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Mitochondrial and thus cellular energetics are highly regulated both thermodynamically and kinetically. Cellular energetics is of prime importance in the regulation of cellular functions since it provides ATP for their accomplishment. However, cellular energetics is not only about ATP production but also about the ability to re-oxidize reduced coenzymes at a proper rate, such that the cellular redox potential remains at a level compatible with enzymatic reactions. However, this parameter is not only difficult to assess due to its dual compartmentation (mitochondrial and cytosolic) but also because it is well known that most NADH in the cells is bound to the enzymes. In this paper, we investigated the potential relevance of mitochondrial quinones redox state as a marker of mitochondrial metabolism and more particularly mitochondrial redox state. We were able to show that Q 2 is an appropriate redox mediator to assess the mitochondrial quinone redox states. On isolated mitochondria, the mitochondrial quinone redox states depend on the mitochondrial substrate and the mitochondrial energetic state (phosphorylating or not phosphorylating). Last but not least, we show that the quinones redox state response allows to better understand the Krebs cycle functioning and respiratory substrates oxidation. Taken together, our results suggest that the quinones redox state is an excellent marker of mitochondrial metabolism., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Metabolic Control Theory (MCT) and Metabolic Control Analysis (MCA) are the two sides, theoretical and experimental, of the measurement of the sensitivity of metabolic networks in the vicinity of a steady state. We will describe the birth and the development of this theory from the first analyses of linear pathways up to a global mathematical theory applicable to any metabolic network. We will describe how the theory, given the global nature of mitochondrial oxidative phosphorylation, solved the problem of what controls mitochondrial ATP synthesis and then how it led to a better understanding of the differential tissue expression of human mitochondrial pathologies and of the heteroplasmy of mitochondrial DNA, leading to the concept of the threshold effect., Competing Interests: Declaration of competing interest The author declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Cancer cells display an altered energy metabolism, which was proposed to be the root of cancer. This early discovery was done by O. Warburg who conducted one of the first studies of tumor cell energy metabolism. Taking advantage of cancer cells that exhibited various growth rates, he showed that cancer cells display a decreased respiration and an increased glycolysis proportional to the increase in their growth rate, suggesting that they mainly depend on fermentative metabolism for ATP generation. Warburg's results and hypothesis generated controversies that are persistent to this day. It is thus of great importance to understand the mechanisms by which cancer cells can reversibly regulate the two pathways of their energy metabolism as well as the functioning of this metabolism in cell proliferation. In this review, we discuss of the origin of the decrease in cell respiratory rate, whether the Warburg effect is mandatory for an increased cell proliferation rate, the consequences of this effect on two major players of cell energy metabolism that are ATP and NADH, and the role of the microenvironment in the regulation of cellular respiration and metabolism both in cancer cell and in yeast., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

One-carbon metabolism (1C-metabolism), also called folate metabolism because the carbon group is attached to folate-derived tetrahydrofolate, is crucial in metabolism. It is at the heart of several essential syntheses, particularly those of purine and thymidylate. After a short reminder of the organization of 1C-metabolism, I list its salient features as reported in the literature. Then, using flux balance analysis, a core model of central metabolism and the flux constraints for an 'average cancer cell metabolism', I explore the fundamentals underlying 1C-metabolism and its relationships with the rest of metabolism. Some unreported properties of 1C-metabolism emerge, such as its potential roles in mitochondrial NADH exchange with cytosolic NADPH, participation in NADH recycling, and optimization of cell proliferation., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

In living cells, growth is the result of coupling between substrate catabolism and multiple metabolic processes that take place during net biomass formation and maintenance processes. During growth, both ATP/ADP and NADH/NAD + molecules play a key role. Cell energy metabolism hence refers to metabolic pathways involved in ATP synthesis linked to NADH turnover. Two main pathways are thus involved in cell energy metabolism: glycolysis/fermentation and oxidative phosphorylation. Glycolysis and mitochondrial oxidative phosphorylation are intertwined through thermodynamic and kinetic constraints that are reviewed herein. Further, our current knowledge of short-term and long term regulation of cell energy metabolism will be reviewed using examples such as the Crabtree and the Warburg effect., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

ROS (superoxide and oxygen peroxide in this paper) play a dual role as signalling molecules and strong oxidizing agents leading to oxidative stress. Their production mainly occurs in mitochondria although they may have other locations (such as NADPH oxidase in particular cell types). Mitochondrial ROS production depends in an interweaving way upon many factors such as the membrane potential, the cell type and the respiratory substrates. Moreover, it is experimentally difficult to quantitatively assess the contribution of each potential site in the respiratory chain. To overcome these difficulties, mathematical models have been developed with different degrees of complexity in order to analyse different physiological questions ranging from a simple reproduction/simulation of experimental results to a detailed model of the possible mechanisms leading to ROS production. Here, we analyse experimental results concerning ROS production including results still under discussion. We then critically review the three models of ROS production in the whole respiratory chain available in the literature and propose some direction for future modelling work.

Protein synthesis and degradation are essential processes that regulate cell status. Because labeling in bulky organs, such as fruits, is difficult, we developed a modeling approach to study protein turnover at the global scale in developing tomato ( Solanum lycopersicum ) fruit. Quantitative data were collected for transcripts and proteins during fruit development. Clustering analysis showed smaller changes in protein abundance compared to mRNA abundance. Furthermore, protein and transcript abundance were poorly correlated, and the coefficient of correlation decreased during fruit development and ripening, with transcript levels decreasing more than protein levels. A mathematical model with one ordinary differential equation was used to estimate translation ( k t ) and degradation ( k d ) rate constants for almost 2,400 detected transcript-protein pairs and was satisfactorily fitted for >1,000 pairs. The model predicted median values of ∼2 min for the translation of a protein, and a protein lifetime of ∼11 d. The constants were validated and inspected for biological relevance. Proteins involved in protein synthesis had higher k t and k d values, indicating that the protein machinery is particularly flexible. Our model also predicts that protein concentration is more strongly affected by the rate of translation than that of degradation., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Genome-scale models of metabolism (GEM) are used to study how metabolism varies in different physiological conditions. However, the great number of reactions involved in GEM makes it difficult to understand these variations. In order to have a more understandable tool, we developed a reduced metabolic model of central carbon and nitrogen metabolism, C2M2N with 77 reactions, 54 internal metabolites, and 3 compartments, taking into account the actual stoichiometry of the reactions, including the stoichiometric role of the cofactors and the irreversibility of some reactions. In order to model oxidative phosphorylation (OXPHOS) functioning, the proton gradient through the inner mitochondrial membrane is represented by two pseudometabolites DPH (∆pH) and DPSI (∆ψ). To illustrate the interest of such a reduced and quantitative model of metabolism in mammalian cells, we used flux balance analysis (FBA) to study all the possible fates of glutamine in metabolism. Our analysis shows that glutamine can supply carbon sources for cell energy production and can be used as carbon and nitrogen sources to synthesize essential metabolites. Finally, we studied the interplay between glucose and glutamine for the formation of cell biomass according to ammonia microenvironment. We then propose a quantitative analysis of the Warburg effect.

