PROTON & ELECTRON TRANSFER & ENERGY COUPLING IN SITE I

宝腾

• 批准号: 2872649
• 负责人: Tomoko None Ohnishi
• 金额: $ 29.95万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-02-01 至 2001-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2872649
• 关键词: Archaea; Escherichia coli; NAD(P)H dehydrogenase; Rhodospirillales; active sites; bioenergetics; cellular respiration; electron spin resonance spectroscopy; electron transport; enzyme complex; enzyme structure; enzyme substrate complex; flavins; free radicals; human tissue; hydrogen transport; iron sulfur protein; mitochondrial DNA; optic nerve disorder; protein structure function; site directed mutagenesis; ubiquinone

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.

描述：NADH- Quinone氧化还原酶（复杂I）是最大的，大多数 详细且最不了解的是转导酶的能量。 它有一个 独特的高质子/电子化学计量学（4-5 H+/2E-）。 自复杂i 编码基因包含一半以上的蛋白质编码基因 线粒体DNA（13分中的7个），大多数线粒体遗传 疾病与复合物的功能障碍有关。因此，研究 复杂的我非常重要。 这个研究项目 特别强调（i）结构功能关系的研究 在复杂的I中，结合了最先进的分子遗传技术和 低温EPR光谱和（ii）复合物I相关线粒体 疾病。 P.I.的小组表明，与长期以来的视图相反， 复合物我由一个大的亲水臂组成 膜（电子喷油器，其中包含一根黄素和几种铁硫 簇）和膜内的疏水部分（质子泵反的反应器 包含三个不同的醌结合位点）。 P.I.的小组提出 一种用于能量耦合机制的新的奎因酮门控质子泵模型 复杂I.将采用以下实验策略：（1） 簇N2和喹酮结合的空间位置的确定 站点； （2）使用使用半喹酮反应的进一步表征 特定的抑制剂，离子载体和解偶联物； （3）识别三个 通过隔离和表征不同的醌结合位点 抗抑制剂的突变体：（4）靶向序列基序的靶向 与位于现场定向的质子通道有共同 诱变； （5）研究复杂的I相关疾病，例如Leber的疾病 遗传性视神经病变和帕金森氏病，使用患者衍生 线粒体在培养的细胞中繁殖。 P.I.将进行合作 这个项目与多学科研究团队，由著名的 分子生物学，线粒体疾病，生物化学和 生物物理学。