Metal Specificity in [NiFe] Hydrogenase Maturation Proteins

The maturation of [NiFe] hydrogenase is a multistep process that requires the participation of accessory proteins to assemble the NiFe(CN)2CO metallocluster at the catalytic center. HypA and HypB are two proteins that are necessary for the efficient incorporation of nickel into the bimetallic active site. The mechanism through which these proteins specifically insert nickel into the hydrogenase metallocluster is unknown. Escherichia coli HypB contains a high-affinity metal-binding site at the N-terminus that binds nickel with a sub-picomolar affinity. We investigated the nickel selectivity of this site by using a small peptide maquette and found that while the high-affinity site binds nickel tightly, it also binds other transition metals with affinities that are in line with the Irving-Williams series. We also examined the G-domain metal-binding site of HypB, and how it is connected to the nucleotide-binding properties of the protein. We find that nickel, but not zinc, is transferred from the low-affinity site of HypB to the N-terminal metal-binding site of HypA, and that this process occurs rapidly when HypB is loaded with GDP. Nickel transfer between proteins is also dependent on a protein-protein complex forming between HypA and HypB, and when that complex is abrogated through mutagenesis, rapid GDP-dependent transfer of nickel is no longer observed. Subsequently, the molecular basis behind the increased rate of nickel transfer was explored. We found that the affinity of HypB for nickel changes based on whether the G-domain is loaded with GDP or a GTP analog, as does the oligomeric state of the protein. Furthermore, GDP-loading of HypB promotes the formation of a HypA-HypB heterodimer, which breaks apart once nickel has been transferred to HypA. These findings shed light on the mechanism of nickel transfer, and help us better understand how nickel is specifically inserted into the active site of [NiFe] hydrogenase.Ph.D