Resonance Raman Spectroscopy of Intact Mitochondria

从猪心脏中分离线粒体，以琥珀酸盐作为呼吸底物时，将呼吸控制比大于3的部分用于拉曼测量。加入琥珀酸盐后，测定了还原线粒体的共振拉曼光谱，显示了细胞色素中血红素的振动光谱。我们在517、576和369 cm^<-1>处观测到新的拉曼波段。它们在^<13>C^<18>O取代时表现出下降。据此，517波段属于V_<Fe-CO>模式，576和369 cm^<-1>波段属于细胞色素c氧化酶中细胞色素a_3 δ_<Fe-C-O>模式。这些频率与相应的溶酶模式相同。这一事实表明细胞色素a_3的血红素口袋结构不受溶解的干扰。在延迟时间0.6 ms时测量还原线粒体与氧反应的时间分辨共振拉曼光谱。在571和804 cm处观察到^<-1>的拉曼谱带，在^<18>O_2取代后，拉曼谱带出现了下降。基于这一结果，我们将571和804 cm^<-1>的波段分别归属于氧化中间体的V_<Fe- o2 >模式和V_<Fe=O>模式。这些中间体在溶解酶和完整线粒体之间的时间演化差异将在下一步进行研究。用时间分辨共振拉曼光谱研究了溶酶在混合价态下与氧的反应。氧化中间体的衰变与氧化中间体的上升是同步发生的，在碱性条件下，氧化中间体的衰变明显慢于中性条件下。这些数据对分析完整线粒体的反应具有重要意义。

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.