PROTON & ELECTRON TRANSFER & ENERGY COUPLING IN SITE I

说明：NADH-苯二酮氧化还原酶(复合体I)是最大、最 对能量转导酶的研究很复杂，也知之甚少。它有一个 独特的高质子/电子化学计量比(4-5H+/2E-)。由于复杂的I 编码基因包括一半以上的蛋白质编码基因 线粒体DNA(13个中的7个)，大多数线粒体基因 疾病与复合体I的功能障碍有关，因此，对 复合体I在医学上具有重要意义。这项研究项目将 重点研究(一)结构-功能关系 在复合体I中，结合了最先进的分子基因技术和 低温EPR波谱，以及(Ii)复合体I相关线粒体 疾病。私家侦探S小组表明，与长期以来的观点相反， 复合体I由一个巨大的亲水性手臂组成，从 膜(含一个黄素和几个铁硫的电子注射器 膜内的疏水部分(质子泵反应堆 包含三个不同的苯醌结合位点)。P.I.S小组提出 一种用于能量耦合机制的新的苯醌门控质子泵模型 复合体I.将采用以下实验策略：(1) 团簇N_2和对苯二酚结合的空间位置的测定 (2)半喹酮参与的反应的进一步表征 特定的抑制剂、离子载体和解偶联剂；(3)鉴定三种 不同的苯醌结合部位的分离和鉴定 抗药突变体：(4)靶向序列基序， 与通过位点定向的含有多肽的质子通道有共同之处 诱变；(5)复杂I相关疾病的研究，如莱伯氏病 遗传性视神经病变和帕金森氏病 线粒体在培养细胞中繁殖。私家侦探将合作 这个项目拥有一个多学科的研究团队，由著名的 分子生物学、线粒体疾病、生物化学和 生物物理学。

DESCRIPTION: NADH-quinone oxidoreductase (Complex I) is the largest, most elaborate and least understood of the energy transducing enzymes. It has a uniquely high proton/electron stoichiometry (4-5 H+/2e-). Since Complex I encoding genes comprise more than half of the protein-encoding genes in mitochondrial DNA (7 out of 13), a majority of mitochondrial genetic diseases are associated with dysfunction of Complex I. Thus, the study of Complex I is of great medical importance. This research project places particular emphasis on the study of (I) the structure-function relationship in Complex I, combining state-of-the-art molecular genetic technology and cryogenic EPR spectroscopy, and (II) Complex I-related mitochondrial diseases. The P.I.'s group has shown that, contrary to a long-held view, Complex I consists of a large hydrophilic arm protruding out from the membrane (electron injector containing one flavin and several iron-sulfur clusters) and a hydrophobic part within the membrane (proton-pumping reactor containing three distinct quinone-binding sites). The P.I.'s group proposes a new quinone-gated proton-pump model for the energy coupling mechanism in Complex I. The following experimental strategies will be employed: (1) Determination of the spatial locations of cluster N2 and of quinone-binding sites; (2) further characterization of semiquinone-involving reactions using specific inhibitors, ionophores, and uncouplers; (3) identification of three different quinone-binding sites by isolation and characterization of inhibitor-resistant mutants: (4) targeting of the sequence motifs, which are in common with proton channel containing polypeptides by site-directed mutagenesis; (5) studies of Complex I-related diseases, such as Leber's Hereditary Optic Neuropathy and Parkinson's disease, using patient derived mitochondria propagated in cultured cells. The P.I. will collaborate on this project with a multi-disciplinary research team, consisting of renowned experts of molecular biology, mitochondrial disease, biochemistry and biophysics.