Articles

Observation of sub-100 ps conformational changes in photolyzed carbonmonoxy-myoglobin probed by time-resolved circular dichroism
- Dartigalongue Thibault
- Hache François
Chemical Physics Letters, Elsevier , 2005, 415 (4-6), pp.313-316 . A time-resolved circular dichroism (CD) experiment is carried out on carbonmonoxy-myoglobin. The CD is measured with a sub-picosecond time resolution after ligand dissociation and the data are interpreted thanks to a classical CD calculation based on the polarizability theory. We observe a decrease of the CD signal in a few picoseconds and a sub-100 ps relaxation towards steady-state deoxy-myoglobin values which we assign to a stress of the proximal histidine which relaxes with the global reorganization of the protein from its liganded geometry to its deliganded one. 2005 Elsevier B.V. All rights reserved (10.1016/j.cplett.2005.09.022)
DOI : 10.1016/j.cplett.2005.09.022
Ability of some plant extracts , traditionally used to treat ciguatera fish poisoning, to prevent the in vitro neurotoxicity produced by sodium channel activators.
- Boydron-Le Garrec Raphaële
- Benoit Evelyne
- Sauviat Martin-Pierre
- J. Lewis Richard
- Molgó Jordi
- Laurent Dominique
Toxicon, Elsevier , 2005, 46(6), pp.625-634 . The effects of 31 plant extracts, which most are traditionally used to treat ciguatera fish poisoning in the Pacific area, were studied on the cytotoxicity of mouse neuroblastoma cells produced by ouabain, veratridine and/or brevetoxin-3 or Pacific ciguatoxin-1. The cell viability was determined using a quantitative colorimetric method. A marked cytotoxicity of seven of the 31 plant extracts studied, was observed. Despite this, these plant extracts were suspected to contain active compound(s) against the cytotoxicity produced by brevetoxin (2 extracts), brevetoxin, ouabain and/or veratridine (3 extracts), or only against that of ouabain and/or veratridine (2 extracts). Among the 24 plant extracts that exhibited by themselves no cytotoxicity, 22 were active against the effect of brevetoxin or against that of both veratridine and brevetoxin. Similar results were obtained when the seven most active plant extracts were reassayed using ciguatoxin instead of brevetoxin. In conclusion, the present work reports the first activity assessment of some plant extracts, achieved in vitro on a quite large scale. The fact that 27 plant extracts were found to exert, in vitro, a protective effect against the action of ciguatoxin and/or brevetoxin, paves the way for finding new active compounds to treat ciguatera fish poisoning, provided these compounds also reverse the effects of sodium channel activators. (10.1016/j.toxicon.2005.07.002)
DOI : 10.1016/j.toxicon.2005.07.002
Nonlinear optical spectroscopy of chiral molecules
- Fischer Peer
- Hache François
Chirality, Wiley , 2005, 17 (8), pp.421 . We review nonlinear optical processes that are specific to chiral molecules in solution and on surfaces. In contrast to conventional natural optical activity phenomena, which depend linearly on the electric field strength of the optical field, we discuss how optical processes that are nonlinear (quadratic, cubic, and quartic) functions of the electromagnetic field strength may probe optically active centers and chiral vibrations. We show that nonlinear techniques open entirely new ways of exploring chirality in chemical and biological systems: The cubic processes give rise to nonlinear circular dichroism and nonlinear optical rotation and make it possible to observe dynamic chiral processes at ultrafast time scales. The quadratic second-harmonic and sum-frequency-generation phenomena and the quartic processes may arise entirely in the electric-dipole approximation and do not require the use of circularly polarized light to detect chirality. They provide surface selectivity and their observables can be relatively much larger than in linear optical activity. These processes also give rise to the generation of light at a new color, and in liquids this frequency conversion only occurs if the solution is optically active. We survey recent chiral nonlinear optical experiments and give examples of their application to problems of biophysical interest. Cop. 2005 Wiley-Liss, Inc. (10.1002/chir.20179)
DOI : 10.1002/chir.20179
Téléphonie cellulaire et cancer: où en est-on? [Cellular phones and cancer: Current status]
- Colonna Anne
