Proton & electron transfer & energy coupling in site I

描述(申请人提供)：在先前拨款的支持下，PI成功地表征了络合物I中几乎所有的铁硫簇合物和蛋白质结合的泛醌。现在，我们准备在这一领域启动一种新的方法。最近，Sazanov的团队成功地确定了从嗜热菌中分离的复合体I的外围部分的X射线晶体结构(3.3A分辨率)。他们的结果与本申请中描述的PI的EPR研究预测的结构完全一致。具体地说，X射线数据表明，直接向蛋白质相关泛醌(指定为SQNf)提供电子的铁-硫团簇N2距离膜结构域界面只有8A。他们还报告说，星系团n2位于附近通向泛醌的短通道附近。所有数据都与PI提出的12A的N_2“-S_(Nf)”距离是一致的。PI发现，在添加解偶联剂后，SQNf信号消失。这种独特的性质不能用经典的化学渗透循环机制来解释。为了证明SQNf信号对膜电位的敏感性，我们制备了由络合物I重组的蛋白脂质体。我们成功地证明了由K-呋喃霉素法诱导的内正膜电位增强了SQNf信号。这一系统将使我们能够应用各种先进的磁共振技术、高频EPR和新开发的弛豫滤波超精细光谱，组合为Reine-Endor或Reine-HYSCORE，以检测门控半醌可能经历的构象变化。我们还将利用现代分子生物学技术对细菌复合体I系统进行膜内质子泵机制的研究。许多线粒体相关遗传病、帕金森病、细胞凋亡和衰老都与复合体I缺陷(S)和/或从复合体I产生活性氧物种(ROS)有关，复合体I是脑和心脏线粒体产生超氧阴离子的主要部位，但关于产生部位(S)尚未达成共识。因此，确定发生部位对于更好地了解这些问题和开发治疗方法至关重要。现在，我们已经为这项研究建立了一个全新的、强大的工具。我们从同一EPR样品中测量了半喹酮、超氧化物-自旋加合物和铁-硫信号。利用这一新策略，我们发现团簇N_2和黄素都能产生超氧化物。我们将巩固这一令人兴奋的发现，并研究其在生理和病理条件下与复杂I功能的相关性。

DESCRIPTION (provided by applicant): With the support of preceding grants, the PI succeeded in characterizing almost all iron-sulfur clusters and protein-bound ubiquinone in complex I. Now, we are ready to launch a new approach in this field. Recently Sazanov's group successfully determined the X-ray crystallographic structure (3.3A resolution) of the peripheral part of complex I isolated from thermophilic bacteria. Their results are fully consistent with the structures predicted by the Pi's EPR studies as described in this application. Specifically, X-ray data indicated that iron-sulfur cluster N2, which directly donates electrons to protein-associated ubiquinone (designated SQNf), is located only 8A from the membrane domain interface. They also reported that the cluster N2 is located close to a nearby short channel leading to ubiquinone. All of their data are consistent with the Pi's proposal of the N2"-"SQNf distance of 12A. The PI found that the SQNf signal disappeared upon addition of uncouplers. This unique property cannot be explained through the classic chemiosmotic loop mechanism. In order to prove that the SQNf signal is sensitive to the membrane potential, we prepared proteoliposomes reconstituted from complex I. We succeeded in demonstrating that the signal is enhanced with an inside-positive membrane potential which was induced by the K-valinomycin method. This system will enable us to apply various advanced magnetic resonance techniques, high frequency EPR and newly developed Relaxation filtered hyperfine spectroscopy, combined as REFINE-ENDOR or REFINE-HYSCORE, in order to detect possible conformational changes that the gating semiquinone may undergo. We will also study the proton pump mechanism within the membrane, using modern molecular biological techniques on bacterial complex I system. Many mitochondria-linked genetic diseases, Parkinson's disease, apoptosis and aging are related with complex I defect(s) and/or generation of reactive oxygen species (ROS) from complex I. Complex I is the major site of superoxide generation in brain and heart mitochondria, but no consensus has been attained regarding the generation site(s). Thus, to identify the generation site is vital for better understanding these problems, and for developing therapeutic managements. Now, we have established an entirely new, powerful tool for this study. We measure signals of semiquinone, superoxide-spin adduct, and iron-sulfur signals from the same EPR sample. Using this new strategy, we found that both cluster N2 and flavin can generate superoxide. We will solidify this exciting finding and study its relevance to complex I function under physiological and pathological conditions.