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A novel mechanism of iron-core formation by Pyrococcus furiosus archaeoferritin, a member of an uncharacterized branch of the ferritin-like superfamily.

Ebrahimi KH, Hagedoorn PL, van der Weel L, Verhaert PD, Hagen WR - J. Biol. Inorg. Chem. (2012)

Bottom Line: Although the function of these iron-storage proteins is constitutive to many organisms to sustain life, the genome of some organisms appears not to encode any of these proteins.Monomers catalyze oxidation of Fe(II) and they store the Fe(III) product as they assemble to form structures comparable to those of 24-meric ferritin.We propose that this mechanism is an alternative method of iron storage by the ferritin-like superfamily of proteins in organisms that lack the regular preassociated 24-meric/12-meric ferritins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.

ABSTRACT
Storage of iron in a nontoxic and bioavailable form is essential for many forms of life. Three subfamilies of the ferritin-like superfamily, namely, ferritin, bacterioferritin, and Dps (DNA-binding proteins from starved cells), are able to store iron. Although the function of these iron-storage proteins is constitutive to many organisms to sustain life, the genome of some organisms appears not to encode any of these proteins. In an attempt to identify new iron-storage systems, we have found and characterized a new member of the ferritin-like superfamily of proteins, which unlike the multimeric storage system of ferritin, bacterioferritin, and Dps is monomeric in the absence of iron. Monomers catalyze oxidation of Fe(II) and they store the Fe(III) product as they assemble to form structures comparable to those of 24-meric ferritin. We propose that this mechanism is an alternative method of iron storage by the ferritin-like superfamily of proteins in organisms that lack the regular preassociated 24-meric/12-meric ferritins.

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Size-exclusion chromatography of Fe(III)-loaded AFR. As-isolated protein was incubated aerobically with different amounts of Fe(II), and after complete oxidation of iron, size-exclusion chromatography was used to measure the formation of multimeric structures. a Chromatogram at 280 nm and b chromatogram at 315 nm. c Chromatogram at 280 and 315 nm of apo-AFR produced after chemical reduction and chelation of iron from AFR that contained 30 Fe(III) ions per monomer. The AFR concentration was 48 μM (monomer). The buffer was 100 mM MOPS pH 7.0 containing 0.1 M NaCl. The flow rate was 0.5 ml/min. V0 indicates the void volume of the column
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Fig7: Size-exclusion chromatography of Fe(III)-loaded AFR. As-isolated protein was incubated aerobically with different amounts of Fe(II), and after complete oxidation of iron, size-exclusion chromatography was used to measure the formation of multimeric structures. a Chromatogram at 280 nm and b chromatogram at 315 nm. c Chromatogram at 280 and 315 nm of apo-AFR produced after chemical reduction and chelation of iron from AFR that contained 30 Fe(III) ions per monomer. The AFR concentration was 48 μM (monomer). The buffer was 100 mM MOPS pH 7.0 containing 0.1 M NaCl. The flow rate was 0.5 ml/min. V0 indicates the void volume of the column

Mentions: A possible operational mechanism for iron oxidation and storage by AFR involves assembly of monomers to form nanocages with a structure similar to that of the ferritin nanocage. To investigate this, monomers were loaded aerobically with different amounts of Fe(II) and size-exclusion chromatography was used to monitor changes in the molecular size of the protein. Chromatograms were recorded at 280 and 315 nm to monitor protein and Fe(III), respectively. Figure 7a and b shows that AFR containing 1 Fe(III) ion per monomer was exclusively monomeric. Addition of one Fe(II) ion per monomer changes the hydrodynamic properties of the protein as the peak of the monomer shifts slightly to a lower molecular weight. Protein containing more than 2 Fe(III) ions per monomer consisted of oligomers with different sizes, and the amount of monomeric protein decreased concomitantly as the amount of Fe(II) added increased. The average size of the oligomers increased as the iron content of the protein increased. Reversibility of the association of monomers and formation of multimers with a ferric mineral core was determined as follows. Multimeric AFR that contained 30 Fe(III) ions per monomer was converted to apo-AFR by chemical reduction and chelation of Fe(II) as described in “Experimental Procedures.” Size-exclusion chromatography of the apoprotein produced in this manner exhibited a single peak (as observed with the as-isolated protein), implying that protein had returned to its original monomeric form as apoprotein (Fig. 7c), and did not exhibit the small fraction of oligomeric structures which had been observed after preparing apoprotein from the as-isolated AFR (Supplementary Fig. 2). The small fraction of oligomeric structures that is observed in Supplementary Fig. 2 is presumably due to the presence of a few Fe(III) ions. The regenerated monomeric protein was able to oxidize Fe(II) again and to assemble into multimers with storage of Fe(III).Fig. 7


A novel mechanism of iron-core formation by Pyrococcus furiosus archaeoferritin, a member of an uncharacterized branch of the ferritin-like superfamily.

