A Dynamic Water Channel Affects O₂ Stability in [FeFe]-Hydrogenases

[FeFe]-hydrogenases are capable of reducing protons at a high rate. However, molecular oxygen (O-2) induces the degradation of their catalytic cofactor, the H-cluster, which consists of a cubane [4Fe4S] subcluster (4Fe(H)) and a unique diiron moiety (2Fe(H)). Previous attempts to prevent O-2-induced damage have focused on enhancing the protein's sieving effect for O-2 by blocking the hydrophobic gas channels that connect the protein surface and the 2Fe(H). In this study, we aimed to block an O-2 diffusion pathway and shield 4Fe(H) instead. Molecular dynamics (MD) simulations identified a novel water channel (W-H) surrounding the H-cluster. As this hydrophilic path may be accessible for O-2 molecules we applied site-directed mutagenesis targeting amino acids along W-H in proximity to 4Fe(H) to block O-2 diffusion. Protein film electrochemistry experiments demonstrate increased O-2 stabilities for variants G302S and S357T, and MD simulations based on high-resolution crystal structures confirmed an enhanced local sieving effect for O-2 in the environment of the 4Fe(H) in both cases. The results strongly suggest that, in wild type proteins, O-2 diffuses from the 4Fe(H) to the 2Fe(H). These results reveal new strategies for improving the O-2 stability of [FeFe]-hydrogenases by focusing on the O-2 diffusion network near the active site.