Ligand binding dynamics in heme-based oxygen sensor FixL studied by ultrafast spectroscopy

International audienceHeme-based oxygen sensors are part of ligand specific two-component regulatory systems. FixL from Bradyrhizobium japonicum is an example of such a sensor. In this protein the binding of oxygen to the heme in the receptor domain causes changes in an associated enzymatic domain that eventually regulates transcription factors. We used femtosecond absorption spectroscopy to investigate the binding dynamics of O2, CO and NO and the characteristics of the first signaling intermediate upon oxygen release with wild type and mutant forms of FixL. In wild-type specifically, the oxygen binding kinetics in FixL are extremely fast and efficient. Within 20 ps, 90% of oxygen is already recombined in a geminate way and only 10% of the dissociated oxygen leaves the heme pocket, showing that the heme pocket acts as an oxygen trap [1]. The heme spectrum after photolysis of oxygen is perturbed compared to the steady state oxygen-binding spectrum, which shows the residual interaction with the dissociated ligand. Arginine 220 is located in the heme pocket and is proposed to stabilize the bound ligand and participate in the signal transduction [2]. Substitution of R220 with isoleucine, glutamic acid and glutamate all had similar effects: It strongly decreased the part of fast oxygen geminate recombination pointing at an important role of this residue in oxygen trapping. Also the residual interaction with dissociated oxygen is lost as witnessed by the less perturbed dissociation spectrum. The steady-state heme conformation and ligand characteristics are similar in FixL, the oxygen sensor, and myoglobin, the oxygen storage protein. In the heme pocket of Mb a histidine can form a hydrogen bond to the ligand. We prepared the R220H mutant of FixL in which the oxygen binding kinetics became biphasic. In addition to the fast phase of oxygen binding appeared a slower, nanosecond component. The amplitude of each phase was about 50% of the total amplitude. The binding of CO also was dramatically affected. In the R220H mutant about 50% of carbon monoxide rebinds geminately with a time constant 2.4 ns, which is observed nor in WT FixL nor in Mb, where only bimolecular binding of CO occurs. The ensemble of our data indicate that R220 plays a key role in transmission of the signal within the heme domain. Preliminary MD simulations support this vie

Nitric oxide (NO) traffic in endothelial NO synthase. Evidence for a new NO binding site dependent on tetrahydrobiopterin?

International audienceNitric oxide (NO) traffic within the reduced ferrous-nitrosyl complex of endothelial nitric-oxide synthase (eNOS) has been studied by ultrafast time-resolved absorption spectroscopy. In the presence of tetrahydrobiopterin, the rate of NO rebinding to the heme upon photodissociation depends on the NO concentration. The time scale of this process, picoseconds to nanoseconds, precludes a diffusion from the solution toward the protein medium, and altogether the data point at a new NO binding site within the protein. Comparison of the kinetics of pterin-bound and -depleted eNOS points out that the existence of this new site depends on the presence of tetrahydrobiopterin. The new non-heme site may act as a "doorstep" to the heme pocket and control NO escape from eNOS

HIV-1 IN alternative molecular recognition of DNA induced by raltegravir resistance mutations

International audienceVirologic failure during treatment with raltegravir, the first effective drug targeting HIV integrase, is associated with two exclusive pathways involving either Q148H/R/K, G140S/A or N155H mutations. We carried out a detailed analysis of the molecular and structural effects of these mutations. We observed no topological change in the integrase core domain, with conservation of a newly identified V-shaped hairpin containing the Q148 residue, in particular. In contrast, the mutations greatly altered the specificity of DNA recognition by integrase. The native residues displayed a clear preference for adenine, whereas the mutant residues strongly favored pyrimidines. Raltegravir may bind to N155 and/or Q148 residues as an adenine bioisoster. This may account for the selected mutations impairing raltegravir binding while allowing alternative DNA recognition by integrase. This study opens up new opportunities for the design of integrase inhibitors active against raltegravir-resistant viruses. Copyright Cop. 2009 John Wiley and Sons, Ltd. Supporting information may be found in the online version of this article

Synchronous photo-initiation of endothelial NO- Synthase activity by a novel nanotrigger addressed to its NADPH site

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Ultrafast Dynamics of Carboxy-Hemoglobin: Two-Dimensional Infrared Spectroscopy Experiments and Simulations

International audienceThis Letter presents a comparison between experimental and simulated 2D mid-infrared spectra of carboxy-hemoglobin in the spectral region of the carbon monoxide stretching mode. The simulations rely on a fluctuating potential energy surface that includes both the effect of heme and the protein surroundings computed from molecular dynamics simulations. A very good agreement between theory and experiment is obtained with no adjustable parameters. The simulations show that the effect of the distal histidine through the hydrogen bond is strong and is directly responsible for the slow decay of the frequency–frequency correlation function on a 10 ps time scale. This study confirms that fluctuations in carboxy-hemoglobin are more inhomogeneous than those in the more frequently studied carboxy-myoglobin. The comparison between simulations and experiments brings valuable information on the complex relation between protein structure and spectral diffusion

Dinoflagellate cysts from the Cretaceous–Paleogene boundary at Ouled Haddou, southeastern Rif, Morocco: biostratigraphy, paleoenvironments and paleobiogeography

A palynological investigation of a section dated by foraminifera, at Ouled Haddou, south-eastern Rifian Corridor, northern Morocco, revealed a rich and well-preserved dinoflagellate cyst assemblage that allowed a palynological separation of Maastrichtian from Danian deposits. The gradual change of the dinoflagellate cyst assemblages and the biostratigraphic resolution attained, suggest that the studied Maastrichtian-Danian section is continuous. The recognition of the latest Maastrichtian and earliest Danian is based on global dinoflagellate cyst events, including the first occurrence of the latest Maastrichtian species Disphaerogena carposphaeropsis, Glaphyrocysta perforata, and Manumiella seelandica, the latest Maastrichtian acme of Manumiella seelandica, and the first occurrence of the earliest Danian markers Carpatella cornuta, Damassadinium californicum, Eisenackia circumtabulata, Membranilarnacia tenella and Senoniasphaera inornata. The Cretaceous-Paleogene boundary is placed above the latest Maastrichtian events, mainly immediately above the acme of M. seelandica and below the earliest Danian events, particularly below the first occurrences of C. cornuta and D. californicum. The biostratigraphic interpretations are based on a comparison with calibrated dinoflagellate cyst ranges from several reference sections, mainly in the Northern Hemisphere middle latitudes. The Cretaceous-Paleogene boundary is not marked by a mass extinction of dinoflagellate cyst species, but shows important changes in the relative abundances of different species or groups of morphologically related species. These changes are paleoenvironmentally controlled. The peridinioid assemblage suggests deposition in a subtropical to warm temperate province. One dinoflagellate cyst species, Phelodinium elongatum, is formally described