Effect of enzyme deficiencies on oxidative phosphorylation: from isolated mitochondria to intact tissues. Theoretical studies. Bernard Korzeniewski Institute of Molecular Biology and Biotechnology, Jagiellonian University, ul. Gronostajowa 7, 30-387 Kraków, Poland * Correspondence: tel. (48 12) 252 63 73. fax. (48 12) 252 69 02, e-mail: benio@mol.uj.edu.pl Keywords: oxidative phosphorylation, mitochondria, enzyme deficiencies, mitochondrial diseases, heteroplasmy, tissue specificity, computer model. Abstract The present article briefly summarizes the theoretical studies made by the authors and co-workers on the effect of inborn enzyme deficiencies on oxidative phosphorylation in intact tissues and on the genesis of mitochondrial diseases. The dynamic computer model of oxidative phosphorylation developed previously allowed to extrapolate experimental data (especially: threshold curves describing the dependence of oxygen consumption and ATP turnover on activities/concentrations of particular oxidative phosphorylation enzymes) obtained for isolated muscle mitochondria in state 3 at saturating oxygen concentrations to more physiological conditions prevailing in intact tissues. In particular, theoretical studies demonstrated that the threshold value of the relative activity/concentration of a given mitochondrial complex, below which a significant decrease in the respiration rate takes place, increases with an increase in energy demand. This fact was proposed as a possible explanation of the tissue specificity of mitochondrial diseases. Additionally, a decreased oxygen concentration was shown to increase the threshold value (and flux control coefficient) for cytochrome oxidase. We subsequently developed a model called binary mitochondria heteroplasmy, in which there are only two subpopulations of mitochondria: one wildtype and one containing only defected molecules of a given enzyme. In this model we show that a defect has a pronounced effect on oxidative phosphorylation, significantly increasing the threshold value. It was also proposed that a parallel activation in the ATP supply-demand system during an increased energy demand significantly lessens the effect of enzyme deficiencies on oxidative phosphorylation (decreases the threshold value). Finally, the necessity of substrate activation may lead to an instability in the system and to appearance of a second threshold, below which respiration suddenly drops to zero, which is equivalent to the energetic death of a cell. Introduction Inborn deficiencies in different oxidative phosphorylation complexes caused by mutations in mtdna may lead to various mitochondrial diseases (Wallace, 1993; Mazat et al., 2001). The characteristic property of these diseases is that essentially no pathological effect is observed when the activity of a given complex decreases to a level that is greater than some threshold value, while a significant disorder in the functioning
of the system takes place when the complex activity drops below this threshold value. At the same time, this value seems to be rather low, lower than 50 % or even 30 % of the wild-type (normal) activity of particular complexes (Mazat et al., 2001). Another characteristic feature of mitochondrial diseases is their tissue specificity, i.e. the fact that, at a given level of enzyme deficiency, the illness is manifested preferentially in some tissues (skeletal muscle, brain for instance), while other tissues (liver, heart for instance) are relatively less affected. The threshold effect in the expression of enzyme deficiencies was discussed in the context of Metabolic Control Analysis and related to the threshold curves representing the dependence of the respiration rate on the complex activity during titration of particular complexes with specific inhibitors in isolated mitochondria in state 3 (maximal oxygen consumption and ATP turnover) at saturating oxygen concentrations (Letellier et al., 1993; Letellier et al., 1994). The threshold curves for particular mitochondrial complexes were next simulated with the use of the version of the computer model of oxidative phosphorylation for isolated skeletal muscle mitochondria (Korzeniewski & Mazat, 1996a). A good agreement between computer simulations and experimental results was observed, suggesting that the model describes correctly largescale changes in fluxes and metabolite concentrations. Nevertheless, the quantitative results concerning the threshold curves (especially: the threshold values) for particular complexes in isolated mitochondria working in state 3 at saturating oxygen concentrations cannot be directly extrapolated to the conditions prevailing in intact tissues in vivo. There is quite a broad set of special conditions (parameter values), regulatory mechanisms and phenomena that are present in intact tissues but absent from the isolated mitochondria system. Theoretical studies made with the use of the previously-developed model of oxidative phosphorylation (Korzeniewski & Mazat, 1996a) helped much to understand better the differences in the behaviour of the system in isolated mitochondria and in more physiological conditions. Theoretical results and discusion The influence of the properties of the system characteristic for intact tissues, but not for isolated mitochondria, was subsequently analyzed in subsequent theoretical studies. First, it was shown that the threshold values of the activity of different complexes are essentially lower in state 3.5 (respiration and ATP turnover intermediate between state 4 and state 3) than in state 3 (Korzeniewski & Mazat, 1996a, Malgat et al. 2000), because most of the control is devoted to ATP consumption (by hexokinase in our model and by myokinase in experiments). The ATP/ADP ratio in state 3.5 much more than the ATP/ADP ratio in state 3 resembles the ATP/ADP ratio in intact tissues, and therefore state 3.5 seems to be more relevant for physiological conditions than state 3. Additionally, the saturating (240 mm) oxygen concentration applied in the isolated mitochondria system is significantly higher than the oxygen concentration present in intact tissues, where, for example in skeletal muscle during prolonged intensive exercise, it may drop to a few mm, or even to a lower value. Theoretical studies demonstrated that a diminished oxygen concentration can significantly increase the threshold value of the activity (concentration) of cytochrome oxidase (complex IV of the respiratory chain) (Korzeniewski & Mazat, 1996a; Korzeniewski & Mazat, 1996b).
During titration of some mitochondrial complex in the above-mentioned experimental studies (Letellier et al., 1993; Letellier et al., 1994), the molecules of such a complex were homogeneously affected in different mitochondria for example, a given dose of an inhibitor blocked 40 % of molecules of some enzyme in each mitochondrion. However, it is likely that a completely different situation takes place in the case of inborn deficiencies in the (subunits of the) respiratory complexes encoded in mitochondrial DNA (mtdna). This is because random segregation of wild-type and mutated mtdna molecules to daughter mitochondria during mitochondria divisions should lead after a few division to generation of two pure mitochondria lines: one wildtype and one mutated (Korzeniewski et al., 2001). In such a case, named binary mitochondria heteroplasmy, all the defected molecules of a given complex will be collected in the mutated subpopulation of mitochondria, what will lead to a complete inactivation of these mitochondria. Computer-aided theoretical studies demonstrated that this fact has a pronounced impact on the expression of a mutation the binary mitochondria heteroplasmy causes a significant increase in the threshold value of each mutated complex (Korzeniewski et al., 2001). Additionally, in this case the threshold value is identical for each complex. On the other hand, enzyme deficiencies caused by mutations in nucleus-encoded subunits are homogeneously distributed among mitochondria and in this aspect they resemble titrations of different complexes with specific inhibitors in isolated mitochondria. Theoretical studies demonstrated also that, in the case of the binary mitochondria heteroplasmy, the threshold value in the amount of wild-type (not containing mutated mtdna molecules and defected complexes) mitochondria depends strongly on the relative energy demand: the greater the energy demand, the higher the threshold value (Korzeniewski et al., 2001). This phenomenon may contribute significantly to the tissue specificity of mitochondria diseases: these diseases are expressed first of all in tissues with high relative energy demand, such as skeletal muscle and brain. It has been demonstrated that there is very little extra capacity of oxidative phosphorylation in intact tissues. Intact heart can use 80-90 % of the maximal capacity of oxidative phosphorylation in isolated heart mitochondria (Motha et al., 1986). In skeletal muscle the maximal respiration rate seems to be even greater than the maximal capacity of oxidative phosphorylation in isolated mitochondria (Tonkonogi & Sahlin, 1997). On the other hand, as it was discussed above, a very significant decrease in the activity/concentration of some mitochondrial complex in the result of an inborn mutation, even to less than 30 % of its normal (wild-type) value, may have no pathological effect on the functioning of an organism. These two sets of experimental data seem to remain in contradiction. This apparent paradox may be resolved using the discussed computer model of oxidative phosphorylation. In isolated mitochondria, the oxidative phosphorylation complexes are activated only indirectly, mostly via an increase in ADP concentration. However, as it was discussed elsewhere (Korzeniewski, 1998), theoretical interpretations of the existing experimental data strongly suggest that (almost) all mitochondrial complexes are directly activated by some external (cytosolic) factor(s), which transmit(s) the signal from neural or hormonal stimulation. Of course, such an activation increases the effective capacity of oxidative phosphorylation in intact tissues in relation to the capacity of this process in isolated mitochondria. Additionally,
as it was demonstrated later, direct activation of mitochondria decreases significantly the threshold value of the amount of the wild-type (not mutated) mitochondria to the value below 30 % of the normal amount of active mitochondria (Korzeniewski, 2002). Thus, the apparent paradox between the small (or none at all) extra capacity of oxidative phosphorylation in isolated mitochondria and the low threshold value of the activity of oxidative phosphorylation in intact tissues, below which a disorder in the functioning of the system appears, vanishes when the parallel activation hypothesis is taken into account. In intact tissues, some ATP must be first used for substrate (glucose, fatty acids) activation in order to produce later much more ATP in glycolysis, tricarboxylate acid cycle and, especially, in oxidative phosphorylation. On the other hand, isolated mitochondria are usually provided with already activated substrates, being intermediate metabolites of tricarboxylate acid cycle or glycolysis, such as succinate, 2- oxoglutarate or pyruvate. The phenomenon of substrate activation causes that, at low ATP concentrations, the system (in particular, the substrate dehydrogenation block) is activated by its own product, ATP. This, in turn, leads to a positive feedback and may cause a instability in the system. Such a destabilizing effect of ATP feedback in glycolysis was discussed by Selkov (1975). Appropriate computer simulations using the version of the model in which the phenomenon of substrate activation was taken into account, actually showed that such a instability may appear in some circumstances (Korzeniewski, submitted for publication). As a result, a