Probing the Interactions of Cytochrome c with Anionic Phospholipid Nanodiscs Using Millisecond Hydrogen–Deuterium Exchange Mass Spectrometry

The interplay between the anionic phospholipid cardiolipin (CL) and cytochrome c (cyt c) holds significance in the early stages of apoptosis. Despite identification of up to four potential sites of interaction between cytochrome c and cardiolipin bearing membranes, the exact mode of interaction remains unexplained, especially given that some of the putative binding surfaces are mutually exclusive. In this study, we utilize millisecond time-resolved electrospray ionization hydrogen-deuterium exchange mass spectrometry (TRESI-HDX-MS) to investigate conformational and dynamic changes in cytochrome c in the presence of various phospholipids (DMPC, POPG, and CL) incorporated into nanodiscs. We observe that, among the proposed binding sites, the adjacent "L"- and "A"-sites exhibited a decrease in deuterium exchange, while the "N" site remained unperturbed, suggesting a specific orientation of cytochrome c with respect to cell membranes upon binding. We also demonstrate that negatively charged phospholipids with physical differences (<i>i.e</i>., POPG and CL) exhibit essentially the same interaction with cytochrome c, supporting the utility of POPG nanodiscs as a model for cytochrome c-membrane interactions.