Mitochondrial Complex IV in Free form Controls Respiratory Channeling Independently of Supercomplex Assembly

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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.

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