Proton pumping mechanism in complex I

描述（由申请人提供）：线粒体氧化磷酸化是真核细胞有效供应ATP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点（络合物I、III和IV）穿过内膜进行易位。复合物I （CI， NADH：泛醌氧化还原酶）为ATP合成提供约40%的质子动力，而CI是活性氧（ROS）生成的主要位点之一，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，如帕金森病。因此，阐明CI机制对于理解这些疾病和制定治疗策略至关重要。然而，由于其巨大的尺寸（约900 kDa）和45个不同亚基的复杂性以及包括多达8个铁硫团簇在内的几个辅助因子，CI一直是研究其机制和功能最具挑战性的分子之一。即使在彼得·米切尔（Peter Mitchell）因其化学渗透理论获得诺贝尔奖30年后，关于CI中电子转移与质子矢量移位之间的关系这一核心基本问题仍未得到解答。本研究的长期目标是阐明CI中氧化还原偶联质子（H+）泵浦机制。此前，PI通过芬吡肟酸酯（一种有效的CI抑制剂，ND5，一种转运体模块膜亚基，参与泛素（UQ）结合和H+易位）的光亲和标记研究取得了重大发现。这促使我们开始了NuoL亚基（大肠杆菌ND5同源物）的突变研究。据预测，在CI中，氧化还原化学通过间接（构象驱动）耦合机制驱动质子易位，但没有可测试的细节。我们最近发现ND5的两个主要但紧密耦合的功能，电子转移（ET）和质子（H+）泵，可以通过新的突变解耦。这有力地表明，CI操作的是一种间接耦合机制。我们假设NuoL（ND5）在CI的间接构象驱动耦合机制中起关键作用。具体目标是：分析一系列新型NuoL突变体ET与H+泵送活性的关系。Aim2。阐明间接氢离子泵耦合机制中重要的构象变化。Aim3。研究其他转运子亚基NuoM（ND4）和NuoN（ND2）在间接耦合中的作用。这个项目将提供一个分子水平的理解间接（构象驱动）耦合过程在CI。

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.