CoChat
Sign Up
Cited by 30Iron Metabolism and DisordersMetalloenzymes and iron-sulfur proteinsPlant Micronutrient Interactions and Effects

Labile Iron Pool of Isolated Escherichia coli Cytosol Likely Includes Fe-ATP and Fe-Citrate but not Fe-Glutathione or Aqueous Fe

Open access
Brawley, Hayley N., Kreinbrink, Alexia C., Hierholzer, Justin D., Vali, Shaik Waseem, Lindahl, Paul A.·2023·Journal of the American Chemical Society
DOI 10.1021/jacs.2c06625PMC PMC9896560
Open this paper in CoChat — free accountOpen PDF

Abstract

High Resolution Image Download MS PowerPoint Slide The existence of labile iron pools (LFePs) in biological systems has been recognized for decades, but their chemical composition remains uncertain. Here, the LFeP in cytosol from Escherichia coli was investigated. Mössbauer spectra of whole vs lysed cells indicated significant degradation of iron-sulfur clusters (ISCs), even using an unusually gentle lysis procedure; this demonstrated the fragility of ISCs. Moreover, the released iron contributed to the non-heme high-spin Fe(II) species in the cell, which likely included the LFeP. Cytosol batches isolated from cells grown with different levels of iron supplementation were passed through a 3 kDa cutoff membrane, and resulting flow-through-solutions (FTSs) were subjected to SEC-ICP-MS. Mössbauer spectroscopy was used to evaluate the oxidation states of standards. FTSs exhibited iron-detected peaks likely due to different forms of Fe-citrate and Fe-nucleotide triphosphate complexes. Fe-Glutathione (GSH) complexes were not detected using physiological concentrations of GSH mixed with either Fe(II) or Fe(III); Fe(II)-GSH was concluded not to be a significant component of the LFeP in E. coli under physiological conditions. Aqueous iron was also not present in significant concentrations in isolated cytosol and is unlikely a major component of the pool. Fe appeared to bind ATP more tightly than citrate, but ATP also hydrolyzed on the timescale of tens of hours. Isolated cytosol contained excess ligands that coordinated the added Fe(II) and Fe(III). The LFeP in healthy metabolically active cells is undoubtedly dominated by the Fe(II) state, but the LFeP is redox-active such that a fraction might be present as stable and soluble Fe(III) complexes especially under oxidatively stressed cellular conditions.

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1021/jacs.2c06625

Cites

1 of 1 references verified0 citation errors · 0 not checkable

Statistics

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Iron quotas in E. coli and B. subtilis are ∼500 μM, predominantly in the reduced Fe2+ state.

    “The iron quotas in the model organisms E. coli and B. subtilis are ∼500 μM, which is predominantly in the reduced Fe2+ (ferrous) state.122−124”

    From Metals in Motion: Understanding Labile Metal Pools in Bacteria (2025) · cites this paper as [124]

  • Recent studies using Mössbauer spectroscopy of anaerobically generated E. coli lysates have defined a low molecular mass, labile pool dominated by Fe2+ complexes with NTPs and citrate, and GSH complexes were not detected.

    “However, recent studies using Mössbauer spectroscopy of anaerobically generated E. coli lysates have defined a low molecular mass, labile pool dominated by Fe2+ complexes with NTPs and citrate, and GSH complexes were not detected.124”

    From Metals in Motion: Understanding Labile Metal Pools in Bacteria (2025) · cites this paper as [124]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Cites

