The cytochrome b p.278Y>C mutation causative of a multisystem disorder enhances superoxide production and alters supramolecular interactions of respiratory chain complexes

Abstract

Cytochrome b is the only mtDNA-encoded subunit of the mitochondrial complex III (CIII), the functional bottleneck of the respiratory chain. Previously, the human cytochrome b missense mutation m.15579A>G, which substitutes the Tyr 278 with Cys (p.278Y>C), was identified in a patient with severe exercise intolerance and multisystem manifestations. In this study, we characterized the biochemical properties of cybrids carrying this mutation and report that the homoplasmic p.278Y>C mutation caused a dramatic reduction in the CIII activity and in CIII-driven mitochondrial ATP synthesis. However, the CI, CI + CIII and CII + CIII activities and the rate of ATP synthesis driven by the CI or CII substrate were only partially reduced or unaffected. Consistent with these findings, mutated cybrids maintained the mitochondrial membrane potential in the presence of oligomycin, indicating that it originated from the respiratory electron transport chain. The p.278Y>C mutation enhanced superoxide production, as indicated by direct measurements in mitochondria and by the imbalance of glutathione homeostasis in intact cybrids. Remarkably, although the assembly of CI or CIII was not affected, the examination of respiratory supercomplexes revealed that the amounts of CIII dimer and III2IV1 were reduced, whereas those of I1III2IVn slightly increased. We therefore suggest that the deleterious effects of p.278Y>C mutation on cytochrome b are palliated when CIII is assembled into the supercomplexes I1III2IVn, in contrast to when it is found alone. These findings underline the importance of supramolecular interactions between complexes for maintaining a basal respiratory chain activity and shed light to the molecular basis of disease manifestations associated with this mutation.

Complex II phosphorylation is triggered by unbalanced redox homeostasis in cells lacking complex III

“…In this study cybrids bearing the m.15649–15,666 microdeletion (I300-P305) in heteroplasmy or homoplasmy (80% or 100% mutation load, respectively) or bearing isogenic wild type (WT) mtDNA [12] and cybrids with the homoplasmic missense mutation m.15579A > G (p.278Y > C) and isogenic WT mtDNA [13] were used. Cells were grown in high glucose (25 mM) Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FBS) (Gibco, Invitrogen, Italy), 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 1 mM sodium-pyruvate, in an incubator with a humidified atmosphere of 5% CO 2 at 37 °C.…”

“…Enzymatic activities of redox proteins were measured at 37 °C by using a dual-wavelength spectrophotometer (V550 Jasco Europe, Italy). The CI (NADH:DB:DCIP oxidoreductase) and the CIII (antimycin A-sensitive ubiquinol: cytochrome c reductase) activities were measured as described in [13]. The CII activity (succinate:DB:DCIP oxidoreductase) was determined in the presence of 60 μM DCIP (λ: 600 nm; ε DCIP : 19.1 mM −1 cm −1 ), 70 μM decyl benzoquinone (DB).…”

“…Samples were loaded on a 4–12% gradient native gel. After electrophoresis, gels were transferred on nitrocellulose membrane and Western blotting was carried out [13] as described above.…”

“…We inquired whether the stimulation of CII activity in vitro was strictly dependent on CIII dysfunction, and if so, whether it required a severe loss of CIII activity only or also the disruption of CIII assembly. For comparison, we studied cybrids bearing a MTCYB missense mutation (m.15579A > G mutation) in homoplasmic state, causing the amino acid substitution p.278Y > C, previously shown to exert strong inhibition of CIII activity without drastic perturbation of its assembly [13]. In addition, we evaluated the CII activation in vivo by measuring the cellular levels of succinate and fumarate, respectively substrate and product of CII, and determined its effect on cell viability under different metabolic stress conditions.…”

Cystic Fibrosis-Related Oxidative Stress and Intestinal Lipid Disorders

“…Indeed, inhibition of CII leads to diminished O 2 -production during the reverse flow of electrons (42). The central CIII of the respiratory chain also seems to contribute to the production of O 2 -through its catalytic active site and during the oxidation of ubiquinone passing through the auto-oxidation stage of the semiubiquinone controlled by a delicate balance among membrane potential, reduced availability, and oxidized forms of ubiquinone and the redox status of CIII (55,65). However, in CF, a more complete investigation is required to obtain a better understanding of mitochondrion contribution, ROS production, and the etiopathology.…”