Evidence for the Crabtree effect was first reported by H. Crabtree in 1929 and is defined as the glucose-induced decrease of cellular respiratory flux. This effect was observed in tumor cells and was not detected in most non-tumor cells. A number of hypotheses on the mechanism underlying the Crabtree effect have been formulated. However, to this day, no consensual mechanism for this effect has been described. In a previous study on isolated mitochondria, we have proposed that fructose-1,6-bisphosphate (F1,6bP), which inhibits the respiratory chain, induces the Crabtree effect. Using whole cells from the yeast Saccharomyces cerevisiae as a model, we show here not only that F1,6bP plays a key role in the process but that glucose-6-phosphate (G6P), a hexose that has an effect opposite to that of F1,6bP on the regulation of the respiratory flux, does as well. Thus, these findings reveal that the Crabtree effect strongly depends on the ratio between these two glycolysis-derived hexose phosphates. Last, in silico modeling of the Crabtree effect illustrated the requirement of an inhibition of the respiratory flux by a coordinated variation of glucose-6-phosphate and fructose-1,6-bisphosphate to fit the respiratory rate decrease observed upon glucose addition to cells. In summary, we conclude that two glycolysis-derived hexose phosphates, G6P and F1,6bP, play a key role in the induction of the Crabtree effect., (© 2018 Rosas Lemus et al.)

Background: The complexity of metabolic networks can make the origin and impact of changes in central metabolism occurring during diseases difficult to understand. Computer simulations can help unravel this complexity, and progress has advanced in genome-scale metabolic models. However, many models produce unrealistic results when challenged to simulate abnormal metabolism as they include incorrect specification and localisation of reactions and transport steps, incorrect reaction parameters, and confounding of prosthetic groups and free metabolites in reactions. Other common drawbacks are due to their scale, making them difficult to parameterise and simulation results hard to interpret. Therefore, it remains important to develop smaller, manually curated models., Results: We present MitoCore, a manually curated constraint-based computer model of human metabolism that incorporates the complexity of central metabolism and simulates this metabolism successfully under normal and abnormal physiological conditions, including hypoxia and mitochondrial diseases. MitoCore describes 324 metabolic reactions, 83 transport steps between mitochondrion and cytosol, and 74 metabolite inputs and outputs through the plasma membrane, to produce a model of manageable scale for easy interpretation of results. Its key innovations include a more accurate partitioning of metabolism between cytosol and mitochondrial matrix; better modelling of connecting transport steps; differentiation of prosthetic groups and free co-factors in reactions; and a new representation of the respiratory chain and the proton motive force. MitoCore's default parameters simulate normal cardiomyocyte metabolism, and to improve usability and allow comparison with other models and types of analysis, its reactions and metabolites have extensive annotation, and cross-reference identifiers from Virtual Metabolic Human database and KEGG. These innovations-including over 100 reactions absent or modified from Recon 2-are necessary to model central metabolism more accurately., Conclusion: We anticipate MitoCore as a research tool for scientists, from experimentalists looking to interpret their data and test hypotheses, to experienced modellers predicting the consequences of disease or using computationally intensive methods that are infeasible with larger models, as well as a teaching tool for those new to modelling and needing a small, manageable model on which to learn and experiment.

Tomato is a model organism to study the development of fleshy fruit including ripening initiation. Unfortunately, few studies deal with the brief phase of accelerated ripening associated with the respiration climacteric because of practical problems involved in measuring fruit respiration. Because constraint-based modelling allows predicting accurate metabolic fluxes, we investigated the respiration and energy dissipation of fruit pericarp at the breaker stage using a detailed stoichiometric model of the respiratory pathway, including alternative oxidase and uncoupling proteins. Assuming steady-state, a metabolic dataset was transformed into constraints to solve the model on a daily basis throughout tomato fruit development. We detected a peak of CO 2 released and an excess of energy dissipated at 40 d post anthesis (DPA) just before the onset of ripening coinciding with the respiration climacteric. We demonstrated the unbalanced carbon allocation with the sharp slowdown of accumulation (for syntheses and storage) and the beginning of the degradation of starch and cell wall polysaccharides. Experiments with fruits harvested from plants cultivated under stress conditions confirmed the concept. We conclude that modelling with an accurate metabolic dataset is an efficient tool to bypass the difficulty of measuring fruit respiration and to elucidate the underlying mechanisms of ripening., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Modelling of metabolic networks is a powerful tool to analyse the behaviour of developing plant organs, including fruits. Guided by our current understanding of heterotrophic metabolism of plant cells, a medium-scale stoichiometric model, including the balance of co-factors and energy, was constructed in order to describe metabolic shifts that occur through the nine sequential stages of Solanum lycopersicum (tomato) fruit development. The measured concentrations of the main biomass components and the accumulated metabolites in the pericarp, determined at each stage, were fitted in order to calculate, by derivation, the corresponding external fluxes. They were used as constraints to solve the model by minimizing the internal fluxes. The distribution of the calculated fluxes of central metabolism were then analysed and compared with known metabolic behaviours. For instance, the partition of the main metabolic pathways (glycolysis, pentose phosphate pathway, etc.) was relevant throughout fruit development. We also predicted a valid import of carbon and nitrogen by the fruit, as well as a consistent CO2 release. Interestingly, the energetic balance indicates that excess ATP is dissipated just before the onset of ripening, supporting the concept of the climacteric crisis. Finally, the apparent contradiction between calculated fluxes with low values compared with measured enzyme capacities suggest a complex reprogramming of the metabolic machinery during fruit development. With a powerful set of experimental data and an accurate definition of the metabolic system, this work provides important insight into the metabolic and physiological requirements of the developing tomato fruits., (© 2014 The Authors The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Victor Henri's great contribution to the understanding of enzyme kinetics and mechanism is not always given the credit that it deserves. In addition, his earlier work in experimental psychology is totally unknown to biochemists, and his later work in spectroscopy and photobiology almost equally so. Applying great rigour to his analysis he succeeded in obtaining a model of enzyme action that explained all of the observations available to him, and he showed why the considerable amount of work done in the preceding decade had not led to understanding. His view was that only physical chemistry could explain the behaviour of enzymes, and that models should be judged in accordance with their capacity not only to explain previously known facts but also to predict new observations against which they could be tested. The kinetic equation usually attributed to Michaelis and Menten was in reality due to him. His thesis of 1903 is now available in English., (Copyright © 2014 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.)

