Oversized ubiquinones as molecular probes for structural dynamics of the ubiquinone reaction site in mitochondrial respiratory complex I
Oversized ubiquinones as molecular probes for structural dynamics of the ubiquinone reaction site in mitochondrial respiratory complex I
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DOI：
10.1074/jbc.ra119.012347
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发表时间：
2020-01
期刊：
影响因子：
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通讯作者：
Shinpei Uno;Takahiro Masuya;K. Shinzawa-Itoh;Jonathan Lasham;Outi Haapanen;T. Shiba;D. Inaoka;Vivek Sharma;M. Murai;H. Miyoshi
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作者：
Shinpei Uno;Takahiro Masuya;K. Shinzawa-Itoh;Jonathan Lasham;Outi Haapanen;T. Shiba;D. Inaoka;Vivek Sharma;M. Murai;H. Miyoshi
NADH-苯醌氧化还原酶(复合体I)将NADH向苯二酚的电子传递与跨膜的质子转移结合在一起。对苯二酚的还原是将能量从对苯二酚的还原部位传递到酶的远程质子泵装置的关键步骤。尽管结构生物学研究已经提出存在一条又长又窄的苯醌通道，但这一通道的生理学相关性仍然存在争议。我们在这里研究了牛心亚线粒体颗粒(SMPS)中的络合物I是否可以催化还原一系列超大的泛醌(OS-UQ)，这些泛素不太可能通过狭窄的通道，因为它们的侧链包括一个巨大的“块”，即∼13Å。我们发现，一些OS-UQ作为来自络合物I的有效电子受体，以与泛醌-2相当的效率接受电子。与此结合的催化还原和质子转移可被不同的醌中心抑制剂完全抑制，这表明OS-UQs的还原发生在泛醌的生理反应中心。值得注意的是，OS-UQ的质子转移效率随其侧链结构的不同而显著不同，这表明OS-UQ的反应特性影响了预测的引发质子转移所需的苯醌反应部位的结构变化。这些结果很难与当前的通道模型相一致；相反，辅酶Q的访问路径可能是开放的，以允许OS-UQ访问反应部位。然而，与在SMPS中观察到的相反，OS-UQ不被重组为脂质体的分离的络合物I催化还原。我们讨论了导致这些相互矛盾的结果的可能原因。
NADH-quinone oxidoreductase (complex I) couples electron transfer from NADH to quinone with proton translocation across the membrane. Quinone reduction is a key step for energy transmission from the site of quinone reduction to the remotely located proton-pumping machinery of the enzyme. Although structural biology studies have proposed the existence of a long and narrow quinone-access channel, the physiological relevance of this channel remains debatable. We investigated here whether complex I in bovine heart submitochondrial particles (SMPs) can catalytically reduce a series of oversized ubiquinones (OS-UQs), which are highly unlikely to transit the narrow channel because their side chain includes a bulky “block” that is ∼13 Å across. We found that some OS-UQs function as efficient electron acceptors from complex I, accepting electrons with an efficiency comparable with ubiquinone-2. The catalytic reduction and proton translocation coupled with this reduction were completely inhibited by different quinone-site inhibitors, indicating that the reduction of OS-UQs takes place at the physiological reaction site for ubiquinone. Notably, the proton-translocating efficiencies of OS-UQs significantly varied depending on their side-chain structures, suggesting that the reaction characteristics of OS-UQs affect the predicted structural changes of the quinone reaction site required for triggering proton translocation. These results are difficult to reconcile with the current channel model; rather, the access path for ubiquinone may be open to allow OS-UQs to access the reaction site. Nevertheless, contrary to the observations in SMPs, OS-UQs were not catalytically reduced by isolated complex I reconstituted into liposomes. We discuss possible reasons for these contradictory results.