Publications

2004

• NO binding and dynamics in reduced heme-copper oxidases aa3 from Paracoccus denitrificans and ba3 from Thermus thermophilus
  
  • Pilet Eric
  • Nitschke W.
  • Rappaport F.
  • Soulimane T.
  • Lambry Jean-Christophe
  • Liebl Ursula
  • Vos Marten H.
  Biochemistry, American Chemical Society, 2004, 43 (44), pp.14118. Cytochrome c oxidase (CcO) has a high affinity for nitric oxide (NO), a property involved in the regulation of respiration. It has been shown that the recombination kinetics of photolyzed NO with reduced CcO from Paracoccus denitrificans on the picosecond time scale depend strongly on the NO/ enzyme stoichiometry and inferred that more than one NO can be accommodated by the active site, already at mildly suprastoichiometric NO concentrations. We have largely extended these studies by monitoring rebinding dynamics from the picosecond to the microsecond time scale, by performing parallel steady-state low-temperature electron paramagnetic resonance (EPR) characterizations on samples prepared similarly as for the optical experiments and comparing them with molecular-modeling results. A comparative study was performed on CcO ba3 from Thermus thermophilus, where two NO molecules cannot be copresent in the active site in the steady state because of its NO reductase activity. The kinetic results allow discrimination between different models of NO-dependent recombination and show that the overall NO escape probability out of the protein is high when only one NO is bound to CcO aa3, whereas strong rebinding on the 15-ns time scale was observed for CcO ba3. The EPR characterizations show similar results for aa3 at substoichiometric NO/enzyme ratios and for ba3, indicating formation of a 6-coordinate heme-NO complex. The presence of a second NO molecule in the aa3 active site strongly modifies the heme-NO EPR spectrum and can be rationalized by a rotation of the Fe-N-O plane with respect to the histidine that coordinates the heme iron. This proposal is supported by molecular-modeling studies that indicate a ~63° rotation of heme-bound NO upon binding of a second NO to the close-lying copper center CuB. It is argued that the second NO binds to CuB. (10.1021/bi0488808)
  DOI : 10.1021/bi0488808
• Electron transfer between hemes in mammalian cytochrome c oxidase
  
  • Pilet Eric
  • Jasaitis Audrius
  • Liebl Ursula
  • Vos Marten H.
  Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences, 2004, 101 (46), pp.16198. Fast intraprotein electron transfer reactions associated with enzymatic catalysis are often difficult to synchronize and therefore to monitor directly in non-light-driven systems. However, in the mitochondrial respiratory enzyme cytochrome oxidase aa(3), the kinetics of the final electron transfer step into the active site can be determined: reverse electron flow between the close-lying and chemically identical hemes a(3) and a can be initiated by flash photolysis of CO from reduced heme a(3) under conditions where heme a is initially oxidized. To follow this reaction, we used transient absorption spectroscopy, with femtosecond time resolution and a time window extending to 4 ns. Comparison of the picosecond heme a(3)-CO photodissociation spectra under different redox states of heme a shows significant spectral interaction between both hemes, a phenomenon complicating the interpretation of spectral studies with low time resolution. Most importantly, we show that the intrinsic electron equilibration, corresponding to a DeltaG(0) of 45-55 meV, occurs in 1.2 +/- 0.1 ns. This is 3 orders of magnitude faster than the previously established equilibration phase of approximate to3 mus, which we suggest to reflect a change in redox equilibrium closely following CO migration out of the active site. Our results allow testing a number of conflicting predictions regarding this reaction between both experimental and theoretical studies. We discuss the potential physiological relevance of fast equilibration associated with this low-driving-force redox reaction. (10.1073/pnas.0405032101)
  DOI : 10.1073/pnas.0405032101
• Intraprotein electron transfer and proton dynamics during photoactivation of DNA photolyase from E. coli: Review and new insights from an "inverse" deuterium isotope effect.
  
  • Byrdin Martin
  • Sartor Valérie
  • Eker André P.M.
  • Vos Marten H.
  • Aubert Corinne
  • Brettel Klaus
  • Mathis Paul
  Biochimica biophysica acta (BBA) - Bioenergetics, Elsevier, 2004, 1655 (12 april), pp.64. We review our work on electron transfer and proton dynamics during photoactivation in DNA photolyase from E. coli and discuss a recent theoretical study on this issue. In addition, we present unpublished data on the charge recombination between the fully reduced FADH- and the neutral (deprotonated) radical of the solvent exposed tryptophan W306. We found a pronounced acceleration with decreasing pH and an inverse deuterium isotope effect (kH/kD=0.35 at pL 6.5) and interpret it in a model of a fast protonation equilibrium for the W306 radical. Due to this fast equilibrium, two parallel recombination channels contribute differently at different pH values: one where reprotonation of the W306 radical is followed by electron transfer from FADH- (electron transfer time constant tet in the order of 10-50 µs), and one where electron transfer from FADH- (tet=25 ms) is followed by reprotonation of the W306 anion. Cop. 2004 Elsevier B.V. All rights reserved. (10.1016/j.bbabio.2003.07.001)
  DOI : 10.1016/j.bbabio.2003.07.001
• CO Vibration as a Probe of Ligand Dissociation and Transfer in Myoglobin
  
