Sakajiri Tetsuya, Nakatsuji Masatoshi, Teraoka Yoshiaki, Furuta Kosuke, Ikuta Katsuya, Shibusa Kotoe, Sugano Eriko, Tomita Hiroshi, Inui Takashi, Yamamura Takaki
Laboratory of Biological Macromolecules, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan.
Faculty of Nutritional Sciences, the University of Morioka, 808 Sunakomi, Takizawa, Iwate 020-0694, Japan.
Metallomics. 2021 Dec 21;13(12). doi: 10.1093/mtomcs/mfab065.
Fe(II) exported from cells is oxidized to Fe(III), possibly by a multicopper ferroxidase (MCF) such as ceruloplasmin (CP), to efficiently bind with the plasma iron transport protein transferrin (TF). As unbound Fe(III) is highly insoluble and reactive, its release into the blood during the transfer from MCF to TF must be prevented. A likely mechanism for preventing the release of unbound Fe(III) is via direct interaction between MCF and TF; however, the occurrence of this phenomenon remains controversial. This study aimed to reveal the interaction between these proteins, possibly mediated by zinc. Using spectrophotometry, isothermal titration calorimetry, and surface plasmon resonance methods, we found that Zn(II)-bound CP bound to iron-free TF (apo-TF) with a Kd of 4.2 μM and a stoichiometry CP:TF of ∼2:1. Computational modeling of the complex between CP and apo-TF predicted that each of the three Zn(II) ions that bind to CP further binds to an acidic amino acid residue of apo-TF to play a role as a cross-linker connecting both proteins. Domain 4 of one CP molecule and domain 6 of the other CP molecule fit tightly into the clefts in the N- and C-lobes of apo-TF, respectively. Upon the binding of two Fe(III) ions to apo-TF, the resulting diferric TF [Fe(III)2TF] dissociated from CP by conformational changes in TF. In human blood plasma, zinc deficiency reduced the production of Fe(III)2TF and concomitantly increased the production of non-TF-bound iron. Our findings suggest that zinc may be involved in the transfer of iron between CP and TF.
从细胞中输出的亚铁离子（Fe(II)）可能被多铜铁氧化酶（MCF）如铜蓝蛋白（CP）氧化为铁离子（Fe(III)），从而有效地与血浆铁转运蛋白转铁蛋白（TF）结合。由于未结合的Fe(III)高度不溶且具有反应性，因此在从MCF转移到TF的过程中必须防止其释放到血液中。防止未结合的Fe(III)释放的一种可能机制是通过MCF和TF之间的直接相互作用；然而，这种现象的发生仍存在争议。本研究旨在揭示这些蛋白质之间可能由锌介导的相互作用。使用分光光度法、等温滴定量热法和表面等离子体共振方法，我们发现结合锌离子（Zn(II)）的CP与无铁转铁蛋白（脱铁转铁蛋白，apo-TF）结合，解离常数（Kd）为4.2 μM，化学计量比CP:TF约为~2:1。CP与apo-TF复合物的计算模型预测，与CP结合的三个Zn(II)离子中的每一个进一步与apo-TF的一个酸性氨基酸残基结合，起到连接两种蛋白质的交联剂作用。一个CP分子的结构域4和另一个CP分子的结构域6分别紧密地嵌入apo-TF的N叶和C叶的裂隙中。当两个Fe(III)离子与apo-TF结合后，生成的双铁转铁蛋白（[Fe(III)2TF]）通过TF的构象变化从CP上解离。在人血浆中，锌缺乏会减少[Fe(III)2TF]的产生，并同时增加非转铁蛋白结合铁的产生。我们的研究结果表明，锌可能参与了铁在CP和TF之间的转移。
