Iron-binding process in the amino- and carboxyl-terminal lobes of ovotransferrin: quantitative studies utilizing single Fe3+-binding mutants.
Iron-binding process in the amino- and carboxyl-terminal lobes of ovotransferrin: quantitative studies utilizing single Fe3+-binding mutants.
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DOI：
10.1021/bi049147j
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发表时间：
2004-08
期刊：
影响因子：
2.9
通讯作者：
Ikuko Okamoto;K. Mizutani;M. Hirose
中科院分区：
文献类型：
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作者：
Ikuko Okamoto;K. Mizutani;M. Hirose
研究了卵转铁蛋白铁配位残基突变体Y191 F和Y 524 F与Fe（3+）的结合特性。吸收光谱和尿素凝胶电泳分别验证了Y191 F和Y 524 F的C-和N-叶上的单一铁结合。一种新开发的竞争性Fe（3+）结合分析，其中等摩尔的Y191 F和Y 524 F与比饱和更少的Fe（3+）混合，使我们能够定量地确定作为N-叶与C-叶的比率（α值）的叶对初始铁进入的偏好。基于动力学模型估计的α值高度依赖于pH;在6.5至9.0的pH范围内，α随pH降低而从2增加至5，具有明显的S形曲线。在差示扫描量热法上，对于Y191 F、Y 524 F和野生型卵转铁蛋白的apo形式，在61 ℃左右观察到单一热转变。然而，Fe（3+）负载突变体在Y191 F中在62.4和82.1摄氏度以及在Y 524 F中在66.4和76.0摄氏度显示出双重转变。根据定义为由对叶上的铁结合诱导的靶叶中的自由能变化的Δ G（AB）值，发现在主要铁结合过程期间诱导了叶间相互作用的显著稳定效应：在无铁C叶中的初级N叶铁结合后，（Δ G（AB），-2.25kcal/mol）和单铁N-叶中的次级C-叶铁结合（Δ G（AB），-6.45kcal/mol）。
Iron-liganding-residue mutants of ovotransferrin, Y191F and Y524F, were investigated for their Fe(3+)-binding properties. The absorption spectrum and urea gel electrophoresis verified the single iron binding on the C- and N-lobes for Y191F and Y524F, respectively. A newly developed competitive Fe(3+)-binding analysis, in which equimolar Y191F and Y524F are mixed with less Fe(3+) than saturation, enabled us to quantitatively determine the lobe preference for initial iron entry as the ratio (alpha value) of N-lobe over C-lobe. The alpha value estimated on the basis of a kinetic model was highly dependent on pH; within a pH range from 6.5 to 9.0, alpha was increased from 2 to 5 on lowering pH with an apparent sigmoid curve. On differential scanning calorimetry, single thermal transition was observed around 61 degrees C for the apo forms of Y191F, Y524F, and wild-type ovotransferrin. The Fe(3+)-loaded mutants, however, showed dual transitions at 62.4 and 82.1 degrees C in Y191F and 66.4 and 76.0 degrees C in Y524F. According to the DeltaG(AB) value that is defined as the free energy change in a target lobe induced by the iron binding on the counter lobe, marked stabilization effects by interlobe interactions were found to be induced during the major iron-binding process: upon the primary N-lobe iron binding in the iron-free C-lobe (DeltaG(AB), -2.25 kcal/mol) and upon the secondary C-lobe iron binding in the monoferric N-lobe (DeltaG(AB), -6.45 kcal/mol).