  • Polack Thomas
  • Ogilvie Jennifer P.
  • Franzen S.
  • Vos Marten H.
  • Joffre Manuel
  • Martin Jean-Louis
  • Alexandrou Antigoni
  Physical Review Letters, American Physical Society, 2004, 93 (1), pp.18102. We report femtosecond visible pump, midinfrared probe, spectrally integrated experiments resolving the dynamics of CO in myoglobin upon photodissociation. Our results show a progressive change in absorption strength of the CO vibrational transition during its transfer from the heme to the docking site, whereas the vibrational frequency change is faster than our time resolution. A phenomenological model gives good qualitative agreement with our data for a time constant of 400 fs for the change in oscillator strength. Density-functional calculations demonstrate that indeed vibrational frequency and absorption strength are not linearly coupled and that the absorption strength varies in a slower manner due to charge transfer from the heme iron to CO (10.1103/PhysRevLett.93.018102)
  DOI : 10.1103/PhysRevLett.93.018102
• Application of the polarizability theory to the calculation of anisotropic circular dichroism spectra
  
  • Hache François
  • Dartigalongue Thibault
  Chemical Physics, Elsevier, 2004, 303 (1-2), pp.197-203. We derive a general expression of the circular dichroism spectra of anisotropically distributed chiral molecules in the polarizability framework by introducing a tensorial rotational strength. Example calculation is carried out on an acridine-substituted Tröger base. It is shown that the circular dichroism is very sensitive to the direction propagation of the light beam. (10.1016/j.chemphys.2004.05.021)
  DOI : 10.1016/j.chemphys.2004.05.021
• Resonant optical rectification in bacteriorhodopsin
  
  • Groma Géza
  • Colonna Anne
  • Lambry Jean-Christophe
  • Petrich Jacob
  • Váró G.
  • Joffre Manuel
  • Vos Marten H.
  • Martin Jean-Louis
  Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences, 2004, 101 (21), pp.7971. The relative role of retinal isomerization and microscopic polarization in the phototransduction process of bacteriorhodopsin is still an open question. It is known that both processes occur on an ultrafast time scale. The retinal trans?cis photoisomerization takes place on the time scale of a few hundred femtoseconds. On the other hand, it has been proposed that the primary light-induced event is a sudden polarization of the retinal environment, although there is no direct experimental evidence for femtosecond charge displacements, because photovoltaic techniques cannot be used to detect charge movements faster than picoseconds. Making use of the known high second-order susceptibility ?(2) of retinal in proteins, we have used a nonlinear technique, interferometric detection of coherent infrared emission, to study macroscopically oriented bacteriorhodopsin-containing purple membranes. We report and characterize impulsive macroscopic polarization of these films by optical rectification of an 11-fs visible light pulse in resonance with the optical transition. This finding provides direct evidence for charge separation as a precursor event for subsequent functional processes. A simple two-level model incorporating the resonant second-order optical properties of retinal, which are known to be a requirement for functioning of bacteriorhodopsin, is used to describe the observations. In addition to the electronic response, long-lived infrared emission at specific frequencies was observed, reflecting charge movements associated with vibrational motions. The simultaneous and phase-sensitive observation of both the electronic and vibrational signals opens the way to study the transduction of the initial polarization into structural dynamics. (10.1073/pnas.0306789101)
  DOI : 10.1073/pnas.0306789101
• Functionalized fluorescent oxide nanoparticles: Artificial toxins for sodium channel targeting and imaging at the single-molecule level
  
