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ATP的主要系统。通过呼吸链的电子转移伴随着质子在三个耦合位点（复合物I，III和IV）跨内膜易位。复合物I（Cl，NADH：泛醌氧化还原酶）为ATP合成提供约40%的质子动力，而Cl是活性氧（ROS）产生的主要位点之一，并且极易受到氧化应激的影响。因此，CI功能障碍涉及多种线粒体疾病，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，CI是研究其机制和功能的最具挑战性的分子之一，因为其巨大的尺寸（~900 kDa）和45个不同亚基和几个辅因子（包括多达8个铁硫簇）的复杂性。自彼得·米切尔因其化学渗透理论获得诺贝尔奖30年以来，CI中电子转移如何与矢量质子易位联系在一起的中心基本问题仍然没有答案。这个建议的长期目标是阐明CI中的氧化还原耦合质子（H+）泵机制。之前，PI从使用唑螨酯（一种有效的CI抑制剂）的光亲和标记研究中获得了重要的发现，即ND 5（转运蛋白模块膜亚基）参与泛醌（UQ）结合和H+易位。这使我们开始了NuoL亚基的突变研究（E。coli ND5 homolog）。据预测，在CI，氧化还原化学驱动质子易位通过间接（构象驱动）耦合机制，但没有可测试的细节。我们最近发现的可能性，两个主要的，但紧密耦合的功能，电子转移（ET）和质子（H+）泵，可以解耦的ND 5中的新突变。这有力地表明，CI操作间接耦合机制。我们假设NuoL（ND 5）是CI中间接构象驱动偶联机制的关键参与者。具体目标是：目标1。分析一系列新的NuoL突变体的ET和H+泵活性之间的关系。目标2。阐明间接H+泵耦合机制的重要构象变化。目标3。研究其他转运蛋白亚基NuoM（ND 4）和NuoN（ND 2）在间接偶联中的参与。这个项目将提供一个分子水平的理解间接（构象驱动）耦合过程中的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.