Mitochondrial cytochrome c accumulation accompanies reduced electron flux through complex IV without enhancing cell sensitivity to apoptosis

Abstract

We show that chronic impairment of mitochondrial respiration is associated with marked accumulation of cytochrome c (Cytc) protein. Using SCO2-deficient HCT116 cells lacking functional cytochrome c oxidase and wild-type cells exposed to sustained hypoxia, we found that substantial mitochondrial Cytc accumulation parallels reduced electron flux through Cytc. SCO2-deficient cells exhibited equally elevated Cytc levels under normoxia (19% O2) and hypoxia (0.1-3% O2). Wild-type cells under sustained hypoxia accumulated Cytc, reaching levels comparable to those in SCO2-deficient cells. This effect was reversible upon reoxygenation. Increased Cytc 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. Cytc 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 Cytc 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 Cytc and an increased Cytc-to-cytochrome b ratio in respiration-deficient cells. Further supporting a stabilisation-based mechanism, the fraction of membrane-unbound ferro-Cytc was decreased in SCO2-deficient cells, consistent with moderate cardiolipin enrichment, which is known to enhance retention of Cytc at the inner mitochondrial membrane. Despite elevated mitochondrial Cytc 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 Cytc accumulation through increased protein stability and membrane retention without enhancing apoptotic sensitivity.