  • Beaurepaire Emmanuel
  • Buissette Valérie
  • Sauviat Martin-Pierre
  • Giaume Domitile
  • Lahlil Khalid
  • Mercuri Antoine
  • Casanova Didier
  • Huignard Arnaud
  • Martin Jean-Louis
  • Gacoin Thierry
  • Boilot Jean-Pierre
  • Alexandrou Antigoni
  Nano Letters, American Chemical Society, 2004, 4 (11), pp.2079-2083. Lanthanide ion-doped oxide nanoparticles were functionalized for use as fluorescent biological labels. These nanoparticles are synthesized directly in water, which facilitates their functionalization, and are remarkably photostable without emission intermittency. Nanoparticles functiqnalized with guanidinium groups act as artificial toxins and specifically target sodium channels. They are individually detectable in live cardiac myocytes, revealing a heterogeneous distribution of sodium channels. Functionalized oxide nanoparticles appear to be a novel tool that is particularly attractive for long-term single-molecule tracking. (10.1021/nl049105g)
  DOI : 10.1021/nl049105g
• Excited-state properties of flavin radicals in flavoproteins: Femtosecond spectroscopy of DNA photolyase, glucose oxidase, and flavodoxin
  
  • Pan Jie
  • Byrdin Martin
  • Aubert Corinne
  • Eker André P. M.
  • Brettel Klaus
  • Vos Marten H.
  Journal of Physical Chemistry B, American Chemical Society, 2004, 108 (28), pp.10160. In isolated, catalytically inactive, DNA photolyase from Escherichia coli (E. coli), the flavin adenine dinucleotide cofactor is in its neutral radical state FAD?. It can be activated by a unique light-induced reduction of the flavin, a process initiated by the formation of the excited-state FAD?*. As the photophysical properties of this state are essentially unknown, we performed a comparative characterization by femtosecond transient absorption spectroscopy of FAD?* in DNA photolyase from E. coli and glucose oxidase from Aspergillus niger, and of the excited neutral radical flavin mononucleotide (FMN?*) in flavodoxin from Desulfovibrio gigas. In contrast to photolyase, in glucose oxidase and flavodoxin no electron-transfer products are observed after selective excitation of the flavin radical. In glucose oxidase, FAD?* decays to the ground state in 59 ± 5 ps, close to the 80-ps intrinsic lifetime of the excited state in photolyase, and we discuss that the intrinsic lifetime of the excited state of flavin radical in protein environment is in the 50-80 ps range. FMN?* in flavodoxin decays much faster (2.3 ± 0.3 ps), possibly because of quenching by formation of a very short-lived (INF 0.7 ps) electron-transfer intermediate. Spectroscopically, the excited state of FAD? in photolyase displays a pronounced spectral feature that is absent in the other systems studied. Further characterization by polarized photoselection experiments identifies the feature as an additional induced absorption band at ~ 550 nm superimposed on the ground-state bleaching signal. In view of the unique U-shape configuration of FAD in photolyase, we suggest it to reflect a flavin-adenine charge-transfer interaction.
• Coherent vibrational climbing in carboxyhemoglobin
  
  • Ventalon Cathie
  • Fraser James M.
  • Vos Marten H.
  • Alexandrou Antigoni
  • Martin Jean-Louis
  • Joffre Manuel
  Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences, 2004, 101 (36), pp.13216-13220. We demonstrate vibrational climbing in the CO stretch of carboxyhemoglobin pumped by midinfrared chirped ultrashort pulses. By use of spectrally resolved pump-probe measurements, we directly observed the induced absorption lines caused by excited vibrational populations up to v = 6. In some cases, we also observed stimulated emission, providing direct evidence of vibrational population inversion. This study provides important spectroscopic parameters on the CO stretch in the strong-field regime, such as transition frequencies and dephasing times up to the v = 6to v = 7 vibrational transition. We measured equally spaced vibrational transitions, in agreement with the energy levels of a Morse potential up to v = 6. It is interesting that the integral of the differential absorption spectra was observed to deviate far from zero, in contrast to what one would expect from a simple one-dimensional Morse model assuming a linear dependence of dipole moment with bond length. (10.1073/pnas.0401844101)
  DOI : 10.1073/pnas.0401844101
• Libration induced stretching mode excitation for pump-probe spectroscopy in pure liquid water
  
  • Amir Wafa
  • Gallot Guilhem
  • Hache François
  The Journal of Chemical Physics, American Institute of Physics, 2004, 121 (16), pp.7908. We developed an experimental approach to study pure liquid water in the infrared and avoid thermal effects. This technique is based on libration induced stretching excitation of water molecules. A direct correspondence between frequencies within the libration and OH stretching bands is demonstrated. Energy diffusion is studied in pure liquid water by measuring wave packet dynamics of OH stretching vibrator with infrared femtosecond spectroscopy. Wave packet dynamics reveals ultrafast energy dynamics and reflects 130 fs intermolecular energy transfer between water vibrators. Energy diffusion is almost two orders of magnitude faster than self diffusion in water. Cop. 2004 American Institute of Physics. (10.1063/1.1800952)
  DOI : 10.1063/1.1800952
• Functional analysis of FAD-dependent thymidylate synthase ThyX from Paramecium bursaria chlorella virus-1
  
