Abstract
Fe(III) storage by ferritin is an essential process of the iron homeostasis machinery. It begins by translocation of Fe(II) from outside the hollow spherical shape structure of the protein, which is formed as the result of self-assembly of 24 subunits, to a di-iron binding site, the ferroxidase center, buried in the middle of each active subunit. The pathway of Fe(II) to the ferroxidase center has remained elusive, and the importance of self-assembly for the functioning of the ferroxidase center has not been investigated. Here we report spectroscopic and metal ion binding studies with a mutant of ferritin from Pyrococcus furiosus (PfFtn) in which self-assembly was abolished by a single amino acid substitution. We show that in this mutant metal ion binding to the ferroxidase center and Fe(II) oxidation at this site was obliterated. However, metal ion binding to a conserved third site (site C), which is located in the inner surface of each subunit in the vicinity of the ferroxidase center and is believed to be the path for Fe(II) to the ferroxidase center, was not disrupted. These results are the basis of a new model for Fe(II) translocation to the ferroxidase center: self-assembly creates channels that guide the Fe(II) ions toward the ferroxidase center directly through the protein shell and not via the internal cavity and site C. The results may be of significance for understanding the molecular basis of ferritin-related disorders such as neuroferritinopathy in which the 24-meric structure with 432 symmetry is distorted.
Highlights
Ferritin has 24 subunits that are self-assembled into a spherical shape structure
We report spectroscopic and metal ion binding studies with a mutant of ferritin from Pyrococcus furiosus (PfFtn) in which self-assembly was abolished by a single amino acid substitution
A Single Amino Acid Substitution at the 3-fold Symmetry Axis Abolished Self-assembly—Based on amino acid substitutions at the 3-fold symmetry axes, where putative channels (3-fold channels) have been proposed to exist, an Fe(II) entry path to the internal cavity of eukaryotic ferritin through 3-fold channels is proposed [29]. To test this possibility in PfFtn, we replaced Arg-117 (Fig. 2A), which is located in the middle of the 3-fold symmetry axis in the putative path of Fe(II) to the internal cavity and more than 16 Å away from the ferroxidase center, with an alanine (R117A) using site-directed mutagenesis (“Experimental Procedures”)
Summary
Ferritin has 24 subunits that are self-assembled into a spherical shape structure. Results: Blocking self-assembly abolished Fe(II) binding and oxidation in the di-iron catalytic center. Fe(III) storage by ferritin is an essential process of the iron homeostasis machinery It begins by translocation of Fe(II) from outside the hollow spherical shape structure of the protein, which is formed as the result of self-assembly of 24 subunits, to a di-iron binding site, the ferroxidase center, buried in the middle of each active subunit. The H and M subunits of eukaryotic ferritin and all subunits of bacterial or archaeal ferritins have three conserved Fe(II) binding sites (1, 9 –11) Two of these sites, denoted A and B (Fig. 1B), are located in the middle of the four-␣-helical bundle of each subunit, and together are named the ferroxidase center, where catalysis of Fe(II) oxidation occurs. The exact route of Fe(II) from outside the protein shell to the catalytic center has not been traced out; based on available data for eukaryotic ferritins (28 –31) and bacterioferritins [32,33,34,35,36], which are structurally similar to ferritin except for a heme group between pairs of subunits, different pathways may be drawn [1] (Fig. 1C)
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