Resonance Raman Investigations of Cytochrome c Conformational Change upon Interaction with the Membranes of Intact and Ca²⁺-Exposed Mitochondria

Abstract

The conformational states of cytochrome c inside intact and Ca(2+)-exposed mitochondria have been investigated using resonance Raman spectroscopy. Intact and swelling bovine heart and rat liver mitochondria were examined with an excitation wavelength (413.1 nm) in resonance with the Soret transition of ferrous cytochrome c. The different b- to c-type cytochrome concentration ratio in mitochondria from two different tissues was used to help assign the Raman spectral components. Resonance Raman spectra were also recorded for mitochondria fractions (supernatants and pellets) obtained from swollen (Ca(2+)-exposed) mitochondria after differential centrifugation. The results illustrate that cytochrome c has an altered vibrational spectrum in solution, in intact, and in swollen mitochondria. When cytochrome c is released from mitochondria, its Raman spectrum becomes identical to that of ferrous cytochrome c in solution. The spectra of mitochondrial pellets indicate that a small amount of structurally modified cytochrome c remains associated with the heavy membrane fraction. Indeed, spectroscopic shifts in the low-frequency fingerprint and the high-frequency marker-band regions suggest that membrane binding leads to a partial opening of the heme pocket and an alteration of the heme thioether bonds. The results support the conclusion that most cytochrome c molecules in mitochondria are membrane-bound and that the cytochrome c structure changes upon binding. Furthermore, changes in the resonance Raman active mode located at 675 cm(-)(1) in the spectra of intact, swollen, and fractionated mitochondria indicate that b-type cytochromes may also undergo structural alterations during mitochondrial swelling and disruption.

Investigations of heme distortion, low-frequency vibrational excitations, and electron transfer in cytochrome c

“…This relationship is consistent with previous cyt c unfolding studies (3,34,35) and with studies (SI Appendix, Fig. S2.7) using live mitochondria (36). , and ν 8 are in excellent agreement among the three proteins.…”

“…Because the ruffling deformation of the heme cofactor is believed to arise primarily from constraints associated with the CXXCH pentapeptide (2, 3), which lies close to the protein-protein binding site (24), the binding event can perturb the CXXCH structure and can lead to changes in the heme ruffling deformation that helps to control the electron transfer process. With regard to this latter point, it is shown (3,36) in SI Appendix, Fig. S2.7 that when cyt c is bound to the living mitochondrial membrane the strong γ 21 mode observed in solution vanishes.…”

Femtosecond broadband stimulated Raman spectroscopy: Apparatus and methods

“…1 -6 Spontaneous Raman scattering can be used to obtain spectra over a ∼1500 cm -1 spectral window, but requires picosecond or longer duration pulses to obtain adequate spectral resolution. 7 A significant benefit of Raman scattering is resonance enhancement, which allows observation of the vibrational spectrum of a specific chromophore in a complex system; 8 however, electronic resonance is often accompanied by fluorescence backgrounds that can easily overwhelm the spontaneous Raman signal. To overcome these problems of time resolution and spectral quality, we have been developing the technique of femtosecond stimulated Raman spectroscopy (FSRS).…”

Monitoring Changes in the Redox State of Myoglobin in Cardiomyocytes by Raman Spectroscopy Enables the Protective Effect of NO Donors to Be Evaluated

“…11 The oxygenated state of myoglobin also acts as a scavenger of cellular NO and, hence, the protein plays a vital role in NO homeostasis, which is particularly important in cardiomyocytes as elevated levels inhibit mitochondrial respiration. 12,13 Non-resonance 14,15 and resonance [16][17][18][19] Raman spectroscopy have been used before in structural imaging of heart tissue 16 and to monitor dynamic changes in both isolated hearts 18 , cardiomyocytes [14][15][16][17] and exposed mitochondria. 19 In our recent work, the cellular mechanisms in ex vivo cardiomyocytes during hypoxia and hyperoxic reoxygenation were studied by Raman spectroscopy using a (non-resonant) excitation wavelength of 488 nm.…”

“…12,13 Non-resonance 14,15 and resonance [16][17][18][19] Raman spectroscopy have been used before in structural imaging of heart tissue 16 and to monitor dynamic changes in both isolated hearts 18 , cardiomyocytes [14][15][16][17] and exposed mitochondria. 19 In our recent work, the cellular mechanisms in ex vivo cardiomyocytes during hypoxia and hyperoxic reoxygenation were studied by Raman spectroscopy using a (non-resonant) excitation wavelength of 488 nm. 15 This excitation wavelength, which lies between the Soret and Q bands for heme proteins, was found to give a strong Raman signal for the pyrrole half-ring symmetric stretch (ν 4 ) of the heme prosthetic group; the notation for the assignment of heme vibrational modes has been described by Spiro and co-workers.…”