  • Graziani Sébastien
  • Xia Y.
  • Gurnon Jr
  • van Etten J.
  • Leduc Damien
  • Skouloubris Stéphane
  • Myllykallio Hannu
  • Liebl Ursula
  Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2004, 279 (52), pp.54340. Sequence analysis of the 330-kb double-stranded DNA genome of Paramecium bursaria chlorella virus-1 revealed an open reading frame A674R that encodes a protein with up to 53% amino acid identity to a recently discovered new class of thymidylate synthases, called ThyX. Unlike the traditional thymidylate synthase, ThyA, that uses methylenetetrahydrofolate (CH(2)H(4)folate) as both a source of the methylene group and the reductant, CH(2)H(4)folate only supplies the methylene group in ThyX-catalyzed reactions. Furthermore, ThyX only catalyzes thymidylate (dTMP) formation in the presence of reduced pyridine nucleotides and oxidized FAD. The distribution and transcription patterns of the a674r gene in Chlorella viruses were examined. The a674r gene was cloned, and the protein was expressed in Escherichia coli. Biochemical characterization of the P. bursaria chlorella virus-1 recombinant ThyX protein indicates that it is more efficient at converting dUMP to dTMP than previously studied ThyX enzymes, thus allowing more detailed mechanistic studies of the enzyme. The ThyX-dUMP complexes with bound FAD function as efficient NAD(P) H oxidases, indicating that dUMP binds to the enzyme prior to NAD(P) H. This oxidation activity is directly linked to FAD reduction. Our results indicate that ThyX-specific inhibitors can be designed that do not affect ThyA enzymes. Finally, a model is proposed for the early stages of ThyX catalysis. (10.1074/jbc.M409121200)
  DOI : 10.1074/jbc.M409121200
• Spectroscopie d'émission infrarouge femtoseconde des protéines photoréceptrices orientées
  
  • Colonna Anne
  • Lambry Jean-Christophe
  • Groma G.I.
  • Martin Jean-Louis
  • Vos Marten H.
  Journal de Physique IV Proceedings, EDP Sciences, 2004, 119, pp.161-162. Ce 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. (10.1051/jp4:2004119037)
  DOI : 10.1051/jp4:2004119037
• Dynamique ultrarapide du proton dans l'eau : étude expérimentale
  
  • Amir Wafa
  • Gallot Guilhem
  • Hache François
  Journal de Physique IV Proceedings, EDP Sciences, 2004, 119, pp.111-112. Nous présentons une étude de la dynamique femtoseconde du proton dans l'eau liquide par spectroscopie vibrationnelle femtoseconde résolue en temps. C'est à ce jour la première observation expérimentale d'une forme limite du proton en solution, et une première étape vers la compréhension du mécanisme de Grotthuss. (10.1051/jp4:2004119015)
  DOI : 10.1051/jp4:2004119015
• Photodissociation of heme distal methionine in ferrous cytochrome c revealed by subpicosecond time-resolved resonance raman spectroscopy
  
  • Cianetti Simona
  • Négrerie Michel
  • Vos Marten H.
  • Martin Jean-Louis
  • Kruglik Sergei G.
  Journal of the American Chemical Society, American Chemical Society, 2004, 126 (43), pp.13932. Cytochrome c (cyt c) is anelectron-transfer heme protein that also binds nitric oxide (NO). In resting cyt c, two endogenous ligands of the heme iron are histidine-18 (His) and methionine-80 (Met) side chains, and NO binding requires the cleavage of one of the axial bonds. Previous femtosecond transient absorption studies suggested the photolysis of either Fe-His or Fe-Met bonds. We aimed at unequivocally identifying the internal side chain that is photodissociated in ferrous cyt c and at monitoring heme structural dynamics, by means of time-resolved resonance Raman (TR3) spectroscopy with ~0.6 ps time resolution. The Fe-His stretching mode at 216 cm-1 has been observed in photoproduct TR3 spectra for the first time for a c-type heme. The same transient mode was observed for a model ferrous cyt c N-fragment (residues 1-56) ligated with two His in the resting state. Our TR3 data reveal that upon ferrous cyt c photoexcitation, (i) distal Met side chain is instantly released, producing a five-coordinated domed heme structure, (ii) proximal His side chain, coupled to the heme, exhibits distortion due to strain exerted by the protein, and (iii) alteration in heme-cysteine coupling takes place along with the relaxation of the protein-induced deformations of the heme macrocycle. Copyright 2004 American Chemical Society. (10.1021/ja046442i)
  DOI : 10.1021/ja046442i
• Laser spectroscopic visualization of hydrogen bond motions in liquid water
  
