E. coli monothiol glutaredoxin GrxD replenishes Fe-S clusters to the essential ErpA A-type carrier under low iron stress.

Iron-sulfur (Fe-S) clusters are required for essential biological pathways, including respiration and isoprenoid biosynthesis. Complex Fe-S cluster biogenesis systems have evolved to maintain an adequate supply of this critical protein cofactor. In Escherichia coli, two Fe-S biosynthetic systems, the "housekeeping" Isc and "stress responsive" Suf pathways, interface with a network of cluster trafficking proteins, such as ErpA, IscA, SufA, and NfuA. GrxD, a Fe-S cluster-binding monothiol glutaredoxin, also participates in Fe-S protein biogenesis in both prokaryotes and eukaryotes. Previous studies in E. coli showed that the ΔgrxD mutation causes sensitivity to iron depletion, spotlighting a critical role for GrxD under conditions that disrupt Fe-S homeostasis. Here, we utilized a global chemoproteomic mass spectrometry (MS) approach to analyse the contribution of GrxD to the Fe-S proteome. Our results demonstrate that 1) GrxD is required for biogenesis of a specific subset of Fe-S proteins under iron-depleted conditions, 2) GrxD is required for cluster delivery to ErpA under iron limitation, 3) GrxD is functionally distinct from other Fe-S trafficking proteins and, 4) GrxD Fe-S cluster binding is responsive to iron limitation. All these results lead to the proposal that GrxD is required to maintain Fe-S cluster delivery to the essential trafficking protein ErpA during iron limitation conditions.

Evidence that a respiratory shield in Escherichia coli protects a low-molecular-mass FeII pool from O2-dependent oxidation.

Iron is critical for virtually all organisms, yet major questions remain regarding the systems-level understanding of iron in whole cells. Here, we obtained Mössbauer and EPR spectra of Escherichia coli cells prepared under different nutrient iron concentrations, carbon sources, growth phases, and O2 concentrations to better understand their global iron content. We investigated WT cells and those lacking Fur, FtnA, Bfr, and Dps proteins. The coarse-grain iron content of exponentially growing cells consisted of iron-sulfur clusters, variable amounts of nonheme high-spin FeII species, and an unassigned residual quadrupole doublet. The iron in stationary-phase cells was dominated by magnetically ordered FeIII ions due to oxyhydroxide nanoparticles. Analysis of cytosolic extracts by size-exclusion chromatography detected by an online inductively coupled plasma mass spectrometer revealed a low-molecular-mass (LMM) FeII pool consisting of two iron complexes with masses of ∼500 (major) and ∼1300 (minor) Da. They appeared to be high-spin FeII species with mostly oxygen donor ligands, perhaps a few nitrogen donors, and probably no sulfur donors. Surprisingly, the iron content of E. coli and its reactivity with O2 were remarkably similar to those of mitochondria. In both cases, a "respiratory shield" composed of membrane-bound iron-rich respiratory complexes may protect the LMM FeII pool from reacting with O2 When exponentially growing cells transition to stationary phase, the shield deactivates as metabolic activity declines. Given the universality of oxidative phosphorylation in aerobic biology, the iron content and respiratory shield in other aerobic prokaryotes might be similar to those of E. coli and mitochondria.

Direct observation of intermediates in the SufS cysteine desulfurase reaction reveals functional roles of conserved active-site residues.

