Protein dynamics affect O 2 -stability of Group B [FeFe]-hydrogenase from Thermosediminibacter oceani

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 cysteine protects H-cluster from O 2 -induced degradation by forming 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. Table of Contents Figure: