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Title: Reactive Oxygen Species Production by Forward and Reverse Electron Fluxes in the Mitochondrial Respiratory Chain}
Article:
Abstract:
Reactive oxygen species( ROS) produced in the mitochondrial respiratory chain( RC) are primary signals that modulate cellular adaptation to environment, and are also destructive factors that damage cells under the conditions of hypoxia/reoxygenation relevant for various systemic diseases or transplantation. The important role of ROS in cell survival requires detailed investigation of mechanism and determinants of ROS production. To perform such an investigation we extended our rule-based model of complex III in order to account for electron transport in the whole RC coupled to proton translocation, transmembrane electrochemical potential generation, TCA cycle reactions, and substrate transport to mitochondria. It fits respiratory electron fluxes measured in rat brain mitochondria fueled by succinate or pyruvate and malate, and the dynamics of NAD+ reduction by reverse electron transport from succinate through complex I. The fitting of measured characteristics gave an insight into the mechanism of underlying processes governing the formation of free radicals that can transfer an unpaired electron to oxygen-producing superoxide and thus can initiate the generation of ROS. Our analysis revealed an association of ROS production with levels of specific radicals of individual electron transporters and their combinations in species of complexes I and III. It was found that the phenomenon of bistability, revealed previously as a property of complex III, remains valid for the whole RC. The conditions for switching to a state with a high content of free radicals in complex III were predicted based on theoretical analysis and were confirmed experimentally. These findings provide a new insight into the mechanisms of ROS production in RC.
Introduction:
Reactive oxygen species( ROS) are side products of electron transport in the mitochondrial respiratory chain, the principal component of energy transformation in mitochondria. ROS generation starts with the formation of a superoxide radical( O2−) as a result of interaction between molecular oxygen and free radicals, e. g. semiquinone( Q−): O2+Q−→O2−+Q[1]. This extremely active compound can be deactivated in cells, mainly through superoxide dismutase[2]. However, H2O2 formed in this process can interact with various intracellular compounds to produce ROS. ROS production serves as a metabolic signal[3]–[5]. However, when released in excess under certain stress conditions such as hypoxia/reoxygenation, ROS can also directly damage cells[6]. This destructive function of the electron transport chain represents the main problem in organ transplantation[7] and in many systemic diseases, as diverse as Parkinson disease[8] and diabetes[9]. The problem can be so great that in some organisms disruption of the electron transport chain can be a positive factor in increasing lifetime[10]. Although electron transport and coupled ROS production have been the focus of intensive research, important details are still not understood. There is currently debate regarding the relative contribution of various sites of the respiratory chain to overall ROS production[11],[12] and the factors that may alter this contribution[13]. The use of specific inhibitors can localize the sites of ROS production, but their contribution under normal and stress conditions without inhibitors in vivo is not clear. It is generally accepted that electron transport from succinate through complex I to NAD+, the phenomenon known as reverse electron transport[14], is important for respiration and ROS production[15],[16]. However, the mechanism of ROS production as a result of electron transfer upstream in the respiratory chain is not understood. Some details of the general mechanism of electron transport, such as the interaction of complex I with quinones that results in translocation of four protons through the membrane and reduction of one ubiquinone molecule per two electrons transported, remain the subject of discussions[17],[18]. Answering these questions will help in understanding the mechanisms of electron transport and coupled ROS production, and will be useful for advances in transplantology and therapy. The solution to such problems requires not only improvements in experimental techniques and new experiments, but also modification of methods for theoretical analysis. Specifically, kinetic modeling, which is an efficient method for investigating complex systems, still needs to be adopted for the mitochondrial respiratory chain. In fact, kinetic modeling in its classical form has been used for analysis of mitochondrial respiration. However, even the most detailed models[19] could consider only simplified scenarios. Huge number of differential equations is necessary to describe the behavior of respiratory complexes, so an automated procedure is required for their construction. Previously we developed a rule-based methodology for the automated