Diffusion-limited REE uptake by eclogite garnets and its consequences for Lu-Hf and Sm-Nd geochronology

Garnets from the Zermatt-Saas Fee eclogites contain narrow central peaks for Lu+Yb+Tm±Er and at least one additional small peak towards the rim. The REE Sm+Eu+Gd+Tb±Dy are depleted in the cores but show one prominent peak close to the rim. These patterns cannot be modeled using Rayleigh fractionation accompanied by mineral breakdown reactions. Instead, the patterns are well explained using a transient matrix diffusion model where REE uptake is limited by diffusion in the matrix surrounding the porphyroblast. Observed profiles are well matched if a roughly linear radius growth rate is used. The secondary peaks in the garnet profiles are interpreted to reflect thermally activated diffusion due to temperature increase during prograde metamorphism. The model predicts anomalously low 176Lu/177Hf and 147Sm/144Nd ratios in garnets where growth rates are fast compared to diffusion of the REE, and these results have important implications for Lu-Hf and Sm-Nd geochronology using garne

Human Cytomegalovirus Entry into Dendritic Cells Occurs via a Macropinocytosis-Like Pathway in a pH-Independent and Cholesterol-Dependent Manner

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that is able to infect fibroblastic, epithelial, endothelial and hematopoietic cells. Over the past ten years, several groups have provided direct evidence that dendritic cells (DCs) fully support the HCMV lytic cycle. We previously demonstrated that the C-type lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) has a prominent role in the docking of HCMV on monocyte-derived DCs (MDDCs). The DC-SIGN/HCMV interaction was demonstrated to be a crucial and early event that substantially enhanced infection in trans, i.e., from one CMV-bearing cell to another non-infected cell (or trans-infection), and rendered susceptible cells fully permissive to HCMV infection. Nevertheless, nothing is yet known about how HCMV enters MDDCs. In this study, we demonstrated that VHL/E HCMV virions (an endothelio/dendrotropic strain) are first internalized into MDDCs by a macropinocytosis-like process in an actin- and cholesterol-dependent, but pH-independent, manner. We observed the accumulation of virions in large uncoated vesicles with endosomal features, and the virions remained as intact particles that retained infectious potential for several hours. This trans-infection property was specific to MDDCs because monocyte-derived macrophages or monocytes from the same donor were unable to allow the accumulation of and the subsequent transmission of the virus. Together, these data allowed us to delineate the early mechanisms of the internalization and entry of an endothelio/dendrotropic HCMV strain into human MDDCs and to propose that DCs can serve as a "Trojan horse" to convey CMV from entry sites to other locations that may favor the occurrence of either latency or acute infection

Purification et caractérisation spectroscopique de cytochrome c oxydases.

