Raman Spectroscopy Reveals Selective Interactions of Cytochrome c with Cardiolipin That Correlate with Membrane Permeability
Permeabilization of the outer mitochondrial membrane is an integral step in apoptosis. The resulting release of proapoptotic signaling proteins leads to cell destruction through activation of the cysteine-aspartic protease (caspase) cascade. However, the mechanism of outer mitochondrial membrane (OMM) permeabilization remains unclear. It was recently shown that cytochrome c can induce pore formation in cardiolipincontaining phospholipid membranes, leading to large dextran and protein permeability. In this work, the interaction of cytochrome c with cardiolipin-containing phospholipid vesicles, serving as models of the OMM, is investigated to probe cytochrome c-induced permeability. Lipid vesicles having either a 1,2-dipalmitoyl-snglycero- 3-phosphocholine (DPPC) or mixed-DPPC/cardiolipin membrane and containing a membrane-impermeable Raman tracer 3-nitrobenzenesulfonate (3-NBS) were optically trapped, translated into a solution containing cytochrome c, and monitored for 3-NBS leakage. Cytochrome-correlated leakage was observed only in cardiolipin-containing vesicles. Structural changes observed in the Raman spectra during permeabilization indicated acyl chain disordering along with decreased intensity of the cardiolipin cis-double-bond stretching modes. When the vesicle-associated cytochrome c Raman spectrum is compared with a spectrum in buffer, heme-resonance bands are absent, indicating loss of Met-80 coordination. To verify selective interactions of cytochrome c with cardiolipin, these experiments were repeated where the DPPC acyl chains were deuterated (D62-DPPC), allowing spectral resolution of the DPPC acyl chain response from that of cardiolipin. Interestingly, D62-DPPC acyl chains were unaffected by cytochrome c accumulation, while cardiolipin showed major changes in acyl chain structure. These results suggest that cytochrome-induced permeabilization proceeds through selective interaction of cytochrome c with cardiolipin, resulting in protein unfolding, where the unfolded form interacts with cardiolipin acyl chains within the bilayer to induce permeability.
- Research Organization:
- Univ. of Utah, Salt Lake City, UT (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC), Basic Energy Sciences (BES)
- Grant/Contract Number:
- FG03-93ER14333
- OSTI ID:
- 1413574
- Alternate ID(s):
- OSTI ID: 1473884; OSTI ID: 1508002
- Journal Information:
- Journal of the American Chemical Society, Journal Name: Journal of the American Chemical Society Vol. 139 Journal Issue: 10; ISSN 0002-7863
- Publisher:
- American Chemical SocietyCopyright Statement
- Country of Publication:
- United States
- Language:
- English
Web of Science
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