Proton & electron transfer & energy coupling in site I

描述（由申请人提供）：在前期资助的支持下，该项目成功表征了复合体i中几乎所有的铁硫簇和蛋白质结合泛醌。现在，我们准备在该领域推出一种新的方法。最近，Sazanov的团队成功地确定了从嗜热细菌中分离出来的复合体I外围部分的x射线晶体结构（3.3A分辨率）。他们的结果与本应用程序中描述的Pi的EPR研究预测的结构完全一致。具体来说，x射线数据表明，直接向蛋白相关泛醌（称为SQNf）提供电子的铁硫簇N2位于距膜结构域界面仅8A处。他们还报告说，簇N2位于附近一个通向泛醌的短通道附近。他们的数据都与Pi提出的N2"-"SQNf距离12A一致。PI发现，加入解耦剂后，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，我们制备了由复合体i重组的蛋白脂质体。我们成功地证明了信号被K-valinomycin方法诱导的膜内正电位增强。该系统将使我们能够应用各种先进的磁共振技术，高频EPR和新开发的弛豫过滤超精细光谱，结合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体I系统进行膜内质子泵机制研究。许多线粒体相关的遗传疾病，如帕金森病、细胞凋亡和衰老都与复合体I缺陷和/或复合体I生成活性氧（ROS）有关。复合体I是脑和心脏线粒体生成超氧化物的主要位点，但关于生成位点尚未达成共识。因此，确定产生部位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。我们测量了来自同一EPR样品的半醌、超氧自旋加合物和铁硫信号。利用这种新策略，我们发现簇N2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合体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.