STRUCTURAL ANALYSIS OF THE MEMBRANE METALLOPROTEIN CYTOCHROME C OXIDASE IN

膜金属蛋白细胞色素C氧化酶的结构分析

• 批准号: 7726019
• 负责人: SHELAGH M FERGUSON-MILLER
• 金额: $ 0.79万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7726019
• 关键词: ATP Synthesis Pathway; Active Sites; Bos taurus; Catalytic Domain; Cattle; Computer Retrieval of Information on Scientific Projects Database; Data Collection; Electrons; Enzymes; Funding; Grant; Heart; Homologous Gene; Hydrogen Bonding; Hydroxyl Radical; Institution; Membrane; Metalloproteins; Metals; Mitochondria; Oxidation-Reduction; Oxygen; Potassium Channel; Production; Protons; Protoporphyrins; Publishing; Reaction; Research; Research Personnel; Resolution; Resources; Rhodobacter sphaeroides; Roentgen Rays; Source; Structure; Tail; United States National Institutes of Health; Water; cytochrome c oxidase; density; heme a3; hydroxyl group; synchrotron radiation

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.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 我们提出的研究涉及从Rhodobacter Sphaeroides（RS）中对细胞色素C氧化物（CCO）进行X射线晶体学分析。 CCO通过提供最终的电子水槽并将氧气减少到水中来催化能量产生的反应，同时将质子转移到整个膜上以形成用于直接ATP合成的电化学梯度。线粒体CCO的机械研究是通过研究其细菌同源物（例如CCO）来制备的。 最近，我们以2.0的氧化形式获得了RSCCO的I-II亚基催化核心的高分辨率晶体结构，并在2.2»分辨率下以二硫代石降低的形式降低了酶的形式。在降低的结构中，看到了血红素A3组的异常位移：整个原生磷脂环以及羟基 - 丝尼基的尾巴被稍微移动并稍微旋转。结果，羟基 - 芬太尼尾巴的OH组与Y288的OH组之间的距离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）。幼兽？血红素A3金属金属距离也更大（4.9€vs. 5.4€），密度归因于活性位点的OH-/H2O，以该结构的氧化形式消失。这些变化在已发表的牛心脏线粒体CCO中未见。 因此，我们需要在X射线数据收集之前，之中和之后确认不同形式的晶体的氧化还原状态，尤其是考虑到同步加速器辐射可以产生光电子以减少氧化物晶体，并且在数据收集期间，氧气仍然可以与还原的晶体接触。需要在线微光照射器14-BM-C，以最终确定晶体的氧化还原状态。该酶在氧化和还原状态的可见光谱区域中提供了不同的光谱峰。