title: Raman Spectroscopy Reveals Selective Interactions of Cytochrome c with Cardiolipin That Correlate with Membrane Permeability.
abstract: 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.
authors.affiliation: Department of Chemistry, University of Utah , 315 South 1400 East, Salt Lake City, Utah 84112, United States.
authors.forename: Jay P
authors.initials: JP
authors.lastname: Kitt
authors.affiliation: Department of Chemistry, University of Utah , 315 South 1400 East, Salt Lake City, Utah 84112, United States.
authors.forename: David A
authors.initials: DA
authors.lastname: Bryce
authors.affiliation: Department of Chemistry, University of Utah , 315 South 1400 East, Salt Lake City, Utah 84112, United States.
authors.forename: Shelley D
authors.identifier: 0000-0002-5788-2249
authors.initials: SD
authors.lastname: Minteer
authors.affiliation: Department of Chemistry, University of Utah , 315 South 1400 East, Salt Lake City, Utah 84112, United States.
authors.forename: Joel M
authors.identifier: 0000-0002-7081-8188
authors.initials: JM
authors.lastname: Harris
mesh_terms: D002308:Cardiolipins / Q000737:chemistry*; D045304:Cytochromes c / Q000737:chemistry*; D051336:Mitochondrial Membranes / Q000737:chemistry*; D008958:Models, Molecular; D010743:Phospholipids / Q000737:chemistry*; D013059:Spectrum Analysis, Raman
chemical_list: D002308:Cardiolipins; D010743:Phospholipids; D045304:Cytochromes c