NfuA, a New Factor Required for Maturing Fe/S Proteins in Escherichia coli under Oxidative Stress and Iron Starvation Conditions

Abstract

Iron/sulfur (Fe/S) proteins are central to the functioning of cells in both prokaryotes and eukaryotes. Here, we show that the yhgI gene, which we renamed nfuA, encodes a two-domain protein that is required for Fe/S biogenesis in Escherichia coli. The N-terminal domain resembles the so-called Fe/S A-type scaffold but, curiously, has lost the functionally important Cys residues. The C-terminal domain shares sequence identity with Nfu proteins. Mössbauer and UV-visible spectroscopic analyses revealed that, upon reconstitution, NfuA binds a [4Fe-4S] cluster. Moreover, NfuA can transfer this cluster to apo-aconitase. Mutagenesis studies indicated that the N-and C-terminal domains are important for NfuA function in vivo. Similarly, the functional importance of Cys residues present in the Nfu-like domain was demonstrated in vivo by introducing Cys3 Ser mutations. In vivo investigations revealed that the nfuA gene is important for E. coli to sustain oxidative stress and iron starvation. Also, combining nfuA with either isc or suf mutations led to additive phenotypic deficiencies, indicating that NfuA is a bona fide new player in Isc-and Suf-dependent Fe/S biogenesis pathways. Taken together, these data demonstrate that NfuA intervenes in the maturation of apoproteins in E. coli, allowing them to acquire Fe/S clusters. By taking into account results from numerous previous transcriptomic studies that had suggested a link between NfuA and protein misfolding, we discuss the possibility that NfuA could act as a scaffold/chaperone for damaged Fe/S proteins.

A Proposed Role for the Azotobacter vinelandii NfuA Protein as an Intermediate Iron-Sulfur Cluster Carrier

“…Such variations in the [Fe-S] protein maturation process in different organisms are probably linked to physiological conditions, most significantly, intracellular redox conditions. Results similar to those reported here using A. vinelandii have also been found for the NfuA protein from E. coli (45).…”

Physical and Functional Interactions of a Monothiol Glutaredoxin and an Iron Sulfur Cluster Carrier Protein with the Sulfur-donating Radical S-Adenosyl-l-methionine Enzyme MiaB

“…A commonly used approach when characterizing FeS cluster scaffold and carrier proteins is to assess their capacity to transfer FeS clusters, intact, to partner carriers or model FeS client apoproteins (8,16,17). Indeed, the in vitro transfer of an FeS cluster from A. vinelandii IscU scaffold protein to the monothiol glutaredoxin A. vinelandii Grx5 and the transfer of an FeS cluster from A. vinelandii Grx-nif (among others) to the A. vinelandii Nif IscA carrier protein have recently been reported (13,36).…”

“…One potential clue as to in vivo GrxD function in E. coli comes from protein-protein interaction data (14). Sequential peptide affinity (SPA) purification of native levels of GrxD-SPA and BolA-SPA affinity-tagged proteins revealed that although GrxD co-purified with only small amounts of BolA, the affinity purification of BolA resulted in the co-purification of GrxD and small quantities of several other proteins including MiaB (14,15) and the recently characterized NfuA (8,16). 3 These data therefore implicated GrxD (and/or BolA) and NfuA in in vivo binding of, and potential FeS cluster transfer to, a client FeS apoprotein in MiaB.…”

“…NfuA is necessary for adaptation to oxidative stress and iron starvation in E. coli and linked to aconitase A maturation in A. vinelandii (16,17). Recent work demonstrated that NfuA acts as an FeS carrier protein that functions outside of defined Suf or Isc biosynthesis systems and is involved in maturation of aconitase B (AcnB) and the NuoG subunit of NADH dehydrogenase in E. coli (8).…”

Bacterial ApbC Protein Has Two Biochemical Activities That Are Required for in Vivo Function

“…Examples include the following: CyaY, an iron-binding protein believed to be involved in iron trafficking and iron delivery (5-7); YggX, an Fe 2ϩ -binding protein that protects the cell from oxidative stress (8,9); ErpA, an alternate A-type [Fe-S] cluster scaffolding protein (10); NfuA, a proposed intermediate [Fe-S] delivery protein (11)(12)(13); YtfE, a protein proposed to be involved in [Fe-S] cluster repair (14,15); and CsdA-CsdE, an alternative cysteine desulferase (16).…”