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Claudia Brocks, Chandan K. Das, Jifu Duan, Shanika Yadav, Ulf-Peter Apfel, Subhasri Ghosh, Eckhard Hofmann, Martin Winkler, Vera Engelbrecht, Lars V. Schäfer, Thomas Happe

• [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.