INVESTIGATION OF SPECTRAL CHANGES OF CYTOCHROME C OXIDASE UPON X-RAY IRRADIATION

X射线照射下细胞色素C氧化酶光谱变化的研究

• 批准号: 8171992
• 负责人: SHELAGH M FERGUSON-MILLER
• 金额: $ 0.73万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171992
• 关键词: Binding; Catalytic Domain; Cattle; Characteristics; Complex; Computer Retrieval of Information on Scientific Projects Database; Cyanides; Data Collection; Enzymes; Funding; Grant; Heart; Institution; Investigation; Mitochondria; Nature; Oxidation-Reduction; Potassium Channel; Proton Pump; Protons; Publishing; Research; Research Personnel; Resolution; Resources; Respiratory Chain; Rhodobacter sphaeroides; Roentgen Rays; Running; Shoulder; Source; Structure; United States National Institutes of Health; cytochrome c oxidase; heme a; heme a3; irradiation; mutant

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), the terminal complex of the respiratory chain. 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, accompanied by opening of the top of a proton input channel (K path). These changes were not seen in the published bovine heart mitochondrial CcO in the reduced form and could be revealing an important aspect of the gating of the proton pump. However, it is critical to know the actual redox state of the enzyme during data collection. In order to investigate the redox states of the different forms of the crystals, we utilized the on-line microspectrophotometer available at BioCARS, 14-BM-C, to observe the spectral characteristics of RsCcO crystals before, during, and after X-ray irradiation. We observed that for the oxidized form of the enzyme, CuA center and heme a became reduced within a couple of minutes of irradiation, and that another spectral peak at 588 nm started to appear. For the reduced form of RsCcO crystals, the crystal remained reduced during the exposure, with a shoulder peak at approximately 635nm formed within a minute of irradiation. In this run, we will be using small, two-subunit crystals of RsCcO in oxidized and reduced states, as well as the cyanide bound form, in order to investigate the nature of the 588nm species. We will also continue our studies on the spectral changes of the K362M mutant crystals, in comparison with those of the wild type enzyme.

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）（呼吸链的末端复合物）中的细胞色素C氧化酶（CCO）进行X射线晶体学分析。 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。在还原结构中，通过打开质子输入通道（K路径）的顶部来完成血红素A3组的异常位移。这些变化在已发表的牛心脏线粒体CCO中没有看到，并且可以揭示质子泵门控的重要方面。但是，在数据收集过程中了解酶的实际氧化还原状态至关重要。 为了研究不同形式的晶体的氧化还原状态，我们利用了在生物剂，14-BM-C的在线微光谱仪，以观察X射线辐照之前，期间和之后的RSCCO晶体的光谱特性。我们观察到，对于酶的氧化形式，CUA中心和血红素A在辐照的几分钟内减少了，并且在588 nm处的另一个光谱峰开始出现。对于RSCCO晶体的还原形式，在暴露期间，晶体保持降低，在辐照的一分钟内形成约635nm的肩峰。在此运行中，我们将在氧化和还原状态以及氰化物结合形式中使用RSCCO的小型，两亚基晶体，以研究588nm物种的性质。与野生型酶相比，我们还将继续研究K362M突变体晶体的光谱变化。