Subunit fusion unlocks rapid in vitro maturation for slowly activating heterodimeric [FeFe]-hydrogenases

Hydrogenases offer a sustainable alternative to noble metals for catalyzing H₂-oxidation and H₂-production. The heterodimeric [FeFe]-hydrogenase of Desulfovibrio desulfuricans ATCC 7757 (DdHydAB) 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 in vitro maturation of the apo-enzyme using a chemically synthesized 2Fe_H cofactor mimic. However, the unusually slow in vitro maturation of DdHydAB raises mechanistic questions and limits its scalability. Through structural and sequence analysis, we identified the cause of this slow maturation and redesigned the enzyme via 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 DdHydAB.