The mitochondrial respiratory chain plays a crucial role in energy metabolism and its dysfunction is implicated in a wide range of human diseases. In order to understand the global expression of local mutations in the rate of oxygen consumption or in the production of adenosine triphosphate (ATP) it is useful to have a mathematical model in which the changes in a given respiratory complex are properly modeled. Our aim in this paper is to provide thermodynamics respecting and structurally simple equations to represent the kinetics of each isolated complexes which can, assembled in a dynamical system, also simulate the behavior of the respiratory chain, as a whole, under a large set of different physiological and pathological conditions. On the example of the reduced nicotinamide adenine dinucleotide (NADH)-ubiquinol-oxidoreductase (complex I) we analyze the suitability of different types of rate equations. Based on our kinetic experiments we show that very simple rate laws, as those often used in many respiratory chain models, fail to describe the kinetic behavior when applied to a wide concentration range. This led us to adapt rate equations containing the essential parameters of enzyme kinetic, maximal velocities and Henri-Michaelis-Menten like-constants (KM and KI) to satisfactorily simulate these data., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A kinetic model combining enzyme activity measurements and subcellular compartmentation was parameterized to fit the sucrose, hexose, and glucose-6-P contents of pericarp throughout tomato (Solanum lycopersicum) fruit development. The model was further validated using independent data obtained from domesticated and wild tomato species and on transgenic lines. A hierarchical clustering analysis of the calculated fluxes and enzyme capacities together revealed stage-dependent features. Cell division was characterized by a high sucrolytic activity of the vacuole, whereas sucrose cleavage during expansion was sustained by both sucrose synthase and neutral invertase, associated with minimal futile cycling. Most importantly, a tight correlation between flux rate and enzyme capacity was found for fructokinase and PPi-dependent phosphofructokinase during cell division and for sucrose synthase, UDP-glucopyrophosphorylase, and phosphoglucomutase during expansion, thus suggesting an adaptation of enzyme abundance to metabolic needs. In contrast, for most enzymes, flux rates varied irrespectively of enzyme capacities, and most enzymes functioned at <5% of their maximal catalytic capacity. One of the major findings with the model was the high accumulation of soluble sugars within the vacuole together with organic acids, thus enabling the osmotic-driven vacuole expansion that was found during cell division., (© 2014 American Society of Plant Biologists. All rights reserved.)

To assess the influence of the environment on fruit metabolism, tomato (Solanum lycopersicum 'Moneymaker') plants were grown under contrasting conditions (optimal for commercial, water limited, or shaded production) and locations. Samples were harvested at nine stages of development, and 36 enzyme activities of central metabolism were measured as well as protein, starch, and major metabolites, such as hexoses, sucrose, organic acids, and amino acids. The most remarkable result was the high reproducibility of enzyme activities throughout development, irrespective of conditions or location. Hierarchical clustering of enzyme activities also revealed tight relationships between metabolic pathways and phases of development. Thus, cell division was characterized by high activities of fructokinase, glucokinase, pyruvate kinase, and tricarboxylic acid cycle enzymes, indicating ATP production as a priority, whereas cell expansion was characterized by enzymes involved in the lower part of glycolysis, suggesting a metabolic reprogramming to anaplerosis. As expected, enzymes involved in the accumulation of sugars, citrate, and glutamate were strongly increased during ripening. However, a group of enzymes involved in ATP production, which is probably fueled by starch degradation, was also increased. Metabolites levels seemed more sensitive than enzymes to the environment, although such differences tended to decrease at ripening. The integration of enzyme and metabolite data obtained under contrasting growth conditions using principal component analysis suggests that, with the exceptions of alanine amino transferase and glutamate and malate dehydrogenase and malate, there are no links between single enzyme activities and metabolite time courses or levels.

Methods and equations for analysing the kinetics of enzyme-catalysed reactions were developed at the beginning of the 20th century in two centres in particular; in Paris, by Victor Henri, and, in Berlin, by Leonor Michaelis and Maud Menten. Henri made a detailed analysis of the work in this area that had preceded him, and arrived at a correct equation for the initial rate of reaction. However, his approach was open to the important objection that he took no account of the hydrogen-ion concentration (a subject largely undeveloped in his time). In addition, although he wrote down an expression for the initial rate of reaction and described the hyperbolic form of its dependence on the substrate concentration, he did not appreciate the great advantages that would come from analysis in terms of initial rates rather than time courses. Michaelis and Menten not only placed Henri's analysis on a firm experimental foundation, but also defined the experimental protocol that remains standard today. Here, we review this development, and discuss other scientific contributions of these individuals. The three parts have different authors, as indicated, and do not necessarily agree on all details, in particular about the relative importance of the contributions of Michaelis and Menten on the one hand and of Henri on the other. Rather than force the review into an unrealistic consensus, we consider it appropriate to leave the disagreements visible., (© 2013 FEBS.)

