Partial purification and characterization of the first hydrogenase isolated from a thermophilic sulfate-reducing bacterium,
Fauque, G., Czechowski M., Berlier Y. M., Lespinat P. A., Legall J., and Moura J. J.
, Biochem Biophys Res Commun, May 15, Volume 184, Number 3, p.1256-60, (1992)
AbstractA soluble [NiFe] hydrogenase has been partially purified from the obligate thermophilic sulfate-reducing bacterium Thermodesulfobacterium mobile. A 17% purification yield was obtained after four chromatographic steps and the hydrogenase presents a purity index (A398 nm/A277 nm) equal to 0.21. This protein appears to be 75% pure on SDS-gel electrophoresis showing two major bands of molecular mass around 55 and 15 kDa. This hydrogenase contains 0.6-0.7 nickel atom and 7-8 iron atoms per mole of enzyme and has a specific activity of 783 in the hydrogen uptake reaction, of 231 in the hydrogen production assay and of 84 in the deuterium-proton exchange reaction. The H2/HD ratio is lower than one in the D2-H+ exchange reaction. The enzyme is very sensitive to NO, relatively little inhibited by CO but unaffected by NO2-. The EPR spectrum of the native hydrogenase shows the presence of a [3Fe-4S] oxidized cluster and of a Ni(III) species.
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)
AbstractNitrate 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.
Periplasmic nitrate reductases and formate dehydrogenases: Biological control of the chemical properties of Mo and W for fine tuning of reactivity, substrate specificity and metabolic role,
Gonzalez, P. J., Rivas M. G., Mota C. S., Brondino C. D., Moura I., and Moura J. J. G.
, Coord Chem Rev, Volume 257, p.315-331, (2013)
Perturbation of membrane dynamics in nerve cells as an early event during bilirubin-induced apoptosis,
Rodrigues, C. M., Sola S., Castro R. E., Laires P. A., Brites D., and Moura J. J.
, J Lipid Res, Jun, Volume 43, Number 6, p.885-94, (2002)
AbstractIncreased 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.
The photochemical reaction between uranyl nitrate and azulene,
Burrows, Hugh D., Cardoso Augusto C., Formosinho Sebastião J., Gil Ana M. P. C., da Miguel Maria Graça M., Barata Belamino, and J.G. Moura José
, Journal of Photochemistry and Photobiology A: Chemistry, Volume 68, Number 3, p.279-287, (1992)
Abstractn/a
The photochemical reaction between uranyl-nitrate and azulene,
Burrows, H. D., Cardoso A. C., Formosinho S. J., Gil Ampc, Miguel M. D., Barata B., and Moura J. J. G.
, Journal of Photochemistry and Photobiology a-Chemistry, Sep 30, Volume 68, Number 3, p.279-287, (1992)
AbstractOn photolysis of solutions of azulene and uranyl nitrate in alcohols, a dark, amorphous precipitate is formed. Various analytical techniques show that this is a mixture of a uranium salt and an organic component, suggested to be polyazulene. The effects of various parameters on the yield of the product have been studied and it is found that oxygen facilitates the reaction. Electron spin resonance studies show that the product is paramagnetic, in agreement with the established ease of oxidation of polyazulene, and suggest that it is formed via electron transfer from azulene to excited uranyl ion, followed by successive dimerizations and deprotonations of radical cation intermediates.
Physico-chemical and Spectroscopic Properties of the Monohemic Cytochrome C552 from Pseudomonas nautica 617,
Saraiva, Lígia M., Fauque Guy, Besson Stéphane, and Moura Isabel
, European Journal of Biochemistry, Volume 224, Number 3, p.1011-1017, (1994)
AbstractA c-type monohemic ferricytochrome c552 (11 kDa) was isolated from the soluble extract of a marine denitrifier, Pseudomonas nautica strain 617, grown under anaerobic conditions with nitrate as final electron acceptor. The NH2-terminal sequence and the amino acid composition of the cytochrome were determined. The heme iron of the cytochrome c552 has histidine-methionine as axial ligands, and a pH-dependent mid-point redox potential, equal to 250 mV at pH 7.6. The presence of methionine was demonstrated by visible, EPR and NMR spectroscopies. The assignment of most of the hemic protons was performed applying two-dimensional NOE spectroscopy (NOESY), and the aromatic region was assigned through two-dimensional correlated spectroscopy (COSY) experiments. The EPR spectrum of the oxidised form of the cytochrome c552 is typical of a low-spin ferric heme.
Protein effects on the electronic structure of the [Fe4S4]2+ cluster in ferredoxin and HiPIP,
Glaser, T., Bertini I., Moura J. J., Hedman B., Hodgson K. O., and Solomon E. I.
