Structural and functional characterization of the hydrogenase-maturation HydF protein

[FeFe] hydrogenase (HydA) catalyzes interconversion between 2H(+) and H-2 at an active site composed of a [4Fe-4S] cluster linked to a 2Fe subcluster that harbors CO, CN- and azapropanedithiolate (adt(2-)) ligands. HydE, HydG and HydF are the maturases specifically involved in the biosynthesis of the 2Fe subcluster. Using ligands synthesized by HydE and HydG, HydF assembles a di-iron precursor of the 2Fe subcluster and transfers it to HydA for maturation. Here we report the first X-ray structure of HydF with its [4Fe-4S] cluster. The cluster is chelated by three cysteines and an exchangeable glutamate, which allows the binding of synthetic mimics of the 2Fe subcluster. [Fe-2(adt)(CO)(4)(CN)(2)](2-) is proposed to be the true di-iron precursor because, when bound to HydF, it matures HydA and displays features in Fourier transform infrared (FTIR) spectra that are similar to those of the native HydF active intermediate. A new route toward the generation of artificial hydrogenases, as combinations of HydF and such biomimetic complexes, is proposed on the basis of the observed hydrogenase activity of chemically modified HydF.