Protein Dynamics Affect O <sub>2</sub> -Stability of Group B [FeFe]-Hydrogenase from <i>Thermosediminibacter oceani</i>
Subhasri Ghosh, Chandan K. Das, S.M. Iftekhar Uddin, Sven T. Stripp, Vera Engelbrecht, M. Winkler, Silke Leimkühler, Claudia Brocks, Jifu Duan, Lars V. Schäfer, Thomas Happe
Abstract
In the pursuit of sustainable “green” energy generation, [FeFe]-hydrogenases have attracted significant attention due to their ability to catalyze hydrogen production. However, the sensitivity of these enzymes to O 2 is a major obstacle for their application as biocatalysts in energy conversion technologies. In the search for an O 2 -stable [FeFe]-hydrogenase, we identified the hydrogenase ToHydA from Thermosediminibacter oceani that belongs to the rarely characterized Group B (M2a) [FeFe]-hydrogenases. Our findings demonstrate that ToHydA exhibits remarkable O 2 -stability, even under prolonged O 2 exposure. By characterizing site-directed mutagenesis variants, we found that the highly conserved proton-transporting active site cysteine residue protects the H-cluster from O 2 -induced degradation by forming the H inact state. The additional cysteine residue in the TSCCCP motif of ToHydA, a feature unique to Group B (M2a) [FeFe]-hydrogenases, enhances the flexibility of that motif and facilitates the formation of the H inact state. Moreover, ToHydA possesses unique features, including the formation of an unusual H inact resting state that distinguishes the enzyme from other [FeFe]-hydrogenases. Our atomistic molecular dynamics simulations reveal a previously unrecognized cluster of hydrophobic residues centered around the proton-transporting cysteine-bearing loop. This structural feature appears to be a common molecular characteristic in hydrogenases that form the O 2 -protected H inact state. By exploiting these molecular features of ToHydA, future research can aim to rationally design hydrogenases that combine high catalytic activity with enhanced O 2 -stability to develop more efficient and durable catalysts.