Komlodi 2018 EBEC2018
|Electron supply to the Q-junction: assessment of mitochondrial respiration, H2O2 flux and the redox state of the Q-pool.|
The coenzyme Q (Q)-junction is a key point of convergent electron flow in the electron transfer system from mt-dehydrogenases via Complex I (CI), from succinate (S) via Complex II, glycerol-3-phosphate (Gp) via mt-glycerophosphate dehydrogenase to Complex III. Deficiency in the Q-junction can impair electron transfer and OXPHOS capacities, while the extent of the inhibition is determined by the nature of the substrates and electron transfer pathways, which results in an altered redox state of the Q-pool . We aimed at investigating the relationship between the Q redox state, H2O2 and O2 fluxes with various fuel substrates.
Mitochondrial respiration and H2O2 production were determined simultaneously by high-resolution respirometry (HRR; Oroboros Instruments, Innsbruck, Austria), whereas the redox state of Q (Qreduced/Qtotal reduced= Qr/Qt) was detected using a three-electrode system [2,3] inserted into the Oroboros Q2k. Experiments were carried out on mitochondria isolated from mouse brain respiring with Gp (20 mM), S (0.2, 10 or 50 mM), or with their combinations, either in the absence (LEAK) or in the presence of saturating ADP (OXPHOS).
We found a linear relationship between Qr/Qt and O2 flux in OXPHOS, between H2O2 flux in LEAK and O2 flux in OXPHOS and all these parameters increased as a function of S concentration both in LEAK and OXPHOS. The highest H2O2 flux was measured using Gp&S50 and Gp&S10 in LEAK, while respiration was highest with the same substrates in OXPHOS. In contrast, Q was most reduced in LEAK with S50. Taken together, combining various substrates had an additive effect both on ROS generation and respiration, but not on Qr/Qt.
In summary, S-evoked H2O2 flux, Qr/Qt and respiration were dependent on S concentration and S&Gp combination: Gp < S0.2 < S10 < S50 and GpS0.2 < GpS10 ~ GpS50. These data suggest that the electron pressure generated by S- and Gp or S&Gp on the Q-junction controls respiration and regulates H2O2 flux by reversed electron transfer through the Q pool to CI.
Affiliations and support
- Komlodi T(1), Hunger M(1), Moore AL(2), Gnaiger E(1,3)
- Oroboros Instruments, Innsbruck, Austria
- Biochemistry Medicine School Life Sciences, Univ Sussex, Brighton, UK
- Daniel Swarovski Research Lab, Mitochondrial Physiology, Dept Visceral, Transplant Thoracic Surgery Medical Univ Innsbruck, Austria. - [email protected]
- Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle: new perspectives of mitochondrial physiology. Int J Biochem Cell Biol 41:1837-45
- Rich PR (1988) Patent of Q-electrode. European Patent no.85900699.1
- Moore AL, Dry IB, Wiskich JT (1988) Measurement of the redox state of the ubiquinone pool in plant mitochondria. FEBS 235:76-80.
Labels: MiParea: Respiration
Stress:Oxidative stress;RONS Organism: Mouse Tissue;cell: Nervous system Preparation: Isolated mitochondria
Regulation: Flux control, Substrate, Q-junction effect Coupling state: LEAK, OXPHOS Pathway: S, Gp HRR: Oxygraph-2k, O2k-Fluorometer Event: Poster MitoEAGLE, AmR