Background: Mitochondrial respiratory efficiency is crucial for sustaining cellular energy production and oxidative balance. It depends not only on the assembly of electron transport chain complexes into supercomplexes, but also on the kinetic competence of their individual components. However, the functional significance of supercomplex assembly remains debated, particularly with regard to whether it ensures effective substrate channeling. Methods: In this study, we investigated the contribution of cytochrome c oxidase (Complex IV, CIV) to NADH-dependent respiratory flux in conditions where CIV is predominantly present in its free form rather than incorporated into the N-respirosome (CI+CIII2+CIV) or the Q-respirosome (CIII2+CIV). Metabolic flux analysis based on cyanide titration was used to assess the control exerted by CIV over the respiratory pathway. Results: Inhibition of CIV caused an exponential decline in both NADH-O2 oxidoreductase and TMPD/ascorbate-O2 oxidoreductase activities. The calculated flux control coefficient identified CIV as the rate-limiting step of NADH-supported respiration. Proteomic analysis further showed that, although both N-respirosomes and Q-respirosomes were present, CIV was predominantly detected in its free, non-assembled form, with only a minor proportion incorporated into supramolecular assemblies. These findings are consistent with the absence of effective kinetic substrate channeling between Complex I, Complex III, and CIV. Conclusions: These findings indicate that the functional state of CIV, rather than SC abundance alone, is a critical determinant of respiratory control and oxidative balance in mitochondria.
- Open Access
- Article
Mitochondrial Complex IV in Free form Controls Respiratory Channeling Independently of Supercomplex Assembly
- Patrycja Anna Glogowski 1,
- Cristina Algieri 2,3,
- Antonia Cugliari 3,
- Maria Cotugno 4,
- Donatella Pietrangelo 5,
- Fabiana Trombetti 3,
- Micaela Fabbri 3,
- Speranza Rubattu 4,5,
- Salvatore Nesci 3,*
Author Information
Received: 27 Mar 2026 | Revised: 19 Apr 2026 | Accepted: 06 May 2026 | Published: 12 May 2026
Abstract
Keywords
mitochondrial respiratory supercomplexes | cytochrome c oxidase | substrate channeling | metabolic flux control analysis
References
- 1.
Vercellino, I.; Sazanov, L.A. The assembly, regulation and function of the mitochondrial respiratory chain. Nat. Rev. Mol. Cell Biol. 2022, 23, 141–161. https://doi.org/10.1038/s41580-021-00415-0.
- 2.
Suomalainen, A.; Nunnari, J. Mitochondria at the crossroads of health and disease. Cell 2024, 187, 2601–2627. https://doi.org/10.1016/j.cell.2024.04.037.
- 3.
Gu, J.; Wu, M.; Guo, R.; et al. The architecture of the mammalian respirasome. Nature 2016, 537, 639–643. https://doi.org/10.1038/nature19359.
- 4.
Wu, M.; Gu, J.; Guo, R.; et al. Structure of Mammalian Respiratory Supercomplex I1III2IV1. Cell 2016, 167, 1598–1609.e10. https://doi.org/10.1016/j.cell.2016.11.012.
- 5.
Vercellino, I.; Sazanov, L.A. Structure and assembly of the mammalian mitochondrial supercomplex CIII2CIV. Nature 2021, 598, 364–367. https://doi.org/10.1038/s41586-021-03927-z.
- 6.
Milenkovic, D.; Blaza, J.N.; Larsson, N.-G.; et al. The Enigma of the Respiratory Chain Supercomplex. Cell Metab. 2017, 25, 765–776. https://doi.org/10.1016/j.cmet.2017.03.009.
- 7.
Vercellino, I.; Sazanov, L.A. SCAF1 drives the compositional diversity of mammalian respirasomes. Nat. Struct. Mol. Biol. 2024, 31, 1061–1071. https://doi.org/10.1038/s41594-024-01255-0.
- 8.
García-Poyatos, C.; Cogliati, S.; Calvo, E.; et al. Scaf1 promotes respiratory supercomplexes and metabolic efficiency in zebrafish. EMBO Rep. 2020, 21, e50287. https://doi.org/10.15252/embr.202050287.
- 9.
Moe, A.; Di Trani, J.; Rubinstein, J.L.; et al. Cryo-EM structure and kinetics reveal electron transfer by 2D diffusion of cytochrome c in the yeast III-IV respiratory supercomplex. Proc. Natl. Acad. Sci. USA 2021, 118, e2021157118. https://doi.org/10.1073/pnas.2021157118.
- 10.
