Purification, characterization and redox properties of hydrogenase from Methanosarcina barkeri (DSM 800),
Fauque, G., Teixeira M., Moura I., Lespinat P. A., Xavier A. V., Dervartanian D. V., Peck, H. D. Jr., Legall J., and Moura J. G.
, Eur J Biochem, Jul 2, Volume 142, Number 1, p.21-8, (1984)
AbstractA soluble hydrogenase from the methanogenic bacterium, Methanosarcina barkeri (DSM 800) has been purified to apparent electrophoretic homogeneity, with an overall 550-fold purification, a 45% yield and a final specific activity of 270 mumol H2 evolved min-1 (mg protein)-1. The hydrogenase has a high molecular mass of approximately equal to 800 kDa and subunits with molecular masses of approximately equal to 60 kDa. The enzyme is stable to heating at 65 degrees C and to exposure to air at 4 degrees C in the oxidized state for periods up to a week. The overall stability of this enzyme is compared with other hydrogenase isolated from strict anaerobic sulfate-reducing bacteria. Ms. barkeri hydrogenase shows an absorption spectrum typical of a non-heme iron protein with maxima at 275 nm, 380 nm and 405 nm. A flavin component, identified as FMN or riboflavin was extracted under acidic conditions and quantified to approximately one flavin molecule per subunit. In addition to this component, 8-10 iron atoms and 0.6-0.8 nickel atom were also detected per subunit. The electron paramagnetic resonance (EPR) spectrum of the native enzyme shows a rhombic signal with g values at 2.24, 2.20 and approximately equal to 2.0. probably due to nickel which is optimally measured at 40 K but still detectable at 77 K. In the reduced state, using dithionite or molecular hydrogen as reductants, at least two types of g = 1.94 EPR signals, due to iron-sulfur centers, could be detected and differentiated on the basis of power and temperature dependence. Center I has g values at 2.04, 1.90 and 1.86, while center II has g values at 2.08, 1.93 and 1.85. When the hydrogenase is reduced by hydrogen or dithionite the rhombic EPR species disappears and is replaced by other EPR-active species with g values at 2.33, 2.23, 2.12, 2.09, 2.04 and 2.00. These complex signals may represent different nickel species and are only observable at temperatures higher than 20 K. In the native preparation, at high temperatures (T greater than 35 K) or in partially reduced samples, a free radical due to the flavin moiety is observed. The EPR spectrum of reduced hydrogenase in 80% Me2SO presents an axial type of spectrum only detectable below 30 K.
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 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)
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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)
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The presence of redox-sensitive nickel in the periplasmic hydrogenase from Desulfovibrio gigas,
Legall, J., Ljungdahl P. O., Moura I., Peck, H. D. Jr., Xavier A. V., Moura J. J., Teixera M., Huynh B. H., and Dervartanian D. V.
, Biochem Biophys Res Commun, May 31, Volume 106, Number 2, p.610-6, (1982)
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