Proton & electron transfer & energy coupling in site I

描述（由申请人提供）：在先前资助的支持下，PI成功地表征了复合物I中几乎所有的铁硫簇和蛋白质结合的泛醌。现在，我们准备在这一领域采取新的做法。最近Sazanov的小组成功地确定了从嗜热菌中分离的复合物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。具体而言，X射线数据表明，铁硫簇N2，它直接捐赠电子的蛋白质相关的泛醌（指定SQNf），位于膜域界面仅8A。他们还报告说，N2簇位于附近一个通往泛醌的短通道附近。他们的所有数据都与Pi提出的N2 "-" SQNf距离为12 A的建议一致。PI发现SQNf信号在加入解偶联剂后消失。这种独特的性质不能通过经典的化学渗透环机制来解释。为了证明SQNf信号对膜电位敏感，我们制备了由复合物I重构的脂蛋白体。我们成功地证明，信号增强与内正膜电位，这是由K-缬氨霉素方法诱导。该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，结合作为REFINE-ENDOR或REFINE-HYSCORE，以检测门控半醌可能发生的构象变化。我们还将利用现代分子生物学技术对细菌复合物I系统进行膜内质子泵机制的研究。许多与帕金森氏病、细胞凋亡和衰老相关的遗传性疾病都与复合物I缺陷和/或复合物I产生活性氧（ROS）有关。复合物I是脑和心脏线粒体中超氧化物生成的主要位点，但关于生成位点尚未达成共识。因此，确定产生位点对于更好地理解这些问题和开发治疗管理至关重要。现在，我们已经为这项研究建立了一个全新的，强大的工具。我们测量信号的半醌，超氧化物自旋加合物，和铁硫信号从同一EPR样品。使用这种新的策略，我们发现团簇N2和黄素都可以产生超氧化物。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合物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.