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

Chertkova, Rita V.; Firsov, Alexander M.; Brazhe, Nadezda A.; Nikelshparg, Evelina I.; Bochkova, Zhanna V.; Bryantseva, Tatyana V.; Semenova, Marina A.; Baizhumanov, Adil A.; Kotova, Elena A.; Kirpichnikov, Mikhail P.; Maksimov, Georgy V.; Antonenko, Yuriy N.; Dolgikh, Dmitry A.

doi:10.3390/biom12050665

Open AccessArticle

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

by

Rita V. Chertkova

^1,*,†,

Alexander M. Firsov

^2,†,

Nadezda A. Brazhe

^3,*,

Evelina I. Nikelshparg

³

,

Zhanna V. Bochkova

^1,3,

Tatyana V. Bryantseva

^1,4,

Marina A. Semenova

¹

,

Adil A. Baizhumanov

³

,

Elena A. Kotova

²,

Mikhail P. Kirpichnikov

^1,4,

Georgy V. Maksimov

^3,5

,

Yuriy N. Antonenko

²

and

Dmitry A. Dolgikh

^1,4

¹

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia

²

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia

³

Biophysics Department, Biological Faculty, Lomonosov Moscow State University, 119234 Moscow, Russia

⁴

Biology Department, Lomonosov Moscow State University, 119899 Moscow, Russia

⁵

Federal State Autonomous Educational Institution of Higher Education “National Research Technological University “MISiS””, 119049 Moscow, Russia

^*

Authors to whom correspondence should be addressed.

^†

These authors contributed equally to this work.

Biomolecules 2022, 12(5), 665; https://doi.org/10.3390/biom12050665

Submission received: 3 April 2022 / Revised: 27 April 2022 / Accepted: 30 April 2022 / Published: 4 May 2022

(This article belongs to the Special Issue State-of-Art in Protein Engineering)

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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.

Keywords: mitochondrial cytochrome c; heme; red Ω-loop of cytochrome c; liposome leakage; cardiolipin; peroxidase activity; resonance Raman spectroscopy; surface-enhanced Raman spectroscopy

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MDPI and ACS Style

Chertkova, R.V.; Firsov, A.M.; Brazhe, N.A.; Nikelshparg, E.I.; Bochkova, Z.V.; Bryantseva, T.V.; Semenova, M.A.; Baizhumanov, A.A.; Kotova, E.A.; Kirpichnikov, M.P.; et al. Multiple Mutations in the Non-Ordered Red Ω-Loop Enhance the Membrane-Permeabilizing and Peroxidase-like Activity of Cytochrome c. Biomolecules 2022, 12, 665. https://doi.org/10.3390/biom12050665

AMA Style

Chertkova RV, Firsov AM, Brazhe NA, Nikelshparg EI, Bochkova ZV, Bryantseva TV, Semenova MA, Baizhumanov AA, Kotova EA, Kirpichnikov MP, et al. Multiple Mutations in the Non-Ordered Red Ω-Loop Enhance the Membrane-Permeabilizing and Peroxidase-like Activity of Cytochrome c. Biomolecules. 2022; 12(5):665. https://doi.org/10.3390/biom12050665

Chicago/Turabian Style

Chertkova, Rita V., Alexander M. Firsov, Nadezda A. Brazhe, Evelina I. Nikelshparg, Zhanna V. Bochkova, Tatyana V. Bryantseva, Marina A. Semenova, Adil A. Baizhumanov, Elena A. Kotova, Mikhail P. Kirpichnikov, and et al. 2022. "Multiple Mutations in the Non-Ordered Red Ω-Loop Enhance the Membrane-Permeabilizing and Peroxidase-like Activity of Cytochrome c" Biomolecules 12, no. 5: 665. https://doi.org/10.3390/biom12050665

APA Style

Chertkova, R. V., Firsov, A. M., Brazhe, N. A., Nikelshparg, E. I., Bochkova, Z. V., Bryantseva, T. V., Semenova, M. A., Baizhumanov, A. A., Kotova, E. A., Kirpichnikov, M. P., Maksimov, G. V., Antonenko, Y. N., & Dolgikh, D. A. (2022). Multiple Mutations in the Non-Ordered Red Ω-Loop Enhance the Membrane-Permeabilizing and Peroxidase-like Activity of Cytochrome c. Biomolecules, 12(5), 665. https://doi.org/10.3390/biom12050665

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

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