Kinetic modeling of H2O2 dynamics in the mitochondria of HeLa cells

Hydrogen peroxide (H2O2) 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 H2O2 signaling are still being elucidated. Mitochondria, a putative source of intracellular H2O2, have recently been demonstrated to be particularly vulnerable to localized H2O2 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 H2O2 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 H2O2 and its reaction partners within individual mitochondria. Time scales ranging from milliseconds to one hour were simulated. We predict that basal, steady-state mitochondrial H2O2 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 H2O2 to the cytosol, the mitochondrial reaction network is expected to control perturbations well up to H2O2 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 H2O2 generation rates. At longer times, substantial efflux of H2O2 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 H2O2 efflux that could reconcile model predictions of Prx3 oxidation states with the experimental observations.


Introduction
Reactive oxygen species (ROS) are a class of chemical species that promote diverse phenotypes depending on intracellular concentration, localization and cumulative dose over time, spanning the gamut from homeostasis to toxicity [1,2]. Among ROS, the behavior of hydrogen peroxide (H 2 O 2 ) most closely resembles that of a classical signaling molecule, based on the specificity of its reactions and its in vivo half-life [3][4][5][6]. Mitochondria are putatively a main intracellular source of H 2 O 2 under basal conditions as a result of the electron transport chain (ETC) and oxidative phosphorylation (OxPhos) [2,7]. This organelle is also hypothesized to be an important site for H 2 O 2 -mediated signaling [8,9].
Previous work in our group has demonstrated that H 2 O 2 perturbations directed to the mitochondrial matrix elicit a marked toxicity in HeLa cells, especially when contrasted against comparable perturbations delivered in the cytosol [10,11]. This toxicity was both concentration-and time-dependent, indicating the importance of a dynamic understanding of the H 2 O 2 reaction network. Building upon our experimental results, we sought to further our mechanistic understanding of mitochondrial H 2 O 2 kinetics by constructing a computational model of the reaction network in this organelle. Detailed molecular mechanisms that connect changes in H 2 O 2 with phenotypic responses such as changes in mitochondrial morphology, mitochondrial permeability transition (MPT), and programmed cell death have not been elucidated. Since these signaling responses occur during excursions in H 2 O 2 concentration from the basal steady state, we expect that establishing a quantitative range that can be connected with phenotypic responses will help inform whether particular cysteine residues are likely to become directly oxidized [12]. Existing models on mitochondrial ROS so far have largely fallen into two categories: detailed kinetic models focusing on fast-respiring cells, such as cardiac cells [13,14], or models that exclude the thioredoxin/peroxiredoxin (Trx/Prx) system [15]. Faster rates of cellular respiration [16] and differing abundances of mitochondrial proteins, which have been reported for differing tissue and cell types [17], may lead to differing steady-state H 2 O 2 concentrations. The Prxs are so abundant and react with H 2 O 2 with such a high secondorder rate constant (10 6 −10 8 M -1 s -1 ) that this antioxidant system cannot be neglected [18,19]. Some additional modeling efforts have focused on the kinetics of species other than H 2 O 2 specifically [20] or on parameter estimation [21]. To our knowledge, this model represents the first kinetic model of the mitochondrial H 2 O 2 reaction network in a transformed cell line, incorporating the most recent quantitative data specific for HeLa cells.
Here, we implement this model to predict basal H 2 O 2 concentrations in HeLa cell mitochondria. We also predict network behavior in response to sustained H 2 O 2 perturbations, including the degree of oxidation of four major antioxidant species present in mitochondria: Prx3, glutathione peroxidase 1 (Gpx1), Prx5, and Gpx4. The mass action kinetics of a network of 30 reactions of 28 chemical species were described using ordinary differential equations and, after parameterization with 30 rate coefficients and species concentrations, solved using MATLAB. Basal mitochondrial H 2 O 2 as well as reaction network response to H 2 O 2 perturbations were predicted. Modeling results were compared with experimental data from redox Western blots of the Prxs using the mitochondrially-localized H 2 O 2 generator D-amino acid oxidase (mito-DAAO). HeLa cells were exposed to a range of D-alanine concentrations, a substrate for mito-DAAO, over time, and Western blots were performed on the cell lysates to observe the change in Prx3 (mitochondrial) and Prx2 (cytosolic) oxidation with the different treatments.

