How the Azadithiolate Ligand Impacts O ₂ -Stability of Group B [FeFe]-Hydrogenase ToHydA

[FeFe]-hydrogenases are metalloenzymes that catalyze the reversible oxidation and production of H ₂ , making them potential candidates for sustainable energy solutions. However, their practical application is restricted by their extreme O ₂ sensitivity, which leads to irreversible active site degradation. A newly characterized Group B hydrogenase, ToHydA from Thermosediminibacter oceani , has exhibited exceptional O ₂ -stability even after longtime exposure to air. In ToHydA, the highly conserved proton-transporting cysteine (C212) safeguards the H-cluster from O ₂ -induced degradation by formation of the H _inact state. In this study, we investigate the effects of replacing the azadithiolate (ADT) ligand of [2Fe] _H with propanedithiolate (PDT), revealing that this substitution prevents the formation of the H _inact and H _trans states observed in ToHydA WT (bearing the ADT ligand). By combining ATR-FTIR spectroscopy and molecular dynamics (MD) simulations, we show that a hydrogen bond between the nitrogen bridgehead of the ADT ligand and the C212 sidechain is crucial for stabilizing these states. The absence of this interaction in ToHydA ^PDT (bearing the PDT ligand) prevents the C212 sidechain from approaching the Fe _d center of [2Fe] _H , thereby reducing H _inact accumulation. Moreover, as-isolated ToHydA ^PDT predominantly exhibits the H _hyd state, which is unusual for [FeFe]-hydrogenases with bound PDT ligand. These findings demonstrate how ligand substitution at the [2Fe] _H site of ToHydA affects the structural dynamics, offering detailed molecular insights into the ligand-dependent modulation of [FeFe]-hydrogenases.