Mass spectrometry studies of demetallation of haemin by recombinant horse L chain apoferritin and its mutant (E 53,56,57,60 Q).
Abstract: An essential difference between eukaryotic ferritins and bacterioferritins is that the latter contain naturally, in vivo haem as Fe-protoporphyrin IX. This haem is located in a hydrophobic pocket along the 2-fold symmetry axes and is liganded by two Met 52. However, in in vivo studies, a cofactor has been isolated in horse spleen apoferritin similar to protoporphyrin IX; in in vitro experiments, it has been shown that horse spleen apoferritin is able to interact with haem. Studies of haemin (Fe(III)-PPIX) incorporation into horse spleen apoferritin have been carried out, which show that the metal free porphyrin is found in a corresponding pocket to haem in bacterioferritins [Précigoux, G., Yariv, J., Gallois, B., Dautant, A., Courseille, C. and Langlois, d'Estaintot B. (1994) A crystallographic study of haem binding to ferritin. Acta Cryst. D 50, 739-743]. A mechanism of demetallation of haemin by L-chain apoferritin was proposed [Crichton, R.R., Soruco, J.A., Roland, F., Michaux, M.A., Gallois, B., Précigoux, G., Mahy, J.P. and Mansuy. (1997) Remarkable ability of horse spleen apoferritin to demetallate hemin and to metallate protoporphyrin IX as a function of pH. J. P. Biochem. 36, 49, 15049-15054]: this involved four Glu residues (53,56,57,60) situated at the entrance of the hydrophobic pocket and appeared to be favoured by acidic conditions. To verify this mechanism, we have mutated these four Glu to Gln and examined demetallation in both acidic and basic conditions. In this paper, we report the mass spectrometry studies of L-chain apoferritin and its mutant incubated with haemin and analysed after different times of incubation: 15 days, 2 months, 6 months, 9 months and 12 months. These studies show that the recombinant L-chain apoferritin and its mutant are able to demetallate haemin to give a hydroxyethyl protoporphyrin IX derivative in a dimeric form [Macieira, S., Martins, B. M. and Huber, R. (2003) Oxygen-dependent coproporphyrinogen IX oxidase from Escherichia coli: one-step purification and biochemical characterization. FEMS. Microbiology Letters 226, 31-37].
Publication Date: 2006-10-24 PubMed ID: 17078950DOI: 10.1016/j.febslet.2006.10.034Google Scholar: Lookup
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Summary
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This study uses mass spectrometry to explore how a protein called L chain apoferritin, sourced from horse spleens, interacts with a molecule known as haemin. Posing the possibility that it might demetallate, or remove the metallic element from, haemin under specific conditions, the research also investigates a mutation of the protein for comparison.
Introduction
- The research stems from the difference between eukaryotic ferritins and bacterioferritins, primarily connected to the fact that bacterioferritins naturally contain haem as Fe-protoporphyrin IX (in the form of haemin, the iron(III) version of protoporphyrin IX).
- By contrast, eukaryotic ferritins, specifically horse spleen apoferritin, have been associated with a molecule similar to protoporphyrin IX.
- Prior studies have also identified that horse spleen apoferritin can interact with haem, meaning it might demetallate haemin.
Methodology
- To test the demetallation hypothesis, the researchers used mass spectrometry to examine the interaction between L chain apoferritin (and its mutant version) with haemin under differing acidic and basic conditions.
- The observation was conducted at several intervals: 15 days, 2 months, 6 months, 9 months, and 12 months.
Results
- The research confirmed that both the protein and its mutant were capable of demetallating haemin, leading to an alternative form known as a hydroxyethyl protoporphyrin IX derivative.
- This derivative was discovered to be a dimer, meaning it’s made up of two identical molecules.
Significance
- This discovery not only confirms a mechanism for the interaction of certain ferritins with haem, but also opens doors to investigating the wider biological implications of this demetallation process.
- The results influence our understanding of iron metabolism in various organisms, and potentially even how these organisms adapt to iron-deficient environments.
Cite This Article
APA
de Val N, Herschbach H, Potier N, Dorsselaer AV, Crichton RR.
(2006).
Mass spectrometry studies of demetallation of haemin by recombinant horse L chain apoferritin and its mutant (E 53,56,57,60 Q).
FEBS Lett, 580(26), 6275-6280.
https://doi.org/10.1016/j.febslet.2006.10.034 Publication
Researcher Affiliations
- Department of Biochemistry, Université Catholique de Louvain, Bâtiment Lavoisier, 1 Place Louis Pasteur, 1348 Louvain-la-Neuve, Belgium. deval@bioc.ucl.ac.be
MeSH Terms
- Animals
- Apoferritins / chemistry
- Apoferritins / genetics
- Crystallography, X-Ray
- Hemin / chemistry
- Horses
- Hydrogen-Ion Concentration
- Mass Spectrometry
- Metals / chemistry
- Mutation, Missense
- Protein Binding
- Protoporphyrins / chemistry
- Recombinant Proteins
- Spleen
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