Contents

📒 Robb 2018

Control of mitochondrial superoxide production by reverse electron transport at complex I1

Sciwheel.

Introduction of RET

  • The production of superxoide at complex I can be driven by reverse electron transport (RET) by a highly reduced coenzyme Q (CoQ) pool and a large protonmotive force ($\Delta p$). https://user-images.githubusercontent.com/40054455/96907147-6d53ed00-14cd-11eb-87d2-2add0c146edd.png

Methods

  • Isolated rat heart mitochondria.
  • Measure H2O2 generation as a function of the membrane potential ($\Delta\Psi$) and of the reduction state of the CoQ and NADH pools. Hydrogen peroxide efflux from mitochondria is proportional to superxoide production.
  • Altering $\Delta\Psi$ with the uncoupler FCCP and also oxidizing the CoQ pool by ectopic expression within heart mitochondria of the alternative oxidase (AOX) from Ciona intestinalis. O2 concentrations are also taken into account
  • $\Delta p$ : TPMP ($\Delta\Psi$ indicator) and $\Delta pH$ as constant
  • NADH: NAD(P)H fluorescence (NADPH pool is much smaller than NADH’s)

Results

Dependence on mitochondrial membrane potential

https://user-images.githubusercontent.com/40054455/96906847-f9b1e000-14cc-11eb-920a-045259e0b7d2.png

  • substrate: 10 mM potassium succinate
  • rotenone ($5 \mu M$) : Q reductase site inhibitior
  • nigericin($1 \mu M$) :K+ / H+ ionophore
  • O2 = $200 \mu M$ (air saturated)
  • Complex I inhibitor rotenone decreased H2O2 production without affecting $\Delta\Psi$ or the Q pool, but oxidation of the NAD(P)H pool => RET
  • Add uncoupler FCCP (decrease $\Delta\Psi$) => H2O2 production decreased
  • Use nigericin (K+ / H+ exchanger) to make matrix pH the same as the environment (7.4) without changing $\Delta p$ (compensatory increase of $\Delta\Psi$)

Dependence on Q redox state

https://user-images.githubusercontent.com/40054455/96906798-eb63c400-14cc-11eb-89fa-82eb4d062a82.png

  • Alternative oxidase (AOX) bypass complex III and IV, oxidizes QH2
    • Not sensitive to cyanide inhibition, but sensitive to N-propyl gallate
  • Oxidation of Q pool by AOX reduces superoxide production

Dependence on oxygen concentration

https://user-images.githubusercontent.com/40054455/96906813-f1f23b80-14cc-11eb-8d60-25860a8bcce6.png

  • A: no direct oxidation of indicator by molecular oxygen. Only levels of hydrogen peroxide could affect the fluorescence
  • Production of superoxide by RET at complex I is proportional (linear) to oxygen concentration
    • FCCP abolished it
    • Different from FET-ROS by rotenone + G/M or antimycin, with a pleatau

Effects of drugs to reduce RET ROS formation

https://user-images.githubusercontent.com/40054455/96906902-0d5d4680-14cd-11eb-8c48-f23687a19064.png

  • MitoQ: a mitochondria-targeted antioxidant based on ubiquinone
  • decylTPP: control compound to correct for nonspecific effects of MitoQ
  • SS31: a peptide
  • CN-POBS: an inhibitor of mitochondrial ROS production by RET at complex I
  • metformin and phenformin (antidiabetics): complex I inhibitor
  • effects of these compounds on RET at complex I may be indirect
    • Decreased $\Delta\Psi$
    • Oxidized NADH and CoQ pool

Thermodynamic force

https://user-images.githubusercontent.com/40054455/96906919-12ba9100-14cd-11eb-8774-c888764bfa6a.png Driving force for one electron transfer from NADH to Q: $$ \Delta E_{h}=E_{h}\left(\frac{\mathrm{NAD}^{+}}{\mathrm{NADH}}\right)-E_{h}\left(\frac{\mathrm{CoQ}}{\mathrm{CoQH}_{2}}\right) $$ Driving force for the full reaction: (NADH-Q vs pumping 4 protons) $$ \Delta G=2 F \Delta E_{h}-4 F \Delta \mathrm{p} $$ Thermodynamic driving force of RET in volts $$ -\Delta G / F=4 \Delta \mathrm{p}-2 \Delta E_{h} $$

https://user-images.githubusercontent.com/40054455/96906939-16e6ae80-14cd-11eb-9702-bcaf1a2294d4.png

  • A compound that only affects ROS production by RET indirectly through altering $\Delta p$ and $\Delta E_h$ would lie on the curve shown
  • In contrast, a compound that directly affected complex I independently of the thermodynamic drivers of RET, would lie below this curve.
  • Nigericin: decrease in H2O2 production was due to its effects on the CoQ pool redox state, hence changing the thermodynamic driving forces for RET
  • Rotenone: not changing the thermodynamic driving forces for RET. Specific inhibitory effects on complex I.
  • The drugs: more likely to be accounted for by their effects on $\Delta p$ and/or $\Delta E_h$ rather than due to specific inhibitory effects on complex I.

Discussion

  • ROS production by RET at complex I is favored by a high $\Delta p$ and a reduced CoQ pool. also highly responsive to small changes in the overall thermodynamic driving force for RET across the complex
  • We favor the FMN site as the source of superoxide production by complex I during RET
    • The penetration of O2 to the CoQ site is difficult to envisage from the structure of complex I. The superoxide anion is thermodynamically unfavorable in the hydrophobic area.
    • the FMN site is well established as a source of superoxide production by rotenone-inhibited complex I. And any superoxide formed at this site is released directly into the aqueous phase.
    • Linear dependence of oxygen concentration: second-order reaction between O2 and FMNH; NAD/NADH has no direct effect.
  • Why ROS formation of RET greater than FET?
    • Midpoint potential of FMN/FMNH = -380mV, compared to NAD/NADH = -335mV
    • RET => relatively more FMNH and more semiquinone radical FMN_dot
  • During RET, NAD/NADH pool is highly reduced and there is no net electron flow from complex I into this pool. https://user-images.githubusercontent.com/40054455/96907125-65944880-14cd-11eb-8ecb-45df69d13795.png
  • the calculation of G / F requires a number of assumptions and combines several technically challenging experimental measurements
  • not considering super complex formation
  • One of the beneficial effects by metformin in vivo maybe due to limiting ROS generation by limiting RET

  1. Robb EL, Hall AR, Prime TA, et al. Control of mitochondrial superoxide production by reverse electron transport at complex I. J Biol Chem. 2018;293(25):9869-9879.PMC6016480 ↩︎