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

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G. K. Vladimirov^1,2,a*, V. E. Remenshchikov¹, A. M. Nesterova^1,2, V. V. Volkov^1,3, and Yu. A. Vladimirov^1,2,4,5

¹Shubnikov Institute of Crystallography, Crystallography and Photonics Federal Scientific Research Centre, Russian Academy of Sciences, 119333 Moscow, Russia

²Institute of Regenerative Medicine, Sechenov First Moscow State Medical University, Ministry of Public Health of Russian Federation, 119991 Moscow, Russia

³Kurchatov National Institute Research Center, 123182 Moscow, Russia

⁴Pirogov Russian National Research Medical University, Ministry of Public Health of Russian Federation, 117997 Moscow, Russia

⁵Lomonosov Moscow State University, Faculty of Fundamental Medicine, 119991 Moscow, Russia

^* To whom correspondence should be addressed.

Received February 20, 2019; Revised March 28, 2019; Accepted April 12, 2019

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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.

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KEY WORDS: apoptosis, cytochrome c, cardiolipin, Cyt–CL complex, hydrophobic medium, dynamic light scattering, small-angle X-ray scattering, molten globule

DOI: 10.1134/S000629791908008X

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