EPR studies with 77Se-enriched (NiFeSe) hydrogenase of Desulfovibrio baculatus. Evidence for a selenium ligand to the active site nickel.
The periplasmic hydrogenase containing equivalent amounts of nickel and selenium plus non-heme iron [NiFeSe) hydrogenase) has been purified from cells of the sulfate reducing bacterium Desulfovibrio baculatus (DSM 1748) grown on a lactate/sulfate medium containing natural Se isotopes and the nuclear isotope, 77Se. Both the 77Se-enriched and unenriched hydrogenases were shown to be free of other hydrogenases and characterized with regard to their Se contents. EPR studies of the reduced nickel signal generated by redox titrations of the enriched and unenriched (NiFeSe) hydrogenases demonstrated that the gx = 2.23 and gy = 2.17 resonances are appreciably broadened by the spin of the 77Se nucleus (I = 1/2). This observation demonstrates unambiguously that the unpaired electron is shared by the Ni and Se atoms and that Se serves as a ligand to the nickel redox center of the (NiFeSe) hydrogenase.
He
S H
SH
Department of Biochemistry, School of Chemical Sciences, University of Georgia, Athens 30602.
Teixeira
M
M
LeGall
J
J
Patil
D S
DS
Moura
I
I
Moura
J J
JJ
DerVartanian
D V
DV
Huynh
B H
BH
Peck
H D
HD
D001665:Binding Sites; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003901:Desulfovibrio / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D006864:Hydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D009532:Nickel / Q000032:analysis*; D012643:Selenium / Q000032:analysis*
D008024:Ligands; D009532:Nickel; C058560:nickel-iron-selenium hydrogenase; D006864:Hydrogenase; D012643:Selenium