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

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