The Increase in the Peroxidase Activity of the Cytochrome C with Substitutions in the Universal Binding Site Is Associated with Changes in the Ability to Interact with External Ligands
Abstract
:1. Introduction
2. Results
2.1. Peroxidase Activity of Mutant CytC Forms
2.2. Effect of Mutations in UBS on Cyanide Binding to CytC
2.2.1. Acceleration of Cyanide Binding to Oxidized Forms of Mutant CytC as an Indicator of Changes in the Strength of Interaction between Heme Iron and S(Met80)
2.2.2. Deceleration of the Decay of the Cyanide Complex with the Reduced form of CytC, Caused by Mutations in UBS, Recorded by the MCD Method
2.3. Molecular Modeling of the Structure of CytC WT and Its Mutant Variants 2Mut, 5Mut, and 8Mut
3. Discussion
4. Materials and Methods
4.1. Mutant Genes Construction
4.2. Expression of the Mutant Genes of CytC, Protein Isolation, and Purification
4.3. Spectrophotometric Assays
4.4. MCD Spectroscopy
4.5. Molecular Dynamics
4.6. Isoelectric Surfaces
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
Appendix A
References
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Type of CytC | WT | 2Mut | 5Mut | 8Mut | Control without CytC |
---|---|---|---|---|---|
µM ferri/min | 1.7 ± 0.1 | 5.6 ± 0.03 | 5.0 ± 0.5 | 6.6 ± 0.2 | 0.77 ± 0.11 |
(TNs-control)/[c] | 0.31 min−1 | 1.61 | 1.41 | 1.95 |
Time, min | 0.3 | 1.2 | 2.1 | 2.9 | 3.7 | 4.5 | 5.3 | 6.1 | 6.9 |
Mut8-CN | 97 | 86.5 | 65.1 | 52 | 44 | 32 | 28.7 | 23 | 19 |
WT-CN | 95 | 75 | 59 | 48.5 | 38 | 30.6 | 26 | 20 | 16 |
Time, min | 7.7 | 8.5 | 9.3 | 10.1 | 10.9 | 11.7 | 12.5 | 13.3 | 14.1 |
Mut8-CN | 18 | 11.9 | 10.7 | 9.4 | 9.4 | 6.8 | 5 | 1.5 | 0.5 |
WT-CN | 13 | 10 | 9.6 | 7.0 | 3.9 | 3.9 | 3 | 0.5 | 0.04 |
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Chertkova, R.V.; Oleynikov, I.P.; Pakhomov, A.A.; Sudakov, R.V.; Semenova, M.A.; Arutyunyan, A.M.; Ptushenko, V.V.; Kirpichnikov, M.P.; Dolgikh, D.A.; Vygodina, T.V. The Increase in the Peroxidase Activity of the Cytochrome C with Substitutions in the Universal Binding Site Is Associated with Changes in the Ability to Interact with External Ligands. Int. J. Mol. Sci. 2024, 25, 8237. https://doi.org/10.3390/ijms25158237
Chertkova RV, Oleynikov IP, Pakhomov AA, Sudakov RV, Semenova MA, Arutyunyan AM, Ptushenko VV, Kirpichnikov MP, Dolgikh DA, Vygodina TV. The Increase in the Peroxidase Activity of the Cytochrome C with Substitutions in the Universal Binding Site Is Associated with Changes in the Ability to Interact with External Ligands. International Journal of Molecular Sciences. 2024; 25(15):8237. https://doi.org/10.3390/ijms25158237
Chicago/Turabian StyleChertkova, Rita V., Ilya P. Oleynikov, Alexey A. Pakhomov, Roman V. Sudakov, Marina A. Semenova, Alexander M. Arutyunyan, Vasily V. Ptushenko, Mikhail P. Kirpichnikov, Dmitry A. Dolgikh, and Tatiana V. Vygodina. 2024. "The Increase in the Peroxidase Activity of the Cytochrome C with Substitutions in the Universal Binding Site Is Associated with Changes in the Ability to Interact with External Ligands" International Journal of Molecular Sciences 25, no. 15: 8237. https://doi.org/10.3390/ijms25158237