Probing the Influence of the Protein Scaffold on H-Cluster Reactivity via Gain-of-Function Studies─Improved H₂ Evolution and O₂ Tolerance through Rational Design of [FeFe] Hydrogenase

[FeFe] hydrogenases make up a structurally diverse family of metalloenzymes that catalyze proton/dihydrogen interconversion. They can be classified into phylogenetically distinct groups denoted A-G, which differ in structure and reactivity. Prototypical Group A hydrogenases have high turnover rates and remarkable energy efficiency. As compared to Group A enzymes, the putatively sensory Group D hydrogenase from <i>Thermoanaerobacter mathranii</i> (<i>Tam</i>HydS) has a thousand-fold lower H₂ evolution rate and a high overpotential requirement to drive catalysis (irreversible) but shows increased inhibitor tolerance. This divergence in structure and activity between hydrogenases makes them ideal models for studying second (active-site environment) and outer (<i>e.g.</i>, substrate transport) coordination sphere effects on metal cofactors. Herein, we generated three <i>Tam</i>HydS-based variants, each mimicking proposed key structural features of Group A hydrogenase: the "active site" (<b>AS</b>), "proton-transfer pathway" (<b>PTP</b>), and "combined" (<b>CM</b> = <b>AS</b> + <b>PTP</b>) variant. A fourth single-point variant, <b>A137C</b>, which introduces a proposed critical cysteine in the active site, was characterized as a reference. No change in isolation resulted in Group A-like behavior; <i>i.e</i>., no positive impact on catalytic performance was observed. The <b>CM</b> variant, however, showed increased H₂ evolution activity but retained the overpotential requirement. Additionally, the <b>CM</b> variant improved the already relatively high stability of <i>Tam</i>HydS against O₂ and CO inhibition. These findings show that activity rates, (ir)reversibility, and susceptibility to gaseous inhibitors are decoupled. Moreover, the results highlight the importance of exploring hydrogenase diversity as a path toward understanding the structural factors that enable the outstanding catalytic properties of [FeFe] hydrogenases.