Model formulation: Baseline model
This model was adapted from our previously published kinetic model of the cytosolic antioxidant network, and consists of a system of first-order ordinary differential equations of the form where C i is the species concentration, t is time and R i is the net reaction for that species [22]. It assumes species concentrations are homogeneous throughout the compartment. The baseline model investigates the steady state conditions in the mitochondria, where the only source of endogenous H 2 O 2 is assumed to be from the ETC due to cellular respiration. For the purposes of this simulation, the rate of H 2 O 2 generation due to OxPhos is assumed to be invariant. As a first approximation, transport of H 2 O 2 between mitochondria and the cytosol is neglected. Release of H 2 O 2 from isolated mitochondria to the surrounding medium has been measurable, but it has not been possible to measure H 2 O 2 efflux from mitochondria to the cytosol in intact cells near basal conditions, perhaps due to insufficient sensitivity of existing analytical techniques. By modeling the reaction network with the assumption that H 2 O 2 efflux is small enough to be neglected and comparing with increasing experimental perturbations to the H 2 O 2 generation rate, we aim to estimate H 2 O 2 generation rates for which this assumption breaks down, motivating the need for a refined model that incorporates H 2 O 2 efflux. The baseline model quickly reaches steady state (less than 1 s), so baseline simulations are carried out to 5 s. The stiff equation solver ode15s in MATLAB was implemented to solve the system of equations.
The main reaction systems that this model captures are the thioredoxin/peroxiredoxin/ thioredoxin reductase (Trx/Prx/TR) and the glutathione/glutathione peroxidase/glutaredoxin (GSH/Gpx/Grx) networks. The Prx isoforms found in the mitochondria are Prx3 and Prx5 [23,24], which are reduced by Trx2 [25,26]. Trx2 also reduces disulfide bonds to protein dithiols [27]. Both Gpx1 and Gpx4 are found in the mitochondria, though at low concentrations in HeLa cells [28]. Grx2 is the most abundant mitochondrial Grx isoform, and is responsible for reducing S-glutathionylated proteins [29][30][31]. While the prior proteins are all considered mitochondrially localized, both GSH and sulfiredoxin (Srx) are generally considered cytosolic molecules that must be imported into the mitochondria [17,[32][33][34]. The mitochondria maintain a large pool of the former, but the latter is only imported based upon a stimulus [33,34]. Catalase is not included because it is not expected to be found in the mitochondria for most cell types, including HeLa cells [17,28,35]. A schematic representation of the reaction networks captured by this model is shown in Fig 1. The reaction rate parameters for mass action or Michaelis-Menten kinetics in Eq. (1) were found in the peer-reviewed literature or derived from published data. The detailed calculations necessary to derive values of some parameters can be found in S1 Appendix. For any cases where mitochondria-specific values could not be located, the cytosolic equivalent was assumed. These parameters are summarized in Table 1. One difference between previously published models and this one is the treatment of Srx. Previous work [22,38] has assumed zeroth-order kinetics with respect to Srx, leading to the following rate law for hyperoxidized Prx3: where k hyperox is the rate of hyperoxidation of Prx3 and k cat is the turnover number reported for Srx by Chang and colleagues [38]. However, overexpression studies have clearly demonstrated an increase in reduction rate of the sulfinic acid with increased Srx concentration [39]. The form of the rate law proposed by Eq (2) fails to capture any dependence on Srx, so we propose a rate law with first-order dependence on Srx as a first approximation: where k' is the estimated second-order rate constant, obtained by dividing 0.18 min -1 , the first order rate constant reported in [38], by the sulfiredoxin concentration used there. These parameters, k hyperox and k' correspond to k 7 and k 8 in Table 1, respectively. Glutathione efflux was treated as a first order reaction and the value was determined via trial-and-error to satisfy the constraint that the total glutathione