, J Am Chem Soc, May 23, Volume 123, Number 20, p.4859-60, (2001)
Abstractn/a
Proteins containing the factor F430 from methanosarcina barkeri and methanobacterium thermoautotrophicum: Isolation and properties,
Moura, Isabel, Moura José J. G., Santos Helena, Xavier Antonio V., Burch Gary, Peck Jr Harry D., and Legall Jean
, Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, Volume 742, Number 1, p.84-90, (1983)
Abstractn/a
Purification and characterization of a tungsten-containing formate dehydrogenase from Desulfovibrio gigas,
Almendra, M. J., Brondino C. D., Gavel O., Pereira A. S., Tavares P., Bursakov S., Duarte R., Caldeira J., Moura J. J., and Moura I.
, Biochemistry, Dec 7, Volume 38, Number 49, p.16366-72, (1999)
AbstractAn air-stable formate dehydrogenase (FDH), an enzyme that catalyzes the oxidation of formate to carbon dioxide, was purified from the sulfate reducing organism Desulfovibrio gigas (D. gigas) NCIB 9332. D. gigas FDH is a heterodimeric protein [alpha (92 kDa) and beta (29 kDa) subunits] and contains 7 +/- 1 Fe/protein and 0.9 +/- 0.1 W/protein. Selenium was not detected. The UV/visible absorption spectrum of D. gigas FDH is typical of an iron-sulfur protein. Analysis of pterin nucleotides yielded a content of 1.3 +/- 0.1 guanine monophosphate/mol of enzyme, which suggests a tungsten coordination with two molybdopterin guanine dinucleotide cofactors. Both Mossbauer spectroscopy performed on D. gigas FDH grown in a medium enriched with (57)Fe and EPR studies performed in the native and fully reduced state of the protein confirmed the presence of two [4Fe-4S] clusters. Variable-temperature EPR studies showed the presence of two signals compatible with an atom in a d(1) configuration albeit with an unusual relaxation behavior as compared to the one generally observed for W(V) ions.
Purification and characterization of three proteins from a halophilic sulfate-reducing bacterium,<i>Desulfovibrio salexigens</i>,
Czechowski, M., Fauque G., Galliano N., Dimon B., Moura I., Moura J. J. G., Xavier A. V., Barato B. A. S., Lino A. R., and Legall J.
, Journal of Industrial Microbiology & Biotechnology, Volume 1, Number 3, p.139-147, (1986)
Abstractn/a
Purification and preliminary characterization of tetraheme cytochrome c3 and adenylylsulfate reductase from the peptidolytic sulfate-reducing bacterium Desulfovibrio aminophilus DSM 12254,
Lopez-Cortes, A., Bursakov S., Figueiredo A., Thapper A. E., Todorovic S., Moura J. J., Ollivier B., Moura I., and Fauque G.
, Bioinorg Chem Appl, p.81-91, (2005)
AbstractTwo proteins were purified and preliminarily characterized from the soluble extract of cells (310 g, wet weight) of the aminolytic and peptidolytic sulfate-reducing bacterium Desulfovibrio (D.) aminophilus DSM 12254. The iron-sulfur flavoenzyme adenylylsulfate (adenosine 5'-phosphosulfate, APS) reductase, a key enzyme in the microbial dissimilatory sulfate reduction, has been purified in three chromatographic steps (DEAE-Biogel A, Source 15, and Superdex 200 columns). It contains two different subunits with molecular masses of 75 and 18 kDa. The fraction after the last purification step had a purity index (A(278nm) / A(388nm)) of 5.34, which was used for further EPR spectroscopic studies. The D. aminophilus APS reductase is very similar to the homologous enzymes isolated from D. gigas and D. desulfuricans ATCC 27774. A tetraheme cytochrome c(3) (His-heme iron-His) has been purified in three chromatographic steps (DEAE- Biogel A, Source 15, and Biogel-HTP columns) and preliminarily characterized. It has a purity index ([A(553nm) - A(570nm)](red) / A(280nm)) of 2.9 and a molecular mass of around 15 kDa, and its spectroscopic characterization (NMR and EPR) has been carried out. This hemoprotein presents similarities with the tetraheme cytochrome c(3) from Desulfomicrobium (Des.) norvegicum (NMR spectra, and N-terminal amino acid sequence).
Purification and Preliminary Characterization of Three C-Type Cytochromes from Pseudomonas Nautica Strain 617,
Saraiva, L. M., Besson S., Moura I., and Fauque G.