Flagged first, then by citations

Research works printed but not linked: 52

  1. Chandrangsu P.; Rensing C.; Helmann J. D. Metal homeostasis and resistance in bacteria. Nat. Rev. Microbiol. 2017, 15, 338– 350. 10.1038/nrmicro.2017.15. 28344348 PMC5963929 · DOI · Paper page
  2. Mettert E. L.; Kiley P. J. How is Fe-S Cluster formation regulated?. Annu. Rev. Microbiol. 2015, 69, 505– 526. 10.1146/annurev-micro-091014-104457. 26488283 PMC7487215 · DOI
  3. Crack J. C.; Le Brun N. E. Biological iron-sulfur clusters: Mechanistic insights from mass spectrometry. Coord. Chem. Rev. 2021, 448, 214171 10.1016/j.ccr.2021.214171. · DOI
  4. Fontecave M.; de Choudens S. O.; Py B.; Barras F. Mechanisms of iron-sulfur cluster assembly: the SUF machinery. J. Biol. Inorg. Chem. 2005, 10, 713– 721. 10.1007/s00775-005-0025-1. 16211402 · DOI
  5. Lindahl P. A.; Moore M. J. Labile Low-Molecular-Mass metal complexes in mitochondria: trials and tribulations of a burgeoning field. Biochemistry 2016, 55, 4140– 4153. 10.1021/acs.biochem.6b00216. 27433847 PMC5049694 · DOI
  6. Jomova K.; Valko M. Advances in metal-induced oxidative stress and human disease. Toxicology 2011, 283, 65– 87. 10.1016/j.tox.2011.03.001. 21414382 · DOI
  7. Konopka K.; Szotor M. Determination of iron in acid-soluble fraction of human erythrocytes. Acta Haematol. 2004, 47, 157– 163. 10.1159/000208510. 4402337 · DOI
  8. Goucher C. R.; Taylor J. F. Compounds of ferric iron with adenosine triphosphate and other nucleoside phosphates. J. Biol. Chem. 1964, 239, 2251– 2255. 10.1016/S0021-9258(20)82227-3. 14209955 · DOI
  9. Bartlett G. R. Iron nucleotides in human and rat red cells. Biochem. Biophys. Res. Commun. 1976, 70, 1063– 1070. 10.1016/0006-291X(76)91010-X. 942428 · DOI
  10. Bakkeren D. L.; De Jeu-Jaspars C. M.; van der Heul C.; van Euk H. G. Analysis of iron-binding components in the low molecular weight fraction of rat reticulocyte cytosol. Int. J. Biochem. 1985, 17, 925– 930. 10.1016/0020-711X(85)90177-6. 4043511 · DOI
  11. Weaver J.; Pollack S. Low-Mr iron isolated from gunea-pig reticulocytes as AMP-Fe and ATP-Fe complexes. Biochem. J. 1989, 261, 787– 792. 10.1042/bj2610787. 2803243 PMC1138900 · DOI
  12. Ma Y.; Abbate V.; Hider R. C. Iron-sensitive fluorescent probes: monitoring intracellular iron pools. Metallomics 2015, 7, 212– 222. 10.1039/C4MT00214H. 25315476 · DOI
  13. Au-Yeung H. Y.; Chan J.; Chantarojsiri T.; Chang C. J. Molecular Imaging of Labile Iron(II) Pools in Living Cells with a Turn-On Fluorescent Probe. J. Am. Chem. Soc. 2013, 135, 15165– 15173. 10.1021/ja4072964. 24063668 PMC3838104 · DOI
  14. Prus E.; Fibach E. Flow cytometry measurement of the labile iron pool in human hematopoietic cells. Cytometry, Part A 2008, 73A, 22– 27. 10.1002/cyto.a.20491. 18044720 · DOI
  15. Iovan D. A.; Jia S.; Chang C. J. Inorganic chemistry approaches to activity-based sensing: from metal sensors to bioorthogonal metal chemistry. Inorg. Chem. 2019, 58, 13546– 13560. 10.1021/acs.inorgchem.9b01221. 31185541 PMC8544879 · DOI
  16. Rothman R. J.; Serroni A.; Farber J. L. Cellular pool of transient ferric iron, chelatable by deferoxamine and distinct from ferritin, that is involved in oxidative cell injury. Mol. Pharmocol. 1992, 42, 703– 710. 1435746
  17. Egyed A.; Saltman P. Iron is maintained as Fe(II) under aerobic conditions in erythroid cells. Biol. Trace Elem. Res. 1984, 6, 357– 364. 10.1007/BF02989243. 24264114 · DOI
  18. Breuer W.; Epsztejn S.; Cabantchik Z. I. Iron acquired from transferrin by K562 cells is delivered into a cytoplasmic pool of chelatable iron(II). J. Biol. Chem. 1995, 270, 24209– 24215. 10.1074/jbc.270.41.24209. 7592626 · DOI · Paper page
  19. Petrat F.; de Groot H.; Sustmann R.; Rauen U. The chelatable iron pool in living cells: a methodically defined quantity. Biol. Chem. 2002, 362, 137– 147. 10.1515/BC.2002.051. 12033438 · DOI
