Showing papers by "Isabel Moura published in 1999"

Purification and characterization of a tungsten-containing formate dehydrogenase from Desulfovibrio gigas.

Abstract: An 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.

Observation of ligand-based redox chemistry at the active site of a molybdenum enzyme

Abstract: The mononuclear molybdenum enzymes all possess one or two molybdopterin cofactors coordinated to the molybdenum through the ditholene motif. Despite this common feature, they exhibit quite diverse functionality. The molybdenum enzymes previously have been described as all involving two-electron redox chemistry at molybdenum, coupled with the transfer of an oxygen atom from water via molybdenum to substrate, or the reverse. While these rules still appear to hold for most molybdenum enzymes, and for their close relatives the tungsten enzymes, it now seems that there are at least some exceptions. The recently discovered tungsten enzyme acetylene hydratase catalyzes a net hydration reaction, rather than a redox one. Very recently it has been shown that formate oxidation to CO{sub 2} by Eschericia coli formate dehydrogenase H (FDH{sub H}) does not involve oxygen atom transfer. This enzyme has also been shown to possess a potentially redox-active selenosulfide ligand to molybdenum, with the selenosulfide sulfur probably being one of the sulfurs of the cofactor dithiolene. The authors present an extended X-ray absorption fine structure (EXAFS) spectroscopic study of the molybdenum site of Desulfovibrio desulfuricans ATCC 27774 formate dehydrogenase (FDH) and show that under reducing conditions the selenosulfide group can be reduced. This is themore » first observation of ligand-based redox chemistry in a molybdenum enzyme.« less

Structural studies by X-ray diffraction on metal substituted desulforedoxin, a rubredoxin-type protein.

Abstract: Desulforedoxin (Dx), isolated from the sulfate reducing bacterium Desulfovibrio gigas, is a small homodimeric (2 x 36 amino acids) protein. Each subunit contains a high-spin iron atom tetrahedrally bound to four cysteinyl sulfur atoms, a metal center similar to that found in rubredoxin (Rd) type proteins. The simplicity of the active center in Dx and the possibility of replacing the iron by other metals make this protein an attractive case for the crystallographic analysis of metal-substituted derivatives. This study extends the relevance of Dx to the bioinorganic chemistry field and is important to obtain model compounds that can mimic the four sulfur coordination of metals in biology. Metal replacement experiments were carried out by reconstituting the apoprotein with In3+, Ga3+, Cd2+, Hg2+, and Ni2+ salts. The In3+ and Ga3+ derivatives are isomorphous with the iron native protein; whereas Cd2+, Hg2+, and Ni2+ substituted Dx crystallized under different experimental conditions, yielding two additional crystal morphologies; their structures were determined by the molecular replacement method. A comparison of the three-dimensional structures for all metal derivatives shows that the overall secondary and tertiary structures are maintained, while some differences in metal coordination geometry occur, namely, bond lengths and angles of the metal with the sulfur ligands. These data are discussed in terms of the entatic state theory.

The solution structure of a [3Fe-4S] ferredoxin: oxidised ferredoxin II from Desulfovibrio gigas.

Abstract: The use of standard 2D NMR experiments in combination with 1D NOE experiments allowed the assignment of 51 of the 58 spin systems of oxidised [3Fe4S] ferredoxin isolated from Desulfovibrio gigas. The NMR solution structure was determined using data from 1D NOE and 2D NOESY spectra, as distance constraints, and information from the X-ray structure for the spin systems not detected by NMR in torsion angle dynamics calculations to produce a family of 15 low target function structures. The quality of the NMR family, as judged by the backbone r.m.s.d. values, was good (0.80 A), with the majority of phi/psi angles falling within the allowed region of the Ramachandran plot. A comparison with the X-ray structure indicated that the overall global fold is very similar in solution and in the solid state. The determination of the solution structure of ferredoxin II (FdII) in the oxidised state (FdIIox) opens the way for the determination of the solution structure of the redox intermediate state of FdII (FdII(int)), for which no X-ray structure is available.

Simple and Complex Iron-Sulfur Proteins in Sulfate Reducing Bacteria

Abstract: Publisher Summary This chapter focuses on simple and complex iron–sulfur-containing proteins isolated from sulfate reducing bacteria (SRB), to review the types and distribution of proteins, metal clusters involved and their association with other centers and cofactors, cluster binding motifs, electronic and magnetic properties of the iron-sulfur clusters present, and cluster interconversions, including heterometal cluster formation The chapter describes the complex structures involving Fe–S basic units and other redox centers (flavin, heme, nickel, and molybdenum) Several three-dimensional crystal structures of simple and complex iron–sulfur-containing proteins isolated from sulfate reducers have been solved The main structural features have been discussed in the chapter They include the simple rubredoxin family ferredoxins, hydrogenase, and AOR In addition, the structures of other electron carriers, such as cytochromes and flavodoxins, have been determined The overall data indicate the wide range of structural solutions adopted in the construction of the metal-containing active sites and were inspiring motifs for the proposal of catalytic mechanisms

Enzymatic Spectrophotometric Determination of Nitrites in Beer

Abstract: A colorimetric assay for nitrite determination in beer based on c-type multiheme enzyme Nitrite reductase (NiR) isolated from Desulfovibrio desulfuricans ATCC 27774, was developed. Using the enzyme in solution, nitrite assay was linear in the 10−8 – 10−2 M range with a detection limit of 10−8 M and a recovery ranging from 90 to 107%. The imprecision ranged from 4 to 10% on the entire calibration curve. With NiR immobilised onto a nylon coil, a flow reactor was developed which showed a narrower linear range (10−5 – 10−2 M) and a higher detection limit (10−5 M) than with the enzyme in solution, but made it possible to reuse the enzyme up to 100 times (50% residual activity). Sample preparation was simple and fast: only degassing and beer dilution by buffer was needed. This enzymatic assay was in good agreement with the results obtained using commercial nitrite determination kits.

Crystallization and preliminary x-ray analysis of a nitrate reductase from Desulfovibrio desulfuricans ATCC 27774.

Abstract: Periplasmic 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.

Spectrophotometric confirmation for oxygen ligation to the reduced haem d 1 of Pseudomonas nautica nitrite reductase

Abstract: Cytochrome cd 1 was discovered in the Gram-negative bacterium Pseudomonas (Ps.) aeruginosa by Horio and coworkers [1]. It was tagged a cytochrome oxidase on the base of its capacity to reduce oxygen. Only a few years later was its main, if not only, physiological function as a nitrite reductase made clear [2]. Numerous articles have since been published on the spectrophotometry of cytochromes cd 1 isolated from various bacteria incuding Ps. aeruginosa (see [3] for an overview) or Ps. nautica [4]. It was earlier believed that the nature of the electron donor was responsible for the considerable discrepancies observed between visible spectra of the same cytochrome cd 1, in the regions of reduced haem d 1 absorption peaks [5]. However, Shimada and Orii [6] demonstrated that an oxygenated-haem d 1 reaction intermediate was the main reason for these discrepancies. Optical spectra of Ps. nautica cytochrome cd 1 recorded under anaerobic conditions still exhibited differences and an investigation was decided to assess and compare respective effects of pH, buffer nature and level of oxygen contamination.