level should not change by more than 5%, just as the total Trx level does not change over the course of the simulations in this work. Species abundances for model initialization were either found in literature, calculated from published datasets, or calculated based on molar balances and rate laws. Species that were found in literature or calculated using published datasets are summarized in Table 2, and species that were derived from molar balances and rate laws are summarized in Table 3. Prx3-SH abundance is given as a range rather than a single value. This is the result of the calculations that are necessary to convert per-cell protein copy numbers from the proteomics dataset in [28] to a per mitochondrion concentration. For these calculations, mitochondrial volume was taken as 0.29 μm 3 [58] and mitochondrial number in a HeLa cell has been reported to range from 383-882 [59]. A total protein density throughout the cell was reported as 2x10 5 mg/L in Table 1. Kinetic parameters. Calculations for parameters that were derived can be found in SI. [28] so we assumed this density was invariant between organelles. Additional details regarding these calculations can be found in S1 Appendix. While all the proteins that were calculated based on the data in [28] produced a range of possible values depending on the number of mitochondria per cell, Prx3-SH was by far the most abundant and has a very high rate constant for reaction with H 2 O 2 . Therefore, we considered the range of Prx3-SH concentrations explicitly while taking the median value for other protein concentrations calculated from [28]. Notably, the abundances of Gpx1 and Gpx4 listed in Table 2, calculated from the proteomics dataset in [28], are much lower than values suggested in previous work with hepatocytes (2 and 1 order of magnitude lower, respectively) [60]. Because several species are initialized by molar balance, the range in Prx3-SH initialization results in several species in Table 3 to initialize differently depending on its concentration. Pr-SH 1x10 -3 [47] Pr-(SH) 2 1.09x10 3 [63] Srx 8.8x10 -3 [28] https://doi.org/10.1371/journal.pcbi.1008202.t002 Table 3. Derived initial species abundances.

Quantifying uncertainty in model predictions
Monte Carlo parameter sampling. Based on the feasible concentration range of Prx3-SH in HeLa mitochondria, we generated 10,000 random samples (shown in S1 Appendix) spread uniformly throughout the feasible space using the following equation [66]: Here, U(0,1) refers to a single uniformly distributed random number in the range of 0 to 1, and C min Prx3À SH is 48 μM and C max Prx3À SH is 110 μM. A set of randomly generated Prx3-SH concentrations was used as initial conditions for implementation of ODE simulations, providing a distribution of predicted steady-state concentrations of each species of interest. Sensitivity analysis. In order to calculate the sensitivity of predicted steady-state H 2 O 2 concentrations and protein redox balances to the values of model parameters used, the finite difference approximation method was used [67]. The sensitivities were calculated using the following equation: where s i is the sensitivity corresponding to parameter k i and C j is the concentration of the species of interest (e.g. H 2 O 2 or Prx3-SS). Parameters were perturbed by 10% to reflect an estimate of typical experimental error, and sensitivities were normalized to adjust for differences in orders of magnitude: Sensitivities of the basal, steady-state model predictions were calculated at 5 s, and here, we report only � s i .

Model formulation: H 2 O 2 perturbation
The second part of this modeling endeavor sought to investigate the effects of a source of H 2 O 2 perturbation, similar to what is introduced by the synthetic biology tool D-amino acid oxidase (DAAO) targeted to the mitochondrial matrix. This was modeled as a constant source term, k DAAO , within the H 2 O 2 rate equation, as depicted in Fig 1. Because we were interested in how the network would respond to perturbations of varying magnitudes, we swept this parameter across a range of values until we reached an upper limit of possible physiological relevance, which we defined as the complete collapse of the Prx3 system. This part of the simulation was carried out to 3600 s (1 hr).

