O ₂ reactions at the six-iron active site (H-cluster) in [FeFe]-hydrogenase

Irreversible inhibition by molecular oxygen (O ₂) complicates the use of [FeFe]-hydrogenases (HydA) for biotechnological hydrogen (H ₂) production. Modification by O ₂ of the active site six-iron complex denoted as the H-cluster ([4Fe4S]-2Fe _H) of HydA1 from the green alga Chlamydomonas reinhardtii was characterized by x-ray absorption spectroscopy at the iron K-edge. In a time-resolved approach, HydA1 protein samples were prepared after increasing O ₂ exposure periods at 0 °C. A kinetic analysis of changes in their x-ray absorption near edge structure and extended X-ray absorption fine structure spectra revealed three phases of O ₂ reactions. The first phase (τ ₁≤4 s) is characterized by the formation of an increased number of Fe-O,C bonds, elongation of the Fe-Fe distance in the binuclear unit (2Fe _H), and oxidation of one iron ion. The second phase (τ ₂ ≈ 15 s) causes a ∼50% decrease of the number of ∼2.7-ÅFe-Fe distances in the [4Fe4S] subcluster and the oxidation of one more iron ion. The final phase (τ ₃ ≤ 1000 s) leads to the disappearance of most Fe-Fe and Fe-S interactions and further iron oxidation. These results favor a reaction sequence, which involves 1) oxygenation at 2Fe _H+ leading to the formation of a reactive oxygen species-like superoxide (O ₂ ^-), followed by 2) H-cluster inactivation and destabilization due to ROS attack on the [4Fe4S] cluster to convert it into an apparent [3Fe4S] ⁺ unit, leading to 3) complete O ₂-induced degradation of the remainders of the H-cluster. This mechanism suggests that blocking of ROS diffusion paths and/or altering the redox potential of the [4Fe4S] cubane by genetic engineering may yield improved O ₂tolerance in [FeFe]-hydrogenase.