The bc1 complex or complex III is a central component of the aerobic respiratory chain in prokaryotic and eukaryotic organisms. It catalyzes the oxidation of quinols and the reduction of cytochrome c, establishing a proton motive force used to synthesize adenosine triphosphate (ATP) by the F1Fo ATP synthase. In eukaryotes, the complex III is located in the inner mitochondrial membrane. The genes coding for the complex III have a dual origin. While cytochrome b is encoded by the mitochondrial genome, all the other subunits are encoded by the nuclear genome. In this review, we compile an exhaustive list of the known human mutations and associated pathologies found in the mitochondrially-encoded cytochrome b gene as well as the fewer mutations in the nuclear genes coding for the complex III structural subunits and accessory proteins such as BCS1L involved in the assembly of the complex III. Due to the inherent difficulties of studying human biopsy material associated with complex III dysfunction, we also review the work that has been conducted to study the pathologies with the easy to handle eukaryotic microorganism, the yeast Saccharomyces cerevisiae. Phenotypes, biochemical data and possible effects due to the mutations are also discussed in the context of the known three-dimensional structure of the eukaryotic complex III. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In nonphotosynthetic organisms, mitochondria are the power plant of the cell, emphasizing their great potentiality for adenosine triphosphate (ATP) synthesis from the redox span between nutrients and oxygen. Also of great importance is their role in the maintenance of the cell redox balance. Even though crystallographic structures of respiratory complexes, ATP synthase, and ATP/adenosine diphosphate (ADP) carrier are now quite well known, the coupling between ATP synthesis and cell redox state remains a controversial issue. In this review, we will present some of the processes that allow a modular coupling between ATP synthesis and redox state. Furthermore, we will present some theoretical approaches of this highly integrated system., (Copyright © 2013 International Union of Biochemistry and Molecular Biology, Inc.)

The respiratory chain is located in the inner membrane of mitochondria and produces the major part of the ATP used by a cell. Cardiolipin (CL), a double charged phospholipid composing ~10-20% of the mitochondrial membrane, plays an important role in the function and supramolecular organization of the respiratory chain complexes. We present an extensive set of coarse-grain molecular dynamics (CGMD) simulations aiming at the determination of the preferential interfaces of CLs on the respiratory chain complex III (cytochrome bc(1), CIII). Six CL binding sites are identified, including the CL binding sites known from earlier structural studies and buried into protein cavities. The simulations revealed the importance of two subunits of CIII (G and K in bovine heart) for the structural integrity of these internal CL binding sites. In addition, new binding sites are found on the membrane-exposed protein surface. The reproducibility of these binding sites over two species (bovine heart and yeast mitochondria) points to an important role for the function of the respiratory chain. Interestingly the membrane-exposed CL binding sites are located on the matrix side of CIII in the inner membrane and thus may provide localized sources of proton ready for uptake by CIII. Furthermore, we found that CLs bound to those membrane-exposed sites bridge the proteins during their assembly into supercomplexes by sharing the binding sites.

An enzyme's activity is the consequence of its structure. The stochastic approach we developed to study the functioning of the respiratory complexes is based upon their 3D structure and their physical and chemical properties. Consequently it should predict their kinetic properties. In this paper we compare the predictions of our stochastic model derived for the complex I with a number of experiments performed with a large range of complex I substrates and products. A good fit was found between the experiments and the prediction of our stochastic approach. We show that, due to the spatial separation of the two half redox reactions (NADH/NAD and Q/QH(2)), the kinetics cannot necessarily obey a simple mechanism (ordered or ping-pong for instance). A plateau in the kinetics is observed at high substrates concentrations, well evidenced in the double reciprocal plots, which is explained by the limiting rate of quinone reduction as compared with the oxidation of NADH at the other end of complex I. Moreover, we show that the set of the seven redox reactions in between the two half redox reactions (NADH/NAD and Q/QH(2)) acts as an electron buffer. An inhibition of complex I activity by quinone is observed at high concentration of this molecule, which cannot be explained by a simple stochastic model based on the known structure. We hypothesize that the distance between the catalytic site close to N2 (iron/sulfur redox center that transfers electrons to quinone) and the membrane forces the quinone/quinol to take several positions in between these sites. We represent these possible positions by an extra site necessarily occupied by the quinone/quinol molecules on their way to the redox site. With this hypothesis, we are able to fit the kinetic experiments over a large range of substrates and products concentrations. The slow rate constants derived for the transition between the two sites could be an indication of a conformational change of the enzyme during the quinone/quinol movement. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012)., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Elementary flux mode analysis is a powerful tool for the theoretical study of metabolic networks. However, when the networks are complex, the determination of elementary flux modes leads to combinatorial explosion of their number which prevents from drawing simple conclusions from their analysis. To deal with this problem we have developed a method based on the Agglomeration of Common Motifs (ACoM) for classifying elementary flux modes. We applied this algorithm to describe the decomposition into elementary flux modes of the central carbon metabolism in Bacillus subtilis and of the yeast mitochondrial energy metabolism. ACoM helps to give biological meaning to the different elementary flux modes and to the relatedness between reactions. ACoM, which can be viewed as a bi-clustering method, can be of general use for sets of vectors with values 0, +1 or -1., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Chronic hypoxia alters mitochondrial energy metabolism. In the heart, oxidative capacity of both ventricles is decreased after 3 weeks of chronic hypoxia. The aim of this study was to evaluate the reversal of these metabolic changes upon normoxia recovery. Rats were exposed to a hypobaric environment for 3 weeks and then subjected to a normoxic environment for 3 weeks (normoxia-recovery group) and compared with rats maintained in a normoxic environment (control group). Mitochondrial energy metabolism was differentially examined in both left and right ventricles. Oxidative capacity (oxygen consumption and ATP synthesis) was measured in saponin-skinned fibers. Activities of mitochondrial respiratory chain complexes and antioxidant enzymes were measured on ventricle homogenates. Morphometric analysis of mitochondria was performed on electron micrographs. In normoxia-recovery rats, oxidative capacities of right ventricles were decreased in the presence of glutamate or palmitoyl carnitine as substrates. In contrast, oxidation of palmitoyl carnitine was maintained in the left ventricle. Enzyme activities of complexes III and IV were significantly decreased in both ventricles. These functional alterations were associated with a decrease in numerical density and an increase in size of mitochondria. Finally, in the normoxia-recovery group, the antioxidant enzyme activities (catalase and glutathione peroxidase) increased. In conclusion, alterations of mitochondrial energy metabolism induced by chronic hypoxia are not totally reversible. Reactive oxygen species could be involved and should be investigated under such conditions, since they may represent a therapeutic target.

