Further, it is suggested that membrane anchoring can be achieved via amphipatic helices.

Branco, LC, Afonso CAM.  2002.  Catalytic asymmetric dihydroxylation of olefins using a recoverable and reusable OsO42- in ionic liquid bmim PF6. Chemical Communications. :3036-3037., Number 24 AbstractWebsite
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Carvalho, AL, Sanz L, Barettino D, Romero A, Calvete JJ, Romao MJ.  2002.  Crystal structure of a prostate kallikrein isolated from stallion seminal plasma: A homologue of human PSA. Journal of Molecular Biology. 322:325-337., Number 2 AbstractWebsite
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Dias, JM, Bonifácio C, Alves T, Moura JJG, Moura I, Romão MJ.  2002.  Crystallization and preliminary X-ray diffraction analysis of two pH-dependent forms of a di-haem cytochrome c peroxidase from Pseudomonas nautica. Acta Crystallographica Section D. 58:697-699., Number 4: Munksgaard International Publishers AbstractWebsite
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Dias, JM, Bonifacio C, Alves T, Moura JJG, Moura I, Romao MJ.  2002.  Crystallization and preliminary X-ray diffraction analysis of two pH-dependent forms of a di-haem cytochrome c peroxidase from Pseudomonas nautica. Acta Crystallographica Section D-Biological Crystallography. 58:697-699. AbstractWebsite
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Raaijmakers, H, Macieira S, Dias JM, Teixeira S, Bursakov S, Huber R, Moura JJG, Moura I, Romao MJ.  2002.  Gene sequence and the 1.8 angstrom crystal structure of the tungsten-containing formate dehydrogenase from Desulfolvibrio gigas. Structure. 10:1261-1272., Number 9 AbstractWebsite
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Branco, LC, Crespo JG, Afonso CAM.  2002.  Highly selective transport of organic compounds by using supported liquid membranes based on ionic liquids. Angewandte Chemie-International Edition. 41:2771-+., Number 15 AbstractWebsite
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Prata, JV, Clemente DTS, Prabhakar S, Lobo AM, Mourato I, Branco PS.  2002.  Intramolecular addition of acyldiazenecarboxylates onto double bonds in the synthesis of heterocycles. JOURNAL OF THE CHEMICAL SOCIETY-PERKIN TRANSACTIONS 1. :513-528., Number 4 Abstract
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Romao, MJ, Cunha CA, Brondino CD, Moura JJ.  2002.  Molybdenum enzymes in reactions involving aldehydes and acids. Met Ions Biol Syst. 39:539-70. AbstractWebsite
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Romao, MJ, Cunha CA, Brondino CD, Moura JJG.  2002.  Molybdenum enzymes in reactions involving aldehydes and acids. Molybdenum and Tungsten: Their Roles in Biological Processes. 39:539-570. AbstractWebsite
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Roque, A, Lodeiro C, Pina F, Maestri M, Ballardini R, Balzani V.  2002.  Photochromic properties of 3-methyl-substituted flavylium salts. European Journal of Organic Chemistry. :2699-2709., Number 16 AbstractWebsite
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Branco, LC, Rosa JN, Ramos JJM, Afonso CAM.  2002.  Preparation and characterization of new room temperature ionic liquids. Chemistry-a European Journal. 8:3671-3677., Number 16 AbstractWebsite
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Branco, LC, Crespo JG, Afonso CAM.  2002.  Studies on the selective transport of organic compounds by using ionic liquids as novel supported liquid membranes. Chemistry-a European Journal. 8:3865-3871., Number 17 AbstractWebsite
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2001
Glaser, T, Bertini I, Moura JJ, Hedman B, Hodgson KO, Solomon EI.  2001.  Protein effects on the electronic structure of the [Fe4S4]2+ cluster in ferredoxin and HiPIP, May 23. J Am Chem Soc. 123:4859-60., Number 20 AbstractWebsite
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Franco, R, Pereira AS, Tavares P, Mangravita A, Barber MJ, Moura I, Ferreira GC.  2001.  Substitution of murine ferrochelatase glutamate-287 with glutamine or alanine leads to porphyrin substrate-bound variants, May 15. Biochemical Journal. 356:217-222. AbstractWebsite

Ferrochelatase (EC 4.99.1.1) is the terminal enzyme of the haem biosynthetic pathway and catalyses iron chelation into the protoporphyrin IX ring. Glutamate-287 (E287) of murine mature ferrochelatase is a conserved residue in all known sequences of ferrochelatase, is present at the active site of the enzyme, as inferred from the Bacillus subtilis ferrochelatase three-dimensional structure, and is critical for enzyme activity. Substitution of E287 with either glutamine (Q) or alanine (A) yielded variants with lower enzymic activity than that of the wild-type ferrochelatase and with different absorption spectra from the wild-type enzyme. In contrast to the wild-type enzyme, the absorption spectra of the variants indicate that these enzymes, as purified, contain protoporphyrin IX. Identification and quantification of the porphyrin bound to the E287-directed variants indicate that approx. 80% of the total porphyrin corresponds to protoporphyrin IX. Significantly, rapid stopped-flow experiments of the E287A and E287Q Variants demonstrate that reaction with Zn2+ results in the formation of bound Zn-protoporphyrin IX, indicating that the endogenously bound protoporphyrin IX can be used as a substrate. Taken together, these findings suggest that the structural strain imposed by ferrochelatase on the porphyrin substrate as a critical step in the enzyme catalytic mechanism is also accomplished by the E287A and E287Q variants, but without the release of the product. Thus E287 in murine ferrochelatase appears to be critical For the catalytic process by controlling the release of the product.

Brito, MA, Brondino CD, Moura JJ, Brites D.  2001.  Effects of bilirubin molecular species on membrane dynamic properties of human erythrocyte membranes: a spin label electron paramagnetic resonance spectroscopy study, Mar 1. Arch Biochem Biophys. 387:57-65., Number 1 AbstractWebsite

Unconjugated bilirubin is a neurotoxic pigment that interacts with membrane lipids. In this study we used electron paramagnetic resonance and the spin labels 5-, 7-, 12-, and 16-doxyl-stearic acid (DSA) to evaluate the depth of the hydrocarbon chain at which interaction of bilirubin preferentially occurs. In addition, we used different pH values to determine the molecular species involved. Resealed right-side-out ghosts were incubated (1-60 min) with bilirubin (3.4-42.8 microM) at pH 7.0, 7.4, and 8.0. Alterations of membrane dynamic properties were maximum after 15 min of incubation with 8.6 microM bilirubin at pH 7.4 and were accompanied by a significant release of phospholipids. Interestingly, concentrations of bilirubin up to 42.8 microM and longer incubations resulted in the elution of cholesterol and further increased that of phospholipids while inducing less structural alterations. Variation of the pH values from 8.0 to 7.4 and 7.0, under conditions of maximum perturbation, led to a change from an increased to a diminished polarity sensed by 5-DSA. Conversely, a progressive enhancement in fluidity was reported by 7-DSA, followed by 12- and 16-DSA. These results indicate that bilirubin while enhancing membrane lipid order at C-5 simultaneously has disordering effects at C-7. Furthermore, recovery of membrane dynamics after 15 min of bilirubin exposure along with the release of lipids is compatible with a membrane adaptive response to the insult. In addition, our data provide evidence that uncharged diacid is the species primarily interacting with the membrane as perturbation is favored by acidosis, a condition frequently associated with hyperbilirubinemia in premature and severely ill infants.

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