Characterizing the Biosynthesis of the [Fe(II)(CN)(CO)₂(cysteinate)]⁻ Organometallic Product of the Radical-SAM Enzyme HydG by EPR and Mössbauer 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 H₂. 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 Mössbauer spectroscopy, revealing an isomer shift of δ = 0.10 mm s^-1 and quadrupole splitting of ΔE_Q = 0.66 mm s^-1. These Mössbauer values are a change from the starting values of δ = 1.15 mm s^-1 and ΔE_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 Mössbauer parameters for the low-spin Fe²⁺ ions in synthetic analogues, such as ⁵⁷Fe 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.