Inhibition of Biocatalysis in [Fe–Fe] Hydrogenase by Oxygen: Molecular Dynamics and Density Functional Theory Calculations

Designing O₂-tolerant hydrogenases is a major challenge in applying [Fe–Fe]­H₂ases for H₂ production. The inhibition involves transport of oxygen through the enzyme to the H-cluster, followed by binding and subsequent deactivation of the active site. To explore the nature of the oxygen diffusion channel for the hydrogenases from <i>Desulfovibrio desulfuricans</i> (<i>Dd</i>) and <i>Clostridium pasteurianum</i> (<i>Cp</i>), empirical molecular dynamics simulations were performed. The dynamic nature of the oxygen pathways in <i>Dd</i> and <i>Cp</i> was elucidated, and insight is provided, in part, into the experimental observation on the difference of oxygen inhibition in <i>Dd</i> and the hydrogenase from <i>Clostridium acetobutylicum</i> (<i>Ca</i>, assumed homologous to <i>Cp</i>). Further, to gain an understanding of the mechanism of oxygen inhibition of the [Fe–Fe]­H₂ase, density functional theory calculations of model compounds composed of the H-cluster and proximate amino acids are reported. Confirmation of the experimentally based suppositions on inactivation by oxygen at the [2Fe]_H domain is provided, validating the model compounds used and oxidation state assumptions, further explaining the mode of damage. This unified approach provides insight into oxygen diffusion in the enzyme, followed by deactivation at the H-cluster