The flitting of electrons in complex I: A stochastic approach

AbstractA 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

Bacterial lipases

Many different bacterial species produce lipases which hydrolyze esters of glycerol with preferably long-chain fatty acids. They act at the interface generated by a hydrophobic lipid substrate in a hydrophilic aqueous medium. A characteristic property of lipases is called interfacial activation, meaning a sharp increase in lipase activity observed when the substrate starts to form an emulsion, thereby presenting to the enzyme an interfacial area. As a consequence, the kinetics of a lipase reaction do not follow the classical Michaelis-Menten model. With only a few exceptions, bacterial lipases are able to completely hydrolyze a triacylglycerol substrate although a certain preference for primary ester bonds has been observed. Numerous lipase assay methods are available using coloured or fluorescent substrates which allow spectroscopic and fluorimetric detection of lipase activity. Another important assay is based on titration of fatty acids released from the substrate. Newly developed methods allow to exactly determine lipase activity via controlled surface pressure or by means of a computer-controlled oil drop tensiometer. The synthesis and secretion of lipases by bacteria is influenced by a variety of environmental factors like ions, carbon sources, or presence of non-metabolizable polysaccharides. The secretion pathway is known for Pseudomonas lipases with P. aeruginosa lipase using a two-step mechanism and P. fluorescens lipase using a one-step mechanism. Additionally, some Pseudomonas lipases need specific chaperone-like proteins assisting their correct folding in the periplasm. These lipase-specific foldases (Lif-proteins) which show a high degree of amino acid sequence homology among different Pseudomonas species are coded for by genes located immediately downstream the lipase structural genes. A comparison of different bacterial lipases on the basis of primary structure revealed only very limited sequence homology. However, determination of the three-dimensional structure of the P. glumae lipase indicated that at least some of the bacterial lipases will presumably reveal a conserved folding pattern called the α/β-hydrolase fold, which has been described for other microbial and human lipases. The catalytic site of lipases is buried inside the protein and contains a serine-protease-like catalytic triad consisting of the amino acids serine, histidine, and aspartate (or glutamate). The Ser-residue is located in a strictly conserved β-εSer-α motif. The active site is covered by a lid-like α-helical structure which moves away upon contact of the lipase with its substrate, thereby exposing hydrophobic residues at the protein's surface mediating the contact between protein and substrate. This movable lid-like α-helix explains at a molecular level the lipase-specific phenomenon of interfacial activation. At least some of the pathogenic bacterial species produce a lipase which has been studied with respect to its role as a virulence factor. Lipases of Propionibacterium acnes and Staphylococcus epidermidis may be involved in colonization and persistence of these bacteria on the human skin. Lipases of S. aureus and P. aeruginosa are produced during the bacterial infection process and, at least in vitro, considerably impair the function of different cell types involved in the human immune response like macrophages or platelets. The present state of knowledge suggests to classify the lipases as important bacterial virulence factors which exert their harmful effects in combination with other bacterial enzymes, in particular the phospholipases C. Most of the steadily increasing interest in bacterial lipases is based on their biotechnological applications which are partly based on their potential to catalyze not only hydrolysis but also synthesis of a variety of industrially valuable products. Optically active compounds, various esters and lactones are among the substances synthesized using bacterial lipases. Recently, an important application emerged with the addition of bacterial lipases to household detergents in order to reduce or even replace synthetic detergent chemicals which pose considerable environmental problems. As a main conclusion, lipases represent an extremely versatile group of bacterial extracellular enzymes that are capable of performing a variety of important reactions, thereby presenting a fascinating field tot future research.

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

Le complexe

Le complexe bcl est central dans l’organisation des oxydations phosphorylantes. Il est largement accepté aujourd’hui que le fonctionnement de ce complexe obéit au schéma décrit par P. Mitchell sous le nom de « cycle Q ». Nous avons montré, en nous appuyant sur un modèle stochastique du transfert des électrons, que la structure de ce complexe permet naturellement l’émergence du cycle Q et minimise les courts-circuits non générateurs d’un gradient de protons transmembranaire. Cette approche fournit un cadre conceptuel général bien adapté à la modélisation des réactions d’oxydoréduction de l’ensemble des complexes de la chaîne respiratoire mitochondrial et de leurs dérèglements pathologiques

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

AbstractUsing a stochastic simulation without any other hypotheses, we recently demonstrated the natural emergence of the modified Mitchell Q-cycle in the functioning of the bc1 complex, with few short-circuits and a very low residence time of the reactive semiquinone species in the Qo site. However, this simple model fails to explain both the inhibition by antimycin of the bc1 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 bL to Qo. With this added hypothesis we obtain a sigmoid inhibition curve due to the fact that when only one antimycin is bound per bc1 dimer, the electron of the inhibited monomer systematically crosses the dimer interface from bL to bL to reduce a quinone or a semiquinone species in the other (free) Qi site. Because this step is not limiting, the activity is unchanged (compared to the activity of the free dimer). Interestingly, this bL–bL pathway is almost exclusively taken in this half-bound antimycin dimer. In the free dimer, the natural faster pathway is bL–bH 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

Modelling mitochondrial ROS production by the respiratory chain

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Neutrophil Metabolic Shift during Their Lifecycle: Impact on Their Survival and Activation

International audiencePolymorphonuclear 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