📒 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.
- binding polynomials
- 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
- 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
- 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
- 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
- The model is hypersensitive to Δp, and at Δp< 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
- 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.