Resonance Raman Spectroscopy of Intact Mitochondria

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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.