1-kHz table-top ultrashort hard x-ray source for time-resolved x-ray protein

International audienceWe describe a compact, reliable, and high-average-power femtosecond x-ray source and its first application to diffraction on protein crystal. The setup relies on a homemade Ti: sapphire system delivering 12 mJ at a 1 kHz repetition rate, associated with a small vacuum chamber especially designed for laser-plasma interaction and x-ray applications. This device allows the generation of 5×109 photons/s/sr at 8 keV and optimized x-ray irradiation of the studied sample, which can be placed close to the source. We present the diffraction pattern of a protein crystal in a divergent beam geometry, which is a first step to a subpicosecond x-ray diffraction experiment

Excited state coherent vibrational motion in deoxymyoglobin

International audienceFemtosecond coherence spectroscopy of deoxymyoglobin has been performed under excitation in the lowest electronic transition (Q band) and probing in the Soret band. Under these excitation conditions, ground state low-frequency vibrational motions are strongly suppressed with respect to Soret excitation. Evidence is presented for a contribution of excited state vibrational dynamics to the coherent response

Primary processes in heme-based sensor proteins

International audienceA wide and still rapidly increasing range of heme-based sensor proteins has been discovered over the last two decades. At the molecular level, these proteins function as bistable switches in which the catalytic activity of an enzymatic domain is altered mostly by binding or dissociation of small gaseous ligands (O2, NO or CO) to the heme in a sensor domain. The initial "signal" at the heme level is subsequently transmitted within the protein to the catalytic site, ultimately leading to adapted expression levels of specific proteins. Making use of the photolability of the heme-ligand bond that mimics thermal dissociation, early processes in this intra-protein signaling pathway can be followed using ultrafast optical spectroscopic techniques; they also occur on timescales accessible to molecular dynamics simulations. Experimental studies performed over the last decade on proteins including the sensors FixL (O2), CooA (CO) and soluble guanylate cyclase (NO) are reviewed with an emphasis on emerging general mechanisms. After heme-ligand bond breaking, the ligand can escape from the heme pocket and eventually from the protein, or rebind directly to the heme. Remarkably, in all sensor proteins the rebinding, specifically of the sensed ligand, is highly efficient. This "ligand trap" property possibly provides means to smoothen the effects of fast environmental fluctuations on the switching frequency. For 6-coordinate proteins, where exchange between an internal heme-bound residue and external gaseous ligands occurs, the study of early processes starting from the unliganded form indicates that mobility of the internal ligand may facilitate signal transfer. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins. Cop. 2013 Elsevier B.V. All rights reserved

Dynamics of nitric oxide in the active site of reduced cytochrome c oxidase aa3

International audienceNitric oxide (NO) is involved in the regulation of respiration by acting as a competitive ligand for molecular oxygen at the binuclear active site of cytochrome c oxidase. The dynamics of NO in and near this site are not well understood. We performed flash photolysis studies of NO from heme a3 in cytochrome c oxidase from Paracoccus denitrificans, using femtosecond transient absorption spectroscopy. The formation of the product state--the unliganded heme a3 ground state--occurs in a similar stepwise manner (period approximately 700 fs) as previously observed for carbon monoxide photolysis from this enzyme and interpreted in terms of ballistic ligand motions in the active site on the subpicosecond time scale [Liebl, U., Lipowski, G., Négrerie, M., Lambry, J.-C., Martin, J.-L., and Vos, M. H. (1999) Nature 401, 181-184]. A fraction (approximately 35% at very low NO concentrations) of the dissociated NO recombines with heme a3 in 200-300 ps. The presence of this recombination phase indicates that a transient bond to the second ligand-binding site, a copper atom (CuB), has a short lifetime or may not be formed. Increasing the NO concentration increases the recombination yield on the hundreds of picoseconds time scale. This effect, unprecedented for heme proteins, implies that, apart from the one NO molecule bound to heme a3, a second NO molecule can be accommodated in the active site, even at relatively low (submicromolar) concentrations. Models for NO accommodation in the active site, based on molecular dynamics energy minimizations are presented. Pathways for NO motion and their relevance for the regulation of respiration are discussed

Spectroscopie d'émission infrarouge femtoseconde des protéines photoréceptrices orientées

