[FeFe]-Hydrogenase Oxygen Inactivation Is Initiated at the H Cluster 2Fe Subcluster

The [FeFe]-hydrogenase catalytic site H cluster is a complex iron sulfur cofactor that is sensitive to oxygen (O<sub>2</sub>). The O<sub>2</sub> sensitivity is a significant barrier for production of hydrogen as an energy source in water-splitting, oxygenic systems. Oxygen reacts directly with the H cluster, which results in rapid enzyme inactivation and eventual degradation. To investigate the progression of O<sub>2</sub>-dependent [FeFe]-hydrogenase inactivation and the process of H cluster degradation, the highly O<sub>2</sub>-sensitive [FeFe]-hydrogenase HydA1 from the green algae Chlamydomonas reinhardtii was exposed to defined concentrations of O<sub>2</sub> while monitoring the loss of activity and accompanying changes in H cluster spectroscopic properties. The results indicate that H cluster degradation proceeds through a series of reactions, the extent of which depend on the initial enzyme reduction/oxidation state. The degradation process begins with O<sub>2</sub> interacting and reacting with the 2Fe subcluster, leading to degradation of the 2Fe subcluster and leaving an inactive [4Fe-4S] subcluster state. This final inactive degradation product could be reactivated <i>in vitro</i> by incubation with 2Fe subcluster maturation machinery, specifically HydF<sup>EG</sup>, which was observed by recovery of enzyme activity.