Proton pumping mechanism in complex I

描述(申请人提供)：线粒体氧化磷酸化是真核细胞有效供应三磷酸腺苷的主要系统。通过呼吸链的电子转移伴随着质子在三个偶联位置(络合物I、III和IV)跨内膜转移。复合体I(CI，NADH：泛醌氧化还原酶)为ATP合成提供了~40%的质子动力，而CI是产生ROS的主要部位之一，极易受到氧化应激的影响。因此，CI功能障碍与多种线粒体疾病有关，包括心力衰竭、2型糖尿病和神经退行性疾病，如帕金森氏病。因此，阐明脑梗塞的发病机制对于了解这些疾病和制定治疗策略是至关重要的。然而，CI是研究其机制和功能的最具挑战性的分子之一，因为其45个不同亚基的大小(~900 kDa)和复杂性，以及包括多达8个铁-硫簇在内的多个辅因子。即使在彼得·米切尔因其化学渗透理论而获得诺贝尔奖30年后，电子转移如何与CI中的矢量质子转移相联系的核心基本问题仍然没有得到回答。这一建议的长期目标是阐明脑梗塞中氧化还原偶联质子(H+)泵的机制。在此之前，PI在与芬吡酯(一种有效的CI抑制剂)的光亲和标记研究中取得了重要的发现，ND5是一个转运体模块膜亚单位，参与了泛醌(UQ)结合和H+转运。这导致我们开始对NuoL亚基(E.ColiND5同源物)进行突变研究。已经预测在CI中，氧化还原化学通过间接(构象驱动)耦合机制驱动质子转移，但没有可测试的细节。我们最近发现，两个主要但紧密耦合的功能，电子转移(ET)和质子(H+)泵，可能会被ND5的新突变解偶联。这有力地表明CI是一种间接耦合机制。我们推测NuoL(ND5)在CI的间接构象驱动偶联机制中起关键作用。具体目标是：Aim1.分析了一系列新的NuoL突变体中ET与H+泵活性的关系。AIM2.阐明构象变化对间接H+泵耦合机制的重要作用。Aim3.调查其他转运蛋白亚基NuoM(ND4)和Nuon(ND2)参与间接偶联的情况。该项目将从分子水平上理解CI中的间接(构象驱动)偶联过程。

DESCRIPTION (provided by applicant): Mitochondrial oxidative phosphorylation is the main system to efficiently supplying ATP for the eukaryotic cell. Electron transfer through the respiratory chain concomitantly translocates protons across the inner membrane at three coupling sites (complex I, III, and IV). Complex I (CI, NADH:ubiquinone oxidoreductase) provides ~40% of proton-motive force for the ATP synthesis, while CI is one of the major sites of reactive oxygen species (ROS) generation and is extremely vulnerable to oxidative stress. Thus CI dysfunction is implicated in a variety of mitochondrial diseases including heart failure, type 2 diabetes, and neurodegenerative diseases such as Parkinson's disease. Therefore, the elucidation of CI mechanisms is crucial for understanding these diseases and developing therapeutic strategies. However, CI has been one of the most challenging molecules to study its mechanisms and functions, because of the gigantic size (~900 kDa) and complexity of 45 different subunits and several cofactors including as many as 8 iron-sulfur clusters. The central fundamental question of how electron transfer is linked to vectorial proton translocation in CI still remains unanswered even 30 years since Peter Mitchell won the Nobel prize for his chemiosmotic theory. The long-term goal of this proposal is to elucidate the redox-coupled proton (H+) pump mechanism in CI. Previously, the PI made significant findings from a photoaffinity labeling study with fenpyroximate (a potent CI inhibitor that ND5, a transporter module membrane subunit, is involved in both ubiquinone(UQ)-binding and H+ translocation. This led us to start mutational study of the NuoL subunit (E. coli ND5 homolog). It has been predicted that in CI, redox chemistry drives proton translocation via an indirect (conformation-driven) coupling mechanism, but there was no testable details. We recently found the possibility that two major but tightly coupled functions, electron transfer (ET) and proton (H+) pump, could be decoupled by novel mutations in ND5. This strongly suggests that CI operates an indirect coupling mechanism. We hypothesize that NuoL(ND5) is the key player in the indirect conformation-driven coupling mechanism in CI. Specific aims are: Aim1. Analyze the relationship between ET and H+ pumping activities in a series of novel NuoL mutants. Aim2. Elucidate conformational changes important for the indirect H+ pump coupling mechanism. Aim3. Investigate the involvement of other transporter subunits NuoM(ND4) and NuoN(ND2) in indirect coupling. This project will provide a molecular level of understanding of indirect (conformation-driven) coupling process in CI.