Iron-sulfur (Fe-S) clusters are necessary for the proper functioning of numerous metalloproteins. Fe-S cluster (Isc) and sulfur utilization factor (Suf) pathways are the key biosynthetic routes responsible for generating these Fe-S cluster prosthetic groups in Escherichia coli Although Isc dominates under normal conditions, Suf takes over during periods of iron depletion and oxidative stress. Sulfur acquisition via these systems relies on the ability to remove sulfur from free cysteine using a cysteine desulfurase mechanism. In the Suf pathway, the dimeric SufS protein uses the cofactor pyridoxal 5'-phosphate (PLP) to abstract sulfur from free cysteine, resulting in the production of alanine and persulfide. Despite much progress, the stepwise mechanism by which this PLP-dependent enzyme operates remains unclear. Here, using rapid-mixing kinetics in conjunction with X-ray crystallography, we analyzed the pre-steady-state kinetics of this process while assigning early intermediates of the mechanism. We employed H123A and C364A SufS variants to trap Cys-aldimine and Cys-ketimine intermediates of the cysteine desulfurase reaction, enabling direct observations of these intermediates and associated conformational changes of the SufS active site. Of note, we propose that Cys-364 is essential for positioning the Cys-aldimine for Cα deprotonation, His-123 acts to protonate the Ala-enamine intermediate, and Arg-56 facilitates catalysis by hydrogen bonding with the sulfhydryl of Cys-aldimine. Our results, along with previous SufS structural findings, suggest a detailed model of the SufS-catalyzed reaction from Cys binding to C-S bond cleavage and indicate that Arg-56, His-123, and Cys-364 are critical SufS residues in this C-S bond cleavage pathway.

Structural Evidence for Dimer-Interface-Driven Regulation of the Type II Cysteine Desulfurase, SufS.

SufS is a type II cysteine desulfurase and acts as the initial step in the Suf Fe-S cluster assembly pathway. In Escherichia coli, this pathway is utilized under conditions of oxidative stress and is resistant to reactive oxygen species. Mechanistically, this means SufS must shift between protecting a covalent persulfide intermediate and making it available for transfer to the next protein partner in the pathway, SufE. Here, we report five X-ray crystal structures of SufS including a new structure of SufS containing an inward-facing persulfide intermediate on C364. Additional structures of SufS variants with substitutions at the dimer interface show changes in dimer geometry and suggest a conserved ß-hairpin structure plays a role in mediating interactions with SufE. These new structures, along with previous HDX-MS and biochemical data, identify an interaction network capable of communication between active-sites of the SufS dimer coordinating the shift between desulfurase and transpersulfurase activities.

Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors.

Heme is an essential cofactor and signaling molecule. Heme acquisition by proteins and heme signaling are ultimately reliant on the ability to mobilize labile heme (LH). However, the properties of LH pools, including concentration, oxidation state, distribution, speciation, and dynamics, are poorly understood. Herein, we elucidate the nature and dynamics of LH using genetically encoded ratiometric fluorescent heme sensors in the unicellular eukaryote Saccharomyces cerevisiae We find that the subcellular distribution of LH is heterogeneous; the cytosol maintains LH at ∼20-40 nM, whereas the mitochondria and nucleus maintain it at concentrations below 2.5 nM. Further, we find that the signaling molecule nitric oxide can initiate the rapid mobilization of heme in the cytosol and nucleus from certain thiol-containing factors. We also find that the glycolytic enzyme glyceraldehyde phosphate dehydrogenase constitutes a major cellular heme buffer, and is responsible for maintaining the activity of the heme-dependent nuclear transcription factor heme activator protein (Hap1p). Altogether, we demonstrate that the heme sensors can be used to reveal fundamental aspects of heme trafficking and dynamics and can be used across multiple organisms, including Escherichia coli, yeast, and human cell lines.

Changes in Protein Dynamics in Escherichia coli SufS Reveal a Possible Conserved Regulatory Mechanism in Type II Cysteine Desulfurase Systems.

