Exploring Interactions between the 49 kDa and ND1 Subunits in Mitochondrial NADH-Ubiquinone Oxidoreductase (Complex I) by Photoaffinity Labeling
Exploring Interactions between the 49 kDa and ND1 Subunits in Mitochondrial NADH-Ubiquinone Oxidoreductase (Complex I) by Photoaffinity Labeling
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DOI：
10.1021/bi200883c
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发表时间：
2011-08-16
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;Mashimo, Yuko;Miyoshi, Hideto
喹唑啉类化合物是牛心脏线粒体中NADH-泛醌氧化还原酶（复合物I）的强抑制剂。使用光反应性喹唑啉、[I-125]AzQ和牛心脏亚线粒体颗粒（SMP），我们先前证明[I-125]AzQ结合在复合物I中的49 kDa和ND 1亚基的界面处;它标记49 kDa亚基的N-末端（Asp 41-Arg 63）区域中的位点，表明该区域接触ND 1亚基[Mural，M.，等人（2009）Biochemistry 48，688-698]。ND 1的标记区域不能被鉴定，因为它是高度疏水的，并且SMPs没有产生足够量的标记蛋白。在这里，我们描述了如何通过[I-125]AzQ的光亲和标记分离的复合物I产生了足够的材料，用于识别ND 1亚基的标记区域。AzQ对分离酶的抑制作用与SMP相当。我们的研究结果表明，ND 1的标记位点在Asp 199和Lys 262之间，最有可能在连接第五和第六跨膜螺旋的第三个基质环中。因此，我们的研究结果揭示了新的信息之间的界面的亲水性和疏水性的结构域的复合物I，该区域被认为是重要的泛醌还原和能量转导。
Quinazolines are strong inhibitors of NADH-ubiquinone oxidoreductase (complex I) from bovine heart mitochondria. Using a photoreactive quinazoline, [I-125]AzQ and bovine heart submitochondrial particles (SMPs), we demonstrated previously that [I-125]AzQ binds at the interface of the 49 kDa and ND1 subunits in complex I; it labeled a site in the N-terminal (Asp41-Arg63) region of the 49 kDa subunit, suggesting that this region contacts the ND1 subunit [Mural, M., et al. (2009) Biochemistry 48, 688-698]. The labeled region of ND1 could not be identified because it is highly hydrophobic, and the SMPs did not yield sufficient amounts of labeled protein. Here, we describe how photoaffinity labeling of isolated complex I by [I-125]AzQ yielded sufficient material for identification of the labeled region of the ND1 subunit. The inhibition of the isolated enzyme by AzQ is comparable to that of SMPs. Our results reveal that the labeled site in ND1 is between Asp199 and Lys262, mostly likely in the third matrix loop that connects the fifth and sixth transmembrane helices. Thus, our results reveal new information about the interface between the hydrophilic and hydrophobic domains of complex I, a region that is thought to be important for ubiquinone reduction and energy transduction.