Multiple Mutations in the Non-Ordered Red Ω-Loop Enhance the Membrane-Permeabilizing and Peroxidase-like Activity of Cytochrome c

Abstract

A key event in the cytochrome c-dependent apoptotic pathway is the permeabilization of the outer mitochondrial membrane, resulting in the release of various apoptogenic factors, including cytochrome c, into the cytosol. It is believed that the permeabilization of the outer mitochondrial membrane can be induced by the peroxidase activity of cytochrome c in a complex with cardiolipin. Using a number of mutant variants of cytochrome c, we showed that both substitutions of Lys residues from the universal binding site for oppositely charged Glu residues and mutations leading to a decrease in the conformational mobility of the red Ω-loop in almost all cases did not affect the ability of cytochrome c to bind to cardiolipin. At the same time, the peroxidase activity of all mutant variants in a complex with cardiolipin was three to five times higher than that of the wild type. A pronounced increase in the ability to permeabilize the lipid membrane in the presence of hydrogen peroxide, as measured by calcein leakage from liposomes, was observed only in the case of four substitutions in the red Ω-loop (M4 mutant). According to resonance and surface-enhanced Raman spectroscopy, the mutations caused significant changes in the heme of oxidized cytochrome c molecules resulting in an increased probability of the plane heme conformation and the enhancement of the rigidity of the protein surrounding the heme. The binding of wild-type and mutant forms of oxidized cytochrome c to cardiolipin-containing liposomes caused the disordering of the acyl lipid chains that was more pronounced for the M4 mutant. Our findings indicate that the Ω-loop is important for the pore formation in cardiolipin-containing membranes.

Interaction of Amphipathic Peptide from Influenza Virus M1 Protein with Mitochondrial Cytochrome Oxidase

“…In the next experiment, we studied the ability of P4, A1, and A4 peptides to form pores in the membrane of the asolectin liposomes loaded with calcein (see [ 54 , 55 ]). The release of calcein from liposomes was followed by the increase in fluorescence caused by the dilution of the dye with the external medium.…”

“…Calcein-loaded liposomes were prepared according to [ 55 ], essentially as described above. After chloroform evaporation, lipids were dispersed in the buffer supplied additionally with 50 mM calcein.…”

The role of cardiolipin and cytochrome c in mitochondrial metabolism of cancer cells determined by Raman imaging: in vitro study on the brain glioblastoma U-87 MG cell line

“…They successfully used a SERS biosensor for the detection of Cyt c in NSCLC. 50 The role of the interaction of Cyt c with CL in metabolism was investigated also by Milorey et al , 45 Chertkova et al , 46 Pletneva's group 47,51 and Vladimirov's group. 52,53 The effect of cardiolipin penetration inside cells was studied also by A.V.…”

“…39 Monitoring the role of Cyt c in cancer detection has been analyzed in numerous successful reports. 2,5,[40][41][42][43][44][45][46][47] With Raman microspectroscopy, N. Brazhe et al investigated the redox status of cytochromes in cardiomyocytes. Their experiment shows that the amount of reduced cytochromes in rod-shaped cardiomyocytes decreases under oxidative stress conditions.…”

Editorial for the Special Issue: “State-of-Art in Protein Engineering”

“…Developing the theme of the membrane-active properties of cytochrome c, Chertkova R. and coworkers [ 1 ] evaluated the ability of amino acid residues from functionally significant regions of cytochrome c to bind to cardiolipin, as well as their membrane-permeable and peroxidase activity. For this purpose, a panel of mutant variants of cytochrome c with multiple substitutions, both in the red Ω-loop and in the universal binding site, was obtained using site-directed mutagenesis.…”