Many essential metalloproteins require iron–sulfur (Fe–S) cluster cofactors for their function. In vivo persulfide formation from l-cysteine is a key step in the biogenesis of Fe–S clusters in most organisms. In Escherichia coli, the SufS cysteine desulfurase mobilizes persulfide from l-cysteine via a PLP-dependent ping-pong reaction. SufS requires the SufE partner protein to transfer the persulfide to the SufB Fe–S cluster scaffold. Without SufE, the SufS enzyme fails to efficiently turn over and remains locked in the persulfide-bound state. Coordinated protein–protein interactions mediate sulfur transfer from SufS to SufE. Multiple studies have suggested that SufE must undergo a conformational change to extend its active site Cys loop during sulfur transfer from SufS. To test this putative model, we mutated SufE Asp74 to Arg (D74R) to increase the dynamics of the SufE Cys51 loop. Amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) analysis of SufE D74R revealed an increase in solvent accessibility and dynamics in the loop containing the active site Cys51 used to accept persulfide from SufS. Our results indicate that the mutant protein has a stronger binding affinity for SufS than that of wild-type SufE. In addition, SufE D74R can still enhance SufS desulfurase activity and did not show saturation at higher SufE D74R concentrations, unlike wild-type SufE. These results show that dynamic changes may shift SufE to a sulfur-acceptor state that interacts more strongly with SufS.

Iron–sulfur (Fe–S) cluster metalloproteins conduct essential functions in nearly all contemporary forms of life. The nearly ubiquitous presence of Fe–S clusters and the fundamental requirement for Fe–S clusters in both aerobic and anaerobic Archaea, Bacteria, and Eukarya suggest that these clusters were likely integrated into central metabolic pathways early in the evolution of life prior to the widespread oxidation of Earth’s atmosphere. Intriguingly, Fe–S cluster-dependent metabolism is sensitive to disruption by oxygen because of the decreased bioavailability of ferric iron as well as direct oxidation of sulfur trafficking intermediates and Fe–S clusters by reactive oxygen species. This fact, coupled with the ubiquity of Fe–S clusters in aerobic organisms, suggests that organisms evolved with mechanisms that facilitate the biogenesis and use of these essential cofactors in the presence of oxygen, which gradually began to accumulate around 2.5 billion years ago as oxygenic photosynthesis proliferated and reduced minerals that buffered against oxidation were depleted. This review highlights the most ancient of the Fe–S cluster biogenesis pathways, the Suf system, which likely was present in early anaerobic forms of life. Herein, we use the evolution of the Suf pathway to assess the relationships between the biochemical functions and physiological roles of Suf proteins, with an emphasis on the selective pressure of oxygen toxicity. Our analysis suggests that diversification into oxygen-containing environments disrupted iron and sulfur metabolism and was a main driving force in the acquisition of accessory Suf proteins (such as SufD, SufE, and SufS) by the core SufB–SufC scaffold complex. This analysis provides a new framework for the study of Fe–S cluster biogenesis pathways and Fe–S cluster-containing metalloenzymes and their complicated patterns of divergence in response to oxygen.

Iron–sulfur (FeS) clusters are inorganic cofactors required for a variety of biological processes. In vivo biogenesis of FeS clusters proceeds via complex pathways involving multiple protein complexes. In the Suf FeS cluster biogenesis system, SufB may be a scaffold for nascent FeS cluster assembly whereas SufA is proposed to act as either a scaffold or an FeS cluster carrier from the scaffold to target apo-proteins. However, SufB can form multiple stable complexes with other Suf proteins, such as SufB2C2 and SufBC2D and the specific functions of these complexes in FeS cluster assembly are not clear. Here we compare the ability of the SufB2C2 and SufBC2D complexes as well as SufA to promote in vitro maturation of the [2Fe2S] ferredoxin (Fdx). We found that SufB2C2 was most proficient as a scaffold for de novo assembly of holo-Fdx using sulfide and iron as freely available building blocks while SufA was best at direct transfer of a pre-formed FeS cluster to Fdx. Furthermore, cluster transfer from [4Fe4S] SufB2C2 or SufBC2D to Fdx will proceed through a SufA intermediate to Fdx if SufA is present. Finally, addition of ATP repressed cluster transfer from [4Fe4S] SufB2C2 to Fdx and from SufBC2D to [2Fe2S] SufA or Fdx. These studies indicate that SufB2C2 can serve as a terminal scaffold to load the SufA FeS cluster carrier for in vitro maturation of [2Fe2S] enzymes like Fdx. This work is the first to systematically compare the cluster transfer rates of a scaffold (SufB) to the transfer rates of a carrier (SufA) under the same conditions to the same target enzyme and is also the first to reconstitute the full transfer pathway (from scaffold to carrier to target enzyme) in a single reaction.This work elucidates the FeS cluster trafficking pathway from SufB to SufA to the target metalloprotein Fdx.Highlights► SufB2C2 acts as a scaffold to enhance Fdx cluster formation. ► SufA acts as an intermediate during FeS cluster transfer. ► ATP suppresses cluster transfer from SufBC2D to SufA. ► SufA does not function as a de novo cluster scaffold.

During oxidative stress in Escherichia coli, the SufABCDSE stress response pathway mediates iron–sulfur (Fe–S) cluster biogenesis rather than the Isc pathway. To determine why the Suf pathway is favored under stress conditions, the stress response SufS–SufE sulfur transfer pathway and the basal housekeeping IscS–IscU pathway were directly compared. We found that SufS–SufE cysteine desulfurase activity is significantly higher than IscS–IscU at physiological cysteine concentrations and after exposure to H2O2. Mass spectrometry analysis demonstrated that IscS–IscU is more susceptible than SufS–SufE to oxidative modification by H2O2. These important results provide biochemical insight into the stress resistance of the Suf pathway.Highlights► We characterize the kinetics of the SufS–SufE sulfur transfer system. ► We directly compare SufS–SufE to the IscS–IscU system from E. coli. ► SufS–SufE are more active at physiological l-cysteine levels than IscS–IscU. ► SufS–SufE activity is more resistant to oxidative stress than IscS–IscU.