The reduction of acetylpyridine adenine dinucleotide by NADH: is it a significant reaction of proton-translocating transhydrogenase, or an artefact?

AbstractTranshydrogenase is a proton pump. It has separate binding sites for NAD+/NADH (on domain I of the protein) and for NADP+/NADPH (on domain III). Purified, detergent-dispersed transhydrogenase from Escherichia coli catalyses the reduction of the NAD+ analogue, acetylpyridine adenine dinucleotide (AcPdAD+), by NADH at a slow rate in the absence of added NADP+ or NADPH. Although it is slow, this reaction is surprizing, since transhydrogenase is generally thought to catalyse hydride transfer between NAD(H) – or its analogues and NADP(H) – or its analogues, by a ternary complex mechanism. It is shown that hydride transfer occurs between the 4A position on the nicotinamide ring of NADH and the 4A position of AcPdAD+. On the basis of the known stereospecificity of the enzyme, this eliminates the possibilities of transhydrogenation (a) from NADH in domain I to AcPdAD+ wrongly located in domain III; and (b) from NADH wrongly located in domain III to AcPdAD+ in domain I. In the presence of low concentrations of added NADP+ or NADPH, detergent-dispersed E. coli transhydrogenase catalyses the very rapid reduction of AcPdAD+ by NADH. This reaction is cyclic; it takes place via the alternate oxidation of NADPH by AcPdAD+ and the reduction of NADP+ by NADH, while the NADPH and NADP+ remain tightly bound to the enzyme. In the present work, it is shown that the rate of the cyclic reaction and the rate of reduction of AcPdAD+ by NADH in the absence of added NADP+/NADPH, have similar dependences on pH and on MgSO4 concentration and that they have a similar kinetic character. It is therefore suggested that the reduction of AcPdAD+ by NADH is actually a cyclic reaction operating, either with tightly bound NADP+/NADPH on a small fraction (<5%) of the enzyme, or with NAD+/NADH (or AcPdAD+/AcPdADH) unnaturally occluded within the domain III site. Transhydrogenase associated with membrane vesicles (chromatophores) of Rhodospirillum rubrum also catalyses the reduction of AcPdAD+ by NADH in the absence of added NADP+/NADPH. When the chromatophores were stripped of transhydrogenase domain I, that reaction was lost in parallel with `normal reverse' transhydrogenation (e.g., the reduction of AcPdAD+ by NADPH). The two reactions were fully recovered upon reconstitution with recombinant domain I protein. However, after repeated washing of the domain I-depleted chromatophores, reverse transhydrogenation activity (when assayed in the presence of domain I) was retained, whereas the reduction of AcPdAD+ by NADH declined in activity. Addition of low concentrations of NADP+ or NADPH always supported the same high rate of the NADH→AcPdAD+ reaction independently of how often the membranes were washed. It is concluded that, as with the purified E. coli enzyme, the reduction of AcPdAD+ by NADH in chromatophores is a cyclic reaction involving nucleotides that are tightly bound in the domain III site of transhydrogenase. However, in the case of R. rubrum membranes it can be shown with some certainty that the bound nucleotides are NADP+ or NADPH. The data are thus adequately explained without recourse to suggestions of multiple nucleotide-binding sites on transhydrogenase

Proton translocating nicotinamide nucleotide transhydrogenase from E. coli. Mechanism of action deduced from its structural and catalytic properties11This review is dedicated to the memory of Professor Lars Ernster.

AbstractTranshydrogenase couples the stereospecific and reversible transfer of hydride equivalents from NADH to NADP+ to the translocation of proton across the inner membrane in mitochondria and the cytoplasmic membrane in bacteria. Like all transhydrogenases, the Escherichia coli enzyme is composed of three domains. Domains I and III protrude from the membrane and contain the binding site for NAD(H) and NADP(H), respectively. Domain II spans the membrane and constitutes at least partly the proton translocating pathway. Three-dimensional models of the hydrophilic domains I and III deduced from crystallographic and NMR data and a new topology of domain II are presented. The new information obtained from the structures and the numerous mutation studies strengthen the proposition of a binding change mechanism, as a way to couple the reduction of NADP+ by NADH to proton translocation and occurring mainly at the level of the NADP(H) binding site

Controle de l'activite de l'ATP synthase des chloroplastiques par lesprotons et implications mecanistiques

SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : TD 81143 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Cholesterol: A modulator of the phagocyte NADPH oxidase activity - A cell-free study

The NADPH oxidase Nox2, a multi-subunit enzyme complex comprising membrane and cytosolic proteins, catalyzes a very intense production of superoxide ions O2•−, which are transformed into other reactive oxygen species (ROS). In vitro, it has to be activated by addition of amphiphiles like arachidonic acid (AA). It has been shown that the membrane part of phagocyte NADPH oxidase is present in lipid rafts rich in cholesterol. Cholesterol plays a significant role in the development of cardio-vascular diseases that are always accompanied by oxidative stress. Our aim was to investigate the influence of cholesterol on the activation process of NADPH oxidase. Our results clearly show that, in a cell-free system, cholesterol is not an efficient activator of NADPH oxidase like arachidonic acid (AA), however it triggers a basal low superoxide production at concentrations similar to what found in neutrophile. A higher concentration, if present during the assembly process of the enzyme, has an inhibitory role on the production of O2•−. Added cholesterol acts on both cytosolic and membrane components, leading to imperfect assembly and decreasing the affinity of cytosolic subunits to the membrane ones. Added to the cytosolic proteins, it retains their conformations but still allows some conformational change induced by AA addition, indispensable to activation of NADPH oxidase

