New insights into the superoxide generation sites in bovine heart NADH-ubiquinone oxidoreductase (Complex I) The significance of protein-associated ubiquinone and the dynamic shifting of generation sites between semiflavin and semiquinone radicals
New insights into the superoxide generation sites in bovine heart NADH-ubiquinone oxidoreductase (Complex I) The significance of protein-associated ubiquinone and the dynamic shifting of generation sites between semiflavin and semiquinone radicals
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DOI：
10.1016/j.bbabio.2010.05.012
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发表时间：
2010-12-01
影响因子：
4.3
通讯作者：
Ohnishi, Tomoko
中科院分区：
文献类型：
--
作者：
Ohnishi, S. Tsuyoshi;Shinzawa-Itoh, Kyoko;Ohnishi, Tomoko
关于纯化的牛心NADH-泛醌氧化还原酶（复合物I）中的超氧化物生成位点仍然存在相当大的分歧，大多数研究人员都同意超氧化物是在黄素位点生成的。这里我们提出一个新的假设，即超氧化物的生成反映了黄酮半醌（半黄素或SF）和半醌（SQ）之间的动态平衡，就像一场通过电子的“拔河比赛”。已在吉川实验室分离出每个复合物 I 有一个蛋白质结合的内源性泛醌（Shinzawa-Itoh 等人生物化学 49 (2010) 487-492）使用这些制剂，我们测量了（i）SF、SQ 和铁硫簇 N2 的 EPR 信号，同时进行低温 EPR 和（ii）使用室温自旋捕获技术和部分乙酰化细胞色素 c 方法的超氧化物产生我们的实验证据(1) 不添加癸基联醌 (DBQ) 不会发生 NADH 的催化氧化 NADH 添加主要产生 SF 并产生超氧化物，如 Kussmaul 和 Hirst 报道 (PNAS 103 (2006) 7607-7612) (2) 在从 NADH 到 DBQ 的催化电子转移过程中，超氧化物生成位点大部分转移到 SQ (3) 醌袋结合抑制剂（鱼藤酮或杀粉蝶素 A）抑制 SQ 的催化形成，增强 SF 的形成并增加总体超氧化物的生成 这表明，如果在病理条件下抑制电子转移，SF 的超氧化物生成将会增加 (C) 2010 Elsevier B V 保留所有权利
Considerable disagreement still exists concerning the superoxide generation sites in the purified bovine heart NADH-ubiquinone oxidoreductase (complex I) Majority of investigators agree that superoxide is generated at the flavin site Here we present a new hypothesis that the generation of superoxide reflects a dynamic balance between the flavosemiquinone (semiflavin or SF) and the semiquinone (SQ) like a "tug-of war" through electrons All preparations of bovine heart complex I which have been isolated at Yoshikawa s laboratory have one protein-bound endogenous ubiquinone per complex I (Shinzawa-Itoh et al Biochemistry 49 (2010) 487-492) Using these preparations we measured (i) EPR signals of the SF the SQ and iron-sulfur cluster N2 simultaneously with cryogenic EPR and (ii) superoxide production with both the room temperature spin-trapping technique and the partially acetylated cytochrome c method Our experimental evidence was (1) without added decylubiquinoni (DBQ) no catalytic oxidation of NADH occurs The NADH addition produced mostly SF and It generated superoxide as reported by Kussmaul and Hirst (PNAS 103 (2006) 7607-7612) (2) During catalytic electron transfer from NADH to DBQ the superoxide generation site was mostly shifted to the SQ (3) A quinone-pocket binding inhibitor (rotenone or piericidin A) inhibits the catalytic formation of the SQ and it enhances the formation of SF and increases the overall superoxide generation This suggests that if electron transfer was inhibited under pathological conditions superoxide generation from the SF would be increased (C) 2010 Elsevier B V All rights reserved