Bulletin du Cancer, Elsevier , 2005, 92 (7), pp.637 . L'évaluation de l'impact des nouvelles technologies sur le corps humain est indispensable pour imposer les normes réglementaires limitant les risques sanitaires. L'apparition et l'évolution des téléphones cellulaires ont été parmi les plus rapides dans l'histoire des innovations. De nombreuses recherches ont été effectuées pour tenter d'évaluer le lien éventuel entre effet sur la santé et téléphonie mobile, au niveau du téléphone cellulaire mais également des stations de base. Cet article présente un aperçu des connaissances en matière de recherche sur l'incidence des ondes radiofréquences. Études épidémiologiques, cellulaires et animales se succèdent mais aucune conclusion définitive n'a pu être établie. Même si certains effets biologiques sur des cellules en culture ont été observés, le lien avec un possible développement de cancer chez l'homme reste encore loin d'être établi. Les études animales montrent pour la plupart des résultats négatifs. Quant aux études épidémiologiques, elles manquent d'un recul suffisant pour pouvoir évaluer l'effet des technologies utilisées aujourd'hui. Face à l'inquiétude des populations, opérateurs de téléphonie mobile, constructeurs et instances gouvernementales financent nombre de projets scientifiques afin de permettre une réglementation adaptée et efficace. [Evaluation of the impact of new technologies on the human body is essential in order to impose regulations to limit health risks. The appearance and evolution of cellular phones have been one of the fastest in the history of innovation. Research reported worldwide has tried to evaluate any potential link between adverse health effects and the mobile phone and its broadcasting stations. This article gives an overview of current research knowledge on the impact of radiofrequency waves on health. Epidemiologic, cellular and animal studies have been carried out, but none of them have reached definitive conclusions. Although some biological effects on cell culture have been observed, their link with human cancer development is far from established. Most of the animal studies show negative results. Epidemiologic studies lack a sufficient perspective to be able to evaluate the effect of evolving technologies used today. High levels of concern by the public have urged mobile phone operators, manufacturers and governmental authorities to finance a number of scientific projects aimed at defining adapted and effective regulations.] Cop. John Libbey Eurotext.
Functionality of nitrated acetylcholine receptor: The two-step formation of nitrotyrosines reveals their differential role in effectors binding
- Négrerie Michel
- Martin Jean-Louis
- Nghiêm H.-O.
FEBS Letters, Wiley , 2005, 579 (12), pp.2643 . The presence of nitrotyrosines is associated with several neurodegenerative pathologies. We evaluated the functionality of the nicotinic acetylcholine receptor possessing nitrotyrosines. The spectrum of the nitrated receptor displays an absorption band characteristic of ortho-nitrophenol. The presence of carbamylcholine in the agonist site prevented the effect of nitration by tetranitromethane in some conditions. The nitration occurred with two discrete steps and pointed out the differential involvement of tyrosines in the binding of acetylcholine and neurotoxin. We concluded that at least two residues involved in agonist binding can be nitrated, which bring similar contributions to the binding energy of the neurotransmitter. Cop. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. (10.1016/j.febslet.2005.03.084)
DOI : 10.1016/j.febslet.2005.03.084
Mid-infrared electric field characterization using a visible charge-coupled-device-based spectrometer
- Kubarych Kevin J.
- Joffre Manuel
- Moore Amy
- Belabas Nadia
- Jonas David
Optics Letters, Optical Society of America - OSA Publishing , 2005, 30 (10), pp.1228-1230 . We characterize ultrashort mid-infrared pulses through upconversion by using the stretched pulses obtained from the uncompressed output of a chirped-pulse amplifier. The power spectrum thus translated into the visible region can be readily measured with a standard silicon CCD camera-based spectrometer. The spectral phase is also characterized by a variant of zero-added-phase spectral phase interferometry for direct electric field reconstruction. This is a general method that provides a multiplex advantage over conventional infrared detector array-based methods. © 2005 Optical Society of America (10.1364/OL.30.001228)
DOI : 10.1364/OL.30.001228
Activationless electron transfer through the hydrophobic core of cytochrome c oxidase
- Jasaitis Audrius
- Rappaport F.
- Pilet Eric
- Liebl Ursula
- Vos Marten H.
Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences , 2005, 102 (31), pp.10882 . Electron transfer (ET) within proteins occurs by means of chains of redox intermediates that favor directional and efficient electron delivery to an acceptor. Individual ET steps are energetically characterized by the electronic coupling V, driving force ΔG, and reorganization energy λ. λ reflects the nuclear rearrangement of the redox partners and their environment associated with the reactions; λ ≈ 700-1,100 meV (1 eV = 1.602 × 10-19 J) has been considered as a typical value for intraprotein ET. In nonphotosynthetic systems, functional ET is difficult to assess directly. However, using femtosecond flash photolysis of the CO-poised membrane protein cytochrome c oxidase, the intrinsic rate constant of the low-ΔG electron injection from heme a into the heme a 3-CuB active site was recently established at (1.4 ns)-1. Here, we determine the temperature dependence of both the rate constant and ΔG of this reaction and establish that this reaction is activationless. Using a quantum mechanical form of nonadiabatic ET theory and common assumptions for the coupled vibrational modes, we deduce that λ is <200 meV. It is demonstrated that the previously accepted value of 760 meV actually originates from the temperature dependence of CuB-CO bond breaking. We discuss that low-ΔG, low-λ reactions are common for efficiently channeling electrons through chains that are buried inside membrane proteins. (10.1073/pnas.0503001102)
DOI : 10.1073/pnas.0503001102
Spectroscopic analysis of keratin endogenous signal for skin multiphoton microscopy: erratum
- Pena Ana-Maria
- Strupler Mathias
- Boulesteix Thierry
- Godeau G.
- Schanne-Klein Marie-Claire
Optics Express, Optical Society of America - OSA Publishing , 2005, 13 (17), pp.6667 . We present corrected versions of the list of authors and of Section 2.1. (10.1364/OPEX.13.006667)
DOI : 10.1364/OPEX.13.006667
Nonlinear spectroscopy of chiral molecules
- Fischer P.
- Hache François
Chirality, Wiley , 2005, 17, pp.421-437 .
Spectroscopic analysis of keratin endogenous signal for skin multiphoton microscopy
- Pena Ana-Maria
- Strupler Mathias
- Boulesteix Thierry
- Schanne-Klein Marie-Claire
Optics Express, Optical Society of America - OSA Publishing , 2005, 13 (16), pp.6268-6274 . We recorded one-photon excited fluorescence (1PEF) and two-photon excited fluorescence (2PEF) spectra of purified keratin from human epidermis, and determined the action cross section of this endogenous chromophore. We used this spectroscopic analysis to analyse multiphoton images of skin biopsies and assign the intrinsic fluorescence signals in the epidermis. We observed a good agreement between in situ and in vitro 2PEF spectra of keratin. This study provides a comprehensive characterization of the 2PEF signal of the keratins from the epidermis, and will be of practical interest for multiphoton imaging of the skin. © 2005 Optical Society of America (10.1364/OPEX.13.006268)
DOI : 10.1364/OPEX.13.006268
Calculation of the circular dichroism spectra of carbon monoxy- and deoxy myoglobin: interpretation of a time-resolved circular dichroism experiment.
- Dartigalongue Thibault
- Hache François
The Journal of Chemical Physics, American Institute of Physics , 2005, 123 (18), pp.184901 . A calculation of the circular dichroism (CD) spectra of carbon monoxy- and deoxy myoglobin is carried out in relation with a time-resolved CD experiment. The calculation is based on the polarizability theory and the parameters are adjusted to fit the experimental absorption and CD spectra. By performing the calculation for intermediate configurations of the protein, we are able to propose an explanation of the CD structure observed on a sub-100 ps time scale. The role of the proximal histidine is, in particular, clearly demonstrated in the first step of the myoglobin relaxation from its liganded to it deliganded form. (10.1063/1.2041467)
DOI : 10.1063/1.2041467
Geminate carbon monoxide rebinding to a c-type haem
- Silkstone G.
- Jasaitis Audrius
- Vos Marten H.
- Wilson M.
