Structural basis of a Ni acquisition cycle for [NiFe] hydrogenase by Ni-metallochaperone HypA and its enhancer

The Ni atom at the catalytic center of [NiFe] hydrogenases is incorporated by a Ni-metallochaperone, HypA, and a GTPase/ATPase, HypB. We report the crystal structures of the transient complex formed between HypA and ATPase-type HypB (HypB_AT) with Ni ions. Transient association between HypA and HypB_AT is controlled by the ATP hydrolysis cycle of HypB_AT, which is accelerated by HypA. Only the ATP-bound form of HypB_AT can interact with HypA and induces drastic conformational changes of HypA. Consequently, upon complex formation, a conserved His residue of HypA comes close to the N-terminal conserved motif of HypA and forms a Ni-binding site, to which a Ni ion is bound with a nearly square-planar geometry. The Ni binding site in the HypAB_AT complex has a nanomolar affinity (K_d = 7 nM), which is in contrast to the micromolar affinity (K_d = 4 μM) observed with the isolated HypA. The ATP hydrolysis and Ni binding cause conformational changes of HypB_AT, affecting its association with HypA. These findings indicate that HypA and HypB_AT constitute an ATP-dependent Ni acquisition cycle for [NiFe]-hydrogenase maturation, wherein HypB_AT functions as a metallochaperone enhancer and considerably increases the Ni-binding affinity of HypA.