  20. Williams R. J. P. Free manganese(II) and iron(II) cations can act as intracellular cell controls. FEBS Lett. 1982, 140, 3– 10. 10.1016/0014-5793(82)80508-5. 7084455 · DOI · Paper page
  21. Thomas F.; Serratrice G.; Béguin C.; Saint Aman E.; Pierre J. L.; Fontecave M.; Laulhère J. P. Calcein as a fluorescent probe for ferric iron – application to iron nutrition in plant cells. J. Biol. Chem. 1999, 274, 13375– 13383. 10.1074/jbc.274.19.13375. 10224100 · DOI
  22. Breuer W.; Epsztejn S.; Cabantchik Z. I. Dynamics of the cytosolic chelatable iron pool of K562 cells. FEBS Lett. 1996, 382, 304– 308. 10.1016/0014-5793(96)00190-1. 8605990 · DOI
  23. Epsztejn S.; Kakhlon O.; Glickstein H.; Breuer W.; Cabantchik Z. I. Fluorescence analysis of the labile iron pool of mammalian cells. Anal. Biochem. 1997, 248, 31– 40. 10.1006/abio.1997.2126. 9177722 · DOI · Paper page
  24. Petrat F.; Rauen U.; De Groot H. Determination of the chelatable iron pool of isolated rat hepatocytes by digital fluorescence microscopy using the fluorescent probe, phen green SK. Hepatology 1999, 29, 1171– 1179. 10.1002/hep.510290435. 10094962 · DOI
  25. Petrat F.; de Groot H.; Sustmann R.; Rauen U. The chelatable iron pool in living cells: a methodically defined quantity. Biol. Chem. 2002, 383, 489– 502. 10.1515/BC.2002.051. 12033438 · DOI
  26. Ma Y.; de Groot H.; Liu Z.; Hider R. C.; Petrat F. Chelation and determination of labile iron in primary hepatocytes by pyridinone fluorescent probes. Biochem. J. 2006, 395, 49– 55. 10.1042/BJ20051496. 16336208 PMC1409684 · DOI
  27. Keyer K.; Imlay J. A. Superoxide accelerates DNA damage by elevating free-iron levels. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 13635– 13640. 10.1073/pnas.93.24.13635. 8942986 PMC19375 · DOI · Paper page
  28. Helmann J. D. Specificity of metal sensing: iron and manganese homeostasis in Bacillus subtilis. J. Biol. Chem. 2014, 289, 28112– 28120. 10.1074/jbc.R114.587071. 25160631 PMC4192466 · DOI · Paper page
  29. Beauchene N. A.; Mettert E. L.; Moore L. J.; Keleş S.; Willey E. R.; Kiley P. J. O2 availability impacts iron homeostasis in Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 12261– 12266. 10.1073/pnas.1707189114. 29087312 PMC5699043 · DOI
  30. Wofford J. D.; Bolaji N.; Dziuba N.; Outten F. W.; Lindahl P. A. Evidence that a respiratory shield in Escherichia coli protects a low-molecular-mass FeII pool from O2-dependent oxidation. J. Biol. Chem. 2019, 294, 50– 62. 10.1074/jbc.RA118.005233. 30337367 PMC6322884 · DOI
  31. Hider R. C.; Kong X. L. Glutathione: a key component of the cytoplasmic labile iron pool. BioMetals 2011, 24, 1179– 1187. 10.1007/s10534-011-9476-8. 21769609 · DOI · Paper page
  32. Hider R. C.; Kong X. Iron speciation in the cytosol: an overview. Dalton Trans. 2013, 42, 3220– 3229. 10.1039/C2DT32149A. 23232973 · DOI
  33. Hider R.; Aviles M. V.; Chen Y.-L.; Latunde-Dada G. O. The Role of GSH in Intracellular Iron Trafficking. Int. J. Mol. Sci. 2021, 22, 1278 10.3390/ijms22031278. 33525417 PMC7865746 · DOI
  34. Brawley H. N.; Lindahl P. A. Low-molecular-mass labile metal pools in Escherichia coli: advances using chromatography and mass spectrometry. J. Biol. Inorg. Chem. 2021, 26, 479– 494. 10.1007/s00775-021-01864-w. 33963934 PMC8205893 · DOI · Paper page
  35. Nguyen T. Q.; Kim J. E.; Brawley H. N.; Lindahl P. A. Chromatographic detection of low-molecular-mass metal complexes in the cytosol of Saccharomyces cerevisiae. Metallomics 2020, 12, 1094– 1105. 10.1039/c9mt00312f. 32301942 PMC7497409 · DOI
  36. Brawley H. N.; Lindahl P. A. Direct detection of the labile nickel pool in Escherichia coli: new perspectives on labile metal pools. J. Am. Chem. Soc. 2021, 143, 18571– 18580. 10.1021/jacs.1c08213. 34723500 PMC8968725 · DOI
