X-ray crystallographic and EPR spectroscopic analysis of HydG, a maturase in [FeFe]-hydrogenase H-cluster assembly

Hydrogenases use complex metal cofactors to catalyze the reversible formation of hydrogen. In [FeFe]-hydrogenases, the H-cluster cofactor includes a diiron subcluster containing azadithiolate, three CO, and two CN ⁻ ligands. During the assembly of the H cluster, the radical S -adenosyl methionine (SAM) enzyme HydG lyses the substrate tyrosine to yield the diatomic ligands. These diatomic products form an enzyme-bound Fe(CO) ₓ(CN) y synthon that serves as a precursor for eventual H-cluster assembly. To further elucidate the mechanism of this complex reaction, we report the crystal structure and EPR analysis of HydG. At one end of the HydG (βα) ₈ triosephosphate isomerase (TIM) barrel, a canonical [4Fe-4S] cluster binds SAM in close proximity to the proposed tyrosine binding site. At the opposite end of the active-site cavity, the structure reveals the auxiliary Fe-S cluster in two states: one monomer contains a [4Fe-5S] cluster, and the other monomer contains a [5Fe-5S] cluster consisting of a [4Fe-4S] cubane bridged by a μ ₂-sulfide ion to a mononuclear Fe ²⁺ center. This fifth iron is held in place by a single highly conserved protein-derived ligand: histidine 265. EPR analysis confirms the presence of the [5Fe-5S] cluster, which on incubation with cyanide, undergoes loss of the labile iron to yield a [4Fe-4S] cluster. We hypothesize that the labile iron of the [5Fe-5S] cluster is the site of Fe(CO) ₓ(CN) y synthon formation and that the limited bonding between this iron and HydG may facilitate transfer of the intact synthon to its cognate acceptor for subsequent H-cluster assembly. Significance Hydrogenases are a source of environmentally benign bioenergy, catalyzing the reversible reduction of protons to form hydrogen. The most active subclass, the [FeFe]-hydrogenases, is dependent on a metallocofactor, the H cluster, which contains iron-bound CO and CN ⁻ ligands. Although the HydG maturase is known to catalytically form a CO- and CN ⁻-bound iron precursor to the H cluster, mechanistic insight into this complex process has been hampered by the lack of structural information about HydG. We now describe the high-resolution crystal structure and EPR analysis of HydG. These results reveal a previously unreported [5Fe-5S] cluster that features a labile iron center proposed to provide the site of formation for a labile Fe(CO) ₂CN synthon, the precursor of the diiron subcluster hydrogenase H cluster.