πŸ“’ Bazil 2014

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



  • 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

  • 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
Model simulations of NADH oxidation rates as a function of DQ compared to data
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
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
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.
Simulated ROS production rate for each redox center under the conditions described in the legend of Figure 5


  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↩︎