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

[FeFe]-hydrogenase catalyzes the reversible reduction of protons to H₂ 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]_H) prior to its transfer to hydrogenase. To delineate the assembly and delivery steps for the 2Fe precursor cluster coordinated to HydF ([2Fe]_F), we have heterologously expressed HydF in the presence of HydE alone (HydF^E) or HydG alone (HydF^G), and characterized the resulting purified HydF^E and HydF^G 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^E is capable of activating HydA^ΔEFG to a limited extent (~ 1% of WT) even though the normal source of CO and CN^- ligands of [2Fe]_H (HydG) was absent. Activation assays performed with HydF^G, in contrast, exhibit no ability to mature HydA^ΔEFG. It appears that in the case of HydF^E, trace diatomics from the cellular environment are incorporated into a [2Fe]_F-like precursor on HydF in the absence of HydG. We conclude that the product of HydE, presumably the dithiomethylamine ligand of [2Fe]_H, is absolutely essential to the activation process, while the diatomic products of HydG can be provided from alternate sources.