Cytochrome c (cyt c) is a heme protein located in the mitochondrial intermembrane space, where it transfers electrons in the respiratory chain. Cyt c also plays an important role in apoptosis by its release to the cytosol. Cyt c associates with the inner mitochondrial membrane by interaction with cardiolipin (CL), which may increase the peroxidase activity of cyt c. Although there have been many studies on the binding character of cyt c to CL-containing membranes, the CL-interaction sites of cyt c have lacked high resolution. Solution NMR is one of the most powerful methods for analyzing molecular interactions in proteins, but it has been difficult to study membrane-associated proteins by solution NMR due to their high molecular weight.
In this study, we used bicelles with a limited number of CL in each bicelle to characterize the interaction of horse cyt c with CL by high-resolution solution NMR spectroscopy. We identified a relatively wide CL-interaction site for cyt c by solution NMR spectroscopy using CL-containing bicelles. The cyt c interaction site with CL was similar to those with Complex III and Complex IV, 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 a dynamic aspect of cyt c for interaction with molecules in mitochondria. Application of bicelles for the characterization of protein−lipid interaction with high-resolution solution NMR spectroscopy may provide us new insights on not only the detailed interaction sites of peripheral membrane proteins but also their dynamics at the membrane surface.