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1. The C-terminal domain of the ferric uptake regulator (Fur) binds a [2Fe–2S] cluster to sense the intracellular free iron content in Escherichia coli

   https://doi.org/10.1007/s10534-023-00517-6  

   Fontenot, Chelsey R.; Ding, Huangen (December 2023, BioMetals)

   Escherichia coli Ferric uptake regulator (Fur) binds a [2Fe-2S] cluster, not a mononuclear iron, when the intracellular free iron content is elevated in E. coli cells. Here we report that the C-terminal domain (residues 83-148) of E. coli Fur (Fur-CTD) is sufficient to bind the [2Fe-2S] cluster in response to elevation of the intracellular free iron content in E. coli cells. Deletion of gene fur in E. coli cells increases the intracellular free iron content and promotes the [2Fe-2S] cluster binding in the Fur-CTD in the cells grown in LB medium under aerobic growth conditions. When the Fur-CTD is expressed in wild type E. coli cells grown in M9 medium supplemented with increasing concentrations of iron, the Fur-CTD also progressively binds a [2Fe-2S] cluster with a maximum occupancy of about 36%. Like the E. coli Fur-CTD, the CTD of the Haemophilus influenzae Fur can also bind a [2Fe-2S] cluster in wild type E. coli cells grown in M9 medium supplemented with increasing concentrations of iron, indicating that binding of the [2Fe-2S] cluster in the C-terminal domain is highly conserved among Fur proteins. The results suggest that the Fur-CTD can be used as a physiological probe to assess the intracellular free iron content in bacteria. 

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2. Mössbauer studies of the redox state of the ferric uptake regulator [2Fe–2S]2+ cluster in Escherichia coli

   https://doi.org/10.1016/j.jinorgbio.2025.112928  

   Fontenot, Chelsey R; Hoepner, Thomas; Xiong, Jin; Ding, Huangen; Popescu, Codrina V (September 2025, Journal of Inorganic Biochemistry)

   Ciurli, S (Ed.)

   The Ferric uptake regulator (Fur) proteins from Haemophilus influenzae and Escherichia coli overexpressed in E. coli cells (MC4100) grown in M9 medium supplemented with 57Fe were studied with Mössbauer spectroscopy. Previous studies have shown that Fur proteins from H. influenzae and E. coli bind a [2Fe-2S]2+ cluster in response to elevation of intracellular free iron content. Here we find that when the [2Fe-2S] 2+ clusters in purified Fur proteins are reduced with dithionite, the reduced clusters are quickly decomposed, forming compounds with two distinct spectral signatures of high spin Fe(II) in tetrahedral and octahedral coordination, respectively. The instability of the reduced [2Fe-2S]1+ cluster in Fur is unique, as the [2Fe-2S]2+ clusters in many other proteins can reversibly undergo one-electron reduction-oxidation. The Mössbauer spectra of whole E. coli cells overexpressing Fur proteins show a quadrupole doublet with the isomer shift of δ1 = 0.28 mm/s and ΔEQ1 = 0.52 mm/s, typical for oxidized [2Fe-2S]2+ clusters and identical with that in the purified Fur protein. The corresponding spectra in large applied magnetic fields show the diamagnetic pattern that unambiguously reveals an exchange-coupled system with a diamagnetic electronic ground state, which confirms its assignment to the oxidized [2Fe-2S]2+ cluster clusters from Fur. No reduced [2Fe-2S]1+ clusters of Fur are observed in the whole-cell E. coli spectra. The Mössbauer spectra of the whole-cell E. coli without the Fur expression do not contain the components associated with the [2Fe-2S]2+ cluster of Fur. 

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3. A VDAC1-mediated NEET protein chain transfers [2Fe-2S] clusters between the mitochondria and the cytosol and impacts mitochondrial dynamics

   https://doi.org/10.1073/pnas.2121491119  

   Karmi, Ola; Marjault, Henri-Baptiste; Bai, Fang; Roy, Susmita; Sohn, Yang-Sung; Darash Yahana, Merav; Morcos, Faruck; Ioannidis, Konstantinos; Nahmias, Yaakov; Jennings, Patricia A.; et al (February 2022, Proceedings of the National Academy of Sciences)

   Mitochondrial inner NEET (MiNT) and the outer mitochondrial membrane (OMM) mitoNEET (mNT) proteins belong to the NEET protein family. This family plays a key role in mitochondrial labile iron and reactive oxygen species (ROS) homeostasis. NEET proteins contain labile [2Fe-2S] clusters which can be transferred to apo-acceptor proteins. In eukaryotes, the biogenesis of [2Fe-2S] clusters occurs within the mitochondria by the iron–sulfur cluster (ISC) system; the clusters are then transferred to [2Fe-2S] proteins within the mitochondria or exported to cytosolic proteins and the cytosolic iron–sulfur cluster assembly (CIA) system. The last step of export of the [2Fe-2S] is not yet fully characterized. Here we show that MiNT interacts with voltage-dependent anion channel 1 (VDAC1), a major OMM protein that connects the intermembrane space with the cytosol and participates in regulating the levels of different ions including mitochondrial labile iron (mLI). We further show that VDAC1 is mediating the interaction between MiNT and mNT, in which MiNT transfers its [2Fe-2S] clusters from inside the mitochondria to mNT that is facing the cytosol. This MiNT–VDAC1–mNT interaction is shown both experimentally and by computational calculations. Additionally, we show that modifying MiNT expression in breast cancer cells affects the dynamics of mitochondrial structure and morphology, mitochondrial function, and breast cancer tumor growth. Our findings reveal a pathway for the transfer of [2Fe-2S] clusters, which are assembled inside the mitochondria, to the cytosol. 