Nesci, S.; Algieri, C.; Trombetti, F.; et al. Two separate pathways underlie NADH and succinate oxidation in swine heart mitochondria: Kinetic evidence on the mobile electron carriers. Biochim. Biophys. Acta Bioenerg. 2023, 1864, 148977. https://doi.org/10.1016/j.bbabio.2023.148977.
- 11.
Rubattu, S.; Di Castro, S.; Schulz, H.; et al. Ndufc2 Gene Inhibition Is Associated With Mitochondrial Dysfunction and Increased Stroke Susceptibility in an Animal Model of Complex Human Disease. J. Am. Heart Assoc. 2016, 5, e002701. https://doi.org/10.1161/JAHA.115.002701.
- 12.
Algieri, C.; Cugliari, A.; Granata, S.; et al. Dextromethorphan Is a Novel Pharmacological Inhibitor of F1FO-ATPase That Targets the Membrane-Embedded Domain Impairing ATP Synthesis and Hydrolysis. Biofactors 2026, 52, e70076. https://doi.org/10.1002/biof.70076.
- 13.
Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248–254. https://doi.org/10.1016/0003-2697(76)90527-3.
- 14.
Cugliari, A.; Algieri, C.; Glogowski, P.A.; et al. The Function and Supramolecular Assembly of the Mitochondrial Respiratory Complexes Underlie Hypertension-Related Stroke Susceptibility in a Model of Complex Human Disease. Biofactors 2025, 51, e70055. https://doi.org/10.1002/biof.70055.
- 15.
Bianchi, C.; Genova, M.L.; Parenti Castelli, G.; et al. The mitochondrial respiratory chain is partially organized in a supercomplex assembly: Kinetic evidence using flux control analysis. J. Biol. Chem. 2004, 279, 36562–36569. https://doi.org/10.1074/jbc.M405135200.
- 16.
Genova, M.L.; Lenaz, G. Functional role of mitochondrial respiratory supercomplexes. Biochim. Biophys. Acta 2014, 1837, 427–443. https://doi.org/10.1016/j.bbabio.2013.11.002.
- 17.
Brzezinski, P.; Moe, A.; Ädelroth, P. Structure and Mechanism of Respiratory III-IV Supercomplexes in Bioenergetic Membranes. Chem. Rev. 2021, 121, 9644–9673. https://doi.org/10.1021/acs.chemrev.1c00140.
- 18.
Cogliati, S.; Frezza, C.; Soriano, M.E.; et al. Mitochondrial cristae shape determines respiratory chain supercomplexes assembly and respiratory efficiency. Cell 2013, 155, 160–171. https://doi.org/10.1016/j.cell.2013.08.032.
- 19.
Hernansanz-Agustín, P.; Enríquez, J.A. Generation of Reactive Oxygen Species by Mitochondria. Antioxidants 2021, 10, 415. https://doi.org/10.3390/antiox10030415.
- 20.
Genova, M.L.; Lenaz, G. The Interplay Between Respiratory Supercomplexes and ROS in Aging. Antioxid. Redox Signal. 2015, 23, 208–238. https://doi.org/10.1089/ars.2014.6214.
- 21.
Murphy, M.P. How mitochondria produce reactive oxygen species. Biochem. J. 2009, 417, 1–13. https://doi.org/10.1042/BJ20081386.
- 22.
Maranzana, E.; Barbero, G.; Falasca, A.I.; et al. Mitochondrial respiratory supercomplex association limits production of reactive oxygen species from complex I. Antioxid. Redox Signal. 2013, 19, 1469–1480. https://doi.org/10.1089/ars.2012.4845.
- 23.
Lenaz, G.; Nesci, S.; Genova, M.L. Understanding differential aspects of microdiffusion (channeling) in the Coenzyme Q and Cytochrome c regions of the mitochondrial respiratory system. Mitochondrion 2024, 74, 101822. https://doi.org/10.1016/j.mito.2023.11.005.
- 24.
Cogliati, S.; Cabrera-Alarcón, J.L.; Enríquez, J.A. Regulation and functional role of the electron transport chain supercomplexes. Biochem. Soc. Trans. 2021, 49, 2655–2668. https://doi.org/10.1042/BST20210460.
- 25.
Ramírez-Camacho, I.; Correa, F.; El Hafidi, M.; et al. Cardioprotective strategies preserve the stability of respiratory chain supercomplexes and reduce oxidative stress in reperfused ischemic hearts. Free Radic. Biol. Med. 2018, 129, 407–417. https://doi.org/10.1016/j.freeradbiomed.2018.09.047.
- 26.
Hinton, A.; Claypool, S.M.; Neikirk, K.; et al. Mitochondrial Structure and Function in Human Heart Failure. Circ. Res. 2024, 135, 372–396. https://doi.org/10.1161/CIRCRESAHA.124.323800.
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