Maturation of the [FeFe]‐Hydrogenase: Direct Transfer of the (κ3‐cysteinate)FeII(CN)(CO)2 Complex B from HydG to HydE

Abstract

Abstract[FeFe]‐hydrogenases efficiently catalyze the reversible oxidation of molecular hydrogen. Their prowess stems from the intricate H‐cluster, combining a [Fe4S4] center with a binuclear iron center ([2Fe]H). In the latter, each iron atom is coordinated by a CO and CN ligand, connected by a CO and an azadithiolate ligand. The synthesis of this active site involves a unique multiprotein assembly, featuring radical SAM proteins HydG and HydE. HydG initiates the transformation of L‐tyrosine into cyanide and carbon monoxide to generate complex B, which is subsequently transferred to HydE to continue the biosynthesis of the [2Fe]H‐subcluster. Due to its instability, complex B isolation for structural or spectroscopic characterization has been elusive thus far. Nevertheless, the use of a biomimetic analogue of complex B allowed circumvention of the need for the HydG protein during in vitro functional investigations, implying a similar structure for complex B. Herein, we used the HydE protein as a nanocage to encapsulate and stabilize the complex B product generated by HydG. Using X‐ray crystallography, we successfully determined its structure at 1.3 Å resolution. Furthermore, we demonstrated that complex B is directly transferred from HydG to HydE, thus not being released into the solution post‐synthesis, highlighting a transient interaction between the two proteins.