Tania Bizouarn

The organization of the membrane domain and its interaction with the NADP(H)-binding site in proton-translocating transhydrogenase from E. coli.

Proton-translocating nicotinamide nucleotide transhydrogenase is a conformationally driven pump which catalyzes the reversibel reduction of NADP(+) by NADH. Transhydrogenases contain three domains, i.e., the hydrophilic NAD(H)-binding domain I and the NADP(H)-binding domain III, and the hydrophobic domain II containing the proton channel. Domains I and III have been separately expressed and characterized structurally by, e.g. X-ray crystallography and NMR. These domains catalyze transhydrogenation in the absence of domain II. However, due to the absence of the latter domain, the reactions catalyzed by domains I and III differ significantly from those catalyzed by the intact enzyme. Mutagenesis of residues in domain II markedly affects the activity of the intact enzyme. In order to resolve the structure-function relationships of the intact enzyme, and the molecular mechanism of proton translocation, it is therefore essential to establish the structure and function of domain II and its interactions with domains I and III. This review describes some relevant recent results in this field of research.

Photophosphorylation at variable ADP concentration but constant ΔpH in lettuce thylakoids: Effect of ΔpH and phosphate on the apparent affinity for ADP

There is a wide variation in the literature of the apparent affinity of membrane-bound F 0 F 1 -ATPases for the substrates of phosphorylation, especially for ADP. Since these measurements are inescapably biased by an increased drop of Δũ H + when the phosphorylating flow rises with substrate concentration, we have developed a method to keep the proton gradient constant when substrates are varied. Light-induced trapping of hexylamine inside lettuce thylakoids delays the ΔpH decrease when the membrane H + -conductivity increases. This was used to measure the initial rate of ATP synthesis just after the addition of variable amounts of ADP, without a significant change of the proton gradient due to the phosphorylating H + -flow. ΔpH was estimated by the quenching of 9-aminoacridine fluorescence, calibrated with the ‘phosphate potential’ ΔG p (in State 4), and the rate of phosphorylation was computed from the medium alkalinization using a glass electrode. Saturation curves (rate vs. ADP concentration) at iso-ΔpH were then obtained, and the magnitude of the proton gradient was also adjusted at different levels by varying the light intensity. An inhibitory effect of high concentrations of ADP on ATP synthesis was noticed in the case of a lowΔpH. Within all the present ΔpH range, phosphate, which raises V max , augments the apparent affinity of the coupling factor for the substrate ADP, i.e. K m is lowered. Finally, the K m for ADP obeys a complex law with the ΔpH increase: first, K m slightly decreases down to some kind of plateau ( K m = 6–7 μM around ΔpH 3.4), then sharply increases ( K m > 20 μM at ΔpH3.5). In the same condition, V max regularly rises, that is V max / K m declines for high ApH values. This cannot be explained by the classical energetic role of the protonmotive force. Rather it could reflect a regulation of substrate affinity by ΔpH, a process also distinct from the well-known ATPase activation by the proton gradient.

trans Arachidonic acid isomers inhibit NADPH-oxidase activity by direct interaction with enzyme components

NADPH-oxidase is an enzyme that represents, when activated, the major source of non-mitochondrial reactive oxygen species. In phagocytes, this production is an indispensable event for the destruction of engulfed pathogens. The functional NADPH-oxidase complex consists of a catalytic membrane flavocytochrome b (Cytb(558)) and four cytosolic proteins p47(phox), p67(phox), Rac and p40(phox). The NADPH-oxidase activity is finely regulated spatially and temporally by cellular signaling events that trigger the translocation of the cytosolic subunits to its membrane partner involving post-translational modifications and activation by second messengers such as arachidonic acid (AA). Arachidonic acid in its natural cis-poly unsaturated form (C20:4) has been described to be an efficient activator of the enzyme in vivo and in vitro. In this work, we examined in a cell-free system whether a change of the natural cis geometry to the trans configuration, which could occur either by diet or be produced by the action of free radicals, may have consequences on the functioning of NADPH-oxidase. We showed the inability of mono-trans AA isomers to activate the NADPH-oxidase complex and demonstrated the inhibitory effect on the cis-AA-induced NADPH oxidase activation. The inhibition is mediated by a direct effect of the mono-trans AA which targets both the membrane fraction containing the cytb(558) and the cytosolic p67(phox). Our results suggest that the loss of the natural geometric feature (cis-AA) induces substantial structural modifications of p67(phox) that prevent its translocation to the complex.

