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.