Oxygen sensitivity of [FeFe]-hydrogenase: a comparative study of active site mimics inside vs. outside the enzyme

[FeFe]-hydrogenase is nature's most efficient proton reducing and H₂-oxidizing enzyme. However, biotechnological applications are hampered by the O₂ sensitivity of this metalloenzyme, and the mechanism of aerobic deactivation is not well understood. Here, we explore the oxygen sensitivity of four mimics of the organometallic active site cofactor of [FeFe]-hydrogenase, [Fe₂(adt)(CO)_6-(CN)] and [Fe₂(pdt)(CO)_6-(CN)] (x = 1, 2) as well as the corresponding cofactor variants of the enzyme by means of infrared, Mössbauer, and NMR spectroscopy. Additionally, we describe a straightforward synthetic recipe for the active site precursor complex Fe₂(adt)(CO)₆. Our data indicate that the aminodithiolate (adt) complex, which is the synthetic precursor of the natural active site cofactor, is most oxygen sensitive. This observation highlights the significance of proton transfer in aerobic deactivation, and supported by DFT calculations facilitates an identification of the responsible reactive oxygen species (ROS). Moreover, we show that the ligand environment of the iron ions critically influences the reactivity with O₂ and ROS like superoxide and H₂O₂ as the oxygen sensitivity increases with the exchange of ligands from CO to CN^-. The trends in aerobic deactivation observed for the model complexes are in line with the respective enzyme variants. Based on experimental and computational data, a model for the initial reaction of [FeFe]-hydrogenase with O₂ is developed. Our study underscores the relevance of model systems in understanding biocatalysis and validates their potential as important tools for elucidating the chemistry of oxygen-induced deactivation of [FeFe]-hydrogenase.