An in vitro reconstitution system to monitor iron transfer to the active site during the maturation of [NiFe]-hydrogenase.

[NiFe]-hydrogenases (Hyds) comprise a small and a large subunit. The latter harbors the biologically unique [NiFe](CN)CO active-site cofactor. The maturation process includes the assembly of the [Fe](CN)CO cofactor precursor, nickel binding, endoproteolytic cleavage of the large subunit, and dimerization with the small subunit to yield active enzyme. The biosynthesis of the [Fe](CN)CO moiety of [NiFe]-Hyd-1 and Hyd-2 occurs on the scaffold complex HybG-HypD (GD), whereas the HypC-HypD complex is specific for the assembly of Hyd-3. The metabolic source and the route for delivering iron to the active site remain unclear. To investigate the maturation process of O-tolerant Hyd-1 from Escherichia coli, we developed an enzymatic in vitro reconstitution system that allows for the synthesis of Hyd-1 using only purified components. Together with this in vitro reconstitution system, we employed biochemical analyses, infrared spectroscopy (attenuated total reflection FTIR), mass spectrometry (MS), and microscale thermophoresis to monitor the iron transfer during the maturation process and to understand how the [Fe](CN)CO cofactor precursor is ultimately incorporated into the large subunit. We demonstrate the direct transfer of iron from Fe-labeled GD complex to the large subunit of Hyd-1. Our data reveal that the GD complex exclusively interacts with the large subunit of Hyd-1 and Hyd-2 but not with the large subunit of Hyd-3. Furthermore, we show that the presence of iron in the active site is a prerequisite for nickel insertion. Taken together, these findings reveal how the [Fe](CN)CO cofactor precursor is transferred and incorporated into the active site of [NiFe]-Hyd.