In the Suf Fe-S cluster assembly pathway, the activity of the cysteine desulfurase, SufS, is regulated by interactions with the accessory sulfotransferase protein, SufE. SufE has been shown to stimulate SufS activity, likely by inducing conformational changes in the SufS active site that promote the desulfurase step and by acting as an efficient persulfide acceptor in the transpersulfuration step. Previous results point toward an additional level of regulation through a "half-sites" mechanism that affects the stoichiometry and affinity for SufE as the dimeric SufS shifts between desulfurase and transpersulfuration activities. Investigation of the covalent persulfide intermediate of SufS by backbone amide hydrogen-deuterium exchange mass spectrometry identified two active site peptides (residues 225-236 and 356-366) and two peptides at the dimer interface of SufS (residues 88-100 and 243-255) that exhibit changes in deuterium uptake upon formation of the intermediate. Residues in these peptides are organized to form a conduit between the two active sites upon persulfide formation and include key cross-monomer interactions, suggesting they may play a role in the half-sites regulation. Three evolutionarily conserved residues at the dimer interface (R92, E96, and E250) were investigated by alanine scanning mutagenesis. Two of the substituted enzymes (E96A and E250A SufS) resulted in 6-fold increases in the value of KSufE, confirming a functional role. Re-examination of the dimer interface in reported crystal structures of SufS and the SufS homologue CsdA identified previously unnoticed residue mobility at the dimer interface. The identification of conformational changes at the dimer interface by hydrogen-deuterium exchange confirmed by mutagenesis and structural reports provides a physical mechanism for active site communication in the half-sites regulation of SufS activity. Given the conservation of the interface interactions, this mechanism may be broadly applicable to type II cysteine desulfurase systems.

Functional Dynamics Revealed by the Structure of the SufBCD Complex, a Novel ATP-binding Cassette (ABC) Protein That Serves as a Scaffold for Iron-Sulfur Cluster Biogenesis.

ATP-binding cassette (ABC)-type ATPases are chemomechanical engines involved in diverse biological pathways. Recent genomic information reveals that ABC ATPase domains/subunits act not only in ABC transporters and structural maintenance of chromosome proteins, but also in iron-sulfur (Fe-S) cluster biogenesis. A novel type of ABC protein, the SufBCD complex, functions in the biosynthesis of nascent Fe-S clusters in almost all Eubacteria and Archaea, as well as eukaryotic chloroplasts. In this study, we determined the first crystal structure of the Escherichia coli SufBCD complex, which exhibits the common architecture of ABC proteins: two ABC ATPase components (SufC) with function-specific components (SufB-SufD protomers). Biochemical and physiological analyses based on this structure provided critical insights into Fe-S cluster assembly and revealed a dynamic conformational change driven by ABC ATPase activity. We propose a molecular mechanism for the biogenesis of the Fe-S cluster in the SufBCD complex.

Recent advances in the Suf Fe-S cluster biogenesis pathway: Beyond the Proteobacteria.

Fe-S clusters play critical roles in cellular function throughout all three kingdoms of life. Consequently, Fe-S cluster biogenesis systems are present in most organisms. The Suf (sulfur formation) system is the most ancient of the three characterized Fe-S cluster biogenesis pathways, which also include the Isc and Nif systems. Much of the first work on the Suf system took place in Gram-negative Proteobacteria used as model organisms. These early studies led to a wealth of biochemical, genetic, and physiological information on Suf function. From those studies we have learned that SufB functions as an Fe-S scaffold in conjunction with SufC (and in some cases SufD). SufS and SufE together mobilize sulfur for cluster assembly and SufA traffics the complete Fe-S cluster from SufB to target apo-proteins. However, recent progress on the Suf system in other organisms has opened up new avenues of research and new hypotheses about Suf function. This review focuses primarily on the most recent discoveries about the Suf pathway and where those new models may lead the field. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.

SufE D74R Substitution Alters Active Site Loop Dynamics To Further Enhance SufE Interaction with the SufS Cysteine Desulfurase.

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.

Communication between binding sites is required for YqjI regulation of target promoters within the yqjH-yqjI intergenic region.

