Ukr.Biochem.J. 2025; Volume 97, Issue 4, Jul-Aug, pp. 43-51

doi: https://doi.org/10.15407/ubj97.04.043

ABTS oxidation reaction as a model of cytochrome c-driven electron transfer

10.09.2025   Uncategorized   No comments

F. Gudratova, A. Aliyeva, S. Mahmudova, K. Gasimov, T. Yusifov*

Institute of Biophysics, Ministry of Science and Education
of the Republic of Azerbaijan, Baku;
*e-mail: tjussifo@ucla.edu

Received: 23 May 2025; Revised: 24 July 2025;
Accepted: 12 September 2025; Available on-line: 17 September2025

Cytochrome c, as an electron carrier within the mitochondria, plays a crucial role in the electron transport chain. To meet the demand for rapid methods that assess the electron transport properties of cytochrome c, we used the electron donor 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonate) (ABTS) as a substrate and suitable spectrophotometric reporter of cytochrome c peroxidase-like activity. ABTS and cytochrome c from bovine were purchased from Sigma-Aldrich Inc. The time course of the cytochrome c-driven ABTS oxidation reaction was studied using H2O2 as a second substrate. It was demonstrated that CytC addition is a prerequisite for the transfer of electrons from ABTS to H2O2. The reaction kinetic analysis with determination of Vmax, Km, kcat, and kcat/Km values for both substrates was performed. Our results demonstrate that the cytochrome c-catalyzed ABTS oxidation reaction can be effectively employed as a model for studying the functional role of cytochrome c in various conditions.

Keywords: , , , ,

References:

