Resonance Raman Spectroscopy of Intact Mitochondria

完整线粒体的共振拉曼光谱

基本信息

批准号：
13640501
负责人：
OGURA Takashi
金额：
$ 1.92万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

Mitochondria was isolated from porcine heart and the fraction that had a respiratory control ratio higher than 3 was used for Raman measurements when succinate was used as respiratory substrate. By addition of succinate, resonance Raman spectra of reduced mitochondria was measured, which exhibited vibrational spectra of hemes in cytochromes. By flushing CO we observed new Raman bands at 517, 576 and 369 cm^<-1>. They showed downshifts upon ^<13>C^<18>O substitution. Accordingly, the band at 517 was assigned to the V_<Fe-CO> mode and the bands at 576 and 369 cm^<-1> to the modes associated with δ_<Fe-C-O> of cytochrome a_3 in cytochrome c oxidase. These frequencies are the same with those of corresponding modes of solubilized enzyme. This fact implies that the heme pocket structure of cytochrome a_3 is not perturbed by solubilization.Time-resolved resonance Raman spectra of the reaction of reduced mitochondria with oxygen were measured at a delay time of 0.6 ms. Raman bands at 571 and 804 cm^<-1> were observed and they showed downshifts upon ^<18>O_2 substitution. Based on the result, we assigned the bands at 571 and 804 cm^<-1> to the V_<Fe-O2> mode of oxygenated and the V_<Fe=O> mode of oxo intermediate, respectively. The difference of the time evolution of these intermediates between solubilized enzyme and intact mitochondria will be examined in the next step.The reaction of solubilized enzyme in the mixed valence state with oxygen was studied with time-resolved resonance Raman spectroscopy. The decay of the oxygenated intermediate took place synchronously with the rise of the oxo intermediate and the decay of the oxygenated intermediate was significantly slower at alkaline pH than at neutral pH. These data for solubilized enzyme are important to analyze the reaction of intact mitochondria.

从猪心脏分离线粒体，当使用琥珀酸作为呼吸底物时，将呼吸控制比高于3的级分用于拉曼测量。通过添加琥珀酸，测量还原线粒体的共振拉曼光谱，其显示振动光谱。通过冲洗 CO，我们在 517、576 和 369 cm^-1 处观察到新的拉曼谱带。 ^ 13 C ^ 18 O取代因此，517处的谱带被指定为V_<Fe-CO>模式，并且576和369cm ^ -1>处的谱带被指定为与δ_<Fe相关的模式。细胞色素c氧化酶中细胞色素a_3的-C-O>这些频率与溶解酶的相应模式的频率相同，这一事实暗示细胞色素a_3的血红素袋结构。在0.6ms的延迟时间测量还原线粒体与氧的反应的时间分辨共振拉曼光谱，观察到571和804cm^-1处的拉曼带，并且它们显示出下移。 <18>O_2 取代根据结果，我们将 571 和 804 cm^<-1> 处的谱带分配给 V_<Fe-O2> 模式。下一步将检查这些中间体在溶解酶和完整线粒体之间的时间演化差异。混合价态溶解酶的反应。用时间分辨共振拉曼光谱研究了含氧中间体的衰变与含氧中间体的上升同步发生，并且含氧中间体的衰变在碱性pH下明显慢于在碱性pH下的衰变。中性 pH 值。溶解酶的这些数据对于分析完整线粒体的反应非常重要。