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 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 Mössbauer spectroscopy, revealing an isomer shift of δ = 0.10 mm s -1 and quadrupole splitting of Δ E Q = 0.66 mm s -1 . These Mössbauer values are a change from the starting values of δ = 1.15 mm s -1 and Δ 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 Mössbauer parameters for the low-spin Fe 2+ ions in synthetic analogues, such as 57 Fe 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.