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.