Kinetic modeling of H2O2 dynamics in the mitochondria of HeLa cells

Abstract

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.

All-optical spatiotemporal mapping of ROS dynamics across mitochondrial microdomainsin situ

“…Moreover, since the concentration of exogenously added H 2 O 2 is likely an over-estimate of intracellular and subcellular accumulation, interpolating local concentrations of H 2 O 2 based on HyPer7 measurements can only provide an upper-bound estimate. Other studies have modeled a 390 to 650-fold difference between extracellular and intracellular H 2 O 2 62,63 and calculated a basal steady-state of 2 to 4 nM in mitochondria 9 . Our experiments never produce a steady-state change approximating greater than 2 μM H 2 O 2 treatment.…”

“…The exact concentration difference between healthy, metabolically signaling ROS and oxidative damage remains unknown. Once generated, ROS can diffuse between mitochondrial compartments and into the cytosol, but the kinetics of this process is only beginning to be characterized 9,10 .…”

“…The exact concentration difference between healthy, metabolically signaling ROS and oxidative damage remains unknown. Once generated, ROS can diffuse between mitochondrial compartments and into the cytosol, but the kinetics of this process is only beginning to be characterized 9,10 . Moreover, the four-decade old hypothesis 11 that ROS releases from the mitochondrial matrix into the cytosol has only minimal experimental evidence 12 .…”

“…Recent advances have bypassed these constraints with targeted expression of H 2 O 2 -generating proteins such as the D-alanine oxidase (DAO) system 24 . These systems provide a high degree of spatial control of H 2 O 2 production but at relatively slow timescales and with limited temporal regulation 9,12 . Furthermore, by producing H 2 O 2 and not superoxide to mimic ROS generation at the ETC, these systems may not fully recapitulate kinetic details of ROS diffusion dynamics.…”

Unique Cellular and Biochemical Features of Human Mitochondrial Peroxiredoxin 3 Establish the Molecular Basis for Its Specific Reaction with Thiostrepton

“…These observations led us to determine which wild-type PRX isoform can form dimers containing some reduced active sites under physiological conditions. For these studies, we used a constant concentration of 100 µM PRX, which is physiologically relevant even if somewhat low for the estimates of PRX3 mitochondrial concentrations (48-125 µM) and slightly higher than the estimated concentration of PRX1 and PRX2 in the cytosol (20-65 µM) [33][34][35]. A recent study using real-time monitoring of the PRX2 oligomerization state confirmed for the first time that PRX2 decamer-dimer oscillations occur and that oxidation favors dimers in cellulo.…”