PROTON & ELECTRON TRANSFER & ENERGY COUPLING IN SIT I

NADH-醌氧化还原酶(复合体I)是线粒体中最大(约1兆道尔顿)、最复杂(43个亚基)的能量传递系统。许多线粒体连锁遗传病已被发现，其中大多数起源于复杂的I缺陷(S)。阐明复合体I的结构-功能关系不仅对生物能量学的研究至关重要，而且对于理解这些疾病的本质以便开发治疗方法也是至关重要的。(1)NADH结合部位、一个FMN分子和大多数低中点电位的铁硫簇合物位于配合物I的亲水海角区域，而铁硫簇合物N2(具有最高的中点氧化还原电位)和三个不同的泛吡喹酮物种位于膜结构域中。我们假设在络合物I中，团簇N_2和这些半喹酮在质子和电子转移中起关键作用。我们发现团簇N_2存在于TYKY或Psst亚基中。这两个亚基至少部分埋在膜内。确定簇合物N_2的亚基位置和配体结构一直是络合物I研究中最重要也是最困难的任务之一。最近，我们开发了具有更简单的细菌复合体I对应物的系统，在其中这两个候选亚基可以被分离。使用这些系统，我们将识别哪个亚基包含簇n2。此外，我们还将利用定点突变技术研究簇N_2的独特功能。(2)Psst亚基(不是TYKY)含有多种复合体I抑制剂的紧密结合位点。我们发现，不同的乌双喹酮对这些抑制剂的反应是不同的。使用不同特异性的Vari9us抑制剂，我们将研究簇合物N2和三个苯醌物种在络合物I中能量偶联机制中的功能作用。(3)我们发现嗜热热菌中的络合物I具有极高的热稳定性，并且其纯化的亚基非常稳定。我们将利用该细菌进行结晶和X射线结晶学研究。(4)我们将通过将最先进的分子基因技术与EPR、Endor、ESEEM和循环伏安等复杂的物理技术相结合，共同努力确定所有重要氧化还原成分的物理化学性质和空间组织。(5)我们开发了一个令人兴奋的细菌模型系统，它允许我们通过进行临床上有意义的点突变来研究线粒体连锁疾病的机制。

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.