Using a stochastic simulation without any other hypotheses, we recently demonstrated the natural emergence of the modified Mitchell Q-cycle in the functioning of the bc(1) complex, with few short-circuits and a very low residence time of the reactive semiquinone species in the Q(o) site. However, this simple model fails to explain both the inhibition by antimycin of the bc(1) complex and the accompanying increase in ROS production. To obtain inhibition, we show that it is necessary to block the return of the electron from the reduced haem b(L) to Q(o). With this added hypothesis we obtain a sigmoid inhibition curve due to the fact that when only one antimycin is bound per bc(1) dimer, the electron of the inhibited monomer systematically crosses the dimer interface from b(L) to b(L) to reduce a quinone or a semiquinone species in the other (free) Q(i) site. Because this step is not limiting, the activity is unchanged (compared to the activity of the free dimer). Interestingly, this b(L)-b(L) pathway is almost exclusively taken in this half-bound antimycin dimer. In the free dimer, the natural faster pathway is b(L)-b(H) on the same monomer. The addition of the assumption of half-of-the-sites reactivity to the previous hypothesis leads to a transient activation in the antimycin titration curve preceding a quasi-complete inhibition at antimycin saturation., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The bc1 complex is a central complex in the mitochondrial respiratory chain. It links the electrons transfer from ubiquinol (or coenzyme Q) to cytochrome c and proton translocation across the inner mitochondrial membrane. It is widely agreed that the "Q-cycle mechanism" proposed by Mitchell correctly describes the bc1 complex working. It is based on an unexpected separation of the two electrons coming from the coenzyme Q bound at the Q0 site of the bc1 complex. Using the stochastic approach of Gillespie and the known spatial structure of bc1 complexes with the kinetic parameters described by Moser and Dutton we demonstrated the natural emergence of the Q-cycle mechanism and the quasi absence of short-circuits in the functional dimer of bc1 complex without the necessity to invoke any additional mechanism. This approach gives a framework which is well adapted to the modelling of all oxido-reduction reactions of the respiratory chain complexes, normal or mutant.

The modelling of OXPHOS (oxidative phosphorylation) in order to integrate all kinetic and thermodynamic aspects of chemiosmotic theory has a long history. We briefly review this history and show how new ways of modelling are required to integrate a local model of the individual respiratory complexes into a global model of OXPHOS and, beyond that, into a reliable overall model of central metabolism.

A stochastic approach based on the Gillespie algorithm is particularly well adapted to describe the time course of the redox reactions that occur inside the respiratory chain complexes because they involve the motion of single electrons between the individual unique redox centres of a given complex. We use this approach to describe the molecular functioning of the peripheral arm of complex I based on its known crystallographic structure and the rate constants of electron tunnelling derived from the Moser and Dutton phenomenological equations. There are several possible electrons pathways but we show that most of them take the route defined by the successive sites and redox centres: NADH+ site-FMN-N3-N1b-N4-N5-N6a-N6b-N2-Q site. However, the electrons do not go directly from NADH towards the ubiquinone molecule. They frequently jump back and forth between neighbouring redox centres with the result that the net flux of electrons through complex I (i.e. net number of electrons reducing a ubiquinone) is far smaller than the number of redox reactions which actually occur. While most of the redox centres are reduced in our simulations the degree of reduction can vary according to the individual midpoint potentials. The high turnover number observed in our simulation seems to indicate that, in the whole complex I, one or several slower step(s) follow(s) the redox reactions involved in the peripheral arm. It also appears that the residence time of FMNH* and SQ* (possible producers of ROS) is low (around 4% and between 1.6% and 5% respectively according to the values of the midpoint potentials). We did not find any evidence for a role of N7 which remains mainly reduced in our simulations. The role of N1a is complex and depends upon its midpoint potential. In all cases its presence slightly decreases the life time of the flavosemiquinone species. These simulations demonstrate the interest of this type of model which links the molecular physico-chemistry of the individual redox reactions to the more global level of the reaction, as is observed experimentally., (Copyright © 2010. Published by Elsevier B.V.)

Diversity of respiratory chain spectrophotometric assays may lead to difficult comparison of results between centers. The French network of mitochondrial diseases diagnostic centers undertook comparison of the results obtained with different protocols in the French diagnostic centers. The diversity of protocols was shown to have striking consequences, which prompted the network to undertake standardization and optimization of the protocols with respect to clinical diagnosis, i.e. high velocity while maintaining linear kinetics relative to time and enzyme concentration. Assays were set up on animal tissues and verified on control human muscle and fibroblasts. Influence of homogenization buffer and narrow range of optimal concentration of phosphate, substrate and tissue were shown. Experimental data and proposed protocols have been posted on a free access website. Their subsequent use in several diagnostic centers has improved consistency for all assays.

Mitochondria play a central role in cellular energetic metabolism. The essential parts of this metabolism are the tricarboxylic acid (TCA) cycle, the respiratory chain and the adenosine triphosphate (ATP) synthesis machinery. Here a simplified model of these three metabolic components with a limited set of differential equations is presented. The existence of a steady state is demonstrated and results of numerical simulations are presented. The relevance of a simple model to represent actual in vivo behavior is discussed.

A stochastic approach based on Gillespie algorithm is particularly well adapted to describe the time course of the redox reactions that occur inside the respiratory chain complexes because they involve the motion of single electrons between individual unique redox centres of a given complex and not populations of electrons and redox centres as usually considered in ordinary differential equations. In this way we approach the molecular functioning of the bc(1) complex based on its known crystallographic structure and the rate constants of electron tunnelling derived from the Moser and Dutton phenomenological equation. The main features of our simulations are the dominant and robust emergence of a Q-cycle mechanism and the near absence of short-circuits in the normal functioning of the bc(1) complex. Thus, in our paper, the Mitchell Q-cycle no longer appears as an a priori hypothesis but arises out of the bc(1) complex structure and of the kinetic laws of redox reactions.

