PROTON & ELECTRON TRANSFER & ENERGY COUPLING IN SIT I

NADH-醌氧化还原酶（复合物I）是最大的（M.W. =约1兆道尔顿）和线粒体中最复杂的（43个亚基）能量转导系统。 已经发现了许多与ESTA相关的遗传疾病，其中大多数起源于复合物I缺陷。 阐明复合物I的结构-功能关系不仅对于生物能量学的研究至关重要，而且对于理解这些疾病的性质，以开发治疗方法也至关重要。 在此基础上，我们将进一步研究以下几个方面：（1）NADH结合位点、一个FMN分子和大多数具有低中点电位的铁硫簇位于复合物I的亲水性岬区。相反，铁硫簇N2（具有最高的中点氧化还原电位）和三个不同的ubisemiquinone物种位于膜域内。 我们推测，簇N2和这些半醌在复合物I中的质子和电子转移中起关键作用。我们发现N2簇位于TYKY或PSST亚基中。 这两个亚基至少部分地埋在膜内。 确定簇N2的亚基位置和配体结构一直是复合物I研究中最重要也是最困难的任务之一。 最近，我们已经开发出了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离。使用这些系统，我们将确定哪些亚基窝藏集群N2。此外，我们将采用定点突变技术研究N2簇的独特功能。 (2)亚基PSST（不是TYKY）含有多种复合物I抑制剂的特异性和紧密结合位点。我们已经发现，不同的泛半醌物种对这些抑制剂的反应不同。 我们将使用不同特异性的抑制剂，研究N2簇和三个醌物种在复合物I的能量耦合机制中的功能作用。(3)我们已经发现，复合物I对应的Thermus thermophilus具有极端的热稳定性，其纯化的亚基是非常稳定的。 我们将使用这种细菌进行结晶和X射线晶体学研究。 (4)我们将确定所有重要的氧化还原组分的物理化学性质和空间组织，通过结合最先进的分子遗传技术与复杂的物理技术，如EPR，ENDOR，ESEEM和循环伏安法作为合作的努力。 (5)我们已经开发了一个令人兴奋的细菌模型系统，它使我们能够通过制造临床上重要的点突变来研究与大肠杆菌相关的疾病的机制。

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.