Thermodynamic Analysis of Ferrous Ion Binding to Escherichia coli Ferritin EcFtnA

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• 作者：Fadi Bou-Abdallah ; Mark R. Woodhall ; Adriá ; n Velá ; zquez-Campoy ; Simon C. Andrews ; and N. Dennis Chasteen
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：October 25, 2005
• 年：2005
• 卷：44
• 期：42
• 页码：13837 - 13846
• 全文大小：160K
• 年卷期：v.44,no.42(October 25, 2005)
• ISSN：1520-4995

文摘

Iron oxidation in the bacterial ferritin EcFtnA from Escherichia coli shows marked differencesfrom its homologue human H-chain ferritin (HuHF). While the amino acid residues that constitute thedinuclear center in these proteins are highly conserved, EcFtnA has a third iron-binding site (C site) inclose proximity to the dinuclear center that is seemingly responsible for these differences. Here, we describethe first thermodynamic study of Fe²⁺ binding to EcFtnA and its variants to determine the location of theprimary ferrous ion-binding sites on the protein and to better understand the role of the third C site in ironbinding. Isothermal titration calorimetric analyses of the wild-type protein reveal the presence of twomain classes of binding sites in the pH range of 6.5-7.5, ascribed to Fe²⁺ binding, first at the A and thenthe B sites. Site-directed mutagenesis of ligands in the A, B, or C sites affects the apparent Fe²⁺-bindingstoichiometries at the unaltered sites. The data imply some degree of inter- and intrasubunit negativecooperative interaction between sites. Unlike HuHF where only the A site initially binds Fe²⁺, both Aand B sites in EcFtnA bind Fe²⁺, implying a role for the C site in influencing the binding of Fe²⁺ at theB site of the di-iron center of EcFtnA. The ITC equations describing a binding model for three classes ofindependent binding sites are reported here for the first time.