Becoming a Peroxidase: Cardiolipin-Induced Unfolding of Cytochrome c

Abstract

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.

The key role played by charge in the interaction of cytochrome c with cardiolipin

“…The difficulties encountered in the study of the cyt c/CL binding process are correlated with several factors, such as the ionic strength of the solution, the membrane curvature, the CL/ cyt c molar ratio at which the binding reaction is followed, which significantly affect the results obtained [7,17,19,45,46]. Moreover, the finding that CL-bound cyt c is present in solution as a heterogeneous ensemble of forms characterized by different heme coordination represents a further complication [15,16,28].…”

“…CL-bound cyt c shows altered tertiary structure, a perturbed heme crevice and the displacement of Met80 from the sixth coordination position of the heme-Fe atom [5][6][7][8][16][17][18][19]. Depending on the experimental conditions, the sixth coordination position of the heme iron remains unbound or is occupied by another side chain (likely a Lys or a His residue) [7,20,21].…”

Defining the Apoptotic Trigger

“…Most studies of this kind have used the exterior surface of liposome membranes to follow the interaction of cytochrome c with cardiolipin. An impressive number have been conducted on the mechanism of this unfolding phase (4,29,45,47,48) and this gross structural perturbation leads to the activation of the peroxidase activity that subsequently initiates critical modifications of cardiolipin (26,28,29,49,50).…”

Disruption of cytochrome c heme coordination is responsible for mitochondrial injury during ischemia

“…Electrostatic interaction between cyt c and CL anchors cyt c to the IMM and optimizes ET between complex III and complex IV [13]. Interestingly, low ATP conditions favor hydrophobic interaction between cyt c and CL [14, 15], which is known to disrupt the Met 80 -Fe ligation, resulting in a pentacoordinated heme Fe [16–19]. The loss of the Met 80 -Fe ligation inhibits ET and converts cyt c from an electron carrier to an oxygenase/peroxidase that can cause CL peroxidation [12, 20–23].…”