  37. Cahill J.; Young R. Phage lysis: multiple genes for multiple barriers. Adv Virus Res 2019, 103, 33– 70. 10.1016/bs.aivir.2018.09.003. 30635077 PMC6733033 · DOI
  38. Lutz R.; Bujard H. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O, and AraC/I1-I2 regulatory elements. Nucleic Acids Res. 1997, 25, 1203– 1210. 10.1093/nar/25.6.1203. 9092630 PMC146584 · DOI
  39. Adam F. I.; Bounds P. L.; Kissner R.; Koppenol W. H. Redox properties and activity of iron-citrate complexes: evidence for redox cycling. Chem. Res. Toxicol. 2015, 28, 604– 614. 10.1021/tx500377b. 25654270 · DOI
  40. Bennett B. D.; Kimball E. H.; Gao M.; Osterhout R.; VanDien S. J.; Rabinowitz J. D. Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli. Nat. Chem. Biol. 2009, 5, 593– 599. 10.1038/nchembio.186. 19561621 PMC2754216 · DOI · Paper page
  41. Mayhew S. G. The redox potential of dithionite and SO2- from equilibrium reactions with flavodoxins, methyl viologen, and hydrogen plus hydrogenase. Eur. J. Biochem. 1978, 85, 535– 547. 10.1111/j.1432-1033.1978.tb12269.x. 648533 · DOI
  42. Cockrell A. L.; Holmes-Hampton G. P.; McCormick S. P.; Chakrabarti M.; Lindahl P. A. Mössbauer and EPR study of iron in vacuoles from fermenting Saccharomyces cerevisiae. Biochemistry 2011, 50, 10275– 10283. 10.1021/bi2014954. 22047049 PMC3226750 · DOI
  43. Walsh M.. J.; Ahner B. A. Determination of stability constants of Cu(I), Cd(II), & Zn(II) complexes with thiols using fluorescent probes. J. Inorg. Biochem. 2013, 128, 112– 123. 10.1016/j.jinorgbio.2013.07.012. 23954481 · DOI
  44. Aliaga M. E.; López-Alarćon C.; Bridi R.; Speisky H. Redox-implications associated with the formation of complexes between copper ions and reduced or oxidized glutathione. J. Inorg. Biochem. 2016, 154, 78– 88. 10.1016/j.jinorgbio.2015.08.005. 26277412 · DOI
  45. Richter Y.; Fischer B. Characterization and elucidation of coordination requirements of adenine nucleotides complexes with Fe(II) ions. Nucleosides, Nucleotides, Nucleic Acids. 2003, 22, 1757– 1780. 10.1081/NCN-120023271. 14533880 · DOI
  46. Philpott C. C.; Patel S. J.; Protchenko O. Management versus miscues in the cytosolic labile iron pool: the varied functions of iron chaperones. Biochim. Biophys. Acta – Mol. Cell Res 2020, 1867, 118830 10.1016/j.bbamcr.2020.118830. 32835748 PMC7484093 · DOI · Paper page
  47. Patel S. J.; Frey A. G.; Palenchar D. J.; Achar S.; Bullough K. Z.; Vashisht A.; Wohlschlegel J. A.; Philpott C. C. A PCBP1-BolA2 chaperone complex delivers iron for cytosolic [2Fe-2S] cluster assembly. Nat. Chem. Biol. 2019, 15, 872– 881. 10.1038/s41589-019-0330-6. 31406370 PMC6702080 · DOI
  48. Hamed M. Y.; Silver J.; Wilson M. T. Studies of the reactions of ferric iron with glutathione and some related thiols. Inorg. Chim. Acta 1983, 78, 1– 11. 10.1016/S0020-1693(00)86480-4. · DOI
  49. Hamed M. Y.; Silver J. Studies of the reactions of ferric iron with glutathione and some related thiols. Part II. Complex formation in the pH range three to seven. Inorg. Chim. Acta 1983, 80, 115– 122. 10.1016/S0020-1693(00)91271-4. · DOI
  50. Hamed M. Y.; Silver J.; Wilson M. T. Studies of the reactions of ferric iron with glutathione and some related thiols. Part III. A study of the iron catalyzed oxidation of glutathione by molecular oxygen. Inorg. Chim. Acta 1983, 80, 237– 244. 10.1016/S0020-1693(00)91289-1. · DOI
  51. Rao K. K.; Evans M. C. W.; Cammack R.; Hall D. O.; Thompson C. L.; Jackson P. J.; Johnson C. E. Mössbauer effect in Rubredoxin. Determination of the hyperfine field of the iron in a simple iron–sulphur protein. Biochem. J. 1972, 129, 1063– 1070. 10.1042/bj1291063. 4348167 PMC1174263 · DOI
  52. Schröder I.; Johnson E.; de Vries S. Microbial ferric ion reductases. FEMS Microbiol. Rev. 2003, 27, 427– 447. 10.1016/S0168-6445(03)00043-3. 12829278 · DOI

