HIGH PRESSURE COOLING OF CYTOCHROME C OXIDASE

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。细胞色素c氧化酶（Cytochrome c oxidase, CcO）是真核生物和许多细菌中电子传递链的末端酶。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，从而提供最终的电子汇。在过去的十年中，已经从牛心脏线粒体和细菌中确定了几种aa3型氧化酶的x射线晶体结构。这种新的丰富的结构信息，结合光谱和定点诱变研究，极大地增强了我们对这种多亚基膜蛋白复合物的结构/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尚未得到解决。虽然我们现在的晶体衍射分辨率为2.35 A，但关键问题是水分子在形成质子转移途径的结构中的排列，以及水在突变形式中的排列变化，因此需要更高质量的衍射数据。据报道，在高压（200Mpa）下低温冷却的蛋白质晶体即使没有任何穿透性的冷冻保护剂也能很好地衍射。因此，在本实验中，细胞色素c氧化酶晶体将采用高压冷却方法处理，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.