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

Abstract

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.

Keywords: Fe−S cluster; SUF helper protein; aconitase; cofactor regeneration; reconstituted cell-free protein synthesis; redox enzymes.

Figure 1

Schematic diagram of the SUF pathway. The SufBC₂D complex acts as a scaffold, receiving the sulfide (S^2–) group from the SufES complex and incorporating Fe²⁺ and/or Fe³⁺ depending on the type of cluster for Fe–S cluster assembly. FADH₂ is used for Fe³⁺ reduction and [4Fe-4S] cluster assembly is driven by ATP hydrolysis. The assembled [4Fe-4S] cluster is then transferred to the SufA, which further transfers the cluster to the recipient proteins.

Figure 2

Anaerobic [4Fe-4S] cluster reconstitution using the SUF system. (A) Cysteine desulfurase activity of the SufES complex was measured with and without SufS or SufE. Free S^2– production was measured based on the time-dependent production of methylene blue (λ_670 nm). The bars represent the range and the data points represent the average from two independent experimental replicates, respectively. (B) Observation of [4Fe-4S] clusters (λ_420 nm) degradation on the reconstituted SufBC₂D complex after a 10 min aeration. (C,D) SUF-mediated [4Fe-4S] assembly was conducted with the oxidized SufBC₂D complex in the (C) presence or (D) absence of SufES under anaerobic conditions.

Figure 3

[4Fe-4S] cluster transfer from mature SufBC₂D complex to apo-AcnA. (A) Apo-AcnA maturation by the SUF system reconstructed under anaerobic and aerobic conditions created by enzymatic O₂-scavenging system (see later section). (B) Anaerobic holo-AcnA synthesis using mature SUF, heat-denatured SUF system, or S^2–. The reconstitution efficiency of holo-AcnA is evaluated as a function of the relative enzymatic activity to that of the fully chemically reconstituted AcnA using an isocitrate dehydrogenase activity assay (see Supporting Methods). The bars represent the standard deviations. The dots and columns represent the individual values and mean from three independent experimental replicates, respectively.

Figure 4

The bifunctional enzyme cascade for FADH₂ regeneration and O₂ scavenge. (A) Schematic diagram showing the bifunctional O₂-scavenging enzyme cascade. (B) FADH₂ production was monitored by a time-dependent decrease in λ_445 nm (0–10 min). The reactions were conducted with and without catalase (CAT), flavin reductase (FRE), or FAD. (C) The anaerobicity of the reaction mixture was evaluated by an oxidation–reduction indicator, resorufin. The bars represent the standard deviation. The dots and columns represent the individual value and mean from three independent experimental replicates, respectively.

Figure 5

The bifunctional O₂-scavenging enzyme cascade enables SUF-mediated [4Fe-4S] cluster assembly under aerobic conditions. (A) The SUF-mediated [4Fe-4S] assembly (based on the increase in λ_420 nm) with and without the O₂-scavenging system under aerobic conditions. (B) The bifunctional O₂-scavenging enzyme cascade protects the [4Fe-4S] cluster from oxidation (based on the decrease in λ_420 nm) under aerobic conditions. The degradation and formation of [4Fe-4S] clusters were monitored by the UV–vis spectrophotometric analysis (280–700 nm).

Figure 6

One-pot de novo synthesis of mature [4Fe-4S] protein under aerobic conditions. (A) Schematic diagram showing one-pot two-step cell-free synthesis of mature [4Fe-4S] cluster bearing AcnA under aerobic conditions. In vitro translation of AcnA mRNA is carried out in a test tube along with the three O₂-scavanging enzymes (Step 1). FeSO₄ and SUF components are further added to drive maturation of the apo-AcnA to its holo-form (Step 2). Other required components such as cysteine (Cys) and ATP are carried over from the PUREfrex reaction. (B) Superfolder GFP (sfGFP) production in the PUREfrex reaction mixtures with and without the O₂-scavenging enzyme cascade after 6 h of incubation. (C) Purification of holo-AcnA free of N-His₆-tag verified by SDS-PAGE analysis. (D) Relative activity of AcnA in the one-pot PURE system with and without the SUF and O₂-scavanging system. The bars represent the standard deviations. The dots and columns represent the individual values and mean from three independent experimental replicates, respectively.