Jorge Lampreia

This communication reports the isolation, purification and characterization of key enzymes involved in dissimilatory sulfate reduction of a sulfate reducing bacterium classified as Desulfovibrio desulfuricans subspecies desulfuricans New Jersey (NCIMB 8313) (Ddd NJ). The chosen strain, originally recovered from a corroding cast iron heat exchanger, was grown in large scale batch cultures. Physico-chemical and spectroscopic studies of the purified enzymes were carried out. These analyses revealed a high degree of similarity between proteins isolated from the DddNJ strain and the homologous proteins obtained from Desulfomicrobium baculatus Norway 4. In view of the results obtained, taxonomic reclassification of Desulfovibrio desulfuricans subspecies desulfuricans New Jersey (NCIMB 8313) into Desulfomicrobium baculatus (New Jersey) is proposed. (C) 1996 Academic Press, Inc.

Tetraheme cytochrome c(3) (13 kDa) and flavodoxin (16 kDa), are small electron transfer proteins that have been used to mimic, in vitro, part of the electron-transfer chain that operates between substract electron donors and respiratory electron accepters partners in Desulfovibrio species (Palma, N., Moura, I., LeGall, J., Van Beeumen, J., Wampler, J., Moura, J. J. G. (1994) Biochemistry 33, 6394-6407). The electron transfer between these two proteins is believed to occur through the formation of a specific complex where electrostatic interaction is the main driving force (Stewart, D., LeGall, J., Moura, I., Moura, J.J.G., Peck, H.D., Xavier, A.V., Weiner, P.K. and Wampler, J.E. (1988) Biochemistry 27, 2444-2450, Stewart, D., LeGall, J., Moura, I., Moura, J.J.G., Peck, H.D., Xavier, A.V., Weiner, P., Wampler, J. (1989) fur. J. Biochem. 185, 695-700). In order to obtain structural information of the pre-complex, a covalent complex between the two proteins was prepared. A water-soluble carbodiimide {[}EDC (1-ethyl-3(3 dimethylaminopropyl) carbodiimide hydrochloride] was used for the cross linking reaction. The reaction was optimized varying a wide number of experimental parameters such as ionic strength, protein and cross linker concentration, and utilization of different cross linkers and reaction time between the crosslinker and proteins. (C) 1999 Academic Press.

Sulphate-reducing bacteria have a wide variety of periplasmic cytochromes involved in electron transfer from the periplasm to the cytoplasm. HmcA is a high molecular mass cytochrome of 550 amino acid residues that harbours 16 c-type heme groups. We report the crystal structure of HmcA isolated from the periplasm of Desulfovibrio gigas. Crystals were grown. using polyethylene glycol 8K and zinc acetate, and diffracted beyond 2.1 angstrom resolution. A multiple-wavelength anomalous dispersion experiment at the iron absorption edge enabled us to obtain good-quality phases for structure solution and model building. DgHmcA has a V-shape architecture, already observed in HmcA isolated from Desulfovibrio vulgaris Hildenborough. The presence of an oligosaccharide molecule covalently bound to an Asn residue was observed in the electron density maps of DgHmcA and confirmed by mass spectrometry. Three modified monosaccharides appear at the highly hydrophobic vertex, possibly acting as an anchor of the protein to the cytoplasmic membrane. (c) 2007 Elsevier Ltd. All rights reserved.

{Nitrite reductase from the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 is a multihaem (type c) membrane-bound enzyme that catalyzes the dissimilatory conversion of nitrite to ammonia. Crystals of the oxidized form of this enzyme were obtained using PEG and CaCl2 as precipitants in the presence of 3-(decylmethylammonium)propane-1-sulfonate and belong to the space group P2(1)2(1)2(1), With unit-cell parameters a = 78.94

The gene encoding cytochrome c nitrite reductase (NrfA) from Desulfovibrio desulfuricans ATCC 27774 was sequenced and the crystal structure of the enzyme was determined to 2.3-Angstrom resolution. In comparison with homologous structures, it presents structural differences mainly located at the regions surrounding the putative substrate inlet and product outlet, and includes a well defined second calcium site with octahedral geometry, coordinated to propionates of hemes 3 and 4, and caged by a loop non-existent in the previous structures. The highly negative electrostatic potential in the environment around hemes 3 and 4 suggests that the main role of this calcium ion may not be electrostatic but structural, namely in the stabilization of the conformation of the additional loop that cages it and influences the solvent accessibility of heme 4. The NrfA active site is similar to that of peroxidases with a nearby calcium site at the heme distal side nearly in the same location as occurs in the class II and class III peroxidases. This fact suggests that the calcium ion at the distal side of the active site in the NrfA enzymes may have a similar physiological role to that reported for the peroxidases.