Abstract

The active site of [FeFe] hydrogenase contains a catalytic binuclear iron subsite coordinated by CN– and CO ligands as well as a unique azadithiolate (adt2–) bridging ligand. It has been established that this binuclear cofactor is synthesized and assembled by three maturation proteins HydE, -F, and -G. By means of in vitro maturation in the presence of 15N- and 13C-labeled tyrosine it has been shown that the CN– and CO ligands originate from tyrosine. The source of the bridging adt2– ligand, however, remains unknown. In order to identify the nitrogen of the bridging amine using HYSCORE spectroscopy and distinguish its spectroscopic signature from that of the CN– nitrogens, we studied three isotope-labeled variants of the H-cluster (15N-adt2–/C14N–, 15N-adt2–/C15N–, and 14N-adt2–/C15N–) and extracted accurate values of the hyperfine and quadrupole couplings of both CN– and adt2– nitrogens. This will allow an evaluation of isotopologues of the H-cluster generated by in vitro bioassembly in the presence of various 15N-labeled potential precursors as possible sources of the bridging ligand.