Bou-Abdallah Fadi, Arosio Paolo, Santambrogio Paolo, Yang Xiaoke, Janus-Chandler Christine, Chasteen N Dennis
Department of Chemistry, University of New Hampshire, Durham, New Hampshire 03824, USA.
Biochemistry. 2002 Sep 17;41(37):11184-91. doi: 10.1021/bi020215g.
Iron deposition within the iron storage protein ferritin involves a complex series of events consisting of Fe(2+) binding, transport, and oxidation at ferroxidase sites and mineralization of a hydrous ferric oxide core, the storage form of iron. In the present study, we have examined the thermodynamic properties of Fe(2+) binding to recombinant human H-chain apoferritin (HuHF) by isothermal titration calorimetry (ITC) in order to determine the location of the primary ferrous ion binding sites on the protein and the principal pathways by which the Fe(2+) travels to the dinuclear ferroxidase center prior to its oxidation to Fe(3+). Calorimetric titrations show that the ferroxidase center is the principal locus for Fe(2+) binding with weaker binding sites elsewhere on the protein and that one site of the ferroxidase center, likely the His65 containing A-site, preferentially binds Fe(2+). That only one site of the ferroxidase center is occupied by Fe(2+) implies that Fe(2+) oxidation to form diFe(III) species might occur in a stepwise fashion. In dilute anaerobic protein solution (3-5 microM), only 12 Fe(2+)/protein bind at pH 6.51 increasing to 24 Fe(2+)/protein at pH 7.04 and 7.5. Mutation of ferroxidase center residues (E62K+H65G) eliminates the binding of Fe(2+) to the center, a result confirming the importance of one or both Glu62 and His65 residues in Fe(2+) binding. The total Fe(2+) binding capacity of the protein is reduced in the 3-fold hydrophilic channel variant S14 (D131I+E134F), indicating that the primary avenue by which Fe(2+) gains access to the interior of ferritin is through these eight channels. The binding stoichiometry of the channel variant is one-third that of the recombinant wild-type H-chain ferritin whereas the enthalpy and association constant for Fe(2+) binding are similar for the two with an average values (DeltaH degrees = 7.82 kJ/mol, binding constant K = 1.48 x 10(5) M(-)(1) at pH 7.04). Since channel mutations do not completely prevent Fe(2+) binding to the ferroxidase center, iron gains access to the center in approximately one-third of the channel variant molecules by other pathways.
铁在铁储存蛋白铁蛋白中的沉积涉及一系列复杂的事件，包括亚铁离子（Fe(2+)）的结合、运输、在铁氧化酶位点的氧化以及水合氧化铁核心（铁的储存形式）的矿化。在本研究中，我们通过等温滴定量热法（ITC）研究了Fe(2+)与重组人H链脱铁铁蛋白（HuHF）结合的热力学性质，以确定蛋白上主要亚铁离子结合位点的位置以及Fe(2+)在氧化为Fe(3+)之前到达双核铁氧化酶中心的主要途径。量热滴定表明，铁氧化酶中心是Fe(2+)结合的主要位点，蛋白上其他位置也存在较弱的结合位点，并且铁氧化酶中心的一个位点，可能是含有His65的A位点，优先结合Fe(2+)。铁氧化酶中心只有一个位点被Fe(2+)占据，这意味着Fe(2+)氧化形成二价铁（III）物种可能是逐步发生的。在稀厌氧蛋白溶液（3 - 5 microM）中，在pH 6.51时只有12个Fe(2+)/蛋白结合，在pH 7.04和7.5时增加到24个Fe(2+)/蛋白。铁氧化酶中心残基的突变（E62K + H65G）消除了Fe(2+)与该中心的结合，这一结果证实了Glu62和His65中的一个或两个残基在Fe(2+)结合中的重要性。在三倍亲水性通道变体S14（D131I + E134F）中，蛋白的总Fe(2+)结合能力降低，这表明Fe(2+)进入铁蛋白内部的主要途径是通过这八个通道。通道变体的结合化学计量是重组野生型H链铁蛋白结合化学计量的三分之一，而Fe(2+)结合的焓和缔合常数在两者中相似，在pH 7.04时的平均值分别为（ΔH° = 7.82 kJ/mol，结合常数K = 1.48 x 10(5) M(-)(1)）。由于通道突变并没有完全阻止Fe(2+)与铁氧化酶中心的结合，大约三分之一的通道变体分子中的铁通过其他途径进入该中心。
