Abstract
HypD and HypC, or its paralogue HybG in Escherichia coli, form the core of the scaffold complex that synthesizes the Fe(CN)(2) CO component of the bimetallic NiFe-cofactor of [NiFe]-hydrogenase. We show here that purified HypC-HypD and HybG-HypD complexes catalyse hydrolysis of ATP to ADP (k(cat) ≅ 0.85·s(-1) ); the ATPase activity of the individual proteins was between 5- and 10-fold lower than that of the complex. Pre-incubation of HypD with ATP was necessary to restore full activity upon addition of HybG. The conserved Cys41 residue on HypD was essential for full ATPase activity of the complex. Together, our data suggest that HypD undergoes ATP-dependent conformational activation to facilitate complex assembly in preparation for substrate reduction.