Kinetic modeling of H(2)O(2) dynamics in the mitochondria of HeLa cells

Hydrogen peroxide (H(2)O(2)) promotes a range of phenotypes depending on its intracellular concentration and dosing kinetics, including cell death. While this qualitative relationship has been well established, the quantitative and mechanistic aspects of H(2)O(2) signaling are still being elucidated. Mitochondria, a putative source of intracellular H(2)O(2), have recently been demonstrated to be particularly vulnerable to localized H(2)O(2) perturbations, eliciting a dramatic cell death response in comparison to similar cytosolic perturbations. We sought to improve our dynamic and mechanistic understanding of the mitochondrial H(2)O(2) reaction network in HeLa cells by creating a kinetic model of this system and using it to explore basal and perturbed conditions. The model uses the most current quantitative proteomic and kinetic data available to predict reaction rates and steady-state concentrations of H(2)O(2) and its reaction partners within individual mitochondria. Time scales ranging from milliseconds to one hour were simulated. We predict that basal, steady-state mitochondrial H(2)O(2) will be in the low nM range (2–4 nM) and will be inversely dependent on the total pool of peroxiredoxin-3 (Prx3). Neglecting efflux of H(2)O(2) to the cytosol, the mitochondrial reaction network is expected to control perturbations well up to H(2)O(2) generation rates ~50 μM/s (0.25 nmol/mg-protein/s), above which point the Prx3 system would be expected to collapse. Comparison of these results with redox Western blots of Prx3 and Prx2 oxidation states demonstrated reasonable trend agreement at short times (≤ 15 min) for a range of experimentally perturbed H(2)O(2) generation rates. At longer times, substantial efflux of H(2)O(2) from the mitochondria to the cytosol was evidenced by peroxiredoxin-2 (Prx2) oxidation, and Prx3 collapse was not observed. A refined model using Monte Carlo parameter sampling was used to explore rates of H(2)O(2) efflux that could reconcile model predictions of Prx3 oxidation states with the experimental observations.