Difference between revisions of "Komlodi 2017 MiPschool Obergurgl"

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{{Abstract
{{Abstract
|title=[[File:KomlodiT.JPG|left|90px|Timea Komlodi]] Electron pressure exerted by convergent succinate- and glycerophosphate pathways to the Q-junction regulate reversed electron transfer to Complex I and H<sub>2</sub>O<sub>2</sub> production.
|title=[[File:KomlodiT.JPG|left|90px|Timea Komlodi]] Electron pressure exerted by convergent succinate- and glycerophosphate-pathways to the Q-junction regulate reversed electron transfer to Complex I and H<sub>2</sub>O<sub>2</sub> production.
|info=[[MITOEAGLE]]
|info=[[MITOEAGLE]]
|authors=Komlodi T, Gnaiger E
|authors=Komlodi T, Gnaiger E
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|event=MiPschool Obergurgl 2017
|event=MiPschool Obergurgl 2017
|abstract=[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MITOEAGLE]]
|abstract=[[Image:MITOEAGLE-logo.jpg|left|100px|link=http://www.mitoglobal.org/index.php/MITOEAGLE|COST Action MITOEAGLE]]
The Q-junction is a key point of convergent electron flow in the electron transfer system (ETS) 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 ETS 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 H<sub>2</sub>O<sub>2</sub> 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 high-resolution respirometry and O2k-Fluorimetry (OROBOROS INSTRUMENTS, Innsbruck) 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 (Ama), myxothiazol (Myx) and malonate (Mna). H<sub>2</sub>O<sub>2</sub> production and O<sub>2</sub> consumption increased as a function of S concentration. The highest rate of H<sub>2</sub>O<sub>2</sub> production and respiration was 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 ETS. In our experiments, Rot inhibited the S and S&Gp-evoked H<sub>2</sub>O<sub>2</sub> generation, but interestingly, Rot did not have any effect on Gp supported H<sub>2</sub>O<sub>2</sub> production in mouse brain mitochondria. The difference of H<sub>2</sub>O<sub>2</sub> production (in the LEAK state) in the presence and absence of Rot yields information on RET to CI through the Q-junction. S-evoked H<sub>2</sub>O<sub>2</sub> 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 < S50 and GpS0.2 < GpS10 ~ GpS50. Thus, control by substrate concentrations and combinations control ETS capacity via reduced electron supply to the Q-junction. In summary, the linear relationship between H<sub>2</sub>O<sub>2</sub> production in the LEAK state and O<sub>2</sub> 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 H<sub>2</sub>O<sub>2</sub> flux in CI.
The Q-junction is a key point of convergent electron flow in the electron transfer system (ETS) 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 ETS 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 H<sub>2</sub>O<sub>2</sub> production according to the contribution of different substrates to the Q-junction and their electron supply capacity. Respiration and H<sub>2</sub>O<sub>2</sub> 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). H<sub>2</sub>O<sub>2</sub> production and O<sub>2</sub> consumption increased as a function of S concentration. The highest H<sub>2</sub>O<sub>2</sub> 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 H<sub>2</sub>O<sub>2</sub> synthesis in the ETS. In our experiments, Rot inhibited the S and S&Gp-evoked H<sub>2</sub>O<sub>2</sub> generation, but interestingly, Rot did not have any effect on Gp supported H<sub>2</sub>O<sub>2</sub> production in mouse brain mitochondria. The difference of H<sub>2</sub>O<sub>2</sub> production (in the LEAK state) in the presence and absence of Rot yields information on RET to CI through the Q-junction. S-evoked H<sub>2</sub>O<sub>2</sub> 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 < S50 and GpS0.2 < GpS10 ~ GpS50. Thus, control by substrate concentrations and combinations control ETS capacity via reduced electron supply to the Q-junction. In summary, the linear relationship between H<sub>2</sub>O<sub>2</sub> production in the LEAK state and O<sub>2</sub> 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 H<sub>2</sub>O<sub>2</sub> flux in CI.
|editor=[[Komlodi T]], [[Kandolf G]]
|editor=[[Komlodi T]], [[Kandolf G]]
|mipnetlab=AT Innsbruck Gnaiger E, AT Innsbruck OROBOROS
|mipnetlab=AT Innsbruck Gnaiger E, AT Innsbruck Oroboros
}}
}}
{{Labeling
{{Labeling
|area=Respiration
|area=Respiration
|diseases=Other
|injuries=Oxidative stress;RONS
|injuries=Oxidative stress;RONS
|organism=Mouse
|organism=Mouse
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:::: Komlodi T(1), Gnaiger E(1,2)
:::: Komlodi T(1), Gnaiger E(1,2)


::::#OROBOROS INTSRUMENTS, Innsbruck, Austria  
::::# Oroboros Instruments, Innsbruck, Austria  
::::#D. Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck
::::# D. Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck

Revision as of 08:18, 13 July 2017

Timea Komlodi
Electron pressure exerted by convergent succinate- and glycerophosphate-pathways to the Q-junction regulate reversed electron transfer to Complex I and H2O2 production.

Link: MITOEAGLE

Komlodi T, Gnaiger E (2017)

Event: MiPschool Obergurgl 2017

COST Action MITOEAGLE

The Q-junction is a key point of convergent electron flow in the electron transfer system (ETS) 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 ETS 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 ETS. In our experiments, Rot inhibited the S and S&Gp-evoked H2O2 generation, but interestingly, Rot did not have any effect on Gp supported H2O2 production in mouse brain mitochondria. 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 < S50 and GpS0.2 < GpS10 ~ GpS50. Thus, control by substrate concentrations and combinations control ETS 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.


Bioblast editor: Komlodi T, Kandolf G O2k-Network Lab: AT Innsbruck Gnaiger E, AT Innsbruck Oroboros


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 


Affiliations

Komlodi T(1), Gnaiger E(1,2)
  1. Oroboros Instruments, Innsbruck, Austria
  2. D. Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck
[email protected]