We revisit an old Russian paper by V.V. Dynnik, R. Heinrich and E.E. Sel'kov (1980a,b) describing: "A mathematical model of carbohydrate energy metabolism. Interaction between glycolysis, the Krebs cycle and the H-transporting shuttles at varying ATPases load". We analyse the model mathematically and calculate the control coefficients as a function of ATPase loads. We also evaluate the structure of the metabolic network in terms of elementary flux modes. We show how this model can respond to an ATPase load as well as to the glucose supply. We also show how this simple model can help in understanding the articulation between the major blocks of energetic metabolism, i.e. glycolysis, the Krebs cycle and the H-transporting shuttles.

The structural analysis of large metabolic networks exhibits a combinatorial explosion of elementary modes. A new method of classification has been developed [called aggregation around common motif (ACoM)], which groups elementary modes into classes with similar substructures. This method is applied to the tricarboxylic acid cycle and metabolite carriers. The analysis of this network evidences a great number of elementary flux modes (204) despite the low number of reactions (23). The ACoM is used to class these elementary modes in a low number of sets (8) with biological meanings.

Intracellular amyloid beta-peptide (A beta) accumulation is considered to be a key pathogenic factor in sporadic Alzheimer's disease (AD), but the mechanisms by which it triggers neuronal dysfunction remain unclear. We hypothesized that gradual mitochondrial dysfunction could play a central role in both initiation and progression of sporadic AD. Thus, we analyzed changes in mitochondrial structure and function following direct exposure to increasing concentrations of A beta(1--42) and A beta(25--35) in order to look more closely at the relationships between mitochondrial membrane viscosity, ATP synthesis, ROS production, and cytochrome c release. Our results show the accumulation of monomeric A beta within rat brain and muscle mitochondria. Subsequently, we observed four different and additive modes of action of A beta, which were concentration dependent: (i) an increase in mitochondrial membrane viscosity with a concomitant decrease in ATP/O, (ii) respiratory chain complexes inhibition, (iii) a potentialization of ROS production, and (iv) cytochrome c release.

The role of some serine/threonine kinases in the regulation of mitochondrial physiology is now well established, but little is known about mitochondrial tyrosine kinases. We showed that tyrosine phosphorylation of rat brain mitochondrial proteins was increased by in vitro addition of ATP and H2O2, and also during in situ ATP production at state 3, and maximal reactive oxygen species production. The Src kinase inhibitor PP2 decreased tyrosine phosphorylation and respiratory rates at state 3. We found that the 39-kDa subunit of complex I was tyrosine phosphorylated, and we identified putative tyrosine-phosphorylated subunits for the other complexes. We also have strong evidence that the FoF1-ATP synthase alpha chain is probably tyrosine-phosphorylated, but demonstrated that the beta chain is not. The tyrosine phosphatase PTP 1B was found in brain but not in muscle, heart or liver mitochondria. Our results suggest that tyrosine kinases and phosphatases are involved in the regulation of oxidative phosphorylation.

Objective: The present study was designed to characterize mitochondrial adaptation to chronic hypoxia (CH) in the rat heart. Mitochondrial energy metabolism was differentially examined in both left and right ventricles since CH selectively triggers pulmonary hypertension and right ventricular hypertrophy., Methods: Rats were exposed to a hypobaric environment for 2 or 3 weeks and compared with rats maintained in a normoxic environment. Oxidative capacity (oxygen consumption and ATP synthesis) was measured in saponin-skinned fibers with glutamate or palmitoyl carnitine as substrates. Enzymatic activities of mitochondrial respiratory chain complexes were measured on tissue homogenates. Morphometric analysis of mitochondria was performed on electron micrographs. Mitochondrial DNA was quantified using Southern blot analysis., Results: Whereas oxidative capacity of both ventricles was decreased following 21 days of CH, oxygen consumption and ATP synthesis was maintained with the glutamate substrate in the right ventricle following 14 days of CH. As for the oxidative capacity, enzyme activities were decreased only in the left ventricle following 14 days of CH and in both ventricles following 21 days of CH. These functional alterations were associated with an increase in numerical density and a decrease in size of mitochondria without a change in volume density in both ventricles. Finally, 21 days of CH also decreased the ratio of mitochondrial DNA to nuclear DNA in both ventricles., Conclusions: CH alters morphometry and function of mitochondria in the heart, but this effect is delayed in the right compared to the left ventricle, suggesting some adaptive processes at the onset of right ventricular hypertrophy.

Objective: To assess mitochondrial metabolism of bladder tissue induced by small-intestinal submucosa (SIS), by comparing the mitochondrial enzyme metabolism in this tissue with that in normal bladder tissue and thus evaluate intracellular normality., Material and Methods: In all, 70 rats were grouped into healthy controls (10), surgical controls with a simple bladder incision (15) and rats treated by partial cystectomy with replacement by the SIS graft (45). At 1, 3 and 6 months the rats were killed, the enzymes of mitochondrial respiratory chain complexes assayed, and the respiration of permeabilized bladder fibres assessed using polarographic analysis., Results: The enzyme activities of control and treated rats at 3 months were identical. The results from the polarographic analysis of respiration were also similar to that in normal tissue apart from a decrease in the number of mitochondria. Histologically, there was complete regeneration at 6 months., Conclusion: After a phase of inflammation the bladder regenerates after a patch is placed. The new tissue has the same enzymatic and histological features as normal bladder tissue.

