STRUCTURAL ANALYSIS OF THE MEMBRANE METALLOPROTEIN CYTOCHROME C OXIDASE IN

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们的研究涉及到细胞色素c氧化酶（CcO）从球形红细菌（RS）的X射线晶体学分析。CcO通过提供最终的电子汇并将氧气还原为水来催化能量产生中至关重要的反应，同时将质子跨膜转移以形成用于直接ATP合成的电化学梯度。研究线粒体CcO的细菌同源物（例如来自RS的CcO）有助于线粒体CcO的机制研究。 我们最近获得了高分辨率晶体结构的I-II亚基催化核心的RsCcO的氧化形式在2.0分辨率，以及连二亚硫酸盐还原形式的酶在2.2分辨率。在还原结构中，血红素a3基团出现了不寻常的位移：整个原卟啉环以及羟基-法呢基尾部发生了轻微的位移和旋转。结果，羟基-法呢基尾部的OH基团和Y288的OH基团之间的距离变得比通常的紧密氢键距离大得多（4.0 <$vs. 2.6 <$），打开了质子输入通道（K路径）的顶部。幼童军？血红素a3金属-金属距离也更大（4.9 <$vs. 5.4 <$），并且在氧化形式的结构中的活性位点处归因于OH-/H2O的密度消失。在已发表的还原形式的牛心脏线粒体CcO中未观察到这些变化。 因此，我们需要在X射线数据收集之前，期间和之后确认不同形式晶体的氧化还原状态，特别是考虑到同步辐射可以产生光电子来还原氧化的晶体，并且在数据收集期间氧气仍然可以与还原的晶体接触。需要BioCARS 14-BM-C的在线显微分光光度计来最终确定晶体的氧化还原状态;酶在氧化和还原状态下在可见光谱区域中给出不同的光谱峰。

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. Our proposed research involves x-ray crystallographic analysis of Cytochrome c oxidase (CcO) from Rhodobacter sphaeroides (Rs). CcO catalyzes the reaction vital in energy production by providing the final electron sink and reducing oxygen to water, while translocating protons across the membrane to form the electrochemical gradient used for direct ATP synthesis. Mechanistic studies of mitochondrial CcO are facilitated by studying its bacterial homologue, eg CcO from Rs. We recently obtained high resolution crystal structures of the I-II subunit catalytic core of the RsCcO in the oxidized form at 2.0 ¿ resolution, as well as the dithionite-reduced form of the enzyme at 2.2 ¿ resolution. In the reduced structure, an unusual displacement of heme a3 group was seen: the entire protoporphyrin ring as well as the hydroxyl-farnesyl tail is shifted and rotated slightly. As a result, the distance between the OH group of the hydroxyl-farnesyl tail and the OH group of Y288 becomes much greater than the usual tight hydrogen bonding distance (4.0 ¿ vs. 2.6 ¿), opening the top of the proton input channel (K path). The CuB ? heme a3 metal-metal distance is also greater (4.9 ¿ vs. 5.4 ¿) and density attributed to OH-/H2O at the active site in the oxidized form of the structure is gone. These changes were not seen in the published bovine heart mitochondrial CcO in the reduced form. Therefore, we need to confirm the redox states of the different forms of the crystals before, during and after X-ray data collection, especially given that the synchrotron radiation could generate photoelectrons to reduce oxidized crystals, and that oxygen could still be in contact with a reduced crystal, during data collection. The on-line microspectrophotometer available at BioCARS, 14-BM-C, is needed to conclusively determine the redox states of the crystal; the enzyme gives distinct spectral peaks in the visible spectrum region in the oxidized and reduced states.