Fe–S clusters are essential cofactors mediating
electron
transfer in respiratory and metabolic networks. However, obtaining
active [4Fe-4S] proteins with heterologous expression is challenging
due to (i) the requirements for [4Fe-4S] cluster assembly, (ii) the
O2 lability of [4Fe-4S] clusters, and (iii) copurification
of undesired proteins (e.g., ferredoxins). Here, we established a
facile and efficient protocol to express mature [4Fe-4S] proteins
in the PURE system under aerobic conditions. An enzyme aconitase and
thermophilic ferredoxin were selected as model [4Fe-4S] proteins
for functional verification. We first reconstituted the SUF system in vitro via a stepwise manner using the recombinant SUF
subunits (SufABCDSE) individually purified from E. coli. Later, the incorporation of recombinant SUF helper proteins into
the PURE system enabled mRNA translation-coupled [4Fe-4S] cluster
assembly under the O2-depleted conditions. To overcome
the O2 lability of [4Fe-4S] Fe–S clusters, an O2-scavenging enzyme cascade was incorporated, which begins
with formate oxidation by formate dehydrogenase for NADH regeneration.
Later, NADH is consumed by flavin reductase for FADH2 regeneration.
Finally, bifunctional flavin reductase, along with catalase, removes
O2 from the reaction while supplying FADH2 to
the SufBC2D complex. These amendments enabled a one-pot,
two-step synthesis of mature [4Fe-4S] proteins under aerobic conditions,
yielding holo-aconitase with a maximum concentration of ∼0.15
mg/mL. This renovated system greatly expands the potential of the
PURE system, paving the way for the future reconstruction of redox-active
synthetic cells and enhanced cell-free biocatalysis.