Comparison of the Size and Properties of the Cytochrome c/Cardiolipin Nanospheres in a Sediment and Non-polar Medium.

Apoptosis, as the major type of programmed cell death, plays an important role in the organism renewal and removal of defective and transformed cells, including cancer cells. One of the earliest apoptotic events is lipid peroxidation in the inner mitochondrial membrane catalyzed by a complex of cytochrome c (CytC) with the mitochondrial phospholipid cardiolipin (CL). It was shown that mixing CytC and CL solutions results in the formation of CytC/CL complexes (Cyt-CL nanospheres) with a diameter of 11-12 nm composed of the molten globule protein molecule and a CL monolayer. Using the methods of dynamic light scattering for the Cyt-CL chloroform solution and small-angle X-ray scattering for the Cyt-CL sediment, it was found that in both cases, Cyt-CL formed nanospheres with a diameter of 8 and 11 nm, which corresponded to the earlier determined lipid/protein ratios of 13-14 and 35-50, respectively. These results showed that the Cyt-CL nanospheres can form not only during crystallization but also in a hydrophobic medium. CytC in the complex exists as a molten globule, as evidenced by the emergence of tryptophan and tyrosine fluorescence (absent in the native protein) due to the Förster resonance transfer of the electron excitation energy onto the heme. At the same time, the coordinate bond between the heme iron and the sulfur atom of methionine 80 in Cyt-CL is disrupted (the absorption band at ~700 nm disappears). Similar disruption of the iron-sulfur bond in Cyt-CL was observed in 50% methanol. These changes were reversible, which corroborates the conclusion on the CytC transition to the molten globule conformation in methanol-containing solutions.

Structure of the complex of cytochrome c with cardiolipin in non-polar environment.

The complex of mitochondrial protein cytochrome c (CytC) with anionic phospholipid cardiolipin (CL) plays a crucial role in the initiation of apoptosis by catalyzing lipid peroxidation in mitochondrial membranes. In our previous papers, we found that CytC and CL mixed in millimolar concentrations form a sediment showing microcrystals composed of nanospheres (Cyt-CL) of 11-12 and 8 nm in diameter. The hypothesis was proposed that Cyt-CL, having hydrophobic shell, may appear inside the membrane lipid bilayer in mitochondria and peroxidase membrane phospholipids so initiating the apoptotic cascade. In this work, Cyt-CL complex dissolved in chloroform or hexane was investigated as a model of the complex in mitochondrial membranes. We used dynamic light scattering method to measure the size of the particles. The analysis of particles size distribution of Cyt-CL in chloroform allows to reveal three dominant diameters of 12.1 ±â€¯1.4, 7.8 ±â€¯1.0, and 4.7 ±â€¯0.7 nm. The first two values are closed to those, earlier obtained with small-angle X-ray scattering method in Cyt-CL microcrystals, 11.1 ±â€¯1.0 and 8.0 ±â€¯0.7 nm. CL extracted in chloroform-methanol forms a real solution of particles with diameter of 0.7 ±â€¯0.1 nm. In methanol-water phase, CL and CL + CytC mixture form particles of 83.7 ±â€¯9.8 and 71.3 ±â€¯11.6 nm, respectively. Apparently, cardiolipin in 50% methanol forms single-layer liposomes regardless of the presence of CytC in the medium. Partial unfolding of CytC in the complex was evidenced by (a) appearance of fluorescence of tyrosine and tryptophan residues and (b) disappearance of the absorption band at 699 nm due to breakdown of heme iron - methionine bond > F⋯S(Met80). In hydrophobic solvent Cyt-CL exhibited quasi-lipoperoxidase and lipoxygenase activity as was shown in kinetic measurements of chemiluminescence enhanced by coumarin C-525, a selective sensitizer of chemiluminescence, associated with reactions of lipid peroxyl radicals. Our data in this model system do not contradict the hypothesis (Vladimirov, Y.A. et al. Biochemistry (Mosc) 78, 1086-1097) that nanospheres of Cyt-CL complex, embedded into the lipid phase of mitochondrial membrane, catalyze lipid peroxidation, thereby initiating apoptosis.