The nickel-responsive transcription factor YqjI represses its own transcription and transcription of the divergent yqjH gene, which encodes a novel ferric siderophore reductase. The intergenic region between the two promoters is complex, with multiple sequence features that may impact YqjI-dependent regulation of its two target promoters. We utilized mutagenesis and DNase I footprinting to characterize YqjI regulation of the yqjH-yqjI intergenic region. The results show that YqjI binding results in an extended footprint at the yqjI promoter (site II) compared to the yqjH promoter (site I). Mutagenesis of in vivo gene reporter constructs revealed that the two YqjI binding sites, while separated by nearly 200 bp, appear to communicate in order to provide full YqjI-dependent regulation at the two target promoters. Thus, YqjI binding at both promoters is required for full repression of either promoter, suggesting that the two YqjI binding sites cooperate to control transcription from the divergent promoters. Furthermore, internal deletions that shorten the total length of the intergenic region disrupt the ability of YqjI to regulate the yqjH promoter. Finally, mutagenesis of the repetitive extragenic palindromic (REP) elements within the yqjH-yqjI intergenic region shows that these sequences are not required for YqjI regulation. These studies provide a complex picture of novel YqjI transcriptional regulation within the yqjH-yqjI intergenic region and suggest a possible model for communication between the YqjI binding sites at each target promoter.

Interplay between oxygen and Fe-S cluster biogenesis: insights from the Suf pathway.

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.

Escherichia coli SufE sulfur transfer protein modulates the SufS cysteine desulfurase through allosteric conformational dynamics.

Fe-S clusters are critical metallocofactors required for cell function. Fe-S cluster biogenesis is carried out by assembly machinery consisting of multiple proteins. Fe-S cluster biogenesis proteins work together to mobilize sulfide and iron, form the nascent cluster, traffic the cluster to target metalloproteins, and regulate the assembly machinery in response to cellular Fe-S cluster demand. A complex series of protein-protein interactions is required for the assembly machinery to function properly. Despite considerable progress in obtaining static three-dimensional structures of the assembly proteins, little is known about transient protein-protein interactions during cluster assembly or the role of protein dynamics in the cluster assembly process. The Escherichia coli cysteine desulfurase SufS (EC 2.8.1.7) and its accessory protein SufE work together to mobilize persulfide from L-cysteine, which is then donated to the SufB Fe-S cluster scaffold. Here we use amide hydrogen/deuterium exchange mass spectrometry (HDX-MS) to characterize SufS-SufE interactions and protein dynamics in solution. HDX-MS analysis shows that SufE binds near the SufS active site to accept persulfide from Cys-364. Furthermore, SufE binding initiates allosteric changes in other parts of the SufS structure that likely affect SufS catalysis and alter SufS monomer-monomer interactions. SufE enhances the initial l-cysteine substrate binding to SufS and formation of the external aldimine with pyridoxal phosphate required for early steps in SufS catalysis. Together, these results provide a new picture of the SufS-SufE sulfur transferase pathway and suggest a more active role for SufE in promoting the SufS cysteine desulfurase reaction for Fe-S cluster assembly.

Facial amphiphilic naphthoic acid-derived antimicrobial polymers against multi-drug resistant gram-negative bacteria and biofilms.

Inspired by the facial amphiphilic nature and antimicrobial efficacy of many antimicrobial peptides, this work reported facial amphiphilic bicyclic naphthoic acid derivatives with different ratios of charges to rings that were installed onto side chains of poly(glycidyl methacrylate). Six quaternary ammonium-charged (QAC) polymers were prepared to investigate the structure-activity relationship. These QAC polymers displayed potent antibacterial activity against various multi-drug resistant (MDR) gram-negative pathogens such as Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii. Polymers demonstrated low hemolysis and high antimicrobial selectivity. Additionally, they were able to eradicate established biofilms and kill metabolically inactive dormant cells. The membrane permeabilization and depolarization results indicated a mechanism of action through membrane disruption. Two lead polymers showed no resistance from MDR-P. aeruginosa and MDR-K. pneumoniae. These facial amphiphiles are potentially a new class of potent antimicrobial agents to tackle the antimicrobial resistance for both planktonic and biofilm-related infections.

Fur and the novel regulator YqjI control transcription of the ferric reductase gene yqjH in Escherichia coli.

