Partial purification and characterization of the first hydrogenase isolated from a thermophilic sulfate-reducing bacterium.
A 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.
Fauque
G
G
Centre d'Océanologie de Marseille, Faculté des Sciences de Luminy, France.
Czechowski
M
M
Berlier
Y M
YM
Lespinat
P A
PA
LeGall
J
J
Moura
J J
JJ
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003901:Desulfovibrio / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D006358:Hot Temperature; D006864:Hydrogenase / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D013816:Thermodynamics
D006864:Hydrogenase