Contents

πŸ“’ Bazil 2014

Determining the origins of superoxide and hydrogen peroxide in the mammalian NADH:ubiquinone oxidoreductase1

Sciwheel

Introduction

  • NADH: ubiquinone oxidoreductase = Complex I
  • The type and origin of ROS produced by mammalian Complex I are controversial: FMN vs IQ site vs Fe-S clusterN2

Material and Methods

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4258523/bin/nihms628859f1.jpg

  • Global thermodynamic consistency is ensured by constraining the reverse rate of each half-reaction. We assume that substrate and product binding only depend on the redox state of the nearest redox center
  • we only consider the redox states for the FMN, Fe-S cluster N2, and SQ.

Reactant binding

  • binding polynomials

Midpoint potentials

  • All midpoint potentials were taken from the literature or directly fit to redox-titration data.

Primary state transitions

  • The state transitions follow a 2e- reduction, 2e- oxidation or 1e-oxidation of the enzyme by NADH, Q or O2, or O2, respectively
  • In order to apply the rapid equilibrium assumption, both the substrate and the product must be present to avoid a mathematical singularity but some conditions are NAD=0, so NAD/NADH are not in rapid equlibrium in this model

Flux expressions

  • The rate of electron input via superoxide or hydrogen peroxide is negligible and can be ignored; however, they are included in the model to maintain thermodynamic consistency

Model Simulations

  • The steady-state equation for the five-state model was analytically solved using MATLAB’s symbolic toolbox
  • parallelized simulated annealing algorithm was used to globally search for feasible parameters before employing a local, gradient-based optimization algorithm

Results and Discussion

  • challenged with a wide array of data from the literature
  • An identifiable parameter is defined as one having high sensitivity and low correlation with other parameters
  • The most sensitive parameters are the ones associated with NADH oxidation and those related to Q reduction rates
  • five parameters are sensitive and relatively uncorrelated with other parameters. These parameters are associated with the minimal set of ROS producing states required to reproduce the data https://user-images.githubusercontent.com/40054455/86616014-d0397b00-bfe7-11ea-984c-79a364be7f63.png

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4258523/bin/nihms628859f2.jpg
Model simulations of NADH oxidation rates as a function of DQ compared to data

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4258523/bin/nihms628859f3.jpg
Model simulations of ROS generation from various sites compared to experimental data

  • the model is captures the NADH dependence of ROS production quite well and also is able to simulate ROS production under RET conditions
  • the model is quite capable of simulating NADH oxidation rates under a wide range of conditions, including physiological ones

Sites of ROS generation

  • The fully reduced FMN in state 2 and the FMN radical in state 1 are responsible for the majority of superoxide at the NADH oxidase site
  • For RET conditions, state 1 produces superoxide from the bound SQ at the Q reductase site. In the reverse mode, superoxide is generated at both the Q reductase and NADH oxidase sites after QH2 is oxidized, with two sites contributing equally.
  • Hydrogen peroxide is produced by the fully reduced FMN in state 3
  • The ability of a SQ to reduce oxygen to for superoxide in Complex I is still debated.
  • There must be a high ΔΨ and a highly reduced Q pool (RET condition) in order for an appreciable amount of SQ to form
  • The model best reproduced the data when superoxide from the Fe-S cluster N2 was excluded.
  • FMN radical is only a significant source of ROS when Q is absent or Q reductase site inhibitors are present. This is consistent with the findings of Kussmaul and Hirst

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4258523/bin/nihms628859f4.jpg
Model simulations of NADH oxidation rates as a function of Q pool redox state at various fixed Ξ”p’s

  • The model is hypersensitive to Ξ”p, and at Ξ”p< 100 mV the rate of NADH oxidation is increasingly insensitive to the Q pool redox state and only depends on the NAD(H) pool redox state
  • we do not have sufficient data to conclusively determine how the energetic cost of proton pumping is distributed in the reaction mechanism

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4258523/bin/nihms628859f5.jpg
Model simulation of ROS stoichiometric coefficient, n, NADH oxidation, and ROS production as a function of ΔΨ, matrix pH, %NADH, and %Q

  • At low ΔΨ and pH, n is small and nearly all of the electrons from NADH reach their intended target, Q. As both ΔΨ and pH increase, not only does n increase, but also, the amount of RET increases as well
  • the model predicts that ROS are produced in greater excess during RET versus FE
  • the fraction of electrons reducing oxygen becomes quite significant when both the NAD(H) and Q pools are highly reduced
  • pumping mechanism that is directly coupled to the reduction of the SQ in the reaction scheme is thermodynamically and kinetically feasible.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4258523/bin/nihms628859f6.jpg
Simulated ROS production rate for each redox center under the conditions described in the legend of Figure 5

Reference


  1. Bazil JN, Pannala VR, Dash RK, Beard DA. Determining the origins of superoxide and hydrogen peroxide in the mammalian NADH:ubiquinone oxidoreductase. Free Radic. Biol. Med. 2014;77:121-129. doi:10.1016/j.freeradbiomed.2014.08.023. PMC4258523↩︎