construction of large systems of differential equations for analysis of 13C isotope tracing experiments in metabolic flux analysis[20]–[22]. We extended this methodology to the mathematical description of multienzyme complexes, specifically mitochondrial respiratory complex III based on a Q-cycle mechanism[23]. A detailed description of complex III operation revealed that in a certain range of parameters complex III has the property of bistability, where two different steady states exist for the same parameters and the system can reach one or the other, depending on its initial state. Perturbations, such as fluctuations in succinate concentrations or temporal hypoxia, can switch the system from low to high ROS producing steady state. Such a switch explains the damaging increase in ROS production on reoxygenation after hypoxia. The prediction of bistability for the mitochondrial respiratory chain was based on analysis of the Q-cycle mechanism for complex III. The contribution of other parts of the respiratory chain and linked processes that provide substrates must affect the properties of the respiratory chain. To study mitochondrial respiration as a whole, we extended the model of complex III[23]. The extended model includes the following elements: a detailed mathematical description of complex I; the stoichiometry of electron transport and proton translocation by the respiratory chain; the transmembrane potential; proton leak; oxidative phosphorylation; the TCA cycle that produces NADH and succinate as substrates for complexes I and III; and the transport of TCA cycle metabolites. The objective of this extension to the whole respiratory chain and linked processes was to create a tool for analysis of the basic behavior of the respiratory chain, in particular under conditions defining different fluxes in the forward and reverse directions. The ultimate aim was to reveal characteristics that have not been measured, such as the content of various free radicals, and thus to provide an insight into the relationship between states of the respiratory chain operation and ROS production.
Discussion:
To construct a detailed mathematical model that accounts for all redox states formed during electron and proton transport in complexes III and I, we used our rule-based methodology for automated construction of large systems of ODE[23]. This model further extends our methodology previously used to model the distribution of 13C isotopes in central metabolism[20]–[22], development of which occasionally coincided in time with that of similar rule-based methodology for signal transduction pathways[32],[33]. For the study of mitochondrial processes our methodology gives a deep insight into the mechanics of respiration and ROS production. Here, rule-based algorithms for mathematical description of mitochondrial respiration coupled to proton translocation and ΔΨ formation was linked to a classical kinetic model that accounts reactions of the TCA cycle, which provides succinate and NADH as substrates for respiration and substrate transport in mitochondria. After fixing the ratios of forward and reverse rate constants for electron transport reactions, free parameters were defined by fitting of forward and reverse electron flows measured under various conditions. High variability of parameters with a good fit to experimental data precluded definition of their values. However, the levels of free radicals calculated in the model showed much less variability. Different sets of parameters with a good fit to experimental data define very similar patterns for free radicals formed in complexes I and III. Thus, the analysis gives a valid insight into the mechanism of respiration and ROS production, even without precise evaluation of the model parameters. A substantial body of experimental data on mitochondrial ROS production cannot be satisfactorily explained within the current experimentally based paradigm. Some of these results were obscure, such as acceleration of succinate-driven ROS production after initial inhibition by rotenone( Figure 3). Others, such as a lower membrane potential in mitochondria fuelled by higher succinate concentration( Figure 9), were even counterintuitive. Calculation for mitochondrial constituents not measurable by current techniques represents a powerful tool for mechanistic explanation of accumulated data and for directing experimental research to test model predictions. A body of evidence indicate that either FMNH[29],[30], or SQ bound to Qn sites of complex I[30], or reduced N2 centers[30],[34],[35] may be a major contributor to ROS production, depending on the tissue, substrate, energy demand and oxygen tension[36],[37]. The simulations revealed correlations between measured ROS production rates and levels calculated for each type of free radical. In this first step of the study we did not assume any explicit link between any specific radical and ROS, but qualitatively compared all of them, taken separately, with measured ROS production. However, the method, which we use, opens a direction for future studies of quantitative contribution