second threshold appears in the threshold curve illustrating the dependence of the respiration rate on the relative activity (concentration) of mitochondria: below a certain threshold value there takes place a sudden drop of the respiration rate to zero and an abrupt collapse of the phosphorylation potential. This irreversible event may serve as an approximate definition of the cell death in the bioenergetic aspect. It was also suggested that, while the normal threshold may be relevant to mild mitochondrial diseases, the instability threshold may be responsible for acute mitochondrial diseases (Korzeniewski, submitted for publication). Theoretical studies showed that the discussed collapse appears only in the case of a low sensitivity of ATP usage to [ATP] or the ATP/ADP ratio. Tissues differ in the sensitivity of their ATP-usage block to [ATP]: in some tissues, like muscle, ATP usage seems to be little sensitive to [ATP], while in other tissues, like liver, ATP usage is much more sensitive to [ATP] (or ATP/ADP). This fact suggests, in the abovediscussed context of substrate activation, that different sensitivity of ATP usage to [ATP] may contribute to the phenomenon of tissue specificity of mitochondrial diseases. Computer simulations suggest also that the parallel activation in the ATP supply-atp demand system lessens the effect of physiological inhibitors (e.g. NO), poisons (KCN, CO, heavy metals, alcohol, chloroform), of decreased oxygen concentration and of substrate shortage on oxidative phosphorylation in vivo (Korzeniewski, 2002). Substrate activation influences significantly the effect of the above-mentioned factors and an increase in energy demand on oxidative phosphorylation (Korzeniewski, submitted for publication). To sum up, computer simulations performed with the use of the discussed model of oxidative phosphorylation (Korzeniewski & Mazat, 1996a) helped to approach gradually the effect of inactivation of mitochondrial complexes on oxidative
phosphorylation in intact tissues, starting from threshold curves of particular complexes titrated with specific inhibitors in state 3 and at saturating oxygen concentration in the isolated mitochondria system (Letellier et al., 1993; Letellier et al., 1994). To do this, several factors were taken into account that can not be easily studied in the experimental way in the isolated mitochondria system, namely: (1) uneven distribution of mutated mtdna molecules (and therefore defective enzyme molecules) among mitochondria (binary mitochondria heteroplasmy); (2) different levels of relative energy demand in different tissues; (3) parallel activation in the ATP supply-atp demand system during an increase in energy demand; (4) lowered (not saturating) oxygen concentration; (5) substrate activation. These theoretical studies show that, although isolated mitochondria titrated with specific inhibitors constitute a good conceptual model of inborn enzyme deficiencies in the qualitative sense, several other factors present in intact tissues may significantly affect the quantitative pattern of the behaviour of the system. The description of the model of oxidative phosphorylation used in the discussed studies is located at the web site: http://www.mol.uj.edu.pl/~benio/ Acknowledgements The author is very grateful to Jean-Pierre Mazat for a stimulating discussion. B.K. was supported by the 6P04A07120 KBN (Polish State Committee for Scientific Research) grant. References Korzeniewski, B., 1998. Regulation of ATP supply during muscle contraction: theoretical studies. Biochem. J. 330, 1189-1195. Korzeniewski, B., 2002. Parallel activation in the ATP supply-demand system lessens the impact of inborn enzyme deficiencies, inhibitors, poisons or substrate shortage on oxidative phosphorylation in vivo. Biophys. Chem. 96: 21-31. Korzeniewski, B., Influence of substrate activation on the effect of enzyme deficiencies, inhibitors, substrate shortage and energy demand on oxidative phosphorylation. [submitted for publication]. Korzeniewski, B. & J.-P. Mazat, 1996a. Theoretical studies on the control of oxidative phosphorylation in muscle mitochondria: application to mitochondrial deficiencies. Biochem. J. 319: 143-148. Korzeniewski, B. & J.-P. Mazat, 1996b. Theoretical studies on control of oxidative phosphorylation in muscle mitochondria at different energy demands and oxygen concentrations. Acta Biotheor. 44: 263-269. Korzeniewski, B., M. Malgat, T. Letellier & J.-P. Mazat, 2001. Effect of binary mitochondria heteroplasmy on respiration and ATP synthesis: implications for mitochondrial diseases. Biochem. J. 357: 835-842. Letellier, T., M. Malgat & J-P Mazat, 1993. Control of oxidative phosphorylation in rat muscle mitochondria: implications for mitochondrial myopathies. Biochim. Biophys. Acta 1141: 58-64. Letellier, T., R. Heinrich, M. Malgat & J.-P. Mazat, 1994. The kinetic basis of threshold effects observed in mitochondrial diseases: a systemic approach. Biochem. J. 302: 171-174. Malgat, M., Letellier, T., Rossignol, R. and Mazat, J.-P., 2000. Metabolic control analysis and threshold effect in oxidative phosphorylation: study in an intermediate state of respiration. 9th International BioThermoKinetics Meeting, Hofmeyr, J.-H. S., Rohwer, J. M. and Snoep, J. L. eds., Stellenbosch University Press Stellenbosch, South Africa pp. 125-134.
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