Catalytic Coupling of Oxidative Phosphorylation, ATP Demand, and Reactive Oxygen Species Generation

Abstract

Competing models of mitochondrial energy metabolism in the heart are highly disputed. In addition, the mechanisms of reactive oxygen species (ROS) production and scavenging are not well understood. To deepen our understanding of these processes, a computer model was developed to integrate the biophysical processes of oxidative phosphorylation and ROS generation. The model was calibrated with experimental data obtained from isolated rat heart mitochondria subjected to physiological conditions and workloads. Model simulations show that changes in the quinone pool redox state are responsible for the apparent inorganic phosphate activation of complex III. Model simulations predict that complex III is responsible for more ROS production during physiological working conditions relative to complex I. However, this relationship is reversed under pathological conditions. Finally, model analysis reveals how a highly reduced quinone pool caused by elevated levels of succinate is likely responsible for the burst of ROS seen during reperfusion after ischemia.

Figure 1

Model diagram. The model consists of descriptors for substrate oxidation, the electron transport chain, oxidative phosphorylation, and the potassium/proton exchanger (KHE). The substrate oxidation descriptor is an empirical function characterizing NADH and FADH₂ production. The electron transport chain consists of NADH-ubiquinone oxidoreductase (CI), succinate dehydrogenase (CII), ubiquinol cytochrome c oxidoreductase (CIII), and cytochrome c oxidase (CIV). The oxidative phosphorylation components are ATP synthase (F₁F_O), adenine nucleotide translocase (ANT), and the inorganic phosphate carrier (P_iC). The number of protons required to produce one ATP molecule (n) by F₁F_O is set to 2.67. An extra proton enters mitochondria through the P_iC and ANT, which raises the H:ATP ratio to 3.67 as seen from outside the mitochondria. To see this figure in color, go online.

Figure 2

Model simulations compared to experimental data from isolated rat heart mitochondria. An extramitochondrial ATPase, apyrase, is titrated into the system to stimulate oxidative phosphorylation. Data and model simulations of key bioenergetics variables (NADH in A, membrane potential in B, cytochrome c²⁺ in C, extra-mitochondrial ADP concentration in D, mitochondrial pH in E, and UQH₂ in F) are shown for low [P_i] (1 mM, blue lines and symbols) and high [P_i] (5 mM, red lines and symbols) conditions. The mitochondrial pH and UQH₂ simulations are model predictions. (F, inset) Steady-state mitochondrial pH values as a function of extramitochondrial [P_i] when the ATPase rate is zero. To see this figure in color, go online.

Figure 3

FCCP titration of mitochondrial energetics. The model is simulated with increasing amounts of FCCP to depolarize the mitochondrial membrane and stimulate respiration. (A) The redox poise of cytochrome c, ubiquinone, and ΔΨ are shown versus FCCP activity. The effect of FCCP is modeled assuming the conductance of protons is linearly related to the FCCP concentration and the proton motive-force. (B) The fractions of reduced cytochrome c, ubiquinone, and NADH versus ΔΨ are shown for the same conditions in (A). (C) The respiration rate is shown versus ΔΨ for the same conditions in (A). Redox poises are computed using the following equation: $E_h=E_m-RT/zF\text{ln}\left({\left[X\right]}_{red}/{\left[X\right]}_{ox}\right)$, where E_m is the midpoint potential for a given redox couple, [X]_red is the concentration of the reduced moiety, and [X]_ox is the concentration of the oxidized moiety. The E_m values are set to 245 mV for the cytochrome c couple and 60 mV for the ubiquinone couple. To see this figure in color, go online.

Figure 4

The OxPhos flux control coefficients for increasing workloads. The flux control coefficients of O₂ consumption by mitochondria under the same conditions as those for Fig. 2 are shown for high P_i (A) and low P_i (B) versus the percent of the maximum respiratory rate. The control coefficients for the independent fluxes: MitoDH, CI, CIII, CIV, proton leak, ANT, and the external ATPase are shown according to the color legend. The dependent fluxes were CII, inorganic phosphate carrier (P_iC), and F₁F_O ATP synthase and are not calculated. For reaction close to equilibrium such as P_iC and F₁F_O ATP synthase, the flux control coefficient is close to zero. To see this figure in color, go online.

Figure 5

Model simulations of ROS steady-state ROS production, scavenging, and concentrations. (Blue lines) Low [P_i] (1 mM) case; (red lines) high [P_i] (5 mM) case. The conditions are as given in Fig. 2. (A) The individual contributions of ROS production by complexes I and III are shown for the apyrase titration. (B) The steady-state ROS production rates are shown versus the apyrase titration. (C) The rate of ROS production versus ΔΨ is shown. (D) The % of electrons that leak to form ROS instead of H₂O is shown versus the apyrase titration. (Inset) Simulation results with a more physiological O₂ concentration of 20 μM. (E and F) The steady-state concentrations of superoxide and H₂O₂ inside the mitochondria are shown versus the apyrase titration. To see this figure in color, go online.

Figure 6

Model simulations of ischemia/reperfusion (I/R). Two I/R simulations are run for the I/R protocol timeline shown. The model parameters are given in Table 1 and the Supporting Material. For the initialization period, the O₂ concentration is set to 30 μM, and the ATPase activity is set to 2 mM/s to produce a moderate workload demand. For the ischemic period, the O₂ concentration is set to 1 nM to reflect the hypoxic conditions during ischemia. For the reperfusion period, the O₂ concentration is reset to its original value for both I/R Simulations 1 and 2. For I/R Simulation 2, the complex II activity and its apparent equilibrium constant are both increased by a factor of 10 to account for an accumulation of succinate that occurs during ischemia (72, 73). In the recovery period, the model parameters are reset to the conditions for the initialization period. The steady-state model output for ΔΨ, fraction of UQH₂, superoxide, H₂O₂, ROS production and O₂ consumption are shown for (A)–(F), respectively. To see this figure in color, go online.