Molecular Dynamics Investigation of O2 Diffusion Mechanisms in the Protective CbA5H Hydrogenase

[FeFe]-hydrogenases are highly efficient biocatalysts for H₂ conversion but are extremely sensitive to O₂. The Clostridium beijerinckii [FeFe]-hydrogenase (CbA5H) displays a unique O₂-protective mechanism, forming an inactive yet rapidly reversible state upon O₂ exposure. Experimental evidence indicates that the TSC-loop enables C367 to complete the electronic configuration of the Fed of the H-cluster. However, the pathways by which O₂ reaches the active site remain unclear. Here, we investigate O₂ diffusion using advanced molecular dynamics simulations (RAMD) to identify possible intraprotein diffusion routes. Our results provide molecular insight into the O₂-protection mechanism of CbA5H and may support protein engineering efforts toward O₂-tolerant [FeFe]-hydrogenases for sustainable H₂ production.