Tuning the H ₂ Production Activity of ToHydA by Molecular Simulation-Informed Protein Engineering

In the context of biohydrogen production, the O₂-stable Group B [FeFe]-hydrogenase from <i>Thermosediminibacter oceani</i> has attracted significant interest due to its distinctive TSCCCP motif near the active site, which contains an additional (third) cysteine residue that is absent in the TSCCP motif of standard Group A hydrogenases. The precise role of the additional cysteine residue in H₂ production has remained an open question. In this study, we sought to contribute to the understanding of this cysteine's role in H₂ production by combining molecular dynamics (MD) simulations, site-directed mutagenesis, biochemical assays, and Fourier-transform infrared (FTIR) spectroscopy. Remarkably, a cysteine-to-serine exchange variant (TSSCCP) demonstrated enhanced H₂ production activity without compromising the O₂-stability of ToHydA, offering new insights into its functional dynamics.