Comparison of model predictions with experimental data
Cell culture. HeLa cells that had previously been transfected by lentivirus to stably express a mitochondrially-targeted D-amino acid oxidase (mito-DAAO) H 2 O 2 generator [10] were maintained in Dulbecco's modified Eagle's medium (DMEM; Lonza), supplemented with 10% fetal bovine serum (FBS; ATCC) at 37˚C in a humidified atmosphere with 5% CO 2 . Cells were passaged approximately every 3 days and were maintained under selective pressure using 6 μg/ mL puromycin (Sigma) until 24 hrs before any experiments.
Analysis of Prx response to mitochondrial H 2 O 2 perturbations. HeLa cells expressing mito-DAAO were seeded at 3.5x10 5 cells/well in 6-well plates~18 hours prior to the start of generation (target confluence~50% at start of experiment). Cells were exposed to 5 μM flavin adenine dinucleotide (FAD; Sigma) and concentrations of D-alanine (Sigma) from 0-25 mM in Roswell Park Memorial Institute 1640 medium (RPMI; Invitrogen) without phenol red. At the end of the H 2 O 2 generation period, cells were washed with ice cold 1x phosphate buffered saline (PBS) and then incubated on ice with 2 mL 100 mM methyl methanethiosulfonate (MMTS; Sigma) for 30 min to block free thiols. Cells were washed twice more with cold PBS, then lysed in 100 μL of lysis buffer (0.5% Triton X-100 (Sigma), 1x HALT protease and phosphatase inhibitor (ThermoFisher), 1x PBS). Lysates were centrifuged on a cooled rotor for 10 min at 10,000xg and the supernatant was collected and stored at -80˚C for further analysis. Western blotting was carried out according to the protocol in [68]. Proteins were separated by non-reducing SDS-PAGE using a pre-cast 12% polyacrylamide stain-free gel (Bio-Rad). Following SDS-PAGE, the gel was activated for 45 s using a ChemiDoc MP (Bio-Rad), then proteins were transferred to a polyvinylidene difluoride (PVDF) membrane for immunoblotting. Blots were blocked using Odyssey blocking buffer (Licor), and incubated with primary antibodies against Prx3 (Abcam, ab73349), Prx2 (R&D Systems, AF3489), and Hsp60 (R&D Systems, Clone# 264233) either overnight at 4˚C or 2 hr at room temperature. Endogenous Hsp60 was used to account for differences in loading. Blots were incubated for 1 hr at room temperature with Licor IRDye secondary antibodies. The ChemiDoc MP system was used to image the blots, then ImageJ was used to quantify the images for densitometry.

Statistical analysis
Analysis of variance (ANOVA) was used to test for trends in the fractional oxidation of the Prx protein, as measured by Western blots. At least three biological replicates per time point were used for trend testing. Post-hoc Tukey's Honest Significant Difference (Tukey-HSD) testing was performed to determine which sample means were different from the control (0 mM D-ala) within each time point.

Model refinement: H 2 O 2 perturbation
An H 2 O 2 efflux reaction was added to represent transport of H 2 O 2 out of the mitochondria and into the cytosol. Monte Carlo parameter sampling using sets of 10,000 random sample points for k DAAO and k efflux were generated in uncertain ranges of these parameters. For high k DAAO values where efflux may be important, the minimum and maximum of k DAAO , or ðk min DAAO ; k max DAAO Þ, were set to (50,100), where the units are μM/s. Two efflux cases were considered, termed low and high. For low k efflux , ðk min efflux ; k max efflux Þ was set to (0, 50), and for high k efflux , ðk min efflux ; k max efflux Þ was set to (50,100), where the units are again μM/s. Equations following the form of (4) above together with uniformly distributed random numbers in the range of 0 to 1 were used to produce sets of parameter values, further described in S1 Appendix, that were used in implementation of ODE simulations.

