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

ABSTRACT In the context of bio-hydrogen production, the O2-stable Group B [FeFe]-hydrogenase from Thermosediminibacter oceani 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. Competing Interest Statement The authors have declared no competing interest.