HIGH PRESSURE COOLING OF CYTOCHROME C OXIDASE

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。细胞色素c氧化酶(CcO)是真核生物和许多细菌中电子转移链的末端酶。它通过接受来自细胞色素c的电子并将它们传递给氧气形成水来提供最终的电子接收器。在过去的十年里，从牛心脏线粒体和细菌中已经确定了几种Aa3型氧化酶的X射线晶体结构。这些新的丰富的结构信息，结合光谱和定点突变研究，极大地提高了我们对这种多亚单位膜蛋白复合体的结构/功能方面的理解。然而，质子的矢量移位及其与电子转移和氧还原的耦合机制仍未得到解决。尽管我们现在有了分辨率为2.35A的晶体，但一个关键的问题是形成质子转移路径的结构中水分子的排列，以及水以突变形式排列的变化，因此需要更高质量的衍射数据。据报道，即使没有任何穿透性的低温保护剂，在高压(200 Mpa)下冷冻的蛋白质晶体也具有很好的衍射性。因此，在本实验中，将对细胞色素C氧化酶晶体进行高压降温处理，并收集高质量的数据集。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cytochrome c oxidase (CcO) is the terminal enzyme in the electron transfer chain in eukaryotes and many bacteria. It provides the final electron sink by accepting electrons from cytochrome c and passing them on to oxygen to form water. Over the past decade, several x-ray crystal structures of aa3-type oxidases have been determined from bovine heart mitochondria and bacteria. This new wealth of structural information, combined with spectroscopic and site-directed mutagenesis studies, has greatly enhanced our understanding of the structure/function aspects of this multisubunit membrane protein complex. However, the mechanism of vectorial translocation of protons and its coupling to electron transfer and oxygen reduction is yet to be solved. Although we now have crystals diffracting to 2.35 A resolution, a key question is the arrangement of water molecules in the structure that form proton transfer pathways, as well as changes in water arrangement in mutant forms, hence the need for higher quality diffraction data. It was reported that protein crystals cryo-cooled at high pressure (200Mpa) diffract excellently even without any penetrating cryoprotectants. So in this experiment, cytochrome c oxidase crystals will be handled by high pressure cooling method and high quality data sets will be collected.