Resonance Raman Spectroscopy of Intact Mitochondria

从猪心分离线粒体，当琥珀酸用作呼吸底物时，将具有呼吸控制比高于3的级分用于拉曼测量。通过加入琥珀酸，测量还原线粒体的共振拉曼光谱，其显示细胞色素中血红素的振动光谱。通过冲洗CO，我们在517、576和369 cm-2处观察到新的拉曼谱带<-1>。它们在^ C^ O取代后显示出降档<13><18>。因此，517处的带被指定为V_<Fe-CO>模式，576和369 cm-3处的带被指定<-1>为与<Fe-C-O>细胞色素c氧化酶中的细胞色素a_3的δ_相关的模式。这些频率与增溶酶的相应模式的频率相同。在延迟时间为0.6 ms时，测量了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-1处观察到拉曼谱带<-1>，它们在^ O_2取代时出现了下移<18>。在此基础上，我们将571和804 cm ~ 2处的谱带分别归属<-1>于含氧化合物的V_<Fe-O2>模和含氧中间体的V_&lt;Fe=O&gt;模。用时间分辨共振拉曼光谱研究了混合价态的溶解酶与氧的反应。含氧中间体的衰减与氧代中间体的上升同步发生，含氧中间体的衰减在碱性pH值下比在中性pH值下明显慢。溶解酶的这些数据对于分析完整线粒体的反应是重要的。

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.