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

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。 我们的研究涉及呼吸链的末端复合体球形红杆菌(Rs)的细胞色素C氧化酶(CcO)的X射线结晶学分析。 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。在简化的结构中，可以看到血红素A3基团的异常位移，并伴随着质子输入通道(K路径)的顶部打开。这些变化在已发表的还原形式的牛心线粒体CcO中没有看到，可能揭示了质子泵门控的一个重要方面。然而，在数据收集过程中了解酶的实际氧化还原状态是至关重要的。 为了研究不同形态RsCcO晶体的氧化还原状态，我们利用BioCARS，14-BM-C的在线显微分光光度计，观察了RsCcO晶体在X射线照射前、期间和之后的光谱特性。我们观察到，对于氧化形式的酶，CUA中心和血红素a在照射后几分钟内变得减少，并在588 nm处开始出现另一个光谱峰。此外，我们发现用于闪冷晶体的冷冻溶液在辐射暴露时似乎也经历了显著的光谱变化，这可能会使研究进一步复杂化。对于还原形式的RsCcO晶体，晶体在曝光过程中保持还原，在照射一分钟内形成约635 nm的肩峰。不幸的是，这些晶体太厚了，无法获得完整的光谱。在这次实验中，我们打算利用野生型和K362M突变体的薄片状晶体，进一步研究这些光谱变化，包括控制低温溶解光谱，以更明确地鉴定酶的氧化还原状态，从而获得更清晰的吸收光谱。

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. Moreover, we found that the cryosolution used to flashcool the crystals appeared to undergo significant spectral changes upon radiation exposure as well, which could further complicate the study. 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. Unfortunately the crystals were too thick to get complete spectra. In this run, we intend to further study these spectral changes inlcuding control cryosolution spectra, to indentify the redox state of the enzyme more definitely, using thin-platelike crystals of wild type and K362M mutant so we can obtain clearer absorption spectra.