PROTON & ELECTRON TRANSFER & ENERGY COUPLING IN SIT I

宝腾

• 批准号: 6694406
• 负责人: Tomoko None Ohnishi
• 金额: $ 36.35万
• 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 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6694406
• 关键词: Escherichia coli; NAD(P)H dehydrogenase; Rhodospirillales; X ray crystallography; bacterial proteins; electron nuclear double resonance spectroscopy; electron spin resonance spectroscopy; electron transport; enzyme activity; gene mutation; heart cell; hydrogen transport; mitochondria; oxidative phosphorylation; semiquinone; site directed mutagenesis; thermodynamics; vitamin K

The NADH-quinone oxidoreductase (complex I) is the largest (M.W. = approximately 1 mega-daltons) and most complicated (43 subunits) energy-transducing system in mitochondria. Many mitochondria-linked genetic diseases have been discovered, and the majority of them originate from a complex I defect(s). The elucidation of the structure-function relationship of complex I is vital not only for the study of bioenergetics, but also for the understanding of the nature of these diseases, in order to develop therapies. Based upon our previous findings, we will extend our studies in the following directions: (1) The NADH-binding site, one FMN molecule, and a majority of iron-sulfur clusters with low midpoint potential are localized in the hydrophilic promontory domain of complex I. In contrast, the iron-sulfur cluster N2 (which has the highest midpoint redox potential) and three distinct ubisemiquinone species are located within the membrane domain. We hypothesized that cluster N2 and these semiquinones play key roles in the proton and electron transfer in complex I. We found that cluster N2 resides in either of TYKY or PSST subunits. Both subunits are at least partially buried within the membrane. Determining the subunit location and ligand structure of cluster N2 has been one of the most important yet difficult tasks in complex I study. Recently, we have developed systems with much simpler bacterial complex I counterparts in which these two candidate subunits can be separated. Using these systems, we will identify which subunit harbors cluster N2. Furthermore, we will study the unique functions of cluster N2 employing site-directed mutagenesis techniques. (2) The subunit PSST (not TYKY) contains a specific and tight binding site for various complex I inhibitors. We have discovered that the distinct ubisemiquinone species respond differently to these inhibitors. Using vari9us inhibitors with different specificity, we will study the functional roles of both cluster N2 and the three quinone species in the energy-coupling mechanism in complex I. (3) We have found that the complex I counterpart in Thermus thermophilus has extreme thermo-stability and that its purified subunits are very stable. We will use this bacterium for crystallization and X-ray crystallographic studies. (4) We will determine physicochemical properties and spatial organization of all important redox components by combining state-of-the-art molecular genetic technology with sophisticated physical techniques such as EPR, ENDOR, ESEEM, and cyclic voltammetry as collaborative efforts. (5) We have developed an exciting bacterial model system, which allows us to study mechanisms of mitochondria-linked diseases by making clinically significant point mutations.

NADH- Quinone氧化还原酶（复合物I）是线粒体中最大的（M.W. =大约1兆 - daltons）和最复杂的（43个亚基）能量转移系统。 已经发现了许多与线粒体关联的遗传疾病，其中大多数源自复杂的I缺陷。 阐明复合物I的结构功能关系不仅对于研究生物能学的研究至关重要，而且对于理解这些疾病的性质至关重要，以开发疗法。 根据我们先前的发现，我们将在以下方向上扩展研究：（1）NADH结合位点，一个FMN分子和大多数具有低中点潜在潜在潜在的铁硫簇的大多数在复合物的亲水性海角域中，相反，在相反的I.中，具有最高的Midpoint N2（其中三个不同的Mentpoine Redox），是在三个不同的中）。 领域。 我们假设群集N2和这些半氨基酮在复合物I中的质子和电子传递中起关键作用。我们发现N2群集驻留在潮流或PSST亚基中。 两个亚基至少部分埋在膜内。 确定群集N2的亚基位置和配体结构一直是复杂I研究中最重要但困难的任务之一。 最近，我们开发了具有更简单的细菌复合物I对应物的系统，其中这两个候选亚基可以分开。使用这些系统，我们将确定哪个亚基港群集N2。此外，我们将研究采用定向诱变技术的N2群集的独特功能。 （2）亚基PSST（非泰国）包含各种复合物I抑制剂的特定且紧密的结合位点。我们已经发现，对这些抑制剂的不同泛氨基氨基酮种类的反应不同。 使用具有不同特异性的vari9us抑制剂，我们将研究群集N2和三种奎因酮物种在复合物I中的能量耦合机制中的功能作用。 我们将使用该细菌进行结晶和X射线晶体学研究。 （4）我们将通过将最先进的分子遗传技术与EPR，ENDOR，ENDOR，ESEEM和环环伏安法与协作努力相结合，确定所有重要氧化还原组件的物理化学特性和空间组织。 （5）我们开发了一个令人兴奋的细菌模型系统，该系统使我们能够通过临床上显着的点突变来研究线粒体连接疾病的机制。