Permeabilization of the outer mitochondrial membrane (OMM) is an integral step in apoptosis. Following OMM permeabilization, cytochrome c (CytC) is released from the mitochondria resulting in activation of the caspase cascade and ultimately cell death. It has been hypothesized that CytC participates in OMM permeabilization via interaction with the inner mitochondrial membrane (IMM) phospholipid cardiolipin, which translocates from the IMM to the OMM just prior to CytC release. Two mechanisms of CytC-induced permeabilization have been proposed: An electrostatic mechanism, wherein positively-charged CytC interacts with negatively-charged CL, and an unfolding mechanism, wherein CytC unfolds into the OMM through interaction with CL resulting in pore formation. In this work, the interaction of CytC with CL and other phospholipids is investigated through Raman spectroscopy of individual mixed phospholipid vesicles. Vesicles comprised of CL (-2 charge) and DPPC (neutral) were formed in a solution of a membrane impermeable Raman tracer (NBS), and Raman spectra monitored on exposure to solutions of CytC. NBS Raman modes disappeared indicating membrane permeabilization. Raman scattering from acyl-chain C-C and C-H vibrations indicate CytC disorders bilayer acyl chains, while C=C vibrations from CL chains indicate CytC does indeed interact with CL. Additionally, CytC resonance Raman modes indicative of native CytC folding disappear during the interaction supporting the unfolding hypothesis. To test the electrostatic hypothesis, mixed POPG (-1 charge) and DPPC vesicles were formed to match the CL charge density in CL bilayers. Surprisingly, CytC <i>also</i> induced permeability to NBS in POPG bilayers, supporting the electrostatic hypothesis. To test these membranes for pore formation (which would be necessary for CytC leakage), similar experiments were carried out, where CytC was used instead of NBS as the tracer. CytC filled vesicles were manipulated into buffer and monitored for CytC leakage. CytC leakage was only observed in CL vesicles, indicating CL is required for pore formation supporting the unfolding hypothesis. It has been suggested that on unfolding, the terminal α-helix of CytC inserts into the membrane, anchoring the protein contributing to pore formation. Current work where the unnatural amino acid p-CN-Phe is incorporated into the CytC structure as an indicator of the local environment of the CytC terminal helix during unfolding will also be presented.

Raman microscopy investigation of cytochrome c-cardiolipin interactions to understand the mechanism of cytochrome c-induced membrane permeabilization proposed to occur during apoptosis

256th National Meeting (2018)

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