Cardoso 2022 Q10 Hamburg

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Cardoso LHD, Donnelly C, Komlodi T, Gnaiger E (2022) Coenzyme Q redox state and respiration in permeabilized HEK 293T cells: coupling and pathway control. Q10 Hamburg.

Link: 10th Conference of the International Coenzyme Q10 Association

Cardoso Luiza HD, Donnelly Chris, Komlodi Timea, Gnaiger Erich (2022)

Event: 10th Conference of the International Coenzyme Q10 Association 2022 Hamburg DE

The Q-junction plays a central role in mitochondrial electron transfer (ET). Multiple pathways converge at the Q-junction and reduce coenzyme Q (Q). Respiratory complexes oxidize different substrates ― such as NADH and succinate ― and transfer electrons to Q followed by oxidation via Complex III and electron transfer to Complex IV and O2. Respiratory rates are modulated by pathway control and coupling control. In oxidative phosphorylation (OXPHOS), ET is coupled to ATP synthesis by the proton circuit through the mitochondrial inner membrane. ET capacity is measured in the noncoupled respiratory state after application of uncouplers, whereas LEAK respiration is assessed in the absence of ADP or after inhibition of ATP synthase activity.

We characterised the influence of pathway and coupling control on the Q redox state using the Oroboros NextGen-O2k with the electrochemical Q-Module, monitoring simultaneously respiration and the reduced Q-fraction in permeabilized HEK 293T cells [1]. The plasma membrane was permeabilized with digitonin. To study pathway control, multiple combinations of substrates (pyruvate, malate, succinate) and inhibitors (rotenone, malonate) were used to supply electrons via the NADH-pathway (N), succinate-pathway (S), or in combination (NS) in the ET state. Alternatively, coupling control was analysed in the S-pathway (succinate and rotenone), varying from LEAK respiration to OXPHOS-, and ET-capacity.

In pathway control, the reduced Q-fraction increased proportionally with increasing respiration, from the N- and S-pathway up to the combined NS-pathway. This reflects the variable push of electrons into the Q-junction exerted by the different pathways upstream of the Q-junction.

In coupling control, the reduced Q-fraction decreased with an increase of respiration. This pattern is opposite to pathway control and can be explained as a pull effect by coupling control downstream of the Q-junction: In the LEAK state, Q is highly reduced by electron transfer into the Q-junction with a minimum pull at low respiration. After stimulation of respiration by ADP, the pull is increased in the OXPHOS state and Q becomes more oxidized. This is more pronounced in the ET state, with Q even more oxidized, when the phosphorylation system does not limit respiration and electron flow exerts a maximum pull effect downstream of the Q-junction. The push and pull effects of pathway and coupling control on the Q-redox state obtained in permeabilized cells are in line with our previous studies on isolated mitochondria. Combined measurements of respiration and the Q-redox state help to understand the complexities of mitochondrial electron transfer at the Q-junction and respiratory control.


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Affiliations and support

Luiza H.D. Cardoso(1)*, Chris Donnelly(1,2), Timea Komlódi(1), Erich Gnaiger(1)
  1. Oroboros Instruments, Innsbruck, Austria; *presenting author – [email protected]
  2. Institute of Sport Sciences, University of Lausanne, Switzerland
This work was part of the NextGen-O2k project, with funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement nº 859770. Chris Donnelly was supported by the Swiss National Science Foundation under grant agreement nº 194964.

References

  1. Komlódi T, Cardoso LHD, Doerrier C, Moore AL, Rich PR, Gnaiger E (2021) Coupling and pathway control of coenzyme Q redox state and respiration in isolated mitochondria. Bioenerg Commun 2021.3. https://doi.org/10.26124/bec:2021-0003