Modelling mitochondrial ROS production by the respiratory chain.

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.

The role of glycolysis-derived hexose phosphates in the induction of the Crabtree 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.

Neutrophil Metabolic Shift during their Lifecycle: Impact on their Survival and Activation.

Polymorphonuclear neutrophils (PMNs) are innate immune cells, which represent 50% to 70% of the total circulating leukocytes. How PMNs adapt to various microenvironments encountered during their life cycle, from the bone marrow, to the blood plasma fraction, and to inflamed or infected tissues remains largely unexplored. Metabolic shifts have been reported in other immune cells such as macrophages or lymphocytes, in response to local changes in their microenvironment, and in association with a modulation of their pro-inflammatory or anti-inflammatory functions. The potential contribution of metabolic shifts in the modulation of neutrophil activation or survival is anticipated even though it is not yet fully described. If neutrophils are considered to be mainly glycolytic, the relative importance of alternative metabolic pathways, such as the pentose phosphate pathway, glutaminolysis, or the mitochondrial oxidative metabolism, has not been fully considered during activation. This statement may be explained by the lack of knowledge regarding the local availability of key metabolites such as glucose, glutamine, and substrates, such as oxygen from the bone marrow to inflamed tissues. As highlighted in this review, the link between specific metabolic pathways and neutrophil activation has been outlined in many reports. However, the impact of neutrophil activation on metabolic shifts' induction has not yet been explored. Beyond its importance in neutrophil survival capacity in response to available metabolites, metabolic shifts may also contribute to neutrophil population heterogeneity reported in cancer (tumor-associated neutrophil) or auto-immune diseases (Low/High Density Neutrophils). This represents an active field of research. In conclusion, the characterization of neutrophil metabolic shifts is an emerging field that may provide important knowledge on neutrophil physiology and activation modulation. The related question of microenvironmental changes occurring during inflammation, to which neutrophils will respond to, will have to be addressed to fully appreciate the importance of neutrophil metabolic shifts in inflammatory diseases.

From in silico to in spectro kinetics of respiratory complex I.

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).

Mitochondrial energetic metabolism-some general principles.

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.

How does antimycin inhibit the bc1 complex? A part-time twin.

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.

The flitting of electrons in complex I: a stochastic approach.

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.

Virtual mitochondrion: towards an integrated model of oxidative phosphorylation complexes and beyond.

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.

[The cytochrome bc1 complex in the mitochondrial respiratory chain functions according to the Q cycle hypothesis of Mitchell: the proof using a stochastic approach?]. / Le complexe bc1 de la chaîne respiratoire mitochondriale fonctionne selon l'hypothèse du cycle Q de Mitchell: la preuve par une approche stochastique?

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 Warburg Effect in Yeast: Repression of Mitochondrial Metabolism Is Not a Prerequisite to Promote Cell Proliferation.

O. Warburg conducted one of the first studies on tumor energy metabolism. His early discoveries pointed out 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. Here, we made use of yeast as a model to study the Warburg effect and its eventual function in allowing an increased ATP synthesis to support cell proliferation. The role of oxidative phosphorylation repression in this effect was investigated. We show that yeast is a good model to study the Warburg effect, where all parameters and their modulation in the presence of glucose can be reconstituted. Moreover, we show that in this model, mitochondria are not dysfunctional, but that there are fewer mitochondria respiratory chain units per cell. Identification of the molecular mechanisms involved in this process allowed us to dissociate the parameters involved in the Warburg effect and show that oxidative phosphorylation repression is not mandatory to promote cell growth. Last but not least, we were able to show that neither cellular ATP synthesis flux nor glucose consumption flux controls cellular growth rate.

Ascorbate maintains a low plasma oxygen level.

In human blood, oxygen is mainly transported by red blood cells. Accordingly, the dissolved oxygen level in plasma is expected to be limited, although it has not been quantified yet. Here, by developing dedicated methods and tools, we determined that human plasma pO2 = 8.4 mmHg (1.1% O2). Oxygen solubility in plasma was believed to be similar to water. Here we reveal that plasma has an additional ascorbate-dependent oxygen-reduction activity. Plasma experimental oxygenation oxidizes ascorbate (49.5 µM in fresh plasma vs < 2 µM in oxidized plasma) and abolishes this capacity, which is restored by ascorbate supplementation. We confirmed these results in vivo, showing that the plasma pO2 is significantly higher in ascorbate-deficient guinea pigs (Ascorbateplasma < 2 µM), compared to control (Ascorbateplasma > 15 µM). Plasma low oxygen level preserves the integrity of oxidation-sensitive components such as ubiquinol. Circulating leucocytes are well adapted to these conditions, since the abundance of their mitochondrial network is limited. These results shed a new light on the importance of oxygen exposure on leucocyte biological study, in regards with the reducing conditions they encounter in vivo; but also, on the manipulation of blood products to improve their integrity and potentially improve transfusions' efficacy.

The loneliness of the electrons in the bc1 complex.

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.

The Fate of Glutamine in Human Metabolism. The Interplay with Glucose in Proliferating Cells.

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.

Structural basis of phospholipase activity of Staphylococcus hyicus lipase.

Staphylococcus hyicus lipase differs from other bacterial lipases in its high phospholipase A1 activity. Here, we present the crystal structure of the S. hyicus lipase at 2.86 A resolution. The lipase is in an open conformation, with the active site partly covered by a neighbouring molecule. Ser124, Asp314 and His355 form the catalytic triad. The substrate-binding cavity contains two large hydrophobic acyl chain-binding pockets and a shallow and more polar third pocket that is capable of binding either a (short) fatty acid or a phospholipid head-group. A model of a phospholipid bound in the active site shows that Lys295 is at hydrogen bonding distance from the substrate's phosphate group. Residues Ser356, Glu292 and Thr294 hold the lysine in position by hydrogen bonding and electrostatic interactions. These observations explain the biochemical data showing the importance of Lys295 and Ser356 for phospholipid binding and phospholipase A1 activity.