PROTON & ELECTRON TRANSFER & ENERGY COUPLING IN SITE I

描述：NADH-醌氧化还原酶（复合物I）是最大的， 能量转换酶中最复杂和最不为人所知的。 它有一个 独特的高质子/电子化学计量比（4-5 H+/2 e-）。 由于复合体I 编码基因包括超过一半的蛋白质编码基因， 线粒体DNA（13个中的7个），大部分线粒体遗传 疾病与复合物I的功能障碍有关。因此，研究 复合物I具有重要的医学意义。 该研究项目将 特别强调研究（一）结构-功能关系 在复合体I中，结合了最先进的分子遗传技术， 低温EPR光谱，和（II）复合物I相关的线粒体 疾病 私家侦探研究小组的研究表明，与长期持有的观点相反， 复合物I由从膜上突出的大亲水臂组成。 膜（电子注入器，含有一种黄素和几种铁硫 簇）和膜内的疏水部分（质子泵送反应器 含有三个不同的醌结合位点）。 私家侦探的小组提出， 一个新的醌门控质子泵模型的能量耦合机制 复合岛本研究将采用以下实验策略：（1） 簇N2和醌结合的空间位置的确定 位点;（2）进一步表征涉及半醌的反应，使用 特异性抑制剂、离子载体和解偶联剂;（3）三种 不同的醌结合位点的分离和表征 耐药突变体：（4）序列基序的靶向， 与含有质子通道的多肽一样， 诱变;（5）复合物I相关疾病的研究，如Leber's 遗传性视神经病变和帕金森病，使用患者来源 线粒体在培养的细胞中增殖。 私家侦探将合作开展 这个项目与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.