Serine is the molecular source of the NH(CH2)2 bridgehead moiety of the in vitro assembled [FeFe] hydrogenase H-cluster.

The active site of [FeFe] hydrogenase, the H-cluster, consists of a canonical [4Fe-4S]H subcluster linked to a unique binuclear [2Fe]H subcluster containing three CO, two CN- and a bridging azadithiolate (adt, NH(CH2S-)2) ligand. While it is known that all five diatomic ligands are derived from tyrosine, there has been little knowledge as to the formation and installation of the adt ligand. Here, by using a combination of a cell-free in vitro maturation approach with pulse electronic paramagnetic resonance spectroscopy, we discover that serine donates the nitrogen atom and the CH2 group to the assembly of the adt ligand. More specifically, both CH2 groups in adt are sourced from the C3 methylene of serine.