The ND1 subunit constructs the inhibitor binding domain in bovine heart mitochondrial complex I
The ND1 subunit constructs the inhibitor binding domain in bovine heart mitochondrial complex I
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DOI：
10.1021/bi7003697
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发表时间：
2007-05-29
期刊：
影响因子：
2.9
通讯作者：
Miyoshi, Hideto
中科院分区：
文献类型：
--
作者：
Murai, Masatoshi;Ishihara, Atsushi;Miyoshi, Hideto
牛复合物 I 中的抑制剂结合结构域被认为是由多亚基构建的，但化学上不同的抑制剂的结合位置如何相互关联仍有待了解。为了深入了解复合物 I 中的抑制剂结合结构域，我们合成了一种光反应性 acetogenin [[I-125](三氟甲基)苯基二氮丙啶基acetogenin，[I-125]TDA]，其中芳基二氮丙啶基团既作为光反应基团又作为γ-内酯环的替代物，γ-内酯环是许多天然 acetogenin 的常见毒基团，并进行光亲和标记以使用牛心脏鉴定标记的亚基软骨下颗粒（SMP）。当 SMP 在 [I-125]TDA 存在下进行 UV 照射时，放射性主要掺入 SDS 凝胶上类似 30 kDa 的条带中。 [I-125]TDA 标记的 SMP 的蓝色非变性凝胶电泳显示，大部分放射性是在复合物 I 中观察到的。在 SDS 凝胶上对复合物 I 的分析显示，放射性主峰位于类似于 30 kDa 处。 [I-125]TDA 标记的复合物 I 与抗牛 ND1 抗体的免疫沉淀表明标记的蛋白质是 ND1 亚基。多种复合物 I 抑制剂，如杀粉粉 A 和鱼藤酮，可有效抑制 [I-125]TDA 与 ND1 的特异性结合，表明它们具有共同的结合域。然而，我们实验室合成的新型复合物I抑制剂Delta lac-acetogenin的抑制效率远低于传统抑制剂。我们的结果明确地揭示了 ND1 亚基构建了抑制剂结合域，尽管该亚基的贡献受到了质疑。此外，本研究证实了我们之前的观点，即 Delta lac-acetogenins 的抑制位点不同于传统抑制剂。
The inhibitor binding domain in bovine complex I is believed to be constructed by multisubunits, but it remains to be learned how the binding positions of chemically diverse inhibitors relate to each other. To get insight into the inhibitor binding domain in complex I, we synthesized a photoreactive acetogenin [[I-125](trifluoromethyl)phenyldiazirinylacetogenin, [I-125]TDA], in which an aryldiazirine group serves as both a photoreactive group and a substitute for the gamma-lactone ring that is a common toxophore of numerous natural acetogenins, and carried out photoaffinity labeling to identify the labeled subunit using bovine heart submitochondrial particles (SMP). When SMP were UV-irradiated in the presence of [I-125]TDA, radioactivity was predominantly incorporated into an similar to 30 kDa band on a SDS gel. Blue native gel electrophoresis of the [I-125]TDA-labeled SMP revealed that the majority of radioactivity was observed in complex I. Analysis of complex I on a SDS gel showed a predominant peak of radioactivity at similar to 30 kDa. Immnoprecipitation of the [I-125]TDA-labeled complex I with anti-bovine ND1 antibody indicated that the labeled protein is the ND1 subunit. A variety of complex I inhibitors such as piericidin A and rotenone efficiently suppressed the specific binding of [I-125]TDA to ND1, indicating that they share a common binding domain. However, the suppression efficiency of Delta lac-acetogenin, a new type of complex I inhibitor synthesized in our laboratory, was much lower than that of the traditional inhibitors. Our results unequivocally reveal that the ND1 subunit constructs the inhibitor binding domain, though the contribution of this subunit has been challenged. Further, the present study corroborates our previous proposition that the inhibition site of Delta lac-acetogenins differs from that of traditional inhibitors.