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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 μ2-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.
Dinis et al. (Tue,) studied this question.