Cytochrome c Oxidase (CcO) membrane protein complexes catalyse the reduction of oxygen that takes place in the respiratory chains of eukaryotes and aerobic bacteria. During this reaction, that requires four protons and four electrons, four additional protons are pumped across the membrane and these participate in the formation of the proton motive force that is required for ATP synthesis. The active site of CcO aa3 contains a high-spin heme, heme a3, and a copper atom, CuB. These cofactors can bind, apart from O2, other diatomic molecules involved in signalling such as NO or CO. My thesis work concerns both CcO-ligand interactions and internal electron transfer (ET), in mitochondrial CcO and in bacterial oxidases aa3 and ba3. These oxidases accommodate four redox centres, heme a3 and CuB as well as heme a (resp. b for ba3) and CuA, involved in ET from the donor, Cytochrome c to the acceptor, the oxygen bound to the active site. The reversible inhibition of CcO by NO is involved in the regulation of respiration. By studying the influence of the NO concentration on the dynamics of NO in the oxidases aa3 from P. denitrificans and ba3 from T. thermophilus, we have demonstrated that multiple ligand interact in the active site. For aa3, geminate recombination of NO, after its photo dissociation from heme a3, occurs in two phases of 200 ps and 20 ns. The amplitude of this phase increases with suprastoichiometric NO concentrations. In contrast, no effect of the NO concentration is observed for Oxidase ba3, where the NO-reductase activity prevents the stable co presence of two NO molecules. Altogether, these results imply that a second NO molecule can be accommodated in/near the active site of CcO aa3 favouring geminate recombination of the first NO molecule, rather than its motion out of the protein. In order to determine the nature of this second NO-binding site, EPR experiments were performed. A spectral change as a function of the NO concentration was only observed for aa3 Oxidase. At low concentrations (CcO: NO <1:1), the signal, very similar to that of ba3, is specific for an NO molecule bound to a histamine coordinated heme. However, at higher concentrations, the spectrum resembles that of a five coordinated nitrosylated heme. As the visible spectrum does not indicate a rupture of the heme-NεHis bond, we propose that the second NO molecule induces a rotation of the heme-bound NO from an orientation parallel to the histamine ring to a more perpendicular one. Such a rotation would disrupt the paramagnetic interaction between the histamine ring and NO. Our interpretation is strengthened by molecular modelling studies of the active site of CcO aa3, with one and two NO molecules bound. These studies indicated that the presence of a second NO molecule, bound to CuB, induces a ~70° rotation of the heme-bound NO. In addition, at high NO concentrations, a signal characteristic of an NO-metal interaction appear that can be attributed to CuB. The assessment of the simultaneous presence of two NO molecules in the active site of CcO aa3, even at low NO concentrations, has implications for understanding the mechanism of CcO inhibition by NO. In order to study the influence of the active site environment modifications on NO dynamics, the amino acid V279 has been substituted. Preliminary studies of strains expressing the mutants indicate modification of their O2 reductase activities. Along a different line, we investigated the reduction kinetics of the active site by heme a. As this reaction occurs too fast to be observed by electron injection into the system, we studied reverse electron transfer. The experiments were performed on mammalian CcO aa3 with heme an oxidised, heme a3 reduced and CO-bound. After the cleavage of the CO-Fe bond by a light pulse, the electrons can equilibrate between the two hemes which have close-lying redox potentials. Spectroscopic ally, we measured that 13 ± 3 % of this transfer takes place in 1.2 ns; this time corresponds to the intrinsic transfer. The fastest rate previously measured for interheme ET in CcO was 3 µs. In the light of our results, this phase can be explained by a modification of the heme a3 redox potential due to CO leaving CuB. The very fast electron exchange determined between the two hemes may help to increase the oxygen trapping efficiency under physiological conditions ([O2] low and weak efflux), by decreasing the duration of the periods where heme a3 is not reduced.Les cytochromes c oxydases (CcO), situées dans les chaînes respiratoires eucaryotes et des bactéries aérobiques, catalysent la réduction de l'oxygène en eau, une réaction qui utilise quatre électrons et quatre protons. Ces complexes protéiques membranaires participent également à la formation de la force protonmotrice nécessaire à la synthèse d'ATP, en pompant quatre protons supplémentaires par molécule de dioxygène réduit. Le site actif des CcO aa3 contient un hème de type a de haut spin, l'hème a3, et un atome de cuivre, le CuB. Ces deux cofacteurs peuvent fixer, outre l'O2, d'autres ligands diatomiques impliqués dans la signalisation telle que le monoxyde d'azote (NO) et le monoxyde de carbone (CO). Les travaux présentés dans ce manuscrit, effectué sur l'oxydase mitochondriale et sur des oxydases bactériennes, aa3 et ba3, concernent à la fois l'interaction de ligands avec la CcO et le transfert interne d'électrons. Les oxydases étudiées possèdent quatre centres redox, l'hème a3 et le CuB ainsi que l'hème a (resp. b pour ba3) et le centre CuA, impliqués dans le transfert d'électron (ET) du donneur, le cytochrome c, à l'accepteur, l'oxygène fixé au site actif. L'inhibition réversible de la CcO par le NO est un processus de régulation de la respiration. En étudiant l'influence de la concentration de NO sur la dynamique du NO au sein des oxydases aa3 de P. denitrificans et ba3 de T. thermophilus nous avons mis en évidence une interaction entre plusieurs ligands dans le site actif. Pour l'oxydase aa3, la recombinaison géminée du NO, après sa photodissociation de l'hème a3, a lieu selon deux phases de 200 ps et 20 ns, dont l'intensité augmente pour les concentrations de NO superstoechiométriques. A l'inverse, aucun effet de la concentration n'est observé pour l'oxydase ba3 où l'activité NO réductase de cette CcO exclut la présence stable de deux molécules de NO dans le site actif. L'ensemble de ces résultats converge vers la présence d'une seconde molécule de NO dans ou à proximité du site actif dans l'oxydase aa3, créant un encombrement favorisant une recombinaison géminée plutôt qu'un cheminement vers l'extérieur de la protéine. Des expériences de spectroscopie RPE ont été effectuées afin de déterminer la nature du second site de fixation du NO. La modification du signal avec la concentration de NO est observée uniquement pour l'oxydase aa3. Si le spectre à basses concentrations (NO: CcO<1:1), très similaire à celui obtenu avec l'oxydase ba3, est caractéristique de la liaison du NO sur un hème ayant pour 5ème ligand une histidine, le spectre enregistré à hautes concentrations est similaire à celui mesuré lorsque NO est lié à un hème sans autre ligand en trans. Comme le spectre visible de l'oxydase n'indique pas une rupture de la liaison Fe-NεHis, nous proposons que la 2nd molécule de NO induise une rotation du NO lié à l'hème depuis une position parallèle au plan de l'histidine à une position perpendiculaire à ce plan, rompant ainsi les interactions paramagnétiques entre l'histidine et le NO. Cette interprétation est renforcée par des modélisations de la structure de l'oxydase aa3 avec un et deux molécules de NO dans le site actif. Elles montrent qu'en effet la présence d'un second NO, lié au CuB dans le site actif, induit une rotation du NO lié à l'hème de ~70°. Par ailleurs, à hautes concentrations de NO, il y a apparition d'un signal caractéristique d'une interaction métal de transition-NO, qui peut être attribué, par élimination, à la liaison CuB-NO. L'établissement de la présence simultanée de deux molécules de NO dans le site actif de la CcO aa3 de P. denitrificans dès les faibles concentrations de NO, ouvrent de nouvelles voies dans la compréhension des mécanismes d'inhibition de l'oxydase par le NO. Afin d'étudier plus en détail l'influence de l'environnement du site actif sur la dynamique du NO dans le site actif, le résidu V279 à été substitué par mutagenèse dirigée. Les premiers résultats sur des souches exprimant les oxydases mutées indiquent des modifications de leur activité O2 réductase. Par ailleurs, nous nous sommes intéressé à la vitesse de réduction du site actif par l'hème a. Cette réaction étant trop rapide pour être visualisée par injection d'électrons, nous avons étudié le flux des électrons en sens inverse. Ces expériences ont été effectuées sur l'oxydase mitochondriale dont l'hème a était oxydé et l'hème a3 réduit et lié une molécule de CO. Après photodissociation du CO de l'hème a3, les électrons se répartissent entre les deux hèmes de potentiel redox proche. Par spectroscopie, nous avons ainsi mesuré que 13 ±3 % de ce transfert s'effectue en 1,2 ns; ce temps correspond au transfert intrinsèque. Des études précédentes avaient montré une phase de 3 µs, considérée jusqu'ici comme la phase la plus rapide du ET entre les hèmes. Au regard de nos résultats, cette phase de ET peut être expliquée par le départ du CO du CuB, ce qui modifierait le potentiel redox de l'hème a3. Le transfert intrinsèque des électrons en 1,2 ns, permettrait à l'oxydase, dans des conditions physiologiques ([O2] faibles et e-peu disponibles) de capturer et de réduire l'oxygène en diminuant la durées des périodes où le site actif n'est pas réduit