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4. Tailoring a Global Iron Regulon to a Uropathogen

   https://doi.org/10.1128/mBio.00351-20  

   Banerjee, Rajdeep; Weisenhorn, Erin; Schwartz, Kevin J.; Myers, Kevin S.; Glasner, Jeremy D.; Perna, Nicole T.; Coon, Joshua J.; Welch, Rodney A.; Kiley, Patricia J. (April 2020, mBio)

   Gottesman, Susan (Ed.)

   ABSTRACT Pathogenicity islands and plasmids bear genes for pathogenesis of various Escherichia coli pathotypes. Although there is a basic understanding of the contribution of these virulence factors to disease, less is known about variation in regulatory networks in determining disease phenotypes. Here, we dissected a regulatory network directed by the conserved iron homeostasis regulator, ferric uptake regulator (Fur), in uropathogenic E. coli (UPEC) strain CFT073. Comparing anaerobic genome-scale Fur DNA binding with Fur-dependent transcript expression and protein levels of the uropathogen to that of commensal E. coli K-12 strain MG1655 showed that the Fur regulon of the core genome is conserved but also includes genes within the pathogenicity/genetic islands. Unexpectedly, regulons indicative of amino acid limitation and the general stress response were also indirectly activated in the uropathogen fur mutant, suggesting that induction of the Fur regulon increases amino acid demand. Using RpoS levels as a proxy, addition of amino acids mitigated the stress. In addition, iron chelation increased RpoS to the same levels as in the fur mutant. The increased amino acid demand of the fur mutant or iron chelated cells was exacerbated by aerobic conditions, which could be partly explained by the O 2 -dependent synthesis of the siderophore aerobactin, encoded by an operon within a pathogenicity island. Taken together, these data suggest that in the iron-poor environment of the urinary tract, amino acid availability could play a role in the proliferation of this uropathogen, particularly if there is sufficient O 2 to produce aerobactin. IMPORTANCE Host iron restriction is a common mechanism for limiting the growth of pathogens. We compared the regulatory network controlled by Fur in uropathogenic E. coli (UPEC) to that of nonpathogenic E. coli K-12 to uncover strategies that pathogenic bacteria use to overcome iron limitation. Although iron homeostasis functions were regulated by Fur in the uropathogen as expected, a surprising finding was the activation of the stringent and general stress responses in the uropathogen fur mutant, which was rescued by amino acid addition. This coordinated global response could be important in controlling growth and survival under nutrient-limiting conditions and during transitions from the nutrient-rich environment of the lower gastrointestinal (GI) tract to the more restrictive environment of the urinary tract. The coupling of the response of iron limitation to increased demand for amino acids could be a critical attribute that sets UPEC apart from other E. coli pathotypes. 

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5. The Cluster Transfer Function of AtNEET Supports the Ferredoxin–Thioredoxin Network of Plant Cells

   https://doi.org/10.3390/antiox11081533  

   Zandalinas, Sara I.; Song, Luhua; Nechushtai, Rachel; Mendoza-Cozatl, David G.; Mittler, Ron (August 2022, Antioxidants)

   NEET proteins are conserved 2Fe-2S proteins that regulate the levels of iron and reactive oxygen species in plant and mammalian cells. Previous studies of seedlings with constitutive expression of AtNEET, or its dominant-negative variant H89C (impaired in 2Fe-2S cluster transfer), revealed that disrupting AtNEET function causes oxidative stress, chloroplast iron overload, activation of iron-deficiency responses, and cell death. Because disrupting AtNEET function is deleterious to plants, we developed an inducible expression system to study AtNEET function in mature plants using a time-course proteomics approach. Here, we report that the suppression of AtNEET cluster transfer function results in drastic changes in the expression of different members of the ferredoxin (Fd), Fd-thioredoxin (TRX) reductase (FTR), and TRX network of Arabidopsis, as well as in cytosolic cluster assembly proteins. In addition, the expression of Yellow Stripe-Like 6 (YSL6), involved in iron export from chloroplasts was elevated. Taken together, our findings reveal new roles for AtNEET in supporting the Fd-TFR-TRX network of plants, iron mobilization from the chloroplast, and cytosolic 2Fe-2S cluster assembly. In addition, we show that the AtNEET function is linked to the expression of glutathione peroxidases (GPXs), which play a key role in the regulation of ferroptosis and redox balance in different organisms. 

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