2025/09/22 by Alexander Marlott, Adrien Pagnier, Eric M. Shepard +9 · 1 voice · 2 citations
Chemistry · Energy · #Electrocatalysts for Energy Conversion #Metal-Catalyzed Oxygenation Mechanisms #Metalloenzymes and iron-sulfur proteins
paper · doi:10.1073/pnas.2517347122
openalex publication_date 2025/09/22 · openalex created_date 2025/10/10 · openalex updated_date 2026/08/01
The [FeFe]-hydrogenase employs an active-site 6Fe H-cluster to catalyze the reversible reduction of protons to H 2 . A [4Fe-4S] subcluster of the H-cluster is synthesized by housekeeping iron-sulfur cluster assembly machinery, and then dedicated hydrogenase maturation enzymes, together with components of the glycine cleavage system, build and deliver a [2Fe] subcluster to generate the full H-cluster. Here, we report that the Escherichia coli iron-sulfur carrier protein NfuA supports in vitro maturation of fully active [FeFe]-hydrogenase, with H 2 production rates comparable to that of the in vivo - matured Chlamydomonas reinhardtii [FeFe]-hydrogenase ( Cr HydA). Inclusion of NfuA in the in vitro maturation process improves its efficacy by delivering the iron essential for formation of the [Fe II (cys)(CN)(CO) 2 ] – synthon at the dangler iron site of the HydG auxiliary cluster. NfuA serves an additional role in reconstituting and maintaining the catalytically essential iron-sulfur clusters on the maturase enzymes HydE, HydF, and HydG. Further inclusion of a high CO affinity myoglobin variant (Mb H64L ) sequesters free CO generated during the maturation process, minimizing formation of the CO-inhibited H ox -CO enzyme state, significantly increasing hydrogenase activity. The addition of NfuA and Mb H64L to the fully defined maturation system thus results in an in vitro [FeFe]-hydrogenase maturation system that generates highly active enzyme while providing insights into factors important to in vivo maturation.