Probing the Influence of the Protein Scaffold on H-Cluster Reactivity via Gain-of-Function Studies─Improved H₂...

[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 Thermoanaerobacter mathranii (TamHydS) 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 (e.g., substrate transport) coordination sphere effects on metal cofactors. Herein, we generated three TamHydS-based variants, each mimicking proposed key structural features of Group A hydrogenase: the "active site" (AS), "proton-transfer pathway" (PTP), and "combined" (CM = AS + PTP) variant. A fourth single-point variant, A137C, 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.e., no positive impact on catalytic performance was observed. The CM variant, however, showed increased H₂ evolution activity but retained the overpotential requirement. Additionally, the CM variant improved the already relatively high stability of TamHydS 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.