title: Self-assembly is prerequisite for catalysis of Fe(II) oxidation by catalytically active subunits of ferritin.
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.
authors.affiliation: From the Department of Biotechnology, Delft University of Technology, 2628 BC Delft, The Netherlands k.h.ebrahimi@tudelft.nl.
authors.forename: Kourosh Honarmand
authors.initials: KH
authors.lastname: Ebrahimi
authors.affiliation: From the Department of Biotechnology, Delft University of Technology, 2628 BC Delft, The Netherlands.
authors.forename: Peter-Leon
authors.initials: PL
authors.lastname: Hagedoorn
authors.affiliation: From the Department of Biotechnology, Delft University of Technology, 2628 BC Delft, The Netherlands w.r.hagen@tudelft.nl.
authors.forename: Wilfred R
authors.initials: WR
authors.lastname: Hagen
mesh_terms: D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D002413:Cations, Divalent; D002570:Ceruloplasmin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005293:Ferritins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007501:Iron / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010084:Oxidation-Reduction; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020027:Pyrococcus furiosus / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D019843:Archaeal Proteins; D002413:Cations, Divalent; D021122:Protein Subunits; D011994:Recombinant Proteins; D005293:Ferritins; D007501:Iron; D002570:Ceruloplasmin
keywords: enzyme kinetics; enzymology; ferritin; ferroxidase; iron metabolism; isothermal titration calorimetry (ITC); protein self-assembly