To Unfold or not to Unfold? Structural Insights of Peroxidase-Active Cardiolipin-Bound Cytochrome c by Solid-State NMR

Cytochrome c (cyt-c) plays a key role in activating intrinsic apoptosis, a closely regulated cell death mechanism. Complete understanding of this process has implications for treatment of neurodegenerative diseases like Huntington's disease, as well as cancer. A pivotal signaling event in this apoptotic pathway is the peroxidation of the mitochondrial lipid cardiolipin (CL) by cyt-c. Cyt-c gains CL-specific lipid peroxidase activity by binding to CL during apoptosis, but exactly what structural changes lead to its proapoptotic activity have remained elusive. We have performed both structural and functional measurements to determine changes in structure and dynamics of cyt-c that accompany the activation of its peroxidase function upon binding to CL. Specifically, we used FTIR and multidimensional magic-angle-spinning (MAS) solid-state NMR (ssNMR) on samples mimicking proapoptotic conditions that trigger cyt-c's peroxidase activity. Comparison to FTIR and solution NMR data for the soluble protein revealed that cyt-c did not experience major unfolding upon membrane binding. We probed the protein-lipid interactions via ssNMR on both the protein and the lipids, and additionally used fluorescence quenching measurements of resonance energy transfer between the cyt-c heme and fluorescently labeled lipids. We conclude that increases in peroxidase activity of peripherally CL-bound cyt-c do not result from extensive unfolding of cyt-c. We instead propose an alternative mechanism in which localized structural changes, rather than global unfolding, in the CL-bound protein may be enough to allow for significant lipid peroxidation function to arise.