A model system for [NiFe] hydrogenase maturation studies: Purification of an active site-containing hydrogenase large subunit without small subunit.
The large subunit HoxC of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha was purified without its small subunit. Two forms of HoxC were identified. Both forms contained iron but only substoichiometric amounts of nickel. One form was a homodimer of HoxC whereas the second also contained the Ni-Fe site maturation proteins HypC and HypB. Despite the presence of the Ni-Fe active site in some of the proteins, both forms, which lack the Fe-S clusters normally present in hydrogenases, cannot activate hydrogen. The incomplete insertion of nickel into the Ni-Fe site provides direct evidence that Fe precedes Ni in the course of metal center assembly.
Winter
Gordon
G
Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Chausseestr. 117, D-10115 Berlin, Germany.
Buhrke
Thorsten
T
Lenz
Oliver
O
Jones
Anne Katherine
AK
Forgber
Michael
M
Friedrich
Bärbel
B
D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D001665:Binding Sites; D020118:Cupriavidus necator / Q000201:enzymology*; D019281:Dimerization; D006859:Hydrogen / Q000378:metabolism; D006864:Hydrogenase / Q000737:chemistry / Q000302:isolation & purification*; D007501:Iron / Q000737:chemistry; D009532:Nickel / Q000737:chemistry; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification
D001426:Bacterial Proteins; D021122:Protein Subunits; D009532:Nickel; D006859:Hydrogen; D007501:Iron; C043296:nickel-iron hydrogenase; D006864:Hydrogenase