Etude de l'assemblage de la NADPH oxydase du phagocyte

La NADPH oxydase du phagocyte est une enzyme impliquée dans la défense immunitaire contre les pathogènes. Après activation du phagocyte, cette enzyme produit des ions superoxyde par réduction du dioxygène par le NADPH. Elle est constituée de quatre sous- unités cytosolubles (p47phox ; p67phox ; p40phox et Rac), et deux membranaires (gp91 ; p22phox). Son activation fait intervenir un processus complexe qui met en jeu des changements d interaction entre les protéines la constituant et qui permet l assemblage des six sous- unités. Afin d obtenir des informations sur les processus d assemblage et d activation, j ai reconstitué le complexe dans un système cell free à l aide de protéines recombinantes pour pouvoir contrôler tous les paramètres. Dans ce travail nous avons comparé les modes d activation de p47phox par phosphorylation, par mutation substitutionelle sérine - aspartate en position S303,S304 et S328 pour mimer la phosphorylation et enfin par addition d acide arachidonique (AA) activateur connu de l enzyme in vitro mais aussi in vivo. Bien qu il ai été montré que ces trois méthodes ouvrent la protéine vers une conformation ayant des propriétés similaires, nous avons trouvé que les effets de ces méthodes d activation sont significativement différents. Ainsi, les changement de conformation observés par dichroisme circulaire, sont dissemblables. Pour p47phox, l addition de AA déstructure la protéine. La phosphorylation induit un déplacement bathochrome des bandes de CD qualitativement similaire, alors que les mutations S-D de p47phox provoquent un déplacement opposé. Pour le complexe p47phox-p67phox l addition d AA destructure le mélange tandis que la mutation induit relativement peu de changement. Nous avons mesuré les constantes de dissociation Kd du complexe p47phox-p67phox. Alors que pour les protéines sauvages , le Kd est faible (4+-2 nM), les mutations de p47phox ainsi que l addition d AA augmentent cette valeur jusqu à environ 50 nM, montrant une diminution de l affinité entre p47phox-p67phox. De même, sur le complexe entier, l effet de la phosphorylation de p47phox est différent de la mutation. Nous avons mesuré les valeurs de EC50 relatives à p67phox pour les différentes formes de p47phox. L activation de p47phox par phosphorylation diminue l EC , alors que les doubles ou triple mutations augmentent sa valeur. Nous avons confirmé que la phosphorylation et la mutation sont insuffisantes pour activer l enzyme. La présence de AA est indispensable pour le fonctionnement du complexe. L ordre de fixation des sous unités cytosoliques semble indifférent mais il faut que tous les composants soient présents lors de l ajout de AA. Enfin, la délétion de p47phox dans la partie C-terminale (aa 343 à 390, domaine d interaction avec p67phox) il n y a plus de formation du dimère mais l enzyme fonctionne normalement. Ces résultats apportent des éléments nouveaux sur le rôle de la dimérisation p47 phox-p67 phox, non indispensable à l activité du système et sur le rôle mineur de la phosphorylation dans l activation de la NADPH oxydase in vitro.The NADPH oxidase of phagocytes is an enzyme involved in the innate defense of organisms against pathogens. After phagocyte activation, this enzyme produces superoxide ions by reduction of dioxygen by NADPH. It is constituted of four cytosolic sub-units (p47phox ; p67phox ; p40phox et Rac) and two membrane proteins (gp91 ; p22phox). Its activation takes place through a complex process that involves protein-protein interaction changes leading to assembly and functionning of the catalytic core. In order to obtain information on this process, I have reconstituted the enzyme in a cell free systeme using recombinant proteins, to be able to fully control all the measurement conditions. In this work, we have compared different activation modes of p47phox i) phosphorylation; ii) substitution serine - aspartate by mutations at positions S303, S304 and S328 to mimic phosphorylation; iii) addition of arachidonic acid (AA), a well known activator molecule in vitro. It has been shown that these three activating methods transform p47phox to an open configuration with similar characteristics. However, we have found that the effects of these methods are significantly different. Indeed, the conformational changes observed by circular dichroism are different. For p47phox, the addition of AA destructures the protein. Its phosphorylation induces a bathochromic displacement of the bands, whereas the mutations S-D lead to an opposite displacement. For the dimer p47phox-p67phox , the addition of AA destructures the proteins while mutations induce hardly no changes. We have measured the dissociation constant Kd of the complex p47phox-p67phox. For wild type proteins, Kd value is low (4+-2 nM), while mutations of p47phox as well as addition of AA increase its value up to 50 nM, showing a decrease of affinity between p47phox and p67phox. Moreover, on the whole complex, the effect of phosphorylation of p47phox is different from mutations. We have shown that the EC50 values relative to p67phox are sensitive to the various modifications of p47phox. Phosphorylation of p47phox decreases EC , while double or triple mutations increase its value. We have confirmed that phosphorylation and mutation are not sufficient to activate the enzyme. The presence of AA is a prerequisite for the functionning of the complex, i.e. production of superoxide. The binding order of the cytosolic proteins seems random but it is necessary that all the components be present during the activation by AA. Finally, deletion of the C terminal part of p47phox (aa 343 to 390, interaction domain with p67phox) leads to the absence of dimer formation but does not affect the enzyme activity. These results bring new information on the role of dimerisation of p47-p67 and on that of phosphorylation in the activation of NADPH oxidase in vitro.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

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

International audienceThe NADPH-oxidase of phagocytic cells is a multicomponent enzyme that generates superoxide. It comprises a membrane flavocytochrome b558 and four cytosolic proteins; p67phox, p47phox, p40phox and Rac. The NADPH-binding site of this complex was shown to be located on the flavocytochrome b558. 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