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.

Automated summary

This study reports the direct observation of an organometallic Fe(II) complex in the hydrogenase active site through Mossbauer spectroscopy, providing insights into its structural characteristics. The results highlight the crucial role of HydG in converting high-spin Fe(II) ions to low-spin organometallic Fe(II) products.