, Biochemical and Biophysical Research Communications, Volume 212, Number 3, p.1088-1097, (1995)
Abstractn/a
Purification, characterization and biological activity of three forms of ferredoxin from the sulfate-reducing bacterium Desulfovibrio gigas,
Bruschi, M., Hatchikian C., Legall J., Moura J. J., and Xavier A. V.
, Biochim Biophys Acta, Nov 9, Volume 449, Number 2, p.275-84, (1976)
AbstractThree forms of ferredoxin FdI, FdI', and FdII have been isolated from Desulfovibrio gigas, a sulfate reducer. They are separated by a combination of DEAE-cellulose and gel filtration chromatographic procedures. FdI and FdI' present a slight difference in isoelectric point which enables the separation of the two forms over DEAE-cellulose, while FdII is easily separated from the two other forms by gel filtration. The three forms have the same amino acid composition and are isolated in different aggregation states. Molecular weight determinations by gel filtration gave values of 18 000 for FdI and FdI' and 24 000 for FdII, whereas a value of 6000 is determined when dissociation is accomplished with sodium dodecyl sulfate. The electronic spectra are different and their ultraviolet-visible absorbance rations are 0.77, 0.87 and 0.68 respectively for FdI, FdI' and FdII. Despite these differences, the physiological activities of the three forms are similar as far as the reduction of sulfite by molecular hydrogen is concerned.
Purification, characterization, and preliminary crystallographic study of copper-containing nitrous oxide reductase from Pseudomonas nautica 617,
Prudencio, M., Pereira A. S., Tavares P., Besson S., Cabrito I., Brown K., Samyn B., Devreese B., Van Beeumen J., Rusnak F., Fauque G., Moura J. J., Tegoni M., Cambillau C., and Moura I.
, Biochemistry, Apr 11, Volume 39, Number 14, p.3899-907, (2000)
AbstractThe aerobic purification of Pseudomonas nautica 617 nitrous oxide reductase yielded two forms of the enzyme exhibiting different chromatographic behaviors. The protein contains six copper atoms per monomer, arranged in two centers named Cu(A) and Cu(Z). Cu(Z) could be neither oxidized nor further reduced under our experimental conditions, and exhibits a 4-line EPR spectrum (g(x)=2.015, A(x)=1.5 mT, g(y)=2.071, A(y)=2 mT, g(z)=2.138, A(z)=7 mT) and a strong absorption at approximately 640 nm. Cu(A) can be stabilized in a reduced EPR-silent state and in an oxidized state with a typical 7-line EPR spectrum (g(x)=g(y)= 2.021, A(x) = A(y)=0 mT, g(z) = 2.178, A(z)= 4 mT) and absorption bands at 480, 540, and approximately 800 nm. The difference between the two purified forms of nitrous oxide reductase is interpreted as a difference in the oxidation state of the Cu(A) center. In form A, Cu(A) is predominantly oxidized (S = (1)/(2), Cu(1.5+)-Cu(1.5+)), while in form B it is mostly in the one-electron reduced state (S = 0, Cu(1+)-Cu(1+)). In both forms, Cu(Z) remains reduced (S = 1/2). Complete crystallographic data at 2.4 A indicate that Cu(A) is a binuclear site (similar to the site found in cytochrome c oxidase) and Cu(Z) is a novel tetracopper cluster [Brown, K., et al. (2000) Nat. Struct. Biol. (in press)]. The complete amino acid sequence of the enzyme was determined and comparisons made with sequences of other nitrous oxide reductases, emphasizing the coordination of the centers. A 10.3 kDa peptide copurified with both forms of nitrous oxide reductase shows strong homology with proteins of the heat-shock GroES chaperonin family.
Purification, crystallization and preliminary X-ray diffraction analysis of adenosine triphosphate sulfurylase (ATPS) from the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774,
Gavel, O. Y., Kladova A. V., Bursakov S. A., Dias J. M., Texeira S., Shnyrov V. L., Moura J. J., Moura I., Romao M. J., and Trincao J.
, Acta Crystallogr Sect F Struct Biol Cryst Commun, Jul 1, Volume 64, Number Pt 7, p.593-5, (2008)
AbstractNative zinc/cobalt-containing ATP sulfurylase (ATPS; EC 2.7.7.4; MgATP:sulfate adenylyltransferase) from Desulfovibrio desulfuricans ATCC 27774 was purified to homogeneity and crystallized. The orthorhombic crystals diffracted to beyond 2.5 A resolution and the X-ray data collected should allow the determination of the structure of the zinc-bound form of this ATPS. Although previous biochemical studies of this protein indicated the presence of a homotrimer in solution, a dimer was found in the asymmetric unit. Elucidation of this structure will permit a better understanding of the role of the metal in the activity and stability of this family of enzymes.