Results
The first quantity investigated was the basal, steady-state concentration of H 2 O 2 in mitochondria, which was predicted to range between 1.8-4.4 nM (Fig 2A). This steady-state concentration showed a strong inverse dependence on the concentration of Prx3 within a mitochondrion. Fig 2A shows that a two-fold increase in Prx3 concentration leads to a twofold decrease in steady state H 2 O 2 concentration. The range of Prx3 concentrations examined in Fig 2A reflects the current state of knowledge of this mitochondrial protein's concentration. Copy numbers of Prx3 proteins per cell have been calculated from pooled lysates of many cells [28], and number of mitochondria per cell have been measured [60], narrowing the likely range of Prx3 concentrations per mitochondrion, and thus basal, steady state H 2 O 2 concentrations within mitochondria, to the ranges that are plotted in 2A. To supplement the ten singlepoint calculations presented in Fig 2A, Monte Carlo parameter sampling within the same range of Prx3 concentrations was used to further investigate the range of steady-state mitochondrial H 2 O 2 concentrations, resulting in the distribution shown in Fig 2B. We next investigated the effect of the total available pool of Prx3 on the dimer fraction of Prx3, calculated as a quantity is often measured experimentally by Western blotting, and at baseline can characterize the variability between different cell types [10,69]. Fig 2C plots this quantity as well as the fractional oxidation of other peroxidases found within the mitochondria. Similar to the basal, steady-state H 2 O 2 concentration, the fractions of oxidized peroxidases all demonstrate an inverse relationship with the total pool of Prx3. Only Prx3 experiences any significant degree of oxidation at baseline, as shown in Fig 2C and 2D and summarized in Table 4. The PLOS COMPUTATIONAL BIOLOGY fractional oxidation in Table 4 represents the dimer fraction for the Prxs and the fraction of Gpxox + GpxSSG for the Gpxs. The basal model predicts that only 2 to 5% of the total Prx3 pool is engaged in maintaining H 2 O 2 at low nM concentrations, leaving a large excess of Prx3-SH. In order to evaluate the impact that parameter uncertainties may have on the model predictions, we performed a sensitivity analysis. The value of � s i can inform us about both the magnitude and direction that changes in a particular parameter will have on the predictions for a given species of interest. For example, a sensitivity of 1 indicates that a 10% increase in the parameter resulted in a 10% increase in the model output, and likewise, a sensitivity of -1 would signify a 10% decrease in the model output. Fig 3 depicts tornado plots of the sensitivities of the predicted basal steady-state concentrations of H 2 O 2 (A), Prx3-SH (B), Prx3-SS (C), and Prx3-SOOH (D) to kinetic parameters within the model. These plots order the parameters from greatest to least effect on the model output. The model prediction for [H 2 O 2 ] was most sensitive to k 1 , the rate of generation of H 2 O 2 by OxPhos, closely followed by the rate constant of oxidation of Prx3-SH, k 6 . Prx3-SS was similarly sensitive to k 1 and was also sensitive to k 10 , the rate constant of reduction of Prx3-SS by Trx2-SH. Prx3-SH was not very sensitive to any single model parameter, and Prx3-SOOH was sensitive to several parameters, especially k 1 . k 1 appeared in all four sensitivity analyses as a top parameter, indicating its importance to all the model predictions. The sensitivity analysis, therefore, pointed to the model's overall dependence on the rate of H 2 O 2 input into the system and the kinetic parameters within the Trx2/ Prx3 pathway.