Ebrahimi KH, Hagedoorn PL, van der Weel L, Verhaert PD, Hagen WR - J. Biol. Inorg. Chem. (2012)

Size-exclusion chromatography of Fe(III)-loaded AFR. As-isolated protein was incubated aerobically with different amounts of Fe(II), and after complete oxidation of iron, size-exclusion chromatography was used to measure the formation of multimeric structures. a Chromatogram at 280 nm and b chromatogram at 315 nm. c Chromatogram at 280 and 315 nm of apo-AFR produced after chemical reduction and chelation of iron from AFR that contained 30 Fe(III) ions per monomer. The AFR concentration was 48 μM (monomer). The buffer was 100 mM MOPS pH 7.0 containing 0.1 M NaCl. The flow rate was 0.5 ml/min. V0 indicates the void volume of the column
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3401498&req=5

Fig7: Size-exclusion chromatography of Fe(III)-loaded AFR. As-isolated protein was incubated aerobically with different amounts of Fe(II), and after complete oxidation of iron, size-exclusion chromatography was used to measure the formation of multimeric structures. a Chromatogram at 280 nm and b chromatogram at 315 nm. c Chromatogram at 280 and 315 nm of apo-AFR produced after chemical reduction and chelation of iron from AFR that contained 30 Fe(III) ions per monomer. The AFR concentration was 48 μM (monomer). The buffer was 100 mM MOPS pH 7.0 containing 0.1 M NaCl. The flow rate was 0.5 ml/min. V0 indicates the void volume of the column
Mentions: A possible operational mechanism for iron oxidation and storage by AFR involves assembly of monomers to form nanocages with a structure similar to that of the ferritin nanocage. To investigate this, monomers were loaded aerobically with different amounts of Fe(II) and size-exclusion chromatography was used to monitor changes in the molecular size of the protein. Chromatograms were recorded at 280 and 315 nm to monitor protein and Fe(III), respectively. Figure 7a and b shows that AFR containing 1 Fe(III) ion per monomer was exclusively monomeric. Addition of one Fe(II) ion per monomer changes the hydrodynamic properties of the protein as the peak of the monomer shifts slightly to a lower molecular weight. Protein containing more than 2 Fe(III) ions per monomer consisted of oligomers with different sizes, and the amount of monomeric protein decreased concomitantly as the amount of Fe(II) added increased. The average size of the oligomers increased as the iron content of the protein increased. Reversibility of the association of monomers and formation of multimers with a ferric mineral core was determined as follows. Multimeric AFR that contained 30 Fe(III) ions per monomer was converted to apo-AFR by chemical reduction and chelation of Fe(II) as described in “Experimental Procedures.” Size-exclusion chromatography of the apoprotein produced in this manner exhibited a single peak (as observed with the as-isolated protein), implying that protein had returned to its original monomeric form as apoprotein (Fig. 7c), and did not exhibit the small fraction of oligomeric structures which had been observed after preparing apoprotein from the as-isolated AFR (Supplementary Fig. 2). The small fraction of oligomeric structures that is observed in Supplementary Fig. 2 is presumably due to the presence of a few Fe(III) ions. The regenerated monomeric protein was able to oxidize Fe(II) again and to assemble into multimers with storage of Fe(III).Fig. 7

Bottom Line: Although the function of these iron-storage proteins is constitutive to many organisms to sustain life, the genome of some organisms appears not to encode any of these proteins.Monomers catalyze oxidation of Fe(II) and they store the Fe(III) product as they assemble to form structures comparable to those of 24-meric ferritin.We propose that this mechanism is an alternative method of iron storage by the ferritin-like superfamily of proteins in organisms that lack the regular preassociated 24-meric/12-meric ferritins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.

ABSTRACT
Storage of iron in a nontoxic and bioavailable form is essential for many forms of life. Three subfamilies of the ferritin-like superfamily, namely, ferritin, bacterioferritin, and Dps (DNA-binding proteins from starved cells), are able to store iron. Although the function of these iron-storage proteins is constitutive to many organisms to sustain life, the genome of some organisms appears not to encode any of these proteins. In an attempt to identify new iron-storage systems, we have found and characterized a new member of the ferritin-like superfamily of proteins, which unlike the multimeric storage system of ferritin, bacterioferritin, and Dps is monomeric in the absence of iron. Monomers catalyze oxidation of Fe(II) and they store the Fe(III) product as they assemble to form structures comparable to those of 24-meric ferritin. We propose that this mechanism is an alternative method of iron storage by the ferritin-like superfamily of proteins in organisms that lack the regular preassociated 24-meric/12-meric ferritins.

Show MeSH
Related in: MedlinePlus