Complexities of complex II: A versatile architecture for respiration

描述（由申请人提供）：配合物II是四种电子转移酶之一，在线粒体有氧呼吸中产生跨膜电化学梯度。它的基因改变或抑制对生物体的生存有重大影响。复合体II超家族可能是最通用的呼吸酶，因为它参与有氧和无氧呼吸和克雷布斯循环。复合体II结构的多用途循环表明，这个褶皱是古老的进化。对这种单一酶的研究可以深入了解富马酸还原、琥珀酸氧化、天冬氨酸氧化、喹啉化学、电子转移和跨膜电化学梯度形成的生物学机制。在初步的研究中，络合物II同源喹啉：富马酸还原酶已经过量生产，结晶和结构确定单独和与喹啉结合位点抑制剂的复合物。此外，已经确定了几个诱变变异体的结构，揭示了意想不到的构象重排，从而深入了解酶的活性。这些初步结果使我们能够对催化反应机理和伴随的构象运动产生一个假设。这为本提案的目的奠定了基础：在目标1中，我们将描述二羧酸结合位点催化的化学细节。为了实现这一目标，我们将与多个小分子共结晶富马酸喹啉还原酶（QFR），并从结构上研究改变催化效率的突变体。在目标2中，我们将描述与催化有关的运动。我们已经把酶的运动分为三种不同的类型——结构域间运动、催化环的运动和全局运动。我们已经开始了一个位点定向突变体的结构表征，它中断了正常的结构域运动。通过计算方法来评估每个捕获态的能量，我们将进一步利用定向自旋标记和电子顺磁共振波谱的组合来表征大的，域间运动。在Aim 3中，我们将研究环境杀菌剂对配合物II催化活性的影响。在这里，我们将几种喹啉结合抑制剂与野生型大肠杆菌QFR和大肠杆菌QFR的变体共结晶，后者具有与人类酶相似的杀菌剂结合特性。我们将进一步利用异源系统表达人类复合体II进行结构研究。

DESCRIPTION (provided by applicant): Complex II is one of the four electron transfer enzymes that generate the transmembrane electrochemical gradient in mitochondrial aerobic respiration. Its genetic alteration or inhibition has a significant effect on survival of the organism. The complex II superfamily is perhaps the most versatile respiratory enzyme since it is involved in aerobic and anaerobic respiration and the Krebs cycle. The recycling of the complex II architecture for multiple purposes suggests that this fold is evolutionary ancient. Examination of this single enzyme can give insight into biological mechanisms of fumarate reduction, succinate oxidation, aspartate oxidation, quinol chemistry, electron transfer, and the formation of the transmembrane electrochemical gradient. In preliminary studies, the complex II homolog quinol:fumarate reductase has been overproduced, crystallized and the structure determined alone and in complex with inhibitors of the quinol-binding site. Further, the structures of several mutagenic variants have been determined, revealing unexpected conformational rearrangements that give insight into enzyme activity. These preliminary results allowed us to generate a hypothesis for the catalytic reaction mechanism and that includes accompanying conformational motion. This sets the stage for the Aims of this proposal: In Aim 1, we will describe the chemical details of catalysis at the dicarboxylate-binding site. To achieve this, we will co-crystallize the quinol:fumarate reductase (QFR) with multiple small molecules and structurally investigate mutants that alter the catalytic efficiency. In Aim 2, we will characterize the motions associated with catalysis. We have already classified enzyme motions into three distinct types - interdomain motions, motions of a catalytic loop, and global motions. We have begun structural characterization of a site-directed mutant that interrupts normal domain motion. Complemented by computational methods that evaluate the energies of each trapped state, we will further characterize the large, interdomain motion using a combination of site-directed spin labeling and electron paramagnetic resonance spectroscopy. In Aim 3, we will investigate the influence of environmental fungicides on the catalytic activity of complex II. Here, we will co-crystallize several quinol-binding inhibitors with both the wild type E. coli QFR and a variant of the E. coli QFR that has fungicide binding properties similar to the human enzyme. We will further work to express human complex II with a heterologous system for structural studies.