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Mutant Cytochrome C as a Potential Detector of Superoxide Generation: Effect of Mutations on the Function and Properties

Cytochrome c (CytC) is a single-electron carrier between complex bc1 and cytochrome c-oxidase (CcO) in the electron transport chain (ETC). It is also known as a good radical scavenger but its participation in electron flow through the ETC makes it impossible to use CytC as a radical sensor. To solve...

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Autores principales: Chertkova, Rita V., Oleynikov, Ilya P., Pakhomov, Alexey A., Sudakov, Roman V., Orlov, Victor N., Semenova, Marina A., Arutyunyan, Alexander M., Ptushenko, Vasily V., Kirpichnikov, Mikhail P., Dolgikh, Dmitry A., Vygodina, Tatiana V.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10528150/
https://www.ncbi.nlm.nih.gov/pubmed/37759538
http://dx.doi.org/10.3390/cells12182316
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author Chertkova, Rita V.
Oleynikov, Ilya P.
Pakhomov, Alexey A.
Sudakov, Roman V.
Orlov, Victor N.
Semenova, Marina A.
Arutyunyan, Alexander M.
Ptushenko, Vasily V.
Kirpichnikov, Mikhail P.
Dolgikh, Dmitry A.
Vygodina, Tatiana V.
author_facet Chertkova, Rita V.
Oleynikov, Ilya P.
Pakhomov, Alexey A.
Sudakov, Roman V.
Orlov, Victor N.
Semenova, Marina A.
Arutyunyan, Alexander M.
Ptushenko, Vasily V.
Kirpichnikov, Mikhail P.
Dolgikh, Dmitry A.
Vygodina, Tatiana V.
author_sort Chertkova, Rita V.
collection PubMed
description Cytochrome c (CytC) is a single-electron carrier between complex bc1 and cytochrome c-oxidase (CcO) in the electron transport chain (ETC). It is also known as a good radical scavenger but its participation in electron flow through the ETC makes it impossible to use CytC as a radical sensor. To solve this problem, a series of mutants were constructed with substitutions of Lys residues in the universal binding site (UBS) which interact electrostatically with negatively charged Asp and Glu residues at the binding sites of CytC partners, bc1 complex and CcO. The aim of this study was to select a mutant that had lost its function as an electron carrier in the ETC, retaining the structure and ability to quench radicals. It was shown that a mutant CytC with substitutions of five (8Mut) and four (5Mut) Lys residues in the UBS was almost inactive toward CcO. However, all mutant proteins kept their antioxidant activity sufficiently with respect to the superoxide radical. Mutations shifted the dipole moment of the CytC molecule due to seriously changed electrostatics on the surface of the protein. In addition, a decrease in the redox potential of the protein as revealed by the redox titrations of 8Mut was detected. Nevertheless, the CD spectrum and dynamic light scattering suggested no significant changes in the secondary structure or aggregation of the molecules of CytC 8Mut. Thus, a variant 8Mut with multiple mutations in the UBS which lost its ability to electron transfer and saved most of its physico-chemical properties can be effectively used as a detector of superoxide generation both in mitochondria and in other systems.
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spelling pubmed-105281502023-09-28 Mutant Cytochrome C as a Potential Detector of Superoxide Generation: Effect of Mutations on the Function and Properties Chertkova, Rita V. Oleynikov, Ilya P. Pakhomov, Alexey A. Sudakov, Roman V. Orlov, Victor N. Semenova, Marina A. Arutyunyan, Alexander M. Ptushenko, Vasily V. Kirpichnikov, Mikhail P. Dolgikh, Dmitry A. Vygodina, Tatiana V. Cells Article Cytochrome c (CytC) is a single-electron carrier between complex bc1 and cytochrome c-oxidase (CcO) in the electron transport chain (ETC). It is also known as a good radical scavenger but its participation in electron flow through the ETC makes it impossible to use CytC as a radical sensor. To solve this problem, a series of mutants were constructed with substitutions of Lys residues in the universal binding site (UBS) which interact electrostatically with negatively charged Asp and Glu residues at the binding sites of CytC partners, bc1 complex and CcO. The aim of this study was to select a mutant that had lost its function as an electron carrier in the ETC, retaining the structure and ability to quench radicals. It was shown that a mutant CytC with substitutions of five (8Mut) and four (5Mut) Lys residues in the UBS was almost inactive toward CcO. However, all mutant proteins kept their antioxidant activity sufficiently with respect to the superoxide radical. Mutations shifted the dipole moment of the CytC molecule due to seriously changed electrostatics on the surface of the protein. In addition, a decrease in the redox potential of the protein as revealed by the redox titrations of 8Mut was detected. Nevertheless, the CD spectrum and dynamic light scattering suggested no significant changes in the secondary structure or aggregation of the molecules of CytC 8Mut. Thus, a variant 8Mut with multiple mutations in the UBS which lost its ability to electron transfer and saved most of its physico-chemical properties can be effectively used as a detector of superoxide generation both in mitochondria and in other systems. MDPI 2023-09-19 /pmc/articles/PMC10528150/ /pubmed/37759538 http://dx.doi.org/10.3390/cells12182316 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Chertkova, Rita V.
Oleynikov, Ilya P.
Pakhomov, Alexey A.
Sudakov, Roman V.
Orlov, Victor N.
Semenova, Marina A.
Arutyunyan, Alexander M.
Ptushenko, Vasily V.
Kirpichnikov, Mikhail P.
Dolgikh, Dmitry A.
Vygodina, Tatiana V.
Mutant Cytochrome C as a Potential Detector of Superoxide Generation: Effect of Mutations on the Function and Properties
title Mutant Cytochrome C as a Potential Detector of Superoxide Generation: Effect of Mutations on the Function and Properties
title_full Mutant Cytochrome C as a Potential Detector of Superoxide Generation: Effect of Mutations on the Function and Properties
title_fullStr Mutant Cytochrome C as a Potential Detector of Superoxide Generation: Effect of Mutations on the Function and Properties
title_full_unstemmed Mutant Cytochrome C as a Potential Detector of Superoxide Generation: Effect of Mutations on the Function and Properties
title_short Mutant Cytochrome C as a Potential Detector of Superoxide Generation: Effect of Mutations on the Function and Properties
title_sort mutant cytochrome c as a potential detector of superoxide generation: effect of mutations on the function and properties
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10528150/
https://www.ncbi.nlm.nih.gov/pubmed/37759538
http://dx.doi.org/10.3390/cells12182316
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