Characterization of the Cytochrome c Membrane-Binding Site Using Cardiolipin-Containing Bicelles with NMR

Cytochrome (cyt) c transports electrons from Complex III to Complex IV in mitochondria. Cyt c is ordinarily anchored to the mitochondrial membrane through interaction with cardiolipin (CL), however its release into the cytosol initiates apoptosis. The cyt c interaction site with CL-containing bicelles was characterized by NMR spectroscopy. Chemical shift perturbations in cyt c signals upon interaction with bicelles revealed that a relatively wide region, which includes the A-site, the CXXCH motif, and the N- and C-terminal helices, and contains multiple Lys residues, interacts cooperatively with CL. The specific cyt c–CL interaction increased with increasing CL molecules in the bicelles. The location of the cyt c interaction site for CL was similar to those for Complex III and Complex IV, thus indicating that cyt c recognizes lipids and partner proteins in a similar way. In addition to elucidating the cyt c membrane-binding site, these results provide insight into the dynamic aspect of cyt c interactions in mitochondria.