Once the baseline was established, we next sought to evaluate the network response to H 2 O 2 perturbations. To clearly show dynamic behavior during a range of perturbations, we fixed the initial concentration of Prx3-SH at 62 μM, one of the ten concentrations examined in Fig 2 and a value that is close to a previous experimental measurement [35]. The magnitude of the perturbation term, k DAAO   trapped as the hyperoxidized isoform, shown in Fig 4D. It is only when the capacity of the Prx3/Trx/TR system was exceeded that other antioxidants were able to kinetically compete and react with H 2 O 2 , as summarized by Table 5, which lists the fractional oxidation for the four major antioxidant species at each perturbation rate, following the same convention as in Table 4. It is important to note that the predicted steady states that result from this H 2 O 2 perturbation analysis are the net effect of the rate of H 2 O 2 generation by OxPhos and the additional k DAAO generation term; a higher OxPhos generation rate would result in a lower k DAAO needed to cause collapse of the Prx3 system. In order to experimentally investigate the trends predicted by the model, we used the genetically-encoded H 2 O 2 generator mito-DAAO, which localizes a H 2 O 2 perturbation to the mitochondrial matrix [10,70]. We varied the concentration of D-alanine (D-ala) substrate the cells were exposed to for up to 1 hr, then probed the Prx3 and Prx2 isoforms using redox Western blots. Prx2 is found in the cytosol and provided a means to assess H 2 O 2 efflux from the mitochondria to the cytosol. The Western blot results are summarized in Fig 5. The experimental data demonstrates consistently higher fractions of oxidized, dimer Prx3 than the model predicts, and this discrepancy is most prominent at high perturbations. Where the model predicts a maximum fractional oxidation of Prx3 to the disulfide-linked dimer form of around 0.5, the experimental data continues to rise monotonically, reaching a fraction of oxidized Prx3 as high as 0.8. Thus, the model over-predicts hyperoxidation. An increase in the concentration of Prx3 during the perturbation could contribute to a lesser degree of oxidation than expected. We examined whether the total amount of Prx3 increased during increased H 2 O 2 generation for up to 1 hour, and found no evidence of increases in total Prx3 abundance (S4 Fig). The Prx2 data demonstrate increased H 2 O 2 flux in the cytosol at certain perturbations after 15 min. At 15 min, while one-way ANOVA testing determined there was a statistically significant trend in Prx3 mean fractional oxidation at the 95% confidence level (P = 0.041), the same  test found the Prx2 means to not differ across D-ala concentrations (P = 0.095) suggesting an undetectable amount of transport at this time scale. However, at subsequent times, both Prx3 and Prx2 oxidation demonstrated significant trends at the 99% confidence level, as determined by one-way ANOVA (P = 0.004 and P = 0.003 for Prx3 and Prx2 at 30 min, P = 1.76 x 10 −5 and P = 2.05 x 10 −5 for Prx3 and Prx2 at 1 hr). This suggests that transport effects may be playing a larger role at these longer times, as Prx2 oxidation becomes increasingly significant. This efflux of H 2 O 2 from the mitochondria to the cytosol may explain, at least in part, why Prx3 did not collapse into the hyperoxidized form after reaching dimer fractions above 0.5 as predicted by a model that neglects H 2 O 2 efflux. Motivated by this experimental evidence, we refined the kinetic model to include an H 2 O 2 efflux reaction that is dependent on the concentration of H 2 O 2 as shown in Fig 6A. The range of values of k DAAO where H 2 O 2 efflux is substantial and the values of k efflux are both uncertain. Monte Carlo parameter sampling was used to explore ranges of both of these variables.  and resulting in dimer fractions that are larger than 0.4. This modelling approach still seems to over-predict hyperoxidation.

Discussion
Our analysis of mitochondrial H 2 O 2 metabolism found that Prx3 is the antioxidant in the mitochondrial H 2 O 2 reaction network that controls the steady state concentration of H 2 O 2 , as has been previously hypothesized [35]. In HeLa cells, we predicted basal H 2 O 2 to be in the range of 2-4 nM. Further, we examined the impact of increasing H 2 O 2 generation rates on the reaction network. Because of the reducing capacity of Prx3, the mitochondrial reaction network is able to control H 2 O 2 perturbations in the low μM/s range without participation from Gpx1, Prx5, and Gpx4. Only at perturbations that cause total saturation of the Prx3 system do we expect oxidation of Gpx1, Prx5, and Gpx4. Thus, under most circumstances, Prx5 and Gpx4 are not expected to react directly with H 2 O 2 , consistent with the peer-reviewed literature describing their other roles. It has been previously reported that, though Prx5 and Gpx4 are able to react with H 2 O 2 , that is not their primary biological function; Prx5 is the putative reductant of reactive nitrogen species and Gpx4 is hypothesized to react with lipid hydroperoxides [23,56,71].
