STRUCTURAL ANALYSIS OF THE MEMBRANE METALLOPROTEIN CYTOCHROME C OXIDASE IN

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们的研究涉及球状红杆菌细胞色素C氧化酶(CcO)的X射线结晶学分析。CCO通过提供最终的电子宿和将氧还原到水中来催化对能量产生至关重要的反应，同时将质子转移到膜上形成用于直接合成ATP的电化学梯度。线粒体CcO的机制研究通过研究其细菌同源物而变得容易，例如来自Rs的CcO。 我们最近获得了RsCcO的I-II亚单位催化核心的高分辨晶体结构，在2.0？分辨率下为氧化形式，以及在2.2？分辨率下为二亚硫酸盐还原形式的酶。在还原的结构中，可以看到血红素A3基团的不寻常的位移：整个原卟啉环以及羟基法尼基的尾巴略有移动和旋转。结果，羟基法尼基尾部的羟基与Y288的羟基之间的距离变得比通常的紧密氢键距离(4.0？vs.2.6？)大得多，打开了质子输入通道的顶部(K路径)。那只幼崽？血红素A3的金属-金属距离也更大(4.9？比5.4？)，氧化形式的结构活性位上的OH-/H2O密度消失了。在已发表的还原形式的牛心线粒体CcO中没有看到这些变化。 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的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被氧化的晶体，并且在数据收集过程中氧可能仍然与被还原的晶体接触。BioCARS提供的在线显微分光光度计14-BM-C需要最终确定晶体的氧化还原状态；该酶在氧化和还原状态下在可见光谱区产生明显的光谱峰。

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.