Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Biochemistry. 2012 Mar 6;51(9):1953-63. doi: 10.1021/bi300047h. Epub 2012 Feb 27.
Using a photoaffinity labeling technique, Nakamaru-Ogiso et al. demonstrated that fenpyroximate, a strong inhibitor of bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I), binds to the ND5 subunit [Nakamaru-Ogiso, E., et al. (2003) Biochemistry 42, 746-754]. Considering that the main body of the ND5 subunit composed of transmembrane helixes 1-15 is located at the distal end of the membrane domain [Efremov, R. G., et al. (2010) Nature 465, 441-445], however, their result may be questionable. Because establishing the number and location of inhibitors and/or quinone binding sites in the membrane domain is necessary to elucidate the function of the enzyme, it is critical to clarify whether there is an additional inhibitor and/or quinone binding site besides the interface between the hydrophilic and membrane domains. We therefore performed photoaffinity labeling experiments using two newly synthesized fenpyroximate derivatives [[(125)I]-4-azidophenyl fenpyroximate ([(125)I]APF) and [(125)I]-3-azido-5-iodobenzyl fenpyroximate ([(125)I]AIF)] possessing a photoreactive azido group at and far from the pharmacophoric core moiety, respectively. Doubled sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that [(125)I]APF and [(125)I]AIF bind to the PSST and 49 kDa subunits, respectively. Careful examination of the fragmentation patterns of the labeled PSST and 49 kDa subunits generated by limited proteolysis indicated that the residues labeled by [(125)I]APF and [(125)I]AIF are located in the Ser43-Arg66 (PSST) and Asp160-Arg174 (49 kDa) regions, respectively, which face the supposed quinone-binding pocket formed at the interface of the PSST, 49 kDa, and ND1 subunits. We conclude that fenpyroximate does not bind to the distal end of the membrane domain but rather resides at the interface between the two domains in a manner such that the pharmacophoric pyrazole ring and side chain of the inhibitor orient toward the PSST and 49 kDa subunits, respectively. This study answers a critical question relating to complex I.
使用光亲和标记技术，Nakamaru-Ogiso 等人证明，法匹拉韦，一种牛心线粒体 NADH-泛醌氧化还原酶（复合物 I）的强抑制剂，与 ND5 亚基结合 [Nakamaru-Ogiso，E.，等人。（2003）生物化学 42，746-754]。然而，考虑到 ND5 亚基的主体由跨膜螺旋 1-15 组成，位于膜域的远端 [Efremov，R.G.，等人。（2010）自然 465，441-445]，他们的结果可能值得怀疑。因为在膜域中建立抑制剂和/或醌结合位点的数量和位置对于阐明酶的功能是必要的，所以关键是要澄清在亲水和膜域之间的界面之外是否存在额外的抑制剂和/或醌结合位点。因此，我们使用两种新合成的法匹拉韦衍生物 [[(125)I]-4-叠氮苯基法匹拉韦([(125)I]APF)和 [(125)I]-3-叠氮-5-碘苄基法匹拉韦([(125)I]AIF)]进行光亲和标记实验，分别在药理学核心部分附近和远处具有光反应性叠氮基团。双重十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示，[(125)I]APF 和 [(125)I]AIF 分别与 PSST 和 49 kDa 亚基结合。通过有限蛋白水解对标记 PSST 和 49 kDa 亚基的片段模式进行仔细检查表明，[(125)I]APF 和 [(125)I]AIF 标记的残基分别位于 Ser43-Arg66(PSST)和 Asp160-Arg174(49 kDa)区域，分别朝向 PSST、49 kDa 和 ND1 亚基形成的假定醌结合口袋。我们得出结论，法匹拉韦不会与膜域的远端结合，而是以抑制剂的药理学吡唑环和侧链分别朝向 PSST 和 49 kDa 亚基的方式位于两个域之间的界面上。这项研究回答了与复合物 I 相关的一个关键问题。