of various radicals of electron transporters, and even specific species of complex I and III, into total ROS production. The similarity between changes in the ROS production rate and in the levels of specific free radicals validates the model and also provides an insight into the mechanism of ROS production. Rotenone inhibition of ROS production in succinate-fueled mitochondria correlated with the free radicals formed in complex I, but not in complex III. Evidently, under the given conditions, reverse electron transport must contribute to free radical formation in complex I, although the net flux reducing NAD+ through complex I exists for only a very limited period of time. In accordance with our previous study that revealed bistability for complex III[23], the extended model confirms the existence of two steady states for the same set of parameters. In one of these states( oxidized), mitochondria can develop a maximal rate of respiration, ΔΨ, and a capacity for ATP synthesis. This is the usual working state. In the presence of pyruvate high succinate concentrations can induce a switch of respiration to the reduced steady state, where lack of electron acceptors strongly restricts electron flow. The levels of free radicals in complex III greatly increase in this state, but decrease in complex I, in contrast. The switch to a more reduced state results in ΔΨ decrease. Indeed, we observed a ΔΨ decrease in isolated mitochondria in conjunction with an increase of succinate concentrations in the presence of pyruvate. Q-cycle mechanism of complex III operation assumes bifurcation of electron flow at Qo site: one electron goes to Rieske center and further to complex IV, and another one reduces cytochrome b. This bifurcation of electron flow underlies the bifurcation between the two steady states. If in some moment the rate of first electron transition to Rieske center is higher than that for the second electron( because cytochrome b is reduced), semiquinones at Qo accumulate, thus preventing Qo liberation, binding and oxidation new molecules of ubiquinol, and thus limiting electron flow. In the case, shown in Figure 9A, greater proton leak resulted in greater transient discrepancy between the two electron flows at the point of bifurcation, which ultimately leaded to more significant inhibition of respiration and deeper descent of ΔΨ. The decrease of ΔΨ, in the case shown in Figure 9B, is relatively small, however, in living cardiomyocytes a much higher decrease of ΔΨ can be observed, accompanied by high ROS production, and associated with mitochondrial permeability transition( MPT)[38]. Although the presented study does not touch a possible link between bistability and MPT, it puts forward some hypotheses, which verification in future can essentially clarify the mechanism of MPT. There are at least two phenomena, which do not find appropriate explanation in terms of current state of knowledge. First, the increase of permeability in this process does not increase electron flux and proton recirculation, as in case of uncouplers. Second, ROS production is high despite the decrease of ΔΨ. If we assume that the switch into the reduced state precedes MPT, both phenomena would find a natural explanation. This hypothesis, although not proved yet, opens avenues for deeper investigation of the MPT mechanisms. The presented new insight into mitochondrial respiration was possible due to the application of novel methodology of modeling that allowed a detailed mathematical description of mitochondrial respiration. The phenomenon of bistability, predicted based on this methodology, has a potential to be a basis of new paradigm for the mechanism of ROS production, which will initiate new research with outcome on academic and practical levels.
Respiration at the level of mitochondria is considered as delivery of electrons and protons from NADH or succinate to oxygen through a set of transporters constituting the respiratory chain ( RC ) . Mitochondrial respiration , dealing with transfer of unpaired electrons , may produce reactive oxygen species ( ROS ) such as O2− and subsequently H2O2 as side products . ROS are chemically very active and can cause oxidative damage to cellular components . The production of ROS , normally low , can increase under stress to the levels incompatible with cell survival; thus , understanding the ways of ROS production in the RC represents a vital task in research . We used mathematical modeling to analyze experiments with isolated brain mitochondria aimed to study relations between electron transport and ROS production . Elsewhere we reported that mitochondrial complex III can operate in two distinct steady states at the same microenvironmental conditions , producing either low or high levels of ROS . Here , this property of bistability was confirmed for the whole RC . The associations between measured ROS production and computed individual free radical levels in complexes I and III were established . The discovered phenomenon of bistability is important as a basis for new strategies in organ transplantation and therapy .