Does the environment around the H-cluster allow coordination of the pendant amine to the catalytic iron center in [FeFe] hydrogenases? Answers from theory

[FeFe] hydrogenases are H-2-evolving enzymes that feature a diiron cluster in their active site (the [2Fe](H) cluster). One of the iron atoms has a vacant coordination site that directly interacts with H-2, thus favoring its splitting in cooperation with the secondary amine group of a neighboring, flexible azadithiolate ligand. The vacant site is also the primary target of the inhibitor O-2. The [2Fe](H) cluster can span various redox states. The active-ready form (H-ox) attains the (FeFeI)-Fe-II state. States more oxidized than H-ox were shown to be inactive and/or resistant to O-2. In this work, we used density functional theory to evaluate whether azadithiolate-to-iron coordination is involved in oxidative inhibition and protection against O-2, a hypothesis supported by recent results on biomimetic compounds. Our study shows that Fe-N(azadithiolate) bond formation is favored for an (FeFeII)-Fe-II active-site model which disregards explicit treatment of the surrounding protein matrix, in line with the case of the corresponding (FeFeII)-Fe-II synthetic system. However, the study of density functional theory models with explicit inclusion of the amino acid environment around the [2Fe](H) cluster indicates that the protein matrix prevents the formation of such a bond. Our results suggest that mechanisms other than the binding of the azadithiolate nitrogen protect the active site from oxygen in the so-called H (ox) (inact) state.