Characterizing the Biosynthesis of the [Fe(II)(CN)(CO)₂(cysteinate)]^- Organometallic Product of the Radical-SAM Enzyme HydG by EPR and Mossbauer Spectroscopy

[FeFe]-hydrogenases employ a catalytic H-cluster, consisting of a [4Fe-4S](H) cluster linked to a [2Fe](H) subcluster with CO, CN- ligands, and an azadithiolate bridge, which mediates the rapid redox interconversion of H+ and H-2. In the biosynthesis of this H-cluster active site, the radical S-adenosyl-l-methionine (radical SAM, RS) enzyme HydG plays the crucial role of generating an organometallic [Fe(II)(CN)(CO)(2)(cysteinate)](-) product that is en route to forming the H-cluster. Here, we report direct observation of this diamagnetic organometallic Fe(II) complex through Mossbauer spectroscopy, revealing an isomer shift of delta = 0.10 mm s(-1) and quadrupole splitting of Delta E-Q = 0.66 mm s(-1). These Mossbauer values are a change from the starting values of delta = 1.15 mm s(-1) and Delta E-Q = 3.23 mm s(-1) for the ferrous "dangler" Fe in HydG. These values of the observed product complex B are in good agreement with Mossbauer parameters for the low-spin Fe2+ ions in synthetic analogues, such as Fe-57 Syn-B, which we report here. These results highlight the essential role that HydG plays in converting a resting-state high-spin Fe(II) to a low-spin organometallic Fe(II) product that can be transferred to the downstream maturase enzymes.