title: Evidence that the specificity of iron incorporation into homopolymers of human ferritin L- and H-chains is conferred by the nucleation and ferroxidase centres.
abstract: Mammalian ferritins are iron-storage proteins made of 24 subunits of two types: the H- and L-chains. L-chains, in contrast with H-chains, lack detectable ferroxidase activity. When ferritins were subjected to iron loading in vitro with increments near the saturation limit of 4000 Fe atoms per molecule, the homopolymers of human H-chains formed insoluble aggregates, caused by non-specific iron hydrolysis, whereas the homopolymers of L-chains remained soluble and incorporated most of the available iron. To analyse the molecular reasons for the difference, Glu-57 and Glu-60, which are conserved and exposed on the cavity of L-chains, were substituted with His, as in H-chains. The double substitution made the L-homopolymers as sensitive as the H-homopolymers to the iron-induced aggregation, whereas the opposite substitution in the H-chain increased homopolymer resistance to the aggregation only marginally. Millimolar concentrations of citrate and phosphate increased iron incorporation in H-homopolymers by reducing non-specific iron hydrolysis, but inhibited that in L-homopolymers by sequestering available iron. The data indicate that the specific iron incorporation into L-homopolymers is mainly due to the iron-nucleation capacity of Glu-57, Glu-60 and other carboxyl groups exposed on the cavity; in contrast, the specificity of iron incorporation into H-homopolymers is related to its ferroxidase activity, which determines rapid Fe(III) accumulation inside the cavity. The finding that ferroxidase centres are essential for the incorporation of iron in the presence of likely candidates of cellular iron transport, such as phosphate and citrate, confirms their importance in ferritin function in vivo.
authors.affiliation: DIBIT Department of Biological and Technological Research, H. San Raffaele Scientific Institute, Milan, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Santambrogio
authors.forename: S
authors.initials: S
authors.lastname: Levi
authors.forename: A
authors.initials: A
authors.lastname: Cozzi
authors.forename: B
authors.initials: B
authors.lastname: Corsi
authors.forename: P
authors.initials: P
authors.lastname: Arosio
mesh_terms: D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D002570:Ceruloplasmin / Q000737:chemistry* / Q000378:metabolism; D002951:Citrates / Q000494:pharmacology; D019343:Citric Acid; D004591:Electrophoresis, Polyacrylamide Gel; D005293:Ferritins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007501:Iron / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000378:metabolism; D010710:Phosphates / Q000494:pharmacology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D012995:Solubility
chemical_list: D000596:Amino Acids; D002951:Citrates; D010455:Peptides; D010710:Phosphates; D011994:Recombinant Proteins; D012710:Serum Albumin, Bovine; D019343:Citric Acid; D005293:Ferritins; D007501:Iron; D002570:Ceruloplasmin; C018279:sodium phosphate