The cytosolic subunit p67phox of the NADPH-oxidase complex does not bind NADPH

The NADPH‐oxidase of phagocytic cells is a multicomponent enzyme that generates superoxide. It comprises a membrane flavocytochrome b 558 and four cytosolic proteins; p67phox, p47phox, p40phox and Rac. The NADPH‐binding site of this complex was shown to be located on the flavocytochrome b 558. However, a number of studies have suggested the presence of another site on the p67phox subunit which is the key activating component. Using several approaches like tryptophan quenching fluorescence measurement, inhibition by 2′,3′‐dialdehyde NADPH, and free/bound NADPH concentration measurements, we demonstrate that no NADPH binds on p67phox, thus definitively solving the controversy on the number and location of the NADPH‐binding sites on this complex.

Titanium Dioxide Nanoparticles Increase Superoxide Anion Production by Acting on NADPH Oxidase

Titanium dioxide (TiO2) anatase nanoparticles (NPs) are metal oxide NPs commercialized for several uses of everyday life. However their toxicity has been poorly investigated. Cellular internalization of NPs has been shown to activate macrophages and neutrophils that contribute to superoxide anion production by the NADPH oxidase complex. Transmission electron micrososcopy images showed that the membrane fractions were close to the NPs while fluorescence indicated an interaction between NPs and cytosolic proteins. Using a cell-free system, we have investigated the influence of TiO2 NPs on the behavior of the NADPH oxidase. In the absence of the classical activator molecules of the enzyme (arachidonic acid) but in the presence of TiO2 NPs, no production of superoxide ions could be detected indicating that TiO2 NPs were unable to activate by themselves the complex. However once the NADPH oxidase was activated (i.e., by arachidonic acid), the rate of superoxide anion production went up to 140% of its value without NPs, this effect being dependent on their concentration. In the presence of TiO2 nanoparticles, the NADPH oxidase produces more superoxide ions, hence induces higher oxidative stress. This hyper-activation and the subsequent increase in ROS production by TiO2 NPs could participate to the oxidative stress development.

Are the Fluorescent Properties of the Cyan Fluorescent Protein Sensitive to Conditions of Oxidative Stress

The modifications induced by reactive oxygen species (ROS) on fluorescent proteins (FPs) may have important implications for live cell fluorescence imaging. Using quantitative γ‐radiolysis, we have studied the ROS‐induced biochemical and photophysical perturbations on recombinant cyan fluorescent protein (CFP). After oxidation by the ˙OH radical, the protein displays a modified RP‐HPLC elution profile, while the CFP fluorescence undergoes pronounced decreases in intensity and lifetime, without changes in its excitation and emission spectra. Meanwhile, the Förster resonant energy transfer (FRET) between the single W57 and the chromophore remains unperturbed. These results rule out a direct oxidation of the CFP chromophore and of W57 as well as major changes in the protein 3D structure, but show that new fluorescent forms associated to a higher level of dynamic quenching have been generated. Thus, strict in situ controls are required when CFP is to be used for FRET studies in situations of oxidative activity, or under strong illumination.

Assembly of phagocyte NADPH oxidase: A concerted binding process?

BACKGROUND The phagocyte NADPH-oxidase is a multicomponent enzyme that generates superoxide anions. It comprises a membrane redox component flavocytochrome b558 and four cytosolic proteins (p67(phox), p47(phox), p40(phox) and Rac) that must assemble to produce an active system. In this work we focused on the spatio-temporal control of the activation process of phagocyte NADPH oxidase. METHODS A wide range of techniques including fast kinetics with a stopped-flow apparatus and various combinations of the activating factors was used to test the order of assembly and the role of the p47(phox)-p67(phox) complex. RESULTS The data presented here are consistent with the absence of a catalytic role of the p47(phox)-p67(phox) interacting state and support the idea of independent binding sites for the cytosolic proteins on the flavocytochrome b558 allowing random binding order. However, the formation of the active complex appears to involve a synergistic process of binding of the activated cytosolic subunits to cytochrome b558. All partners should be in the vicinity for optimal assembly, a delay or the absence of one of the partners in this process seems to lead to a decrease in the efficiency of the catalytic core. CONCLUSION AND GENERAL SIGNIFICANCE The activation and assembly of the NADPH oxidase components have to be achieved simultaneously for the formation of an efficient and optimal enzyme complex. This mechanism appears to be incompatible with continuous fast exchanges of the cytosolic proteins during the production of superoxide ion in the phagosome.

