Conformational properties of cardiolipin-bound cytochrome c.

Interactions of cytochrome c (cyt c) with cardiolipin (CL) are important for both electron transfer and apoptotic functions of this protein. A sluggish peroxidase in its native state, when bound to CL, cyt c catalyzes CL peroxidation, which contributes to the protein apoptotic release. The heterogeneous CL-bound cyt c ensemble is difficult to characterize with traditional structural methods and ensemble-averaged probes. We have employed time-resolved FRET measurements to evaluate structural properties of the CL-bound protein in four dansyl (Dns)-labeled variants of horse heart cyt c. The Dns decay curves and extracted Dns-to-heme distance distributions P(r) reveal a conformational diversity of the CL-bound cyt c ensemble with distinct populations of the polypeptide structures that vary in their degree of protein unfolding. A fraction of the ensemble is substantially unfolded, with Dns-to-heme distances resembling those in the guanidine hydrochloride-denatured state. These largely open cyt c structures likely dominate the peroxidase activity of the CL-bound cyt c ensemble. Site variations in P(r) distributions uncover structural features of the CL-bound cyt c, rationalize previous findings, and implicate the prime role of electrostatic interactions, particularly with the protein C terminus, in the CL-induced unfolding.

Multifaceted effects of ATP on cardiolipin-bound cytochrome c.

Using a collection of dye-labeled cytochrome c (cyt c) variants, we identify transformations of the heterogeneous cardiolipin (CL)-bound cyt c ensemble with added ATP. Distributions of dye-to-heme distances P(r) from time-resolved fluorescence resonance energy transfer show that ATP decreases the population of largely unfolded cyt c conformers, but its effects are distinct from those of a simple salt. The high peroxidase activity of CL-bound cyt c with added ATP suggests binding interactions that favor protein structures with the open heme pocket. Although ATP weakens cyt c-CL binding interactions, it also boosts the apoptosis-relevant peroxidase activity of CL-bound cyt c.

Becoming a peroxidase: cardiolipin-induced unfolding of cytochrome c.

Interactions of cytochrome c (cyt c) with a unique mitochondrial glycerophospholipid cardiolipin (CL) are relevant for the protein's function in oxidative phosphorylation and apoptosis. Binding to CL-containing membranes promotes cyt c unfolding and dramatically enhances the protein's peroxidase activity, which is critical in early stages of apoptosis. We have employed a collection of seven dansyl variants of horse heart cyt c to probe the sequence of steps in this functional transformation. Kinetic measurements have unraveled four distinct processes during CL-induced cyt c unfolding: rapid protein binding to CL liposomes; rearrangements of protein substructures with small unfolding energies; partial insertion of the protein into the lipid bilayer; and extensive protein restructuring leading to "open" extended structures. While early rearrangements depend on a hierarchy of foldons in the native structure, the later process of large-scale unfolding is influenced by protein interactions with the membrane surface. The opening of the cyt c structure exposes the heme group, which enhances the protein's peroxidase activity and also frees the C-terminal helix to aid in the translocation of the protein through CL membranes.