Iron acquisition in aerobic habitats is complicated by the low solubility of ferric hydroxides. Siderophores that bind ferric iron with high affinity are used to mobilize iron. The reduction of ferric iron to the ferrous form can be coupled to the release of iron from siderophores. Iron is also stored intracellularly as a ferric mineral in proteins, such as ferritin, and must be reduced during release. In Escherichia coli, the yqjH gene encodes a putative ferric siderophore reductase that is also part of the Fur regulon. Here we show that YqjH has ferric reductase activity and is required for iron homeostasis in E. coli. Divergently transcribed from yqjH is the yqjI gene, which encodes a novel member of the winged-helix family of transcriptional regulators and also contains an N-terminal extension similar to the Ni(2+)-binding C-terminal tail of SlyD. Deletion of yqjI leads to constitutive high-level activity of the yqjH and yqjI promoters. Purified YqjI binds inverted repeat target sequences within the yqjH and yqjI promoters. We also observed that YqjI-dependent transcriptional repression is reduced when cells are exposed to elevated nickel levels, resulting in increased expression of yqjH and yqjI. YqjI binding to nickel or iron reduces YqjI DNA-binding activity in vitro. Furthermore, we found that elevated nickel stress levels disrupt iron homeostasis in E. coli and that deletion of yqjH increases nickel toxicity. Our results suggest that the YqjI protein controls expression of yqjH to help maintain iron homeostasis under conditions (such as elevated cellular nickel levels) that disrupt iron metabolism.

Ni-NTA Affinity Chromatography to Characterize Protein-Protein Interactions During Fe-S Cluster Biogenesis.

Affinity chromatography can be repurposed to provide useful information about the specific partner protein(s) to which a protein of interest may bind as well as the relative binding affinity of that partner protein for the protein of interest. Here, we provide a protocol for an Ni-NTA affinity chromatography assay that may be utilized to uncover insightful information about the nature of protein-protein interactions during iron-sulfur (Fe-S) cluster biogenesis reactions.

SufD and SufC ATPase activity are required for iron acquisition during in vivo Fe-S cluster formation on SufB.

In vivo biogenesis of Fe-S cluster cofactors requires complex biosynthetic machinery to limit release of iron and sulfide, to protect the Fe-S cluster from oxidation, and to target the Fe-S cluster to the correct apoenzyme. The SufABCDSE pathway for Fe-S cluster assembly in Escherichia coli accomplishes these tasks under iron starvation and oxidative stress conditions that disrupt Fe-S cluster metabolism. Although SufB, SufC, and SufD are all required for in vivo Suf function, their exact roles are unclear. Here we show that SufB, SufC, and SufD, coexpressed with the SufS-SufE sulfur transfer pair, purify as two distinct complexes (SufBC(2)D and SufB(2)C(2)) that contain Fe-S clusters and FADH(2). These studies also show that SufC and SufD are required for in vivo Fe-S cluster formation on SufB. Furthermore, while SufD is dispensable for in vivo sulfur transfer, it is absolutely required for in vivo iron acquisition. Finally, we demonstrate for the first time that the ATPase activity of SufC is necessary for in vivo iron acquisition during Fe-S cluster assembly.

Fe-S cluster biogenesis by the bacterial Suf pathway.

Biogenesis of iron-sulfur (FeS) clusters in an essential process in living organisms due to the critical role of FeS cluster proteins in myriad cell functions. During biogenesis of FeS clusters, multi-protein complexes are used to drive the mobilization and protection of reactive sulfur and iron intermediates, regulate assembly of various FeS clusters on an ATPase-dependent, multi-protein scaffold, and target nascent clusters to their downstream protein targets. The evolutionarily ancient sulfur formation (Suf) pathway for FeS cluster assembly is found in bacteria and archaea. In Escherichia coli, the Suf pathway functions as an emergency pathway under conditions of iron limitation or oxidative stress. In other pathogenic bacteria, such as Mycobacterium tuberculosis and Enterococcus faecalis, the Suf pathway is the sole source for FeS clusters and therefore is a potential target for the development of novel antibacterial compounds. Here we summarize the considerable progress that has been made in characterizing the first step of mobilization and protection of reactive sulfur carried out by the SufS-SufE or SufS-SufU complex, FeS cluster assembly on SufBC2D scaffold complexes, and the downstream trafficking of nascent FeS clusters to A-type carrier (ATC) proteins. Cell Biology of Metals III edited by Roland Lill and Mick Petris.

