Molecular architecture of UQH2:cyt c2 oxidoreductase

描述（由申请人提供）：我们建议进一步研究bc 1复合物（UQH 2：cyt c2氧化还原酶）的机制，以了解其在细胞衰老中的作用，以及其作为药物和害虫控制试剂靶标的功能。后者取决于对醌模拟物的不同敏感性，所述醌模拟物用作抗疟疾药物、杀真菌剂、杀虫剂、除草剂等，在不同的物种。这些酶是所有主要呼吸和光合途径的核心，直接负责生物圈约30%的能量转换。生物学中的这种核心重要性提供了一种内在的兴趣，直接关系到我们对细胞生理学，能量转换机制和维护的理解。光合细菌为研究医学上重要的线粒体复合体提供了一个模型系统。bc 1复合物的催化核心在整个肠道-细菌分离中是高度保守的，反应机制基本上是相同的。在细菌系统中，功能和结构之间的相互作用可以更容易地研究，因为该系统可以被光照激活，在10秒的时间尺度内启动周转。此外，细菌系统易于通过特异性诱变进行分子工程改造。这项资助支持的研究为我们理解这些复合物的功能做出了重大贡献。我们利用35年的分析功能的经验，通过多管齐下的方法探索机制，利用分子工程，生物物理分析，X射线晶体学结构研究，通过光谱学详细分析局部结构，并通过计算机模拟建模之间的协同作用。从这些研究中出现的复合物的分子结构提供了目前这种复杂性的分子机器的最详细的描述之一。晶体学结构的可用性激发了人们的兴趣，并为我们提出的修改的Q循环提供了强有力的支持，这被普遍接受为潜在的机制。然而，这些结构也引发了一些有趣的问题，主要涉及意想不到的动态特征，包括大规模的结构域运动，以及更微妙的局部分子芭蕾舞，允许快速周转而不破坏旁路反应。在这项提案中，我们解决了一些更具争议性的问题，包括机制的特点，最大限度地减少生产的破坏性活性氧的前体物质，导致细胞窒息。该建议是继续研究代谢的关键酶之一，bc 1复合物（泛氢醌-细胞色素c氧化还原酶）。线粒体通过代谢物的氧化为细胞提供动力，使用呼吸链将电子传递给O2。bc 1复合物是该链的中心酶。它进化过程中的一个设计缺陷使这种复合物能够产生损害细胞的有害氧自由基。我们在球形红细菌（Rhodobacter sphaeroides）中研究了相同的酶，球形红细菌是一种光合细菌，接近线粒体的细菌祖先。由于该酶可以通过光合机制被激活，因此更容易研究快速的单周转动力学，从而探索其机制。细菌系统已成为这种重要酶的标准实验模型。通过了解这一机制，我们希望了解破坏性的自由基是如何产生的，以及进化是如何微调这一机制以使这种反应最小化的。这种复合物也是抗疟疾药物、杀真菌剂和杀虫剂的靶标，在医学和农业中都很重要。

DESCRIPTION (provided by applicant): We propose to further investigate the mechanism of the bc1 complex (UQH2:cyt c2 oxidoreductase) in order to understand its role in cellular aging, and its function as the target for drugs and pest-control reagents. These latter depend on differential sensitivities to quinone-mimics that act as anti-malarial drugs, fungicides, pesti- cides, herbicides, etc., in different species. These enzymes are at the core of all major respiratory and photosynthetic pathways, and are directly responsible for about 30% of the energy conversion of the biosphere. This central importance in biology provides an intrinsic interest, relating directly to our understanding of cellular physiology, energy conversion mechanisms, and maintenance. The photosynthetic bacteria provide a model system for studying the medically important mitochondrial complex. The catalytic core of the bc1 complex is highly conserved across the mitochondrial-bacterial divide, and the reaction mechanism is essentially the same. In the bacterial system, the interplay between function and structure can be more easily studied because the system can be activated by illumination, initiating turnover in the 10 <s time scale. In addition, the bacte- rial system is readily amenable to molecular engineering through specific mutagenesis. The research supported by this grant has contributed substantially to our understanding of how these complexes function. We take advantage of 35 years of experience in assaying function to explore the mechanism through a multi-pronged approach exploiting the synergy between molecular engineering, biophysical assay, structural studies by X-ray crystallography, detailed analysis of local structure through spectroscopy, and modeling through computer simulation. The molecular architecture of the complex that is emerging from these studies provides one of the most detailed descriptions of a molecular machine of this complexity currently available. The availability of crystallographic structures has stimulated much interest, and has provided strong support for the modified Q- cycle we proposed, which is generally accepted as the underlying mechanism. However, the structures have also provoked some interesting questions, mainly relating to unexpected dynamic features, including a large scale domain movement, and a more subtle local molecular ballet that allows rapid turnover without damaging bypass reactions. In this proposal, we address some of the more controversial issues, including features of the mechanism that minimize production of precursors of the damaging reactive oxygen species that lead to cellular suffocation. The proposal is for continuation of work on one of the key enzymes of metabolism, the bc1 complex (ubihydroquinone -cytochrome c oxidoreductase). Mitochondria power the cell through oxidation of metabolites, using the respiratory chain to pass electrons to O2. The bc1 complex is the central enzyme of the chain. A design defect from its evolutionary past has left this complex with an ability to generate damaging oxygen radicals that harm the cell. We study the same enzyme in Rhodobacter sphaeroides, a photosynthetic bacterium close to the bacterial ancestor of the mitochondria. Because the enzyme can be activated through the photosynthetic machinery, it is much easier to study rapid, single-turnover kinetics, and hence to probe the mechanism. The bacterial system has become a standard experimental model for this important enzyme. By understanding the mechanism, we hope to understand how the damaging radicals are generated, and how evolution has fined-tuned the mechanism so as to minimize this reaction. The complex is also a target for anti- malarial drugs, and for fungicides and pesticides, important both in medicine and agriculture.