Komlodi 2017 MiPschool Obergurgl
Event: MiPschool Obergurgl 2017
The Q-junction is a key point of convergent electron flow in the electron transfer-pathway (ET-pathway) primarily from NADH-linked substrates and mt-dehydrogenases via Complex I (CI), succinate (S) via CII (succinate dehydrogenase), F-type substrates and FA oxidation via electron-transferring flavoprotein Complex (CETF), glycerophosphate (Gp) via glycerophosphate dehydrogenase Complex (CGpDH), and to a minor extend from dihydroorotate (DHO) via dihydroorotate dehydrogenase (DHOD) and choline (CHO) via choline dehydrogenase (CHODH). Downstream of the Q-junction electrons are transferred to Complex III (CIII), cytochrome c oxidase and finally to oxygen. Deficiency in the Q-junction can impair ET-pathway and OXPHOS capacities, but the extent of the inhibition is controlled by the substrate contributions and the involved pathways towards the Q-junction. We hypothesize that in the absence of NADH-linked substrates, reversed electron transfer (RET) to CI can be driven not only from S, but from other branches into the Q-junction according to their pathway capacities. The aim of the present study is to determine the stimulation of H2O2 production according to the contribution of different substrates to the Q-junction and their electron supply capacity. Respiration and H2O2 production were determined simultaneously by O2k-Fluorimetry (Oroboros Instruments, Innsbruck, Austria) in the absence and presence of ADP (LEAK and OXPHOS states), and the absence and presence of the CI inhibitor rotenone, selectively blocking RET. Experiments were carried out on isolated mouse brain mitochondria respiring with Gp (20 mM), S (0.2, 10 and 50 mM), or with their combinations. The following protocol was used throughout the experiments: substrate, +/- ADP, +/- rotenone (Rot), antimycin A (Ama), myxothiazol (Myx) and malonate (Mna). H2O2 production and O2 consumption increased as a function of S concentration. The highest H2O2 and respiratory fluxes were observed with Gp and S50 or S10 added together in the absence of Rot. These findings support the notion that the combined administration of substrates has an additive effect both on ROS generation and respiration. It is generally accepted that RET supported by S or Gp is blocked by addition of Rot by preventing the transfer of electrons in CI between iron-sulfur cluster N-2 and ubiquinone. Therefore, Rot can be used for the determination of the site of H2O2 synthesis in the ET-pathway. In our experiments, Rot inhibited the S, the Gp and S&Gp-evoked H2O2 generation. The difference of H2O2 production (in the LEAK state) in the presence and absence of Rot yields information on RET to CI through the Q-junction. S-evoked H2O2 production in the LEAK state and OXPHOS capacity (ADP-saturated flux) were highly dependent on S concentration and S&Gp combination: Gp20 < S0.2 ~ S10 < GpS0.2 < GpS10 < S50 < GpS50. Thus, control by substrate concentrations and combinations control ET-pathway capacity via reduced electron supply to the Q-junction. In summary, the linear relationship between H2O2 production in the LEAK state and O2 consumption in the OXHOS state establishes the concept that electron pressure generated by the linear S- and Gp-pathways or the convergent SGp-pathway on the Q-junction drives RET into CI and thus regulates H2O2 flux in CI.
- Komlodi T(1), Gnaiger E(1,2)
- Oroboros Instruments, Innsbruck, Austria
- D. Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck
Labels: MiParea: Respiration
Stress:Oxidative stress;RONS Organism: Mouse Tissue;cell: Nervous system Preparation: Isolated mitochondria Enzyme: Complex I, Complex II;succinate dehydrogenase Regulation: Flux control, Inhibitor, Q-junction effect, Redox state, Substrate Coupling state: LEAK, OXPHOS Pathway: N, S, Gp, Other combinations, ROX HRR: Oxygraph-2k, O2k-Fluorometer Event: A1, Oral