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Biochemistry. 2003 May 27;42(20):6149-58.

Resonance Raman investigations of cytochrome c conformational change upon interaction with the membranes of intact and Ca2+-exposed mitochondria.

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Physics Department and Center for Interdisciplinary Research on Complex Systems, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts 02115, USA.


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.

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