Zhdanov, A.;Brazhe, N.;Nikelshparg, E.;Power, L.;Lewis, P.;Silva, P.;Wouw, M.;O\'Connor, P.;Cryan, J.;Sosnovtseva, O.;Andreev, D.;Yordanova, M.;Baranov, P.;Dmitriev, R.;Papkovsky, D.

We show that chronic impairment of mitochondrial respiration is associated with marked accumulation of cytochrome c (Cyt c ) protein. Using SCO2-deficient HCT116 cells lacking functional cytochrome c oxidase and wild-type cells exposed to sustained hypoxia, we found that substantial mitochondrial Cyt c accumulation parallels reduced electron flux through Cyt c . SCO2-deficient cells exhibited equally elevated Cyt c levels under normoxia (19% O 2 ) and hypoxia (0.1-3% O 2 ). Wild-type cells under sustained hypoxia accumulated Cyt c , reaching levels comparable to those in SCO2-deficient cells. This effect was reversible upon reoxygenation. Increased Cyt c protein levels were also observed in other cell models, including primary cortical neurons cultured under chronic hypoxia and in cerebral cortex tissue from hypoxia-exposed mice. Cyt c accumulation occurred independently of CYCS transcription, mRNA translation, HIF activation, ROS production and changes in mitochondrial network. Pharmacological inhibition of complex III was likewise accompanied by increased Cyt c levels, whereas mitochondrial uncoupling had no effect, suggesting that impaired electron transfer rather than membrane depolarisation per se underlies this association. Raman spectroscopy revealed enrichment of reduced Cyt c and an increased Cyt c -to-cytochrome b ratio in respiration-deficient cells. Further supporting a stabilisation-based mechanism, the fraction of membrane-unbound ferro-Cyt c was decreased in SCO2-deficient cells, consistent with moderate cardiolipin enrichment, which is known to enhance retention of Cyt c at the inner mitochondrial membrane. Despite elevated mitochondrial Cyt c content, SCO2-deficient cells were less susceptible to apoptosis induced by intermittent hypoxia or dichloroacetate. Together, these findings indicate that reduced electron flux through complex IV is associated with Cyt c accumulation through increased protein stability and membrane retention without enhancing apoptotic sensitivity.