title: Dinuclear center of ferritin: studies of iron binding and oxidation show differences in the two iron sites.
abstract: The ferroxidase activity of human ferritin has previously been associated with a diiron site situated centrally within the four-helix bundle of H-type chains (HuHF). However, direct information about the site of Fe(II) binding has been lacking, and events between Fe(II) binding and its oxidation have not previously been studied. A sequential stopped-flow assay has now been developed to enable the dissection of binding and oxidation. It depends on the ability of 1,10-phenanthroline to complex protein-bound Fe(II) and to distinguish it from the more immediately available free Fe(II). This approach, aided by the use of site-directed variants, indicates that in HuHF and the non-heme ferritin of Escherichia coli the first 48 Fe(II) atoms/molecule added are bound and oxidized at the dinuclear centers. At a constant iron concentration, the rate of Fe(II) oxidation was maximal for additions of 2 Fe(II) atoms/subunit, consistent with a two-electron oxidation of the Fe(II) pair. Although, at low Fe(II)/protein ratios, no cooperativity in Fe(II) binding was observed; a preferred order of binding was deduced [Fe(II) binding first at site A and then at site B]. Binding of Fe(II) at both sites was essential for fast oxidation. Modification of site A ligands resulted in slow iron binding and slow oxidation. Modification of site B did not prevent Fe(II) binding at site A but greatly reduced its oxidation rate. These differences may mean that dioxygen is initially bound to Fe(II) at site B.
authors.affiliation: Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, U.K.
authors.forename: A
authors.initials: A
authors.lastname: Treffry
authors.forename: Z
authors.initials: Z
authors.lastname: Zhao
authors.forename: M A
authors.initials: MA
authors.lastname: Quail
authors.forename: J R
authors.initials: JR
authors.lastname: Guest
authors.forename: P M
authors.initials: PM
authors.lastname: Harrison
mesh_terms: D001665:Binding Sites; D005293:Ferritins / Q000737:chemistry* / Q000378:metabolism*; D005973:Glutamine; D006801:Humans; D007501:Iron / Q000032:analysis / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010618:Phenanthrolines / Q000494:pharmacology; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors
chemical_list: D010618:Phenanthrolines; D011994:Recombinant Proteins; D005973:Glutamine; D005293:Ferritins; D007501:Iron; D008239:Lysine; C025205:1,10-phenanthroline