Caracterisations structurales des sites actifs des centres reactionnels de plantes superieures par diffusion Raman de resonance

SIGLEINIST T 70669 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Opposite Movement of the External Gate of a Glutamate Transporter Homolog upon Binding Cotransported Sodium Compared with Substrate

Recently, a new model for glutamate uptake by glutamate transporters was proposed based on crystal structures of the bacterial glutamate transporter homologue Glt(Ph). It was proposed that hairpin two (HP2) functions as the extracellular gate and that Na(+) and glutamate binding closes HP2, thereby allowing for the translocation of the glutamate binding pocket across the membrane. However, the conformation of HP2 in the apo state and the Na(+) bound state is unknown. We here use double site-directed spin-labeling electron paramagnetic resonance spectroscopy on the bacterial transporter Glt(Ph) from Pyrococcus Horikoshi to examine conformational changes in HP2. Surprisingly, the cotransported substrates Na(+) and aspartate induce opposite movements of HP2. We find that in the apo state HP2 is in a similar conformation as in the aspartate-bound closed state. Na(+) binding to the apo state opens HP2, while the subsequent binding of aspartate closes HP2. Our findings show that Na(+) binding opens and stabilizes the extracellular gate thereby allowing for amino acid substrate binding. In contrast, in the absence of Na(+) and aspartate, HP2 closes, thereby suggesting a potential mechanism for the translocation of the empty binding pocket necessary to complete the transport cycle. The finding that physiological Na(+) concentrations stabilize the open HP2 state would ensure that the outward facing conformation of the transporter is maintained in physiological solutions and ensure that glutamate transporters are ready to quickly bind glutamate released from glutamatergic synapses

Opposite Movement of the External Gate of a Glutamate Transporter Homolog upon Binding Cotransported Sodium Compared with Substrate

Recently, a new model for glutamate uptake by glutamate transporters was proposed based on crystal structures of the bacterial glutamate transporter homolog Glt Ph . It was proposed that hairpin two (HP2) functions as the extracellular gate and that Na + and glutamate binding closes HP2, thereby allowing for the translocation of the glutamate binding pocket across the membrane. However, the conformation of HP2 in the apo state and the Na + bound state is unknown. We here use double site-directed spin-labeling electron paramagnetic resonance spectroscopy on the bacterial transporter Glt Ph from Pyrococcus horikoshi to examine conformational changes in HP2. Surprisingly, the cotransported substrates Na + and aspartate induce opposite movements of HP2. We find that in the apo state, HP2 is in a similar conformation as in the aspartate-bound closed state. Na + binding to the apo state opens HP2, whereas the subsequent binding of aspartate closes HP2. Our findings show that Na + binding opens and stabilizes the extracellular gate, thereby allowing for amino acid substrate binding. In contrast, in the absence of Na + and aspartate, HP2 closes, suggesting a potential mechanism for the translocation of the empty binding pocket necessary to complete the transport cycle. The finding that physiological Na + concentrations stabilize the open HP2 state would ensure that the outward-facing conformation of the transporter is maintained in physiological solutions and that glutamate transporters are ready to quickly bind glutamate released from glutamatergic synapses

Carboxylate as the Protonation Site in (Peroxo)diiron(III) Model Complexes of Soluble Methane Monooxygenase and Related Diiron Proteins

Dioxygen activation by carboxylate-bridged diiron enzymes is involved in essential biological processes ranging from DNA synthesis and hydrocarbon metabolism to cell proliferation.1-3 The carboxylate-bridged diiron superfamily of proteins includes ribonucleotide reductase (RNR),4 Δ9 desaturase,5 bacterial multicomponent monooxygenases (BMMs),6,7 and most recently human deoxyhypusine hydroxylase (hDOHH).3 In all of these systems, the O2 reduction step proceeds through a (peroxo)- diiron(III) intermediate in which the resulting peroxo ligand is proposed to bridge two iron atoms in a μ-1,2 or μ-η2η2 coordination mode.8-10 Extensive studies of soluble methane monooxygenase (sMMO), a BMM family member that oxidizes methane to methanol, reveal that the generation and activation of Fe2O2 units requires protons.11,12 Given the complexity of protein environments, identifying the sites involved in such proton translocation processes and their effect on O2 activation is not a trivial undertaking.National Institute of General Medical Sciences (U.S.) (grant GM032134)National Institute of General Medical Sciences (U.S.) (grant GM74785

Versatile Reactivity of a Solvent-Coordinated Diiron(II) Compound: Synthesis and Dioxygen Reactivity of a Mixed-Valent Fe [superscript II] Fe [superscript III] Species

