One-Pot De Novo Synthesis of [4Fe-4S] Proteins Using a Recombinant SUF System under Aerobic Conditions

Fe-S clusters are essential cofactors mediatingelectrontransfer in respiratory and metabolic networks. However, obtainingactive [4Fe-4S] proteins with heterologous expression is challengingdue to (i) the requirements for [4Fe-4S] cluster assembly, (ii) theO(2) lability of [4Fe-4S] clusters, and (iii) copurificationof undesired proteins (e.g., ferredoxins). Here, we established afacile and efficient protocol to express mature [4Fe-4S] proteinsin the PURE system under aerobic conditions. An enzyme aconitase andthermophilic ferredoxin were selected as model [4Fe-4S] proteinsfor functional verification. We first reconstituted the SUF system in vitro via a stepwise manner using the recombinant SUFsubunits (SufABCDSE) individually purified from E. coli. Later, the incorporation of recombinant SUF helper proteins intothe PURE system enabled mRNA translation-coupled [4Fe-4S] clusterassembly under the O-2-depleted conditions. To overcomethe O-2 lability of [4Fe-4S] Fe-S clusters, an O-2-scavenging enzyme cascade was incorporated, which beginswith formate oxidation by formate dehydrogenase for NADH regeneration.Later, NADH is consumed by flavin reductase for FADH(2) regeneration.Finally, bifunctional flavin reductase, along with catalase, removesO(2) from the reaction while supplying FADH(2) tothe SufBC(2)D 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 & SIM;0.15mg/mL. This renovated system greatly expands the potential of thePURE system, paving the way for the future reconstruction of redox-activesynthetic cells and enhanced cell-free biocatalysis.