Cited by

Sorted by citations

Authors
Brawley, H. N., Kreinbrink, A. C., Hierholzer, J. D., Vali, S. W., & Lindahl, P. A.
Affiliations
Texas A&M University (US)
Corresponding author
Lindahl, Paul A.
ORCID
Venue
Journal of the American Chemical Society
Publisher
American Chemical Society (ACS)
Published
2023-01-20
Volume
145
Issue
4
Pages
2104-2117
ISSN
  • 0002-7863 (ISSN-L)
  • 1520-5126
DOI
10.1021/jacs.2c06625
PMC
PMC9896560
OpenAlex
W4317543103
Cited by
30
References
1
Citations by year
2026: 6 · 2025: 11 · 2024: 6 · 2023: 7
FWCI
5.90
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of the American Chemical Society (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
Funders
  • Welch Foundation (A1170)
  • Division of Molecular and Cellular Biosciences (MCB-1817389)
  • National Institute of General Medical Sciences (GM127021)
Topics
  • Health Sciences › Medicine › Hematology › Iron Metabolism and Disorders
  • Physical Sciences › Energy › Renewable Energy, Sustainability and the Environment › Metalloenzymes and iron-sulfur proteins
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Micronutrient Interactions and Effects
Keywords
labile iron pool, iron-sulfur clusters, iron-citrate complex, non-heme iron, high-spin Fe(II), Mössbauer spectroscopy, iron oxidation state, cellular iron homeostasis, oxidative stress
MeSH terms
Adenosine Triphosphate, Citrates, Cytosol, Escherichia coli, Ferrous Compounds, Glutathione, Iron, Spectroscopy, Mossbauer, Citric Acid

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review