Alanine Charge Screening Demonstrates Cytochrome C Unfolding on Cardiolipin Membrane Surfaces

Abstract

Cytochrome c (Cytc) has been shown to play an important role in signaling during apoptosis. In the initial stages, positively charged regions of Cytc interact electrostatically with negatively charged, cardiolipin (CL) on the inner mitochondrial membrane. In the presence of reactive oxygen species, Cytc demonstrates increased peroxidase activity and a propensity to oxidize CL. Cytc dissociate from the oxidized CL freeing it to form the apoptosome in the cytoplasm and initiate apoptosis. The exact mechanism for this initial electrostatic interaction and the role of structure and conformation of Cytc have been extensively debated. Using heme Soret band circular dichroism (CD), fluorescence (Trp59) and UV-visible spectroscopies, in coordination with alanine charge screening, we investigate the initial electrostatic cytochrome c/cardiolipin interaction.To date, several cardiolipin interaction sites have been proposed on cytochrome c. At pH 8, we are able to isolate and study the cytochrome c/cardiolipin interaction at anionic binding site A with 100% cardiolipin vesicles. Using alanine charge screening (Lys→Ala mutations), we investigated the role of lysines 72, 73, 86 and 87 in site A during electrostatic cardiolipin binding. Optimized titration techniques were utilized that generate reproducible quantitative data that can then be fit to extract the binding contribution to cardiolipin for each amino acid investigated. Our data indicate that an initial binding occurs at lipid to protein, L/P, ratios below 5. However, neither Trp59 fluorescence nor Soret band CD is strongly sensitive to this early binding event making quantitative evaluation of this interaction difficult. At L/P ratios >20 a binding event consistent with a conformational rearrangement on the surface of the CL vesicle occurs. Our data indicate that one lysine from either side of Site A (Lys72/73 versus Lys86/87) is important for both the cooperativity of and L/P ratio needed for this conformational rearrangement on the liposome surface.