A model that neglects efflux of H 2 O 2 from the mitochondria to the cytosol predicts a great deal of hyperoxidation of Prx3 at moderate to large perturbation rates. This is inconsistent with experimental observations of monotonically increasing dimeric Prx3-SS in redox Western blots as a function of increasing H 2 O 2 generation rates (Fig 5). If hyperoxidation of Prx3 became prevalent at a particular increased H 2 O 2 generation rate, it would be evidenced experimentally by a decrease in the intensity of the dimeric Prx3-SS band. This behavior was observed for Prx2 ( Fig 5F). Prx3 is known to be less prone to hyperoxidation as compared with Prx2, as it has faster resolution kinetics of disulfide formation [44,72]. One limitation to accurately predicting Prx3 hyperoxidation is that the reduction kinetics of the sulfinic acid form of Prx3 have not been well characterized, nor the dynamics of Srx import into and export from the mitochondria. In addition, the reduction of Srx itself is still poorly understood [73]. More careful quantitative analyses of the kinetics governing this reaction pathway will improve our understanding of the dynamics of hyperoxidation. However, the largest contributor to the inconsistency of the model's predictions with experimental data at large perturbation rates arises from neglecting efflux. Redox Western blots of mitochondrial and cytosolic Prx isoforms showed that while efflux of H 2 O 2 from the mitochondria to the cytosol wasn't detectable at 15 minutes, it became increasing important at longer times over the range of perturbations studied.
Physiologically, a variety of interesting reactions within and across the mitochondrial membranes may occur over the range of perturbation rates we studied, including aquaporin or other pore-mediated diffusion of H 2 O 2 into the cytosol and even possible depolarization of the mitochondrial membrane caused by the mitochondrial permeability transition (MPT) [74][75][76]. The molecular details of these and many other stress responses within mitochondria are not precisely understood. The compartment-specific perturbation tool used here and others that are complementary [77] may provide a means to better understand redox metabolism in this important organelle.
In a study of isolated mitochondria [40], Treberg et al. calculated H 2 O 2 consumption rates and estimated steady state mitochondrial H 2 O 2 concentrations of � 484 ± 28 nM. In our present study of mitochondria within cells, the kinetic model says that the consumption rate of H 2 O 2 is expected to be the same at steady-state as the generation rates of H 2 O 2 via OxPhos and the DAAO system. With the concentration of Prx3 is taken as 62 μM within mitochondria, we calculated steady-state H 2 O 2 concentrations of 3.4-230 nM with a generation rate from OxPhos of 4 μM/s and 0 � k DAAO � 47 μM/s. Notably, our range in intact cells, even with extreme k DAAO perturbations, is lower than the upper bound for isolated mitochondria. Our prediction of basal, steady-state concentrations of 2-4 nM H 2 O 2 in the mitochondria of HeLa cells, with the range dependent on Prx3 concentrations from 48-110 μM, is also lower than the previously predicted value of 40 nM [20]. This previous estimate was derived using parameters for a faster respiring cell type, which would produce more H 2 O 2 through OxPhos, perhaps leading to higher basal H 2 O 2 concentrations. Our findings suggest the utility of measuring Prx oxidation as a marker of H 2 O 2 concentrations. Other groups have pointed out that the Prxs could be informative biomarkers for certain cancers [78,79]. This model corroborates that idea, and demonstrates not only a relationship between Prx oxidation and H 2 O 2 perturbation, but also Prx oxidation and the total available pool. Moving forward, this model can be used as a general framework for understanding mitochondrial H 2 O 2 clearance, and it can be parametrized to match other cells and tissues as data become available.