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.

Key Role of Cytochrome C for Apoptosis Detection Using Raman Microimaging in an Animal Model of Brain Ischemia with Insulin Treatment

“…36,38 More specifically, the oxidation is catalyzed by a cardiolipin-specific peroxidase activity of cardiolipin-bound CytC, which may even occur before the release of proapoptotic factors. 39 The interplay between CytC and cardiolipin has been recently studied also by Raman spectroscopy, 40 evidencing how CytC can induce permeabilization of the mithocondrial membrane which in turn starts the apoptosis process. In these mentioned cases, it is the CytC bound to membrane and/or membrane constituent (i.e., cardiolipin) which plays an important role in cell death and/or cell oxidative damage.…”

Redox‐State‐Mediated Regulation of Cytochrome c Release in Apoptosis Revealed by Surface‐Enhanced Raman Scattering on Nickel Substrates

“…The peroxidized CL with other hydroperoxides would inhibit the activity of Cyt co xidase, [41] leading to the accumulation of Fe 2+ Cyt ci nt he mitochondrial intermembrane.A tt he outer mitochondrial membrane,s elective interaction of Fe 2+ Cyt c with CL induces permeabilization and pore formation with Bcl-2 family proteins,which stimulate the release of Cyt cinto the cytoplasm. [7,35] Ther eleased Cyt ci nc ytoplasm includes both the ferrous state and the ferric state as revealed by our recent study. [37] In summary,weinvestigated the interactions between CL and Cyt ci nb oth oxidized and reduced states by SERS.T he results demonstrated that the interaction between Fe 2+ Cyt c and CL caused arelatively larger conformational alteration of the protein than that of Fe 3+ Cyt c, on the basis of which the binding of Fe 2+ Cyt ct oC Lc an induce much higher peroxidase activity at the inner membrane and higher permeability of the outer mitochondrial membranes.O ur study revealed that Fe 2+ Cyt cp robably plays am ore important role in the early events of apoptosis as further evidenced by the in situ investigation of the Cyt cr elease on the Ni substrates.T hus, we believe that the redox states of Cyt cregulate and trigger the release of Cyt cf rom mitochondria into cytosol.…”

“…[6] Am ore recent study revealed that the Cyt cs electively interacted with CL, changing the permeability and thus facilitated the release of Cyt cinto the cytosol. [7] Theinteractions between ferric (Fe 3+ ) Cyt ca nd CL-containing liposomes are extensively investigated by optical spectroscopy,t ime-resolved fluorescence resonance energy transfer and fluorescence correlation spectroscopy, [8] surface-enhanced infrared spectroelectrochemistry, [9,10] and visible circular dichroism (CD). [11] Ther esults obtained thus indicated that that the interaction of Fe 3+ Cyt c with CL leads to substantial unfolding,h eme exposure,a nd replacement of Met axial ligand with aH is imidazole group.…”

An optimal surface concentration of pure cardiolipin deposited onto glassy carbon electrode promoting the direct electron transfer of cytochrome-c

“…[2] The specific and selective interaction between cardiolipin and cytochrome-c is not well understood and has been assigned to a combination of electrostatic and hydrophobic effects, hydrogen bonding and/or the formation of a cardiolipin/cytochrome-c complex. [2,[6][7][8][9][10][11][12][15][16][17][18] The electrochemical behavior of redox proteins such as cytochrome-c, a water-soluble haemoprotein involved in the respiratory chain of mitochondria, has been extensively reported since 1977. [19][20][21][22] This model redox protein has often been studied at lipid-modified electrodes because the detection of its electroactivity is seldom possible at bare electrode surfaces even with large scanning potential range.…”

“…after three cyclic voltammograms. Thus, the immobilization of cytochrome-c onto the modified glassy carbon electrode surface is assigned to specific and selective interactions between cytochrome-c and cardiolipin as documented in the literature [2,[6][7][8][9]15]. The electron transfer rate constant k ET of cytochrome-c immobilized on cardiolipin-modified glassy carbon electrode has been estimated through numerical simulations[51, 52] of the cyclic voltammograms and amounts to 1.5 ± 0.5 s -1 .…”