Phosphatidylserine exposure in platelets is required for normal haemostasis and is also a hallmark of apoptosis. It results from activation of a phospholipid scramblase, which has been shown to be differently stimulated by Ca(2+)-influx and during apoptosis, thus suggesting that mitochondria may be involved in phosphatidylserine exposure in platelets. It is also well known that local anaesthetics can expose phosphatidylserine in platelets and affect the mitochondrial metabolism in other cells. Thus, the present study was undertaken to evaluate the specific involvement of mitochondria in phosphatidylserine exposure in platelets. For this purpose, we stimulated phosphatidylserine exposure by local anaesthetics and avoided any external Ca(2+)-influx by performing all experiments in the absence of added Ca(2+). We report that phosphatidylserine exposure, induced by the lipophilic local anaesthetics dibucaine and tetracaine, was accompanied by depolarization of the mitochondrial membrane, cytochrome c release, calpain-processing of caspases 9 and 3 to active enzymes, as well as a prolonged increase in both cytosolic and mitochondrial Ca(2+) concentrations. In contrast, in the absence of extracellular Ca(2+), the Ca(2+)-ionophore A23187 induced a smaller transient increase in both cytosolic and mitochondrial Ca(2+) concentrations, but did not induce any other phenomena, nor phosphatidylserine exposure. However, phosphatidylserine exposure and depolarization induced by dibucaine still occurred in spite of inhibition of intracellular Ca(2+) elevation. Thus we conclude that phosphatidylserine exposure in platelets is associated with mitochondrial apoptotic-like events. Therefore, we propose that mitochondria engagement in an apoptotic pathway in platelets could lead to PS exposure without the participation of Ca(2+).

Long-term preservation of muscle mitochondria for consequent functional analysis is an important and still unresolved challenge in the clinical study of metabolic diseases and in the basic research of mitochondrial physiology. We here present a method for cryopreservation of mitochondria in various muscle types including human biopsies. Mitochondrial function was analyzed after freeze-thawing permeabilized muscle fibers using glycerol and dimethyl sulfoxide as cryoprotectant. Using optimal freeze-thawing conditions, high rates of adenosine 5(')-diphosphate-stimulated respiration and high respiratory control were observed, showing intactness of mitochondrial respiratory function after cryopreservation. Measurement of adenosine 5(')-triphosphate (ATP) formation showed normal rates of ATP synthesis and ATP/O ratios. Intactness of the outer mitochondrial membrane and functional coupling between mitochondrial creatine kinase and oxidative phosphorylation were verified by respiratory cytochrome c and creatine tests. Simultaneous confocal imaging of mitochondrial flavoproteins and nicotinamide adenine dinucleotide revealed normal intracellular arrangement and metabolic responses of mitochondria after freeze-thawing. The method therefore permits, after freezing and long-term storage of muscle samples, mitochondrial function to be estimated and energy metabolism to be monitored in situ. This will significantly expand the scope for screening and exchange of human biopsy samples between research centers, thus providing a new basis for functional analysis of mitochondrial defects in various diseases.

The enzymatic activities of threonine pathway in Escherichia coli are sensitive to pollutants such as cadmium, copper and mercury, which, even at low concentration, can substantially decrease or even block the pathway at several steps. Our aim was to investigate the complex effects on a metabolic pathway of such general enzyme inhibitors with several sites of action, using a previously developed computer simulation of the pathway. For this purpose, the inhibition parameters were experimentally determined and incorporated in the model. The calculation of the flux control coefficient distribution between the five steps of the threonine pathway showed that control remains shared between the three first steps under most inhibition conditions. Response coefficient analysis shows that the inhibition of aspartate semialdehyde dehydrogenase is quantitatively dominant in most circumstances.

The study of mitochondrial diseases has revealed dramatic variability in the phenotypic presentation of mitochondrial genetic defects. To attempt to understand this variability, different authors have studied energy metabolism in transmitochondrial cell lines carrying different proportions of various pathogenic mutations in their mitochondrial DNA. The same kinds of experiments have been performed on isolated mitochondria and on tissue biopsies taken from patients with mitochondrial diseases. The results have shown that, in most cases, phenotypic manifestation of the genetic defect occurs only when a threshold level is exceeded, and this phenomenon has been named the 'phenotypic threshold effect'. Subsequently, several authors showed that it was possible to inhibit considerably the activity of a respiratory chain complex, up to a critical value, without affecting the rate of mitochondrial respiration or ATP synthesis. This phenomenon was called the 'biochemical threshold effect'. More recently, quantitative analysis of the effects of various mutations in mitochondrial DNA on the rate of mitochondrial protein synthesis has revealed the existence of a 'translational threshold effect'. In this review these different mitochondrial threshold effects are discussed, along with their molecular bases and the roles that they play in the presentation of mitochondrial diseases.

Amyloid deposits and neurofibrillary tangles (NFT) are the two hallmarks that characterize Alzheimer's disease (AD). In order to find the molecular partners of these degenerating processes, we have developed antibodies against insoluble AD brain lesions. One clone, named AD46, detects only NFT. Biochemical and histochemistry analyses demonstrate that the labeled protein accumulating in the cytosol of Alzheimer degenerating neurons is the alpha-chain of the ATP synthase. The cytosolic accumulation of the alpha-chain of ATP synthase is observed even at early stages of neurofibrillary degenerating process. It is specifically observed in degenerating neurons, either alone or tightly associated with aggregates of tau proteins, suggesting that it is a new molecular event related to neurodegeneration. Overall, our results strongly suggest the implication of the alpha-chain of ATP synthase in neurofibrillary degeneration of AD that is illustrated by the cytosolic accumulation of this mitochondrial protein, which belongs to the mitochondrial respiratory system. This regulatory subunit of the respiratory complex V of mitochondria is thus a potential target for therapeutic and diagnostic strategies.

Background: Adaptation to chronic exposure to hypoxia alters energy metabolism in the heart, particularly in the left ventricle, which undergoes a loss in oxidative capacity. Highly lipophilic local anesthetics interfere with mitochondrial energy metabolism. The purpose of this study was to compare the effects of bupivacaine on mitochondrial energy metabolism in heart of rats subjected to normoxic or hypoxic environments., Methods: Male Wistar rats (n = 10) were subjected to hypobaric hypoxia (simulated altitude = 5,000 m, 380 mmHg) for 2 weeks. Control rats (n = 10) were maintained in an ambient normoxic environment. Mitochondrial metabolism (oxygen consumption and adenosine triphosphate synthesis) was assessed using saponin-skinned ventricular fibers. Bupivacaine (0-5 mM) was tested on both left and right ventricles of normoxic or hypoxic heart., Results: In animals exposed to hypobaric hypoxia for 14 days, cardiac mass significantly increased, and the right-to-left ventricular ratio was approximately twofold (0.48 +/- 0.11 vs. 0.22 +/- 0.04, P < 0.05). Oxygen consumption and adenosine triphosphate synthesis were significantly lower in the hypoxic left ventricles but not in the right ones. The uncoupling effect of bupivacaine was more pronounced in the left ventricle from hypoxic heart than in the right ventricle; the bupivacaine-induced decrease in the adenosine triphosphate synthesis rate and in the adenosine triphosphate-to-oxygen ratio was significantly greater in the hypoxic left ventricle than in the normoxic one., Conclusions: Chronic hypoxia impairs cardiac energy metabolism in left ventricles and enhances the depressant effects of bupivacaine on mitochondrial functions.