Purification et caractérisation spectroscopique de cytochrome c oxydases

PALAISEAU-Polytechnique (914772301) / SudocSudocFranceF

Ultrafast spectroscopy of electron transfer between hemes in bovine heart cytochrome c oxidase

International audienceIn cytochrome c oxidase, reduction of molecular oxygen in the active site requires transfer of electrons from cytochrome c towards heme a3. This occurs via a chain of redox intermediates. The dynamics of the final step in this chain, reduction of heme a3 by the chemically identical heme a, cannot be resolved in direct external electron injection experiments. Instead electron redistribution can be monitored upon photodissociation of CO from the mixed-valence (MV) enzyme-CO complex. The published value for the equilibration time in the mitochondrial enzyme is f3 As [1]. However, a detailed spectral analysis of the microsecond data (by Wikstro¨m's group) predicted a substantial additional equilibration phase on a much faster time scale [2]. This prediction was contested (by Brzezinski's group) on the basis of nanosecond experiments [3]. We have performed transient absorption experiments with femtosecond resolution and a time window up to 4 ns under selective excitation of heme a3 in the alpha band (595 nm) starting from the fully reduced (a2+a32+-CO) and MV (a3+a32+-CO) COcomplexes. In the MV complex only, a significant spectral evolution with a nanosecond time constant was observed both in alpha and in Soret spectral regions that can be fully ascribed to electron equilibration (a3+a32+a2+a33+). We suggest that the intrinsic time constant of reduction of heme a3 by heme a is on this time scale. The result will be discussed in the framework of conflicting theoretical predictions. An additional remarkable observation is that the CO-dissociation spectrum from heme a3 depends on the redox state of heme a. This finding adds to the notion that can in principle not be treated as spectrally independent entities. This observation may relate to the strong variation in spectral decompositions using various methods

Accommodation of NO in the active site of mammalian and bacterial cytochrome c oxidase aa3

International audienceFollowing different reports on the stoichiometry and configuration of NO binding to mammalian and bacterial reduced cytochrome c oxidase aa3 (CcO), we investigated NO binding and dynamics in the active site of beef heart CcO as a function of NO concentration, using ultrafast transient absorption and EPR spectroscopy. We find that in the physiological range only one NO molecule binds to heme a3, and time-resolved experiments indicate that even transient binding to CuB does not occur. Only at very high (∼ 2 mM) concentrations a second NO is accommodated in the active site, although in a different configuration than previously observed for CcO from Paracoccus denitrificans [E. Pilet, W. Nitschke, F. Rappaport, T. Soulimane, J.-C. Lambry, U. Liebl and M.H. Vos. Biochemistry 43 (2004) 14118-14127], where we proposed that a second NO does bind to CuB. In addition, in the bacterial enzyme two NO molecules can bind already at NO concentrations of ∼ 1 μM. The unexpected differences highlighted in this study may relate to differences in the physiological relevance of the CcO-NO interactions in both species