International audienceCe travail a pour but l'étude de la dynamique primaire des protéines et de leur rôle dans la fonction du complexe protéique. La technique utilisée est une spectroscopie femtoseconde non linéaire ( $\chi ^{(2)}$) dans le domaine moyen infrarouge. L'ensemble des mouvements de charge induits par une excitation ultra-brève (~11fs) d'un échantillon de membranes orientées de bactériorhodopsine, un analogue bactérien de la protéine photoréceptrice du système visuel chez les mammifères, donne lieu à une émission directionnelle infrarouge (redressement optique). Les caractéristiques de cette émission (fréquence, phase, amplitude, direction) reflètent la réponse électronique et vibrationnelle de la molécule

Sub-picosecond Raman spectrometer for time-resolved studies of structural dynamics in heme proteins

International audienceWe describe a pump-probe Raman spectrometer based on a femtosecond Ti:sapphire laser, an optical parametric generator and two optical parametric amplifiers for time-resolved studies, with emphasis on the structural dynamics in heme proteins. The system provides a 100-fs pump pulse tunable in the range 500-600 nm and a transform-limited sub-picosecond probe pulse tunable in the range 390-450 nm. The spectrometer has spectral (25 cm(-1)) and temporal (similar to 0.7 ps) resolutions which constitute an effective compromise for identifying transient heme protein species and for following their structural evolution by spontaneous Raman scattering in the time range 0.5 ps to 2 ns. This apparatus was applied to time-resolved studies of a broad range of heme proteins, monitoring the primary dynamics of photoinduced heme coordination state and structural changes, its interaction with protein side-chains and diatomic gaseous ligands, as well as heme vibrational cooling. The treatment of transient Raman spectra is described in detail, and the advantages and shortcomings of spontaneous resonance Raman spectroscopy for ultrafast heme proteins studies are discussed. We demonstrate the efficiency of the constructed spectrometer by measuring Raman spectra in the sub-picosecond and picosecond time ranges for the oxygen-storage heme protein myoglobin and for the oxygen-sensor heme protein FixLH in interaction with the diatomic gaseous ligands CO, NO, and O-2. Copyright (C) 2010 John Wiley and Sons, Ltd

Selective addressing of a novel nanotrigger to the NADPH site of the endothelial NO-Synthase and synchronous photo-initiation of the catalysis

International audienc

Structural changes and picosecond to second dynamics of cytochrome c in interaction with nitric oxide in ferrous and ferric redox states

International audienceApart from its role in electron transfer, mitochondrial cytochrome c also plays a role in apoptosis and is subject to nitrosylation. The cleavage of the Fe–Met80 bond plays a role in several processes including the release of Cyt c from mitochondria or increase of its peroxidase activity. Nitrosylation of Cyt c precludes the reformation of the disrupted Fe–Met80 bond and was shown to occur during apoptosis. These physiological properties are associated with a conformational change of the heme center of Cyt c. Here, we demonstrate that NO binding induces pronounced heme conformational changes in the six-coordinate Cyt c–NO complex. Equilibrium and time-resolved Raman data reveal that the heme structural conformation depends both on the nature of the distal iron ligand (NO or Met80) and on the Fe2+ or Fe3+ heme redox state. Upon nitrosylation, the heme ruffling distortion is greatly enhanced for ferrous Cyt c. Contrastingly, the initial strong heme distortion in native ferric Cyt c almost disappears after NO binding. We measured the heme coordination dynamics in the picosecond to second time range and identified Met80 and NO rebinding phases using time-resolved Raman and absorption spectroscopies. Dissociation of NO instantly produces 5-coordinate heme with a domed structure which continues to rearrange within 15 ps, while the initial ruffling distortion disappears. The rates of Cyt c–NO complex formation measured by transient absorption are kon = 1.81 × 106 M−1 s−1 for ferric Cyt c and 83 M−1 s−1 for ferrous Cyt c. After NO dissociation and exit from the heme pocket, the rebinding of Met80 to the heme iron takes place 6 orders of magnitude more slowly (3–5 μs) than Met80 rebinding in the absence of NO (5 ps). Altogether, these data reveal the structural and dynamic properties of Cyt c in interaction with nitric oxide relevant for the molecular mechanism of apoptosis