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

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.

Авторы: Chertkova Rita V., Oleynikov Ilya P., Pakhomov Alexey A., Sudakov Roman V., Semenova Marina A., Arutyunyan Alexander M., Ptushenko Vasily V., Kirpichnikov Mikhail P., Dolgikh Dmitry A., Vygodina Tatiana V.
Журнал: International Journal of Molecular Sciences
Том: 25
Номер: 15
Год издания: 2024
Издательство: MDPI
Местоположение издательства: Basel, Switzerland
Номер статьи: 8237
DOI: 10.3390/ijms25158237
Аннотация: Cytochrome c (CytC), a one-electron carrier, transfers electrons from complex bc1 tocytochrome c oxidase (CcO) in the electron-transport chain. Electrostatic interaction with the partners,complex bc1 and CcO, is ensured by a lysine cluster near the heme forming the Universal Binding Site(UBS).We constructed three mutant variants of mitochondrial CytC with one (2Mut), four (5Mut), andfive (8Mut) Lys->Glu substitutions in the UBS and some compensating Glu->Lys substitutions at theperiphery of the UBS for charge compensation. All mutants showed a 4–6 times increased peroxidaseactivity and accelerated binding of cyanide to the ferric heme of CytC. In contrast, decomposition ofthe cyanide complex with ferrous CytC, as monitored by magnetic circular dichroism spectroscopy,was slower in mutants compared to WT. Molecular dynamic simulations revealed the increase in thefluctuations of Cα atoms of individual residues of mutant CytC compared to WT, especially in theΩ-loop (70–85), which can cause destabilization of the Fe. . .S(Met80) coordination link, facilitationof the binding of exogenous ligands cyanide and peroxide, and an increase in peroxidase activity.It was found that only one substitution K72E is enough to induce all these changes, indicating thesignificance of K72 and the Ω-loop (70–85) for the structure and physiology of mitochondrial CytC. Inthis work, we also propose using a ferro-ferricyanide buffer as a substrate to monitor the peroxidaseactivity of CytC. This new approach allows us to determine the rate of peroxidase activity at moderate(200 μM) concentrations of H2O2 and avoid complications of radical formation during the reaction.
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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 Ligandsстатья

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