Pinpoint Chemical Modification of the Quinone-Access Channel of Mitochondrial Complex I via a Two-Step Conjugation Reaction
Pinpoint Chemical Modification of the Quinone-Access Channel of Mitochondrial Complex I via a Two-Step Conjugation Reaction
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DOI：
10.1021/acs.biochem.7b00612
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发表时间：
2017-08-15
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：
Masuya, Takahiro;Murai, Masatoshi;Miyoshi, Hideto
我们先前表明，具有罗丹明荧光团的大体积环应变环炔直接与并入（经由配体导向的甲苯磺酰基化学）到牛心脏亚线粒体颗粒中的复合物I的49 kDa亚基中的Asp 160中的叠氮基反应（经由应变促进的点击化学）[Masuya，T.，等人（2014）Biochemistry 53，7816-7823]。这种两步缀合可能是一种有前途的技术，通过各种分子探针对复合物I中的醌通道进行特定的化学修饰，这将导致研究酶的新方法。然而，由于环张力环炔的反应性通常较高，它们也与线粒体蛋白中的其他亲核氨基酸反应，导致显著的不期望的副反应。为了最大限度地减少副反应并实现49 kDa Asp 160的精确精确化学修饰，我们研究了两步缀合反应的最佳化学标签对。我们发现，而不是应变促进点击化学，狄尔斯-阿尔德环加成的一对环丙烯纳入49 kDa的Asp 160（通过配体定向甲苯磺酰基化学）和外部添加的四嗪是更有效的定点修饰。过量的醌位点抑制剂不干扰环丙烯和四嗪之间的狄尔斯-阿尔德环加成。这些结果沿着先前的发现（上文引用）强烈地表明，与通过X射线晶体学和单粒子低温电子显微镜研究建模的预测的醌进入通道相反，通道是开放的或经历大的结构重排以允许大的配体进入49 kDa Asp 160附近。
We previously showed that a bulky ring-strained cycloalkyne possessing a rhodamine fluorophore directly reacts (via strain-promoted click chemistry) with the azido group incorporated (via ligand-directed tosyl chemistry) into Asp160 in the 49 kDa subunit of complex I in bovine heart submitochondrial particles [Masuya, T., et al. (2014) Biochemistry 53, 7816-7823]. This two-step conjugation may be a promising technique for specific chemical modifications of the quinone-access channel in complex I by various molecular probes, which would lead to new methodologies for studying the enzyme. However, because the reactivities of ring-strained cycloalkynes are generally high, they also react with other nucleophilic amino acids in mitochondrial proteins, resulting in significant undesired side reactions. To minimize side reactions and achieve precise pinpoint chemical modification of 49 kDa Asp160, we investigated an optimal pair of chemical tags for the two-step conjugation reaction. We found that instead of strain-promoted click chemistry, Diels-Alder cycloaddition of a pair of cyclopropene incorporated into 49 kDa Asp160 (via ligand-directed tosyl chemistry) and externally added tetrazine is more efficient for the pinpoint modification. An excess of quinone-site inhibitors did not interfere with Diels-Alder cycloaddition between the cyclopropene and tetrazine. These results along with the previous findings (cited above) strongly suggest that in contrast to the predicted quinone-access channel modeled by X-ray crystallographic and single-particle cryo-electron microscopic studies, the channel is open or undergoes large structural rearrangements to allow bulky ligands into the proximity of 49 kDa Asp160.