Subunit fusion unlocks rapid <i>in vitro</i> maturation for slowly activating heterodimeric [FeFe]-hydrogenases.

Abstract

Hydrogenases offer a sustainable alternative to noble metals for catalyzing H₂-oxidation and H₂-production. The heterodimeric [FeFe]-hydrogenase of <i>Desulfovibrio desulfuricans</i> ATCC 7757 (<i>Dd</i>HydAB) is most promising due to its exceptional catalytic activity and high-yield heterologous expression of its apo-form. Scalable production of the holo-form relies on <i>in vitro</i> maturation of the apo-enzyme using a chemically synthesized 2Fe_H cofactor mimic. However, the unusually slow <i>in vitro</i> maturation of <i>Dd</i>HydAB raises mechanistic questions and limits its scalability. Through structural and sequence analysis, we identified the cause of this slow maturation and redesigned the enzyme <i>via</i> subunit fusion, inserting short peptide linkers near the active site. This modification facilitates the rearrangement of a critical locking element after cofactor uptake, increasing the maturation rate by up to 41-fold without compromising catalytic performance. Our findings elucidate a key step in the plug-lock-lid mechanism underlying maturation and promote the industrial applicability of <i>Dd</i>HydAB.