Expression of functional mammal flavocytochrome b(558) in yeast: comparison with improved insect cell system.

Activity of phagocyte NADPH-oxidase relies on the assembly of five proteins, among them the transmembrane flavocytochrome b(558) (Cytb(558)) which consists of a heterodimer of the gp91(phox) and p22(phox) subunits. The Cytb(558) is the catalytic core of the NADPH-oxidase that generates a superoxide anion from oxygen by using a reducing equivalent provided by NADPH via FAD and two hemes. We report a novel strategy to engineer and produce the stable and functional recombinant Cytb(558) (rCytb(558)). We expressed the gp91(phox) and p22(phox) subunits using the baculovirus insect cell and, for the first time, the highly inducible Pichia pastoris system. In both hosts, the expression of the full-length proteins reproduced native electrophoretic patterns demonstrating that the two polypeptides are present and, that gp91(phox) undergoes co-translational glycosylation. Spectroscopic analyses showed that the rCytb(558) displayed comparable spectral properties to neutrophil Cytb(558). In contrast to rCytb(558) produced in the insect cells with higher yield, the enzyme expressed in yeast displayed a superoxide dismutase-sensitive NADPH-oxidase activity, indicating a superoxide generation activity. It was also blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium (DPI). As in neutrophil NADPH-oxidase, activation occurred by the interactions with the soluble regulatory subunits suggesting comparable protein-protein contact patterns. We focus on the stability and function of the protein during solubilisation and reconstitution into liposomes. By comparing oxidase activities in different membrane types, we confirm that the lipid-protein environment plays a key role in the protein function.

The effect of energy-transfer inhibitors on the photophosphorylation parameters in lettuce thylakoids and the question of the Km (ADP) variation with membrane energization

Abstract In lettuce thylakoids illuminated with continuous light, buffering of ΔpH variations by internal accumulation of hexylamine was used to measure the initial rate of photophosphorylation upon ADP addition, keeping ΔpH constant whilst ADP concentration was changed (variable vectorial H + efflux). Then, the ΔpH was adjusted to another, constant, value and a similar concentration curve was traced. At high ΔpH (above 3.5), the apparent Michaelis constant for ADP was found between 15 and 40 μM, increasing with the proton gradient. Tentoxin, an irreversible F 1 inhibitor, did not change this apparent affinity for ADP, whereas phloridzin, a reversible F 1 inhibitor, lowered the K m . DCCD and venturicidin, two F 0 inhibitors, unexpectedly decreased the K m for ADP. These results are compatible in principle with the existence of a diffusion barrier in the unstirred layer covering the membrane, which limits the access of ADP to ATP-synthases at high turnover rates. This would lower the actual ADP concentration around the enzymes and raise the apparent K m , which is based on concentration known in the bulk phase. However, considering the quantitative effects observed, an additional hypothesis is that the protonic activation of the enzyme is not, as usually believed, an all-or-nothing process, but involves different functional states with different affinities for the substrate ADP.

Recombinant form of mammalian gp91(phox) is active in the absence of p22(phox).

The flavocytochrome b558 of the phagocyte NADPH oxidase complex comprises two membrane proteins, a glycosylated gp91phox and a non-glycosylated p22phox. Gp91phox contains all of the redox carriers necessary to reduce molecular oxygen to superoxide using NADPH. The capacity of gp91phox to produce superoxide in the absence of its membrane partner p22phox has been little studied. In the present study, we have generated in Pichia pastoris for the first time an active form of bovine gp91phox able to carry out the entire NADPH oxidase activity in the absence of p22phox. Collected information on the maturation and the activity of the recombinant gp91phox and the participation of individual cytosolic subunits in the active complex allowed us to propose, in the absence of p22phox, an unconventional stabilized complex compared with the heterodimer.