H-cluster assembly intermediates built on HydF by the radical SAM enzymes HydE and HydG.

[FeFe]-hydrogenase catalyzes the reversible reduction of protons to H at a complex metallocofactor site, the H-cluster. Biosynthesis of this active-site H-cluster requires three maturation enzymes: the radical S-adenosylmethionine enzymes HydE and HydG synthesize the nonprotein ligands, while the GTPase HydF provides a scaffold for assembly of the 2Fe subcluster of the H-cluster ([2Fe]) prior to its transfer to hydrogenase. To delineate the assembly and delivery steps for the 2Fe precursor cluster coordinated to HydF ([2Fe]), we have heterologously expressed HydF in the presence of HydE alone (HydF) or HydG alone (HydF), and characterized the resulting purified HydF and HydF using UV-visible, EPR, and FTIR spectroscopies and biochemical assays. The iron-sulfur clusters on HydF are modified by co-expression with HydE or HydG, as evidenced by the changes in the visible, EPR, and FTIR spectral features. Further, biochemical assays show that HydF is capable of activating HydA to a limited extent (~ 1% of WT) even though the normal source of CO and CN ligands of [2Fe] (HydG) was absent. Activation assays performed with HydF, in contrast, exhibit no ability to mature HydA. It appears that in the case of HydF, trace diatomics from the cellular environment are incorporated into a [2Fe]-like precursor on HydF in the absence of HydG. We conclude that the product of HydE, presumably the dithiomethylamine ligand of [2Fe], is absolutely essential to the activation process, while the diatomic products of HydG can be provided from alternate sources.