Ferrous Ion Binding to Recombinant Human H-Chain Ferritin. An Isothermal Titration Calorimetry Study
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- 作者：Fadi Bou-Abdallah ; Paolo Arosio ; Paolo Santambrogio ; Xiaoke Yang ; Christine Janus-Chandler ; and N. Dennis Chasteen
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：September 17, 2002
- 年：2002
- 卷：41
- 期：37
- 页码：11184 - 11191
- 全文大小：154K
- 年卷期：v.41,no.37(September 17, 2002)
- ISSN：1520-4995
文摘
Iron deposition within the iron storage protein ferritin involves a complex series of eventsconsisting of Fe2+ binding, transport, and oxidation at ferroxidase sites and mineralization of a hydrousferric oxide core, the storage form of iron. In the present study, we have examined the thermodynamicproperties of Fe2+ binding to recombinant human H-chain apoferritin (HuHF) by isothermal titrationcalorimetry (ITC) in order to determine the location of the primary ferrous ion binding sites on the proteinand the principal pathways by which the Fe2+ travels to the dinuclear ferroxidase center prior to its oxidationto Fe3+. Calorimetric titrations show that the ferroxidase center is the principal locus for Fe2+ bindingwith weaker binding sites elsewhere on the protein and that one site of the ferroxidase center, likely theHis65 containing A-site, preferentially binds Fe2+. That only one site of the ferroxidase center is occupiedby Fe2+ implies that Fe2+ oxidation to form diFe(III) species might occur in a stepwise fashion. In diluteanaerobic protein solution (3-5 
M), only 12 Fe2+/protein bind at pH 6.51 increasing to 24 Fe2+/proteinat pH 7.04 and 7.5. Mutation of ferroxidase center residues (E62K+H65G) eliminates the binding ofFe2+ to the center, a result confirming the importance of one or both Glu62 and His65 residues in Fe2+binding. The total Fe2+ binding capacity of the protein is reduced in the 3-fold hydrophilic channel variantS14 (D131I+E134F), indicating that the primary avenue by which Fe2+ gains access to the interior offerritin is through these eight channels. The binding stoichiometry of the channel variant is one-third thatof the recombinant wild-type H-chain ferritin whereas the enthalpy and association constant for Fe2+binding are similar for the two with an average values (
H
= 7.82 kJ/mol, binding constant K = 1.48× 105 M-1 at pH 7.04). Since channel mutations do not completely prevent Fe2+ binding to the ferroxidasecenter, iron gains access to the center in approximately one-third of the channel variant molecules byother pathways.
M), only 12 Fe2+/protein bind at pH 6.51 increasing to 24 Fe2+/proteinat pH 7.04 and 7.5. Mutation of ferroxidase center residues (E62K+H65G) eliminates the binding ofFe2+ to the center, a result confirming the importance of one or both Glu62 and His65 residues in Fe2+binding. The total Fe2+ binding capacity of the protein is reduced in the 3-fold hydrophilic channel variantS14 (D131I+E134F), indicating that the primary avenue by which Fe2+ gains access to the interior offerritin is through these eight channels. The binding stoichiometry of the channel variant is one-third thatof the recombinant wild-type H-chain ferritin whereas the enthalpy and association constant for Fe2+binding are similar for the two with an average values (H = 7.82 kJ/mol, binding constant K = 1.48× 105 M-1 at pH 7.04). Since channel mutations do not completely prevent Fe2+ binding to the ferroxidasecenter, iron gains access to the center in approximately one-third of the channel variant molecules byother pathways.