Computational Modeling Analysis of Kinetics of Fumarate Reductase Activity and ROS Production during Reverse Electron Transfer in Mitochondrial Respiratory Complex II.

Reverse electron transfer in mitochondrial complex II (CII) plays an important role in hypoxia/anoxia, in particular, in ischemia, when the blood supply to an organ is disrupted and oxygen is not available. A computational model of CII was developed in this work to facilitate the quantitative analysis of the kinetics of quinol-fumarate reduction as well as ROS production during reverse electron transfer in CII. The model consists of 20 ordinary differential equations and 7 moiety conservation equations. The parameter values were determined at which the kinetics of electron transfer in CII in both forward and reverse directions would be explained simultaneously. The possibility of the existence of the "tunnel diode" behavior in the reverse electron transfer in CII, where the driving force is QH2, was tested. It was found that any high concentrations of QH2 and fumarate are insufficient for the appearance of a tunnel effect. The results of computer modeling show that the maximum rate of succinate production cannot provide a high concentration of succinate in ischemia. Furthermore, computational modeling results predict a very low rate of ROS production, about 50 pmol/min/mg mitochondrial protein, which is considerably less than 1000 pmol/min/mg protein observed in CII in forward direction.

Mathematical Modeling of ROS Production and Diode-like Behavior in the SDHA/SDHB Subcomplex of Succinate Dehydrogenases in Reverse Quinol-Fumarate Reductase Direction.

Succinate dehydrogenase (SDH) plays an important role in reverse electron transfer during hypoxia/anoxia, in particular, in ischemia, when blood supply to an organ is disrupted, and oxygen is not available. It was detected in the voltammetry studies about three decades ago that the SDHA/SDHB subcomplex of SDH can have such a strong nonlinear property as a "tunnel-diode" behavior in reverse quinol-fumarate reductase direction. The molecular and kinetic mechanisms of this phenomenon, that is, a strong drop in the rate of fumarate reduction as the driving force is increased, are still unclear. In order to account for this property of SDH, we developed and analyzed a mechanistic computational model of reverse electron transfer in the SDHA/SDHB subcomplex of SDH. It was shown that a decrease in the rate of succinate release from the active center during fumarate reduction quantitatively explains the experimentally observed tunnel-diode behavior in SDH and threshold values of the electrode potential of about -80 mV. Computational analysis of ROS production in the SDHA/SDHB subcomplex of SDH during reverse electron transfer predicts that the rate of ROS production decreases when the tunnel-diode behavior appears. These results predict a low rate of ROS production by the SDHA/SDHB subcomplex of SDH during ischemia.

Computational Modeling Analysis of Generation of Reactive Oxygen Species by Mitochondrial Assembled and Disintegrated Complex II.

Reactive oxygen species (ROS) function as critical mediators in a broad range of cellular signaling processes. The mitochondrial electron transport chain is one of the major contributors to ROS formation in most cells. Increasing evidence indicates that the respiratory Complex II (CII) can be the predominant ROS generator under certain conditions. A computational, mechanistic model of electron transfer and ROS formation in CII was developed in the present study to facilitate quantitative analysis of mitochondrial ROS production. The model was calibrated by fitting the computer simulated results to experimental data obtained on submitochondrial particles (SMP) prepared from bovine and rat heart mitochondria upon inhibition of the ubiquinone (Q)-binding site by atpenin A5 (AA5) and Complex III by myxothiazol, respectively. The model predicts that only reduced flavin adenine dinucleotide (FADH2) in the unoccupied dicarboxylate state and flavin semiquinone radical (FADH•) feature the experimentally observed bell-shaped dependence of the rate of ROS production on the succinate concentration upon inhibition of respiratory Complex III (CIII) or Q-binding site of CII, i.e., suppression of succinate-Q reductase (SQR) activity. The other redox centers of CII such as Fe-S clusters and Q-binding site have a hyperbolic dependence of ROS formation on the succinate concentration with very small maximal rate under any condition and cannot be considered as substantial ROS generators in CII. Computer simulation results show that CII disintegration (which results in dissociation of the hydrophilic SDHA/SDHB subunits from the inner membrane to the mitochondrial matrix) causes crucial changes in the kinetics of ROS production by CII that are qualitatively and quantitatively close to changes in the kinetics of ROS production by assembled CII upon inhibition of CIII or Q-binding site of CII. Thus, the main conclusions from the present computational modeling study are the following: (i) the impairment of the SQR activity of CII resulting from inhibition of CIII or Q-binding site of CII and (ii) CII disintegration causes a transition in the succinate-dependence of ROS production from a small-amplitude sigmoid (hyperbolic) shape, determined by Q-binding site or [3Fe-4S] cluster to a high-amplitude bell-shaped kinetics with a shift to small subsaturated concentrations of succinate, determined by the flavin site.

Hysteresis and bistability in the succinate-CoQ reductase activity and reactive oxygen species production in the mitochondrial respiratory complex II.

The mitochondrial respiratory Complex II (CII) is one of key enzymes of cell energy metabolism, linking the tricarboxylic acid (TCA) cycle and the electron transport chain (ETC). CII reversibly oxidizes succinate to fumarate in the TCA cycle and transfers the electrons, produced by this reaction to the membrane quinone pool, providing ubiquinol QH2 to ETC. CII is also known as a generator of reactive oxygen species (ROS). It was shown experimentally that succinate can serve as not only a substrate in the forward succinate-quinone oxidoreductase (SQR) direction, but also an enzyme activator. Molecular and kinetic mechanisms of this property of CII are still unclear. In order to account for activation of CII by succinate in the forward SQR direction, we developed and analyzed a computational mechanistic model of electron transfer and ROS formation in CII. It was found that re-binding of succinate to the unoccupied dicarboxylate binding site when FAD is reduced with subsequent oxidation of FADH2 creates a positive feedback loop in the succinate oxidation. The model predicts that this positive feedback can result in hysteresis and bistable switches in SQR activity and ROS production in CII. This requires that the rate constant of re-binding of succinate has to be higher than the rate constant of the initial succinate binding to the active center when FAD is oxidized. Hysteresis and bistability in the SQR activity and ROS production in CII can play an important physiological role. In the presence of hysteresis with two stable branches with high and low SQR activity, high SQR activity is maintained even with a very strong drop in the succinate concentration, which may be necessary in the process of cell functioning in stressful situations. For the same reason, a high stationary rate of ROS production in CII can be maintained at low succinate concentrations.