Permeabilization of the outer mitochondrial membrane (OMM) is a key step in apoptosis leading to release of cytochrome c (CytC) activating the apoptotic caspase cascade and resulting in cell-death. It has been suggested that CytC participates in the permeabilization process through interaction with cardiolipin (CL), an inner mitochondrial membrane phospholipid which translocates to the OMM preceding CytC release. Two mechanisms of permeabilization have been proposed: One is electrostatic, wherein positively-charged CytC interacts with negatively (-2) charged CL inducing curvature and pore-formation. The other is CytC insertion into the membrane where CytC unfolds and interacts with CL to induce an inverse phospholipid phase and pore formation. In this work, CytC-induced permeabilization of phospholipid vesicle membranes of varying composition is investigated via Raman spectroscopy. Vesicles comprising 80mol% POPC (16:0-18:1 phosphocholine, neutral) and 20mol% POPG (16:0-18:1 phosphoglycerol, -1 charge) or 90mol% POPC and 10mol% CL were formed in a solution of membrane-impermeable small molecule tracer nitrobenzenesulfonate (NBS). Vesicles were optically trapped, translated into CytC solutions and monitored for NBS leakage. Leakage was observed in POPG <i>and</i> CL vesicles, supporting the electrostatic interaction hypothesis. Interestingly, disappearance of resonance Raman bands associated with Met-80 coordination of the heme is observed in both cases indicating protein unfolding. However, in CL, distinct changes are observed in the phospholipid acyl-chain C=C modes which are not observed in POPG, suggesting interaction of CytC with CL occurs deeper in the membrane. Additionally, small-molecule permeabilization can occur without pore formation, typically through boundaries between ordered and disordered phases; this type of permeabilization would not allow the much larger CytC to traverse the membrane. To determine the nature of permeabilization in each case, the same experiments were conducted where CytC was used as the “tracer.” CytC-filled vesicles were translated into buffer and monitored for leakage. CytC leakage was only observed in CL vesicles, indicating pores that are sufficiently large for CytC translocation were formed. Despite protein unfolding in both cases, only CL membranes allow for CytC leakage which proceeds through large pore formation induced through interaction with CytC deep in the CL membrane in support of a membrane-insertion hypothesis.<br/>

Confocal Raman microscopy investigation of cytochrome c-phospholipid interactions which induce permeability in mixed phospholipid vesicle membranes

255th National Meeting (2018)

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