Cytochrome c Complexes with Cardiolipin Monolayer Formed under Different Surface Pressure

The formation of the complex of cytochrome c (Cytc) with a phospholipid cardiolipin (CL) in mitochondria is a crucial event in apoptosis development. There are two viewpoints on the structure of the complex. (1) Cytc is bound on the surface of the lipid bilayer. (2) The complex is a hydrophobic nanoparticle Cytc-CL formed by Cytc molten globule, covered by CL monolayer.1 In the present work, we attempted to bridge the gap between these two structures. We investigated the interaction between Cytc and Langmuir monolayers of CL. The surface pressure increase during incorporation of Cytc into CL monolayer obeys the equation: π = π₀ + Δπ∞[1 – exp(−βt)], where β is pseudo-first-order rate constant of Cytc binding, directly proportional to the initial Cytc concentration c₀. Parameters Δπ∞ and the rate β measured in different conditions were virtually equal for natural bovine CL and peroxidation-resistant tetraoleoyl CL in all experiments. Surface area–surface pressure isotherms of Cytc alone and in combination with a CL monolayer were similar in shape. Apparently, the protein exposes hydrophilic groups to the water phase and hydrophobic to the air or to the hydrocarbon chains of CL. The 30% ethanol dramatically accelerated the adsorption of Cytc on the water surface. The protein–lipid surface films showed, in compression–expansion cycles, that hysteresis loops were observed always when Cytc present, reproducible in repeating cycles. Taken together, our data show that when incorporated in a lipid monolayer or after adsorption on the water–air interface, Cytc undergoes conformational transition. In that, one part of the globule sphere becomes predominantly hydrophobic and the other, hydrophilic and charged (“stratified” Cytc). We hypothesize that in CL-containing bilayer membranes, Cytc incorporation into the lipid monolayer would result in membrane folding with subsequent formation of either catalytically reactive “bubbles” inside the bilayer, formed by Cytc-CL, or the appearance of hydrophilic pores. The role of lipid peroxidation catalyzed by Cytc-CL in the appearance of pores and apoptosis is also discussed.