A new, DMF-coordinated, preorganized diiron compound [Fe[subscript 2](N-Et-HPTB)(DMF)[subscript 4]](BF[subscript 4])[subscript 3] (1) was synthesized, avoiding the formation of [Fe(N-Et-HPTB)](BF4)2 (10) and [Fe2(N-Et-HPTB)(μ-MeCONH)](BF[subscript 4])[subscript 2] (11), where N-Et-HPTB is the anion of N,N,N′,N′-tetrakis[2-(1-ethylbenzimidazolyl)]-2-hydroxy-1,3-diaminopropane. Compound 1 is a versatile reactant from which nine new compounds have been generated. Transformations include solvent exchange to yield [Fe[subscript 2](N-Et-HPTB)(MeCN)[subscript 4]](BF[subscript 4])[subscript 3] (2), substitution to afford [Fe[subscript 2](N-Et-HPTB)(μ-RCOO)](BF[subscript 4])[subscript 2] (3, R = Ph; 4, RCOO = 4-methyl-2,6-diphenyl benzoate]), one-electron oxidation by (Cp[subscript 2]Fe)(BF[subscript 4]) to yield a Robin–Day class II mixed-valent diiron(II,III) compound, [Fe[subscript 2](N-Et-HPTB)(μ-PhCOO)(DMF)[subscript 2]](BF[subscript 4])[subscript 3] (5), two-electron oxidation with tris(4-bromophenyl)aminium hexachloroantimonate to generate [Fe[subscript 2](N-Et-HPTB)Cl[subscript 3](DMF)](BF[subscript 4])[subscript 2] (6), reaction with (2,2,6,6-tetramethylpiperidin-1-yl)oxyl to form [Fe[subscript 5](N-Et-HPTB)[subscript 2](μ-OH)[subscript 4](μ-O)(DMF)[subscript 2]](BF[subscript 4])[subscript 4] (7), and reaction with dioxygen to yield an unstable peroxo compound that decomposes at room temperature to generate [Fe[subscript 4](N-Et-HPTB)2(μ-O)[subscript 3](H[subscript 2]O)[subscript 2]](BF[subscript 4])·8DMF (8) and [Fe[subscript 4](N-Et-HPTB)[subscript 2](μ-O)[subscript 4]](BF[subscript 4])[subscript 2] (9). Compound 5 loses its bridging benzoate ligand upon further oxidation to form [Fe[subscript 2](N-Et-HPTB)(OH)[subscript 2](DMF)[subscript 2]](BF[subscript 4])[subscript 3] (12). Reaction of the diiron(II,III) compound 5 with dioxygen was studied in detail by spectroscopic methods. All compounds (1–12) were characterized by single-crystal X-ray structure determinations. Selected compounds and reaction intermediates were further examined by a combination of elemental analysis, electronic absorption spectroscopy, Mössbauer spectroscopy, EPR spectroscopy, resonance Raman spectroscopy, and cyclic voltammetry.National Institute of General Medical Sciences (U.S.) (Grant GM032134)Alexander von Humboldt-Stiftung (Postdoctoral Fellowship

Genotype 1 hepatitis C virus envelope features that determine antiviral response assessed through optimal covariance networks.

The poor response to the combined antiviral therapy of pegylated alfa-interferon and ribavarin for hepatitis C virus (HCV) infection may be linked to mutations in the viral envelope gene E1E2 (env), which can result in escape from the immune response and higher efficacy of viral entry. Mutations that result in failure of therapy most likely require compensatory mutations to achieve sufficient change in envelope structure and function. Compensatory mutations were investigated by determining positions in the E1E2 gene where amino acids (aa) covaried across groups of individuals. We assessed networks of covarying positions in E1E2 sequences that differentiated sustained virological response (SVR) from non-response (NR) in 43 genotype 1a (17 SVR), and 49 genotype 1b (25 SVR) chronically HCV-infected individuals. Binary integer programming over covariance networks was used to extract aa combinations that differed between response groups. Genotype 1a E1E2 sequences exhibited higher degrees of covariance and clustered into 3 main groups while 1b sequences exhibited no clustering. Between 5 and 9 aa pairs were required to separate SVR from NR in each genotype. aa in hypervariable region 1 were 6 times more likely than chance to occur in the optimal networks. The pair 531-626 (EI) appeared frequently in the optimal networks and was present in 6 of 9 NR in one of the 1a clusters. The most frequent pairs representing SVR were 431-481 (EE), 500-522 (QA) in 1a, and 407-434 (AQ) in 1b. Optimal networks based on covarying aa pairs in HCV envelope can indicate features that are associated with failure or success to antiviral therapy