In airway myocytes, like in many cells, Ca(2+) signaling is controlled by inositol 1,4,5-trisphosphate (InsP(3)) via InsP(3) receptors (InsP(3)R) located in the sarco-endoplasmic reticulum. Three types of InsP(3)R exist, labeled Types 1, 2, and 3, which differ in their gating kinetics. We analyze a possible impact of the different gating kinetics of Type 1 and Type 3 InsP(3)R on the time course of cytosolic Ca(2+) concentration in tracheal smooth muscle cells upon agonist stimulation. Previous experimental data in rat tracheal myocytes showed that upon gradually increased stimulation with acetylcholine (ACh), a contractile agonist that acts via InsP(3) production, signal spikes, several spikes with declining maxima, and sustained oscillations appear. Our model reproduces the time courses of cytosolic Ca(2+) measured in tracheal myocytes. Moreover, by postulating slight variations in the model parameters which determine the total number of receptors expressed and the ratio between Type 1 and Type 3 InsP(3)R, it offers an explanation to the experimental observation of qualitatively different responses of cells within a presumably homogeneous tissue.

Both experimental and theoretical studies of metabolism are likely to relate to a segment that has been isolated for analytical purposes. In practice, it will be embedded in the whole of cellular metabolism. Thus, it is necessary to consider how conclusions about the control of an isolated pathway may be modified in this wider context where the input and output metabolites are considered as variables of cellular metabolism. Here, we analyse the effect of expanding a linear metabolic pathway by adding an extra input or an extra output. In particular, we analyse the effect of the elasticities of the extra steps on control coefficients. We derive matrix algebraic relationships for obtaining flux and concentration control coefficients from expressions depending on these extra elasticities and on parameters (elasticities and control coefficients) of the original pathway. These equations can be shown in certain cases to be generalized versions of earlier rescaling relationships and to be related to top-down analysis, but also apply where the new variable metabolite of the expanded pathway is an effector of more than one step of the original pathway. We use our relationships to analyse the dependence or independence of control coefficients upon these extra elasticities for the published analyses of the pathway of mammalian serine biosynthesis (Fell & Snell, 1988) and Escherischia coli threonine biosynthesis (Chassagnole et al., 2001). The same analysis can be applied to determine whether the transport reactions of substrates and products of a pathway in and out of a cell need to be included in estimations of the control coefficients of the enzymes., (Copyright 2002 Elsevier Science Ltd. All rights reserved.)

Inside the eukaryotic cell, mitochondria are internal organelles of prokaryotic origin, responsible for energy supply in the cell. The control of the mitochondrial ATP production is a complex problem with different patterns according to different tissues and organs. Our aim is to continue to develop the modelling of oxidative phosphorylation in different tissues, to model other parts of mitochondrial metabolism and to include this virtual mitochondria in a virtual cell. In constructing the complete metabolic map of mitochondria, we will take advantage of the sequenced genomes of eukaryotic organism (10-15% of the yeast genome concerns mitochondria).

In order to determine the effect of chronic and acute stress on muscle mitochondrial metabolism, two strains of rats were selected on the basis of their different hypothalamo-pituitary-adrenal (HPA) axis responses to different stressors [Spontaneous Hypertensive Rats (SHR) and Lewis rats]. For 8 weeks animals were stressed by daily exposure to either a novel environment (SHR: n=16, Lewis: n=16) or forced exercise (SHR: n=16, Lewis: n=16). An unstressed group was left undisturbed (SHR: n=5, Lewis: n=5). Half of the stressed animals (n=32) were submitted to an acute stress (1-h immobilization). The mitochondrial responses of plantaris muscle [cytochrome-c-oxidase (COX), citrate synthase and succinate dehydrogenase activities, the latter two being measured as indices of functional mitochondrial amount] in the presence of different physiological plasma corticosterone (CORT) concentrations were analyzed. The novel environment and forced exercise stress induced different levels of plasma CORT which were negatively correlated with the amount of functional mitochondria in the plantaris muscle. Therefore, a chronic intermittent stress is able to induce an increase in plasma CORT which may be related to deleterious changes in muscle mitochondrial metabolism. Lastly, the acute stress was not associated with a decrease in functional mitochondria but with an increase in COX activity. This suggests that the relationship between CORT and muscle mitochondrial metabolism depends both on the level and duration of endogenous glucocorticoids exposure.

Respiratory-chain-complex subunits in mitochondria are encoded by nuclear or mitochondrial DNA. This property might have profound implications for the phenotypic expression of mutations affecting oxidative phosphorylation complexes. The aim of this paper is to study the importance of the origin of the mutation (nuclear or mitochondrial) on the expression of mitochondrial defects. We have therefore developed theoretical models illustrating three mechanisms of nuclear or mitochondrial DNA mutation giving rise to a deficiency in the respiratory-chain complex: (1) a partial deficiency, homogeneously distributed in all of the mitochondria; (2) a complete deficiency, only affecting some of the mitochondria ('binary mitochondrial heteroplasmy'); and (3) a partial deficiency, affecting only some of the mitochondria. We show that mutations affecting oxidative phosphorylation complexes will be expressed in different ways depending on their origins. Although the expression of nuclear or mitochondrial mutations is evidence of a biochemical threshold, we demonstrate that the threshold value depends on the origin and distribution of the mutation (homogeneous or not) and also on the energy demand of the tissue. This last prediction has been confirmed in an experimental model using hexokinase for the simulation of the energy demand and a variation in mitochondrial concentration. We also emphasize the possible role of 'binary mitochondrial heteroplasmy' in the expression of mitochondrial DNA mutations and thus the importance of the origin of the deficit (mutation) for the diagnosis or therapy of mitochondrial diseases.