Jean-Pierre Mazat

Evidence for Cardiolipin Binding Sites on the Membrane-Exposed Surface of the Cytochrome bc(1)

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.

Control of oxidative phosphorylations in yeast mitochondria. Role of the phosphate carrier

This work describes the control of ATP synthesis and O2 consumption as a function of external inorganic phosphate (Pi) concentration at steady states which are characterised by a high external (ADPATP) ratio. As a function of external Pi concentration both fluxes vary closely with each other and have a biphasic behaviour characterised by a rapid increase below 2 mM and a slow increase above this concentration. Nevertheless, the control of the fluxes is not always the same. The adenine nucleotide carrier exhibits no control in either flux whatever the external Pi concentration is. Cytochrome c oxidase is always a controlling step in both fluxes. At low Pi concentration, the proton leak controls both fluxes, but at high Pi concentration Pi transport takes the place of proton reentry as the other main controlling step. The steep threshold in external Pi concentration for which the exchange between the controlling steps occurs strongly depends on the proton permeability of the membrane. In ATP synthesis flux, proton permeability exerts a negative control (branched pathway) although the control is positive on the O2 consumption flux. In accordance with the summation theorem, this property implies that the controls must be very different at some other steps of the two fluxes.

Modelling central metabolic fluxes by constraint-based optimization reveals metabolic reprogramming of developing Solanum lycopersicum (tomato) fruit.

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.

Tryptophanyl-tRNA synthetase from beef pancreas. Spectroscopic analysis of the stoichiometry of formation of the enzyme-tryptophanyl-adenylate complex

The dimeric enzyme tryptophanyl-tRNA synthetase from beef pancreas catalyses the stoichiometric formation of one mole of tryptophanyl-adenylate per subunit. This formation is associated with optical changes (absorbance, fluorescence, optical rotation) and is confirmed by analytical ultracentrifugation. An equal amplitude of the change is observed for each adenylation site at pH 8.0, 25 degrees C, regardless of the optical method used. The formation of two tryptophanyl adenylates per dimer corresponds to a molar absorbance change delta epsilon 291 = 12000 +/- 500 cm-1 M-1, to a fluorescence quenching of 24 per cent at 340 nm and to a variation in optical rotation of 6 per cent at 313 nm. The circular dichroic band of the adenosine moiety of ATP is strongly increased. The addition of sodium pyrophosphate to the tryptophanyl-adenylate-enzyme complex restores the absorbance and fluorescence amplitude observed prior to the addition of ATP to the enzyme. Magnesium ions are necessary to the reaction. A pertubation of the environment of both the protein and the substrates (tryptophan and ATP) have to be taken into account to explain the magnitude of the observed changes.

Mitochondrial energetic metabolism—some general principles

Summary

Isolation and characterization of an uncoupler-resistant mutant of Saccharomyces cerevisiae.

SummaryOne mutant resistant to carbonylcyanide m-chlorophenylhydrazone (CCCP), an uncoupler of oxidative phosphorylation, was isolated in Saccharomyces cerevisiae.Genetic analysis showed that a single nuclear gene is responsible for increased resistance; this gene was dominant.The mutant showed cross-resistance or collateral sensitivity to several chemically-unrelated inhibitors (cycloheximide, dinitrophenol, tributhyltin chloride, chloramphenicol).The resistance of the mutant is related to a decreased uptake of CCCP which is not expressed in glucose-starved cells. It was shown that glucose induced a CCCP efflux which was more efficient in the mutant than in the wild-type cells. This effect was correlated to a greater acidification of the internal pH by glucose addition in the mutant cells.It was proposed that resistance was not due to a change of permeability of the plasmic membrane itself but to the change of internal pH which determines the extent of accumulation of weak acids or bases.

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

An enzymes 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).