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

Cytochrome (cyt)c transports electrons from ComplexIII to ComplexIV in mitochondria. Cytc is ordinarily anchored to the mitochondrial membrane through interaction with cardiolipin (CL), however its release into the cytosol initiates apoptosis. The cytc interaction site with CL-containing bicelles was characterized by NMR spectroscopy. Chemical shift perturbations in cytc 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 cytc-CL interaction increased with increasing CL molecules in the bicelles. The location of the cytc interaction site for CL was similar to those for ComplexIII and ComplexIV, thus indicating that cytc recognizes lipids and partner proteins in a similar way. In addition to elucidating the cytc membrane-binding site, these results provide insight into the dynamic aspect of cytc interactions in mitochondria.