  1. Marques HM. Electron transfer in biological systems. J Biol Inorg Chem. 2024;29(7-8):641-683. PubMed, PubMed, CrossRef
  2. Imai M, Saio T, Kumeta H, Uchida T, Inagaki F, Ishimori K. Investigation of the redox-dependent modulation of structure and dynamics in human cytochrome c. Biochem Biophys Res Commun. 2016;469(4):978-984. PubMed, CrossRef
  3. Hsu CP, Hammarström L, Newton MD. 65 years of electron transfer. J Chem Phys. 2022;157(2):020401. PubMed, CrossRef
  4. Goldman AD, Weber JM, LaRowe DE, Barge LM. Electron transport chains as a window into the earliest stages of evolution. Proc Natl Acad Sci USA. 2023;120(34):e2210924120. PubMed, PubMed, CrossRef
  5. Ahmad M, Wolberg A, Kahwaji CI. Biochemistry, Electron Transport Chain. In: StatPearls. Treasure Island (FL): StatPearls Publishing; 2023. PubMed
  6. Morse PT, Arroum T, Wan J, Pham L, Vaishnav A, Bell J, Pavelich L, Malek MH, Sanderson TH, Edwards BFP, Hüttemann M. Phosphorylations and acetylations of cytochrome c control mitochondrial respiration, mitochondrial membrane potential, energy, ROS, and apoptosis. Cells. 2024;13(6):493.
    PubMed, PubMed, CrossRef
  7. Zhou Z, Arroum T, Luo X, Kang R, Lee YJ, Tang D, Hüttemann M, Song X. Diverse functions of cytochrome c in cell death and disease. Cell Death Differ. 2024 Apr;31(4):387-404.
    PubMed, PubMed, CrossRef
  8. Santucci R, Sinibaldi F, Cozza P, Polticelli F, Fiorucci L. Cytochrome c: An extreme multifunctional protein with a key role in cell fate. Int J Biol Macromol. 2019;136:1237-1246. PubMed, CrossRef
  9. Sofi S, Mehraj U, Jan N, Almilaibary A, Ahmad I, Ahmad F, Ahmad Mir M. Clinicopathological significance and expression pattern of Bcl2 in breast cancer: a comprehensive in silico and in vitro study. Saudi J Biol Sci. 2024;31(2):103916. PubMed, PubMed, CrossRef
  10. Hüttemann M, Doan JW, Goustin AS, Sinkler C, Mahapatra G, Shay J, Liu J, Elbaz H, Aras S, Grossman LI, Ding Y, Zielske SP, Malek MH, Sanderson TH, Lee I. Regulation of CytC in respiration, apoptosis, neurodegeneration and cancer: The good, the bad and the ugly. Hauppauge, NY, USA: Nova Science Publishers, Inc.; 2014:1-38.
  11. Li F, Srinivasan A, Wang Y, Armstrong RC, Tomaselli KJ, Fritz LC. Cell-specific induction of apoptosis by microinjection of cytochrome c. Bcl-xL has activity independent of cytochrome c release. J Biol Chem. 1997;272(48):30299-30305. PubMed, CrossRef
  12. Zhivotovsky B, Orrenius S, Brustugun OT, Døskeland SO. Injected cytochrome c induces apoptosis. Nature. 1998;391(6666):449-450. PubMed, CrossRef
  13. Delinois LJ, De León-Vélez O, Vázquez-Medina A, Vélez-Cabrera A, Marrero-Sánchez A, Nieves-Escobar C, Alfonso-Cano D, Caraballo-Rodríguez D, Rodriguez-Ortiz J, Acosta-Mercado J, Benjamín-Rivera JA, González-González K, Fernández-Adorno K, Santiago-Pagán L, Delgado-Vergara R, Torres-Ávila X, Maser-Figueroa A, Grajales-Avilés G, Miranda Méndez GI, Santiago-Pagán J, Nieves-Santiago M, Álvarez-Carrillo V, Griebenow K, Tinoco AD. Cytochrome c: using biological insight toward engineering an optimized anticancer biodrug. Inorganics (Basel). 2021;9(11):83. PubMed, PubMed, CrossRef
  14. Alshehri B. Cytochrome c and cancer cell metabolism: A new perspective. Saudi Pharm J. 2024;32(12):102194. PubMed, PubMed, CrossRef
  15. Tomášková N, Varhač R, Lysáková V, Musatov A, Sedlák E. Peroxidase activity of cytochrome c in its compact state depends on dynamics of the heme region. Biochim Biophys Acta Proteins Proteom. 2018;1866(11):1073-1083. PubMed, CrossRef
  16. Shin KS, Lee YJ. Purification and characterization of a new member of the laccase family from the white-rot basidiomycete Coriolus hirsutus. Arch Biochem Biophys. 2000;384(1):109-115. PubMed, CrossRef
  17. Bruice TC, Benkovic SJ. Chemical basis for enzyme catalysis. Biochemistry. 2000;39(21):6267-6274. PubMed, CrossRef
  18. Vlasova II. Peroxidase activity of human hemoproteins: keeping the fire under control. Molecules. 2018;23(10):2561. PubMed, PubMed, CrossRef
  19. Chertkova RV, Brazhe NA, Bryantseva TV, Nekrasov AN, Dolgikh DA, Yusipovich AI, Sosnovtseva O, Maksimov GV, Rubin AB, Kirpichnikov MP. New insight into the mechanism of mitochondrial cytochrome c function. PLoS One. 2017;12(5):e0178280. PubMed, PubMed, CrossRef
  20. Pérez-Mejías G, Olloqui-Sariego JL, Guerra-Castellano A, Díaz-Quintana A, Calvente JJ, Andreu R, De la Rosa MA, Díaz-Moreno I. Physical contact between cytochrome c1 and cytochrome c increases the driving force for electron transfer. Biochim Biophys Acta Bioenerg. 2020;1861(12):148277. PubMed, CrossRef
  21. Berndtsson J, Kohler A, Rathore S, Marin-Buera L, Dawitz H, Diessl J, Kohler V, Barrientos A, Büttner S, Fontanesi F, Ott M. Respiratory supercomplexes enhance electron transport by decreasing cytochrome c diffusion distance. EMBO Rep. 2020;21(12):e51015. PubMed, PubMed, CrossRef
  22. Ferri T, Poscia A, Ascoli F, Santucci R. Direct electrochemical evidence for an equilibrium intermediate in the guanidine-induced unfolding of cytochrome c. Biochim Biophys Acta. 1996;1298(1):102-108. PubMed, CrossRef
  23. González-Arzola K, Díaz-Quintana A, Bernardo-García N, Martínez-Fábregas J, Rivero-Rodríguez F, Casado-Combreras MÁ, Elena-Real CA, Velázquez-Cruz A, Gil-Caballero S, Velázquez-Campoy A, Szulc E, Gavilán MP, Ayala I, Arranz R, Ríos RM, Salvatella X, Valpuesta JM, Hermoso JA, De la Rosa MA, Díaz-Moreno I. Nucleus-translocated mitochondrial cytochrome c liberates nucleophosmin-sequestered ARF tumor suppressor by changing nucleolar liquid-liquid phase separation. Nat Struct Mol Biol. 2022;29(10):1024-1036. PubMed, CrossRef
  24. Zhou C, Zhang J, Ying W. Mitochondrial electron transport chain inhibition suppresses LPS-induced inflammatory responses via TREM1/STAT3 pathway in BV2 microglia. bioRxiv [preprint]. 2019. CrossRef
  25. Wen Q, Zhang X, Cai J, Yang PH. A novel strategy for real-time and in situ detection of cytochrome c and caspase-9 in Hela cells during apoptosis. Analyst. 2014;139(10):2499-2506. PubMed, CrossRef
  26. Barczyk K, Kreuter M, Pryjma J, Booy EP, Maddika S, Ghavami S, Berdel WE, Roth J, Los M. Serum cytochrome c indicates in vivo apoptosis and can serve as a prognostic marker during cancer therapy. Int J Cancer. 2005;116(2):167-173. PubMed, CrossRef
  27. Pessoa J. Cytochrome c in cancer therapy and prognosis. Biosci Rep. 2022;42(12):BSR20222171. PubMed, PubMed, CrossRef
  28. Bose D, Aggarwal S, Das D, Narayana C, Chakrabarti A. Erythroid spectrin binding modulates peroxidase and catalase activity of heme proteins. IUBMB Life. 2022;74(5):474-487. PubMed, CrossRef
  29. Wu LB, Du KJ, Nie CM, Gao SQ, Wen GB, Tan X, Lin YW. Peroxidase activity enhancement of myoglobin by two cooperative distal histidines and a channel to the heme pocket. J Mol Catal B Enzym. 2016;134(Pt B):367-371. 30. De Lauzon S, Quilez R, Lion L, Desfosses B, Desfosses B, Lee I, Sari MA, Benkovic SJ, Mansuy D, Mahy JP. Active site topology of artificial peroxidase-like hemoproteins based on antibodies constructed from a specifically designed ortho-carboxy-substituted tetraarylporphyrin. Eur J Biochem. 1998;257(1):121-130. PubMed, CrossRef
  30. de Oliveira FK, Santos LO, Buffon JG. Mechanism of action, sources, and application of peroxidases. Food Res Int. 2021;143:110266. PubMed, CrossRef
  31. Chen K, Arnold FH. Engineering new catalytic activities in enzymes. Nat Catal. 2020;3:203-213. CrossRef
  32. Mathews CK, van Holde KE, Ahern KG. Biochemistry. 3rd ed. Upper Saddle River, NJ: Prentice Hall; 1999. 1200 p.
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