IscR controls iron-dependent biofilm formation in Escherichia coli by regulating type I fimbria expression.

Biofilm formation is a complex developmental process regulated by multiple environmental signals. In addition to other nutrients, the transition metal iron can also regulate biofilm formation. Iron-dependent regulation of biofilm formation varies by bacterial species, and the exact regulatory pathways that control iron-dependent biofilm formation are often unknown or only partially characterized. To address this gap in our knowledge, we examined the role of iron availability in regulating biofilm formation in Escherichia coli. The results indicate that biofilm formation is repressed under low-iron conditions in E. coli. Furthermore, a key iron regulator, IscR, controls biofilm formation in response to changes in cellular Fe-S homeostasis. IscR regulates the FimE recombinase to control expression of type I fimbriae in E. coli. We propose that iron-dependent regulation of FimE via IscR leads to decreased surface attachment and biofilm dispersal under iron-limiting conditions.

The SufBCD Fe-S scaffold complex interacts with SufA for Fe-S cluster transfer.

Iron-sulfur clusters are key iron cofactors in biological pathways ranging from nitrogen fixation to respiration. Because of the toxicity of ferrous iron and sulfide to the cell, in vivo Fe-S cluster assembly transpires via multiprotein biosynthetic pathways. Fe-S cluster assembly proteins traffic iron and sulfide, assemble nascent Fe-S clusters, and correctly transfer Fe-S clusters to the appropriate target metalloproteins in vivo. The Gram-negative bacterium Escherichia coli contains a stress-responsive Fe-S cluster assembly system, the SufABCDSE pathway, that functions under iron starvation and oxidative stress conditions that compromise Fe-S homeostasis. Using a combination of protein-protein interaction and in vitro Fe-S cluster assembly assays, we have characterized the relative roles of the SufBCD complex and the SufA protein during Suf Fe-S cluster biosynthesis. These studies reveal that SufA interacts with SufBCD to accept Fe-S clusters formed de novo on the SufBCD complex. Our results represent the first biochemical evidence that the SufBCD complex within the Suf pathway functions as a novel Fe-S scaffold system to assemble nascent clusters and transfer them to the SufA Fe-S shuttle.

Native Escherichia coli SufA, coexpressed with SufBCDSE, purifies as a [2Fe-2S] protein and acts as an Fe-S transporter to Fe-S target enzymes.

Iron-sulfur (Fe-S) clusters are versatile biological cofactors that require biosynthetic systems in vivo to be assembled. In Escherichia coli, the Isc (iscRSUA-hscBA-fdx-iscX) and Suf (sufABCDSE) pathways fulfill this function. Despite extensive biochemical and genetic analysis of these two pathways, the physiological function of the A-type proteins of each pathway (IscA and SufA) is still unclear. Studies conducted in vitro suggest two possible functions for A-type proteins, as Fe-S scaffold/transfer proteins or as iron donors during cluster assembly. To resolve this issue, SufA was coexpressed in vivo with its cognate partner proteins from the suf operon, SufBCDSE. Native SufA purified anaerobically using this approach was unambiguously demonstrated to be a [2Fe-2S] protein by biochemical analysis and UV-vis, Mossbauer, resonance Raman, and EPR spectroscopy. Furthermore, native [2Fe-2S] SufA can transfer its Fe-S cluster to both [2Fe-2S] and [4Fe-4S] apoproteins. These results clearly show that A-type proteins form Fe-S clusters in vivo and are competent to function as Fe-S transfer proteins as purified. This study resolves the contradictory results from previous in vitro studies and demonstrates the critical importance of providing in vivo partner proteins during protein overexpression to allow correct biochemical maturation of metalloproteins.