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.

这个子项目是许多利用 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。 我们提出的研究涉及细胞色素c氧化酶（CcO）从红细菌sphaeroides（Rs），呼吸链的终端复合物的X射线晶体学分析。 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，观察到血红素a3基团的不寻常位移，伴随着质子输入通道（K路径）顶部的打开。这些变化在已发表的还原形式的牛心脏线粒体CcO中未观察到，并且可能揭示了质子泵门控的重要方面。然而，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，我们利用在线显微分光光度计可在BioCARS，14-BM-C，观察RsCcO晶体的光谱特性之前，期间和之后的X射线照射。我们观察到，对于酶的氧化形式，CuA中心和血红素a在照射的几分钟内被还原，并且在588 nm处开始出现另一个光谱峰。对于还原形式的RsCcO晶体，晶体在暴露期间保持还原，在照射的一分钟内在约635 nm处形成肩峰。在这次运行中，我们将使用氧化态和还原态的RsCcO的小型双亚基晶体，以及氰化物结合形式，以研究588 nm物质的性质。我们还将继续研究K362 M突变晶体的光谱变化，与野生型酶的光谱变化进行比较。