Impact of Cardiolipin and Phosphatidylcholine Interactions on the Conformational Ensemble of Cytochrome c.

Primarily known for its function in the electron transport chain, cytochrome c (Cyt c) also plays a critical role in the initiation of mitochondrially induced apoptosis through specific interactions with cardiolipin (CL), a negatively charged phospholipid found in the inner mitochondrial membrane. In this work, we study the conformational dynamics of Cyt c in the presence of CL and phosphatidylcholine (PC) phospholipids also present in the mitochondrial membrane to better understand how these interactions might drive transformation to the peroxidase-active protein. Using ion mobility mass spectrometry and millisecond hydrogen-deuterium exchange mass spectrometry, we demonstrate heterogeneity in the lipid-bound ensemble, with zwitterionic (PC) phospholipids inducing destabilization of residues necessary for peroxidase coordination, and increased dynamics on the proximal face of the heme binding pocket. In contrast to what might be expected from classical models for CL-driven Cyt c peroxidase activation, interactions with CL are shown to rigidify heme coordination. To reconcile this observation with the well-supported view that CL is linked to peroxidase activation, we propose a mechanism in which CL stabilizes the conformational transition between the peroxidase-active and inactive forms.