Competition studies in horse spleen ferritin probed by a kinetically inert inhibitor, [Cr(TREN)(H(2)O)(OH)](2+), and a highly luminescent Tb(III) reagent.
Abstract: The ability of ferritin as an Fe(II) detoxifier and Fe(III) storage protein is limited by its ability to recognize and incorporate Fe(II), which is then oxidized and mineralized at internal protein sites. The Cr(III) amine complex [Cr(N(CH(2)CH(2)NH(2))(3)(H(2)O)(OH)](2+) [abbreviated as Cr(TREN)] is a kinetically inert inhibitor of iron incorporation and mineralization in ferritin. Unlike other inhibitors, Cr(TREN) can only exchange its two aqua/hydroxy ligands. Competition studies between Cr(TREN) and Tb(III) binding have been performed in horse spleen ferritin (HoSF) to probe uptake of Fe(II). From these studies, we propose that Cr(TREN) inhibits Fe(II) uptake by obstructing the routes of metal uptake and by disrupting the early recognition events at the protein surface that precede metal ion uptake. Using an improved luminescence approach to quantify Tb(III) binding to the protein, we demonstrate that Tb(III) cannot interfere with Cr(TREN) binding to ferritin, but that Cr(TREN) dramatically inhibits Tb(III) binding. We show that bound Tb(III) serves as a reliable reporter for Cr(TREN) binding, as the latter efficiently quenches the Tb(III) luminescence via inter-ion energy transfer. Two types of Cr(TREN) binding sites were successfully distinguished from these competition experiments. A common Tb(III)/Cr(TREN) site was identified with stoichiometry of approximately 0.6 equivalents of metal cation per ferritin subunit. We propose that the sites along the three-fold channels and the ferroxidase sites are common binding sites for Tb(III) and Cr(TREN). The remaining Cr(TREN) (2.4 equivalents of metal ions/subunit) does not compete with Tb(III) but rather blocks Tb(III) access into the cavity and decreases the protein's affinity for Tb(III).
Publication Date: 2002-10-17 PubMed ID: 12459915DOI: 10.1007/s00775-002-0409-4Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
- Research Support
- U.S. Gov't
- P.H.S.
Summary
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The research focused on studying the properties of ferritin, a protein in horse spleens, in terms of its ability to detoxify and store iron and especially how the use of two complexes, Cr(TREN) and Tb(III), influence its behavior.
Role of Ferritin and its Challenges
- Ferritin in horses is responsible for detoxifying Fe(II) – a form of iron – and successfully storing Fe(III) – another form of iron.
- However, this protein’s effectiveness is hindered by its capacity to recognize and incorporate Fe(II) which is then oxidized and mineralized at specific areas within the protein.
Experiment with the Cr(TREN) Complex
- The researchers used a Cr(III) amine complex known as Cr(TREN), which is a kinetically inert inhibitor.
- This particular inhibitor interferes with iron’s incorporation and mineralization processes in ferritin. Unlike other inhibitors, Cr(TREN) can only exchange its two aqua/hydroxy ligands.
- Cr(TREN) is suggested to hinder the uptake of Fe(II) by obstructing the uptake channels of the metal and destabilizing the early recognition events at the protean surface that are prerequisite to ion uptake.
Competition Studies of Cr(TREN) and Tb(III)
- In an attempt to investigate the method of Fe(II) uptake, competition studies were carried out by binding Cr(TREN) and Tb(III) within horse spleen ferritin (HoSF).
- It was revealed that Cr(TREN) dramatically inhibits the binding of Tb(III). However, Tb(III) does not impact the binding of Cr(TREN) to ferritin.
- Tb(III) when bound serves as a reliable reporter for the binding of Cr(TREN) as it efficiently extinguishes Tb(III) luminescence via inter-ion energy transfer.
- From these experiments, it was evident that there are two types of binding sites for Cr(TREN).
- One common site identified was Tb(III)/Cr(TREN) with an estimated stoichiometry of roughly 0.6 equivalents of metal cation per ferritin subunit. This shared site points at the possible similarity between the paths along the three-fold channels and the ferroxidase sites for both Tb(III) and Cr(TREN).
- The remaining Cr(TREN), an approximate calculation of 2.4 equivalents of metal ions/subunit, does not compete with Tb(III). Instead, it obstructs Tb(III)’s access into the cavity and reduces the protein’s affinity for Tb(III).
Cite This Article
APA
Barnés CM, Petoud S, Cohen SM, Raymond KN.
(2002).
Competition studies in horse spleen ferritin probed by a kinetically inert inhibitor, [Cr(TREN)(H(2)O)(OH)](2+), and a highly luminescent Tb(III) reagent.
J Biol Inorg Chem, 8(1-2), 195-205.
https://doi.org/10.1007/s00775-002-0409-4 Publication
Researcher Affiliations
- Department of Chemistry, University of California, Berkeley, CA 94720, USA.
MeSH Terms
- Animals
- Binding Sites
- Binding, Competitive
- Ferritins / antagonists & inhibitors
- Ferritins / chemistry
- Ferritins / metabolism
- Ferrous Compounds / metabolism
- Horses
- Luminescent Measurements
- Models, Molecular
- Organometallic Compounds / chemistry
- Organometallic Compounds / metabolism
- Organometallic Compounds / pharmacology
- Protein Binding
- Protein Subunits
- Spectrometry, Fluorescence / methods
- Spleen / chemistry
- Terbium / chemistry
- Terbium / metabolism
- Terbium / pharmacology
Grant Funding
- DK32999 / NIDDK NIH HHS
- DK57814 / NIDDK NIH HHS
Citations
This article has been cited 2 times.- Bou-Abdallah F, Zhao G, Biasiotto G, Poli M, Arosio P, Chasteen ND. Facilitated diffusion of iron(II) and dioxygen substrates into human H-chain ferritin. A fluorescence and absorbance study employing the ferroxidase center substitution Y34W.. J Am Chem Soc 2008 Dec 31;130(52):17801-11.
- Gray HB. Biological inorganic chemistry at the beginning of the 21st century.. Proc Natl Acad Sci U S A 2003 Apr 1;100(7):3563-8.
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