Chemical and NADH-induced, ROS-dependent, cross-linking between subunits of complex I from Escherichia coli and Thermus thermophilus
Chemical and NADH-induced, ROS-dependent, cross-linking between subunits of complex I from Escherichia coli and Thermus thermophilus
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DOI：
10.1021/bi801160u
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发表时间：
2008-09-30
期刊：
影响因子：
2.9
通讯作者：
Sazanov, Leonid A.
中科院分区：
文献类型：
--
作者：
Berrisford, John M.;Thompson, Christopher J.;Sazanov, Leonid A.
呼吸链的复合物I将电子从NADH转移到泛醌，并与质子跨膜的移位相结合。两种替代耦合机制正在讨论中，氧化还原驱动或构象驱动。使用“零长度”交联剂和分离的亲水性结构域的复合物I从大肠杆菌和嗜热栖热菌，我们表明，在NADH的存在下，亚基之间的交联模式的变化显着。以前用完整纯化的E. coli和牛复合物I。这表明，在还原与NADH，类似的构象变化可能会发生在完整的酶和分离的亲水性结构域（可用于晶体学研究）。在完整的E. coli复合物1中，疏水亚基NuoA和NuoJ之间的交联在NADH的存在下被废除，表明构象变化延伸到膜结构域，可能作为偶联机制的一部分。出乎意料的是，在不存在任何化学交联剂的情况下，复合物I与NADH的孵育导致亚基Nqo 4（NuoCD）和Nqo 6（NuoB）之间以及Nqo 6和Nqo 9之间的共价交联。它们的形成取决于氧的存在，因此可能是通过活性氧（ROS）诱导交联的氧化损伤的结果。此外，观察到这些亚基（以及Nqo 3）的ROS和金属离子依赖性蛋白水解。Fe-S簇N2在亚基Nqo 4和Nqo 6之间配位，并可能参与这些过程。我们的观察表明，氧化损伤复合物I在体内可能不仅包括侧链修饰，但也蛋白质交联和降解。
Complex I of respiratory chains transfers electrons from NADH to ubiquinone, coupled to the translocation of protons across the membrane. Two alternative coupling mechanisms are being discussed, redox-driven or conformation-driven. Using "zero-length" cross-linking reagent and isolated hydrophilic domains of complex I from Escherichia coli and Thermus thermophilus, we show that the pattern of cross-links between subunits changes significantly in the presence of NADH. Similar observations were made previously with intact purified E. coli and bovine complex I. This indicates that, upon reduction with NADH, similar conformational changes are likely to occur in the intact enzyme and in the isolated hydrophilic domain (which can be used for crystallographic studies). Within intact E. coli complex 1, the cross-link between the hydrophobic subunits NuoA and NuoJ was abolished in the presence of NADH, indicating that conformational changes extend into the membrane domain, possibly as part of a coupling mechanism. Unexpectedly, in the absence of any chemical cross-linker, incubation of complex I with NADH resulted in covalent cross-links between subunits Nqo4 (NuoCD) and Nqo6 (NuoB), as well as between Nqo6 and Nqo9. Their formation depends on the presence of oxygen and so is likely a result of oxidative damage via reactive oxygen species (ROS) induced cross-linking. In addition, ROS- and metal ion-dependent proteolysis of these subunits (as well as Nqo3) is observed. Fe-S cluster N2 is coordinated between subunits Nqo4 and Nqo6 and could be involved in these processes. Our observations suggest that oxidative damage to complex I in vivo may include not only side-chain modifications but also protein cross-linking and degradation.