Complexities of complex II: A versatile architecture for respiration

描述(申请人提供)：复合体II是在线粒体有氧呼吸中产生跨膜电化学梯度的四种电子转移酶之一。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超家族可能是用途最广泛的呼吸酶，因为它参与有氧和无氧呼吸以及克雷布斯循环。复杂的II建筑的多种用途的循环利用表明，这个褶皱是进化古老的。对这一单一酶的检测可以深入了解富马酸还原、琥珀酸氧化、天冬氨酸氧化、对苯二酚化学、电子转移以及跨膜电化学梯度的形成的生物学机制。在初步研究中，已经过量生产和结晶了化合物II的同系物喹酚：富马酸还原酶，并确定了单独和与喹酚结合部位的抑制剂的混合物的结构。此外，已经确定了几个突变变体的结构，揭示了意想不到的构象重排，从而深入了解了酶的活性。这些初步结果使我们能够产生一个关于催化反应机制的假说，其中包括伴随的构象运动。这为本提案的目标奠定了基础：在目标1中，我们将描述二元酸结合部位催化的化学细节。为了实现这一点，我们将使喹酚：富马酸还原酶(QFR)与多个小分子共结晶，并从结构上研究改变催化效率的突变体。在目标2中，我们将描述与催化相关的运动。我们已经将酶的运动分为三种不同的类型--域间运动、催化环运动和全局运动。我们已经开始研究能够干扰正常结构域运动的定点突变体的结构特征。辅以计算方法来评估每个俘获态的能量，我们将使用位置定向自旋标记和电子顺磁共振光谱的组合来进一步表征大的、域间运动。在目标3中，我们将研究环境杀菌剂对化合物II催化活性的影响。在这里，我们将把几种喹酚结合抑制剂与野生型大肠杆菌QFR和具有类似于人类酶的杀菌剂结合特性的变异型大肠杆菌QFR共结晶。我们将进一步致力于用异源系统表达人类复合体II以进行结构研究。

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.