Characterization of the Inhibitor Binding Site in Mitochondrial NADH-Ubiquinone Oxidoreductase by Photoaffinity Labeling Using a Quinazoline-Type Inhibitor
Characterization of the Inhibitor Binding Site in Mitochondrial NADH-Ubiquinone Oxidoreductase by Photoaffinity Labeling Using a Quinazoline-Type Inhibitor
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DOI：
10.1021/bi8019977
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发表时间：
2009-02-03
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;Sekiguchi, Koji;Miyoshi, Hideto
牛心脏线粒体复合物I（NADH-泛醌氧化还原酶）的不同抑制剂被认为共享一个共同的大结合域，部分重叠的网站，虽然目前还不清楚这些结合位点如何相互关联。为了获得对复合物1中的抑制剂结合结构域的新见解，我们合成了光反应性叠氮喹唑啉{[I-125]-6-叠氮基-4-（4-碘苯乙氨基）喹唑啉，[I-125]AzQ），其中光不稳定的叠氮基被引入到toxophoric喹唑啉环中以允许特异性交联，并使用牛心脏亚线粒体颗粒进行了光亲和标记研究。通过肽质量指纹图谱和免疫印迹分析光交联蛋白，发现[I-125]AzQ特异性结合49 kDa和ND 1亚基，频率接近4：1。交联完全阻断过量的其他抑制剂，如丙酮和fenpyroximate。在ADP/ATP载体和3-羟基丁酸脱氢酶中也检测到相当大的交联，尽管它与这两种蛋白质的功能障碍无关。用V8-蛋白酶对[I-125] AzQ标记的49 kDa亚基进行部分蛋白水解，并对所得肽进行N-末端测序，结果表明，由[I-125]AzQ交联的氨基酸残基位于序列区Thr 25-Glu 143（118个氨基酸）内。此外，通过V8蛋白酶、赖氨酰内肽酶或胰蛋白酶对[I-125] AzQ标记的49 kDa亚基进行彻底消化产生的片段模式的检查强烈表明，交联残基位于Asp 41-Arg 63（23个氨基酸）区域内。本研究首次揭示了复合物I在亚亚基水平上的抑制剂结合位点。
The diverse inhibitors of bovine heart mitochondrial complex I (NADH-ubiquinone oxidoreductase) are believed to share a common large binding domain with partially overlapping sites, though it remains unclear how these binding sites relate to each other. To obtain new insight into the inhibitor binding domain in complex 1, we synthesized a photoreactive azidoquinazoline {[I-125]-6-azido-4-(4-iodophenethylamino)quinazoline, [I-125]AzQ), in which a photolabile azido group was introduced into the toxophoric quinazoline ring to allow specific cross-linking, and carried out a photoaffinity labeling study using bovine heart submitochondrial particles. Analysis of the photo-cross-linked proteins by peptide mass fingerprinting and immunoblotting revealed that [I-125]AzQ specifically binds to the 49 kDa and ND1 subunits with a frequency of similar to 4:1. The cross-linking was completely blocked by excess amounts of other inhibitors such as acetogenin and fenpyroximate. Considerable cross-linking was also detected in the ADP/ATP carrier and 3-hydroxybutyrate dehydrogenase, though it was not associated with dysfunction of the two proteins. The partial proteolysis of the [I-125]AzQ-Iabeled 49 kDa subunit by V8-protease and N-terminal sequencing of the resulting peptides revealed that the amino acid residue cross-linked by [I-125]AzQ is within the sequence region Thr25-Glu143 (118 amino acids). Furthermore, examination of fragment patterns generated by exhaustive digestion of the [I-125]AzQ-labeled 49 kDa subunit by V8-protease, lysylendopeptidase, or trypsin strongly suggested that the cross-linked residue is located within the region Asp41-Arg63 (23 amino acids). The present study has revealed, for the first time, the inhibitor binding site in complex I at the sub-subunit level.