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Title: 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 pro-apoptotic 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 cardiolipin-containing 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-sn-glycero-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 resolutionmore » 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.« less
Authors:
 [1] ;  [1] ; ORCiD logo [1] ; ORCiD logo [1]
  1. Univ. of Utah, Salt Lake City, UT (United States). Dept. of Chemistry
Publication Date:
Grant/Contract Number:
FG03-93ER14333
Type:
Published Article
Journal Name:
Journal of the American Chemical Society
Additional Journal Information:
Journal Volume: 139; Journal Issue: 10; Journal ID: ISSN 0002-7863
Publisher:
American Chemical Society (ACS)
Research Org:
Univ. of Utah, Salt Lake City, UT (United States)
Sponsoring Org:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
OSTI Identifier:
1413574
Alternate Identifier(s):
OSTI ID: 1473884

Kitt, Jay P., Bryce, David A., Minteer, Shelley D., and Harris, Joel M.. Raman Spectroscopy Reveals Selective Interactions of Cytochrome $c$ with Cardiolipin That Correlate with Membrane Permeability. United States: N. p., Web. doi:10.1021/jacs.7b00238.
Kitt, Jay P., Bryce, David A., Minteer, Shelley D., & Harris, Joel M.. Raman Spectroscopy Reveals Selective Interactions of Cytochrome $c$ with Cardiolipin That Correlate with Membrane Permeability. United States. doi:10.1021/jacs.7b00238.
Kitt, Jay P., Bryce, David A., Minteer, Shelley D., and Harris, Joel M.. 2017. "Raman Spectroscopy Reveals Selective Interactions of Cytochrome $c$ with Cardiolipin That Correlate with Membrane Permeability". United States. doi:10.1021/jacs.7b00238.
@article{osti_1413574,
title = {Raman Spectroscopy Reveals Selective Interactions of Cytochrome $c$ with Cardiolipin That Correlate with Membrane Permeability},
author = {Kitt, Jay P. and Bryce, David A. and Minteer, Shelley D. and Harris, Joel M.},
abstractNote = {Permeabilization of the outer mitochondrial membrane is an integral step in apoptosis. The resulting release of pro-apoptotic 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 cardiolipin-containing 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-sn-glycero-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.},
doi = {10.1021/jacs.7b00238},
journal = {Journal of the American Chemical Society},
number = 10,
volume = 139,
place = {United States},
year = {2017},
month = {3}
}