  • Bratos S.
  • Leicknam J.-C.
  • Pommeret S.
  • Gallot Guilhem
  Journal of Molecular Structure, Elsevier, 2004, 708 (1-3), pp.197-203. Ultrafast pump-probe experiments are described permitting a visualization of molecular motions in diluted HDO/D2O solutions. The experiments were realized in the mid-infrared spectral region with a time resolution of 150 fs. They were interpreted by a careful theoretical analysis, based on the correlation function approach of statistical mechanics. Combining experiment and theory, stretching motions of the OH?O bonds as well as HDO rotations were 'filmed' in real time. It was found that molecular rotations are the principal agent of hydrogen bond breaking and making in water. Recent literatures covering the subject, including molecular dynamics simulations, are reviewed in detail. Cop. 2004 Elsevier B.V. All rights reserved. (10.1016/j.molstruc.2004.06.036)
  DOI : 10.1016/j.molstruc.2004.06.036
• Functional evidence for active site location of tetrameric thymidylate synthase X at the interphase of three monomers
  
  • Leduc Damien
  • Graziani Sébastien
  • Lipowski Gérard
  • Marchand C.
  • Le Maréchal P.
  • Liebl Ursula
  • Myllykallio Hannu
  Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences, 2004, 101 (19), pp.7252-7257. Little is known about the catalytic mechanism of the recently discovered ThyX family of flavin-dependent thymidylate synthases that are required for thymidylate (deoxythymidine 5'-monophosphate) synthesis in a large number of microbial species. Using a combination of site-directed mutagenesis and biochemical measurements, we have identified several residues of the Helicobacter pylori ThyX protein with crucial roles in ThyX catalysis. By providing functional evidence that the active site(s) of homotetrameric ThyX proteins is formed by three different subunits, our findings suggest that ThyX proteins have evolved through multimerization of inactive monomers. Moreover, because the active-site configurations of ThyX proteins, present in many human pathogenic bacteria, and of human thymidylate synthase ThyA are different, our results will aid in the identification of compounds specifically inhibiting microbial growth. (10.1073/pnas.0401365101)
  DOI : 10.1073/pnas.0401365101
• Dynamic regulation of the inducible nitric-oxide synthase by NO - Comparison with the endothelial isoform
  
  • Gautier Clément
  • Négrerie Michel
  • Wang Zq
  • Lambry Jean-Christophe
  • Stuehr Dennis
  • Collin Fabrice
  • Martin Jean-Louis
  • Slama-Schwok Anny
  Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2004, 279 (6), pp.4358. We studied by ultrafast time-resolved absorption spectroscopy the geminate recombination of NO to the oxygenase domain of the inducible NO synthase, iNOSoxy, and to mutated proteins at position Trp-457. This tryptophan interacts with the tetrahydrobiopterin cofactor BH4, and W457A/F mutations largely reduced the catalytic formation of NO. BH4 decreases the rate of NO rebinding to the ferric iNOSoxy compared with that measured in its absence. The pterin has a larger effect on W457A/F than on the WT protein by increasing NO release from the protein. Therefore, BH4 raises the energy barrier for NO recombination to the mutated proteins in contrast with our observations on eNOS (SlamaSchwok, A., Negrerie, M., Berka, V., Lambry, J: C., Tsai, A.-L., Vos, M., and Martin, J.-L. (2002) J. Biol. Chem. 277, 7581-7586). Thus, we show a differential effect of BH4 on NO release from eNOS and iNOS. Compared with the position of this residue in the BH4-repleted enzyme, simulations of the NO dissociation dynamics point out at a swing of Trp-457 toward the missing pterin in the absence of BH4. NO geminate-rebinding data show a more efficient NO release from eNOS than from iNOS once NO is formed. Consistently, NO produced by iNOS is regulated by its ferric nitrosyl complex in contrast with eNOS. We show that the small enhancement of the NO geminate recombination rate in W457A/F compared with that in the WT enzyme cannot explain the decrease of NO yield because of the mutation; the major effect of the mutation thus arises from an uncoupled catalysis (Wang, Z. Q., Wei, C. C., Ghosh, S., Meade, A. L., Hemann, C., Hille, R., and Stuehr, D. J. (2001) Biochemistry 40, 12819-12825). (10.1074/jbc.M305048200)
  DOI : 10.1074/jbc.M305048200