In vitro maturation of fully active [FeFe]-hydrogenase in a defined system including the iron carrier NfuA

The [FeFe]-hydrogenase employs an active-site 6Fe H-cluster to catalyze the reversible reduction of protons to H₂. 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 <i>Escherichia coli</i> iron-sulfur carrier protein NfuA supports in vitro maturation of fully active [FeFe]-hydrogenase, with H₂ production rates comparable to that of the in vivo<i>-</i>matured <i>Chlamydomonas reinhardtii</i> [FeFe]-hydrogenase (<i>Cr</i>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.