Dalton Transactions, Royal Society of Chemistry , 2005, 21 (7), pp.3489 . A chemically modified form of cytochrome c (cyt. c), termed carboxymethyl cytochrome c (cm cyt. c), possesses a vacant sixth coordination site to the haem iron that is available to bind external ligands. We present data on the rapid flash photolysis of CO from the ferrous haem iron of cm cyt. c and describe the kinetics and spectral transitions that accompany the recombination. This was achieved using 30-femtosecond laser pulses and a white light continuum to monitor spectral transitions. Whereas the photo-dissociation quantum yield is close to 1, the yield of CO escape from the protein (the apparent quantum yield, φ) relative to myoglobin (φ = 1) is small due to rapid geminate recombination of CO. On ligand photo-dissociation the haem undergoes a spin-state transition from low-spin ferrous CO bound to penta-coordinate high-spin. Subsequently the system reverts to the CO bound form. The data were fitted with a minimum number of exponentials using global analysis. Recombination of CO with the haem iron of cm cyt. c is multiphasic (τ = 16 ps, 120 ps and 1 ns), involving three spectrally distinct components. The fraction of haem (0.11) not recombining with CO within 4 ns is similar to the value of φ (0.12) measured on the same preparation by the "pulse method" (M. Brunori, G. Giacometti, E. Antonini and J. Wyman, Proc. Natl. Acad. Sci. USA, 1973, 70, 3141-3144, ). This implies that no further geminate recombination occurs at t > 4 ns. This unusually efficient CO-haem geminate recombination indicates the sterically hindered ("caged") nature of the distal haem pocket in cm cyt. c from which it is difficult for CO to escape. The large geminate phase may be contrasted with the behaviour of myoglobin in which geminate recombination is small. This is in general agreement with the well-documented extensive structural dynamics in myoglobin that allow ligand passage, and a higher structural rigidity in cyt. c imposed by the restraints of minimising reorganisation energy for electron transfer (M. Brunori, D. Bourgeois and D. Vallone, J. Struct. Biol., 2004, 147, 223-234, ). The high pH ferrous form of cm cyt. c is a low-spin species having a lysine bound to the central iron atom of the haem (M. Brunori, M. Wilson and E. Antonini, J. Biol. Chem., 1972, 247, 6076-6081; G. Silkstone, G. Stanway, P. Brzezinski and M. Wilson, Biophys. Chem., 2002, 98, 65-77, ). This high pH (pH 8) form of deoxy cm cyt. c undergoes photo-dissociation of lysine (although the proximal histidine is possible) after photo-excitation. Recombination occurs with a time constant (τ) of 7 ps. This is similar to that observed for the geminate rebinding of the Met80 residue in native ferrous cyt. c (τ 6 ps) following its photo-dissociation (S. Cianetti, M. Negrerie, M. Vos, J.-L. Martin and S. Kruglik, J. Am. Chem. Soc., 2004, 126, 13932-13933; W. Wang, X. Ye, A. Demidov, F. Rosca, T. Sjodin, W. Cao, M. Sheeran and P. Champion, J. Phys. Chem., 2000, 104, 10789-10801, ). (10.1039/B508183C)
DOI : 10.1039/B508183C
Retinal isomerization dynamics in dry bacteriorhodopsin films
- Colonna Anne
- Groma Geza I.
- Vos Marten H.
Chemical Physics Letters, Elsevier , 2005, 415 (1-3), pp.69-73 . The primary photoprocesses in neutral and acid forms of oriented dried bacteriorhodopsin films were investigated by femtosecond absorption spectroscopy. The excitation energy dependence of the signals was used to distinguish photochemistry from processes involving photophysics of photocycle intermediates. Both the kinetics and the quantum yield of all-trans excited state decay by retinal photoisomerization and subsequent J → K transition were found to be very similar as in hydrated environments. Therefore, unlike slower photocycle phases, communication of the retinal with the environment does not play a role in retinal isomerization. Our results are important for understanding recent nonlinear optical applications of such films. (10.1016/j.cplett.2005.08.132)
DOI : 10.1016/j.cplett.2005.08.132
Electrical activity alterations induced by chronic absorption of lindane (gamma-hexachlorocyclohexane) trace concentrations in adult rat heart
- Sauviat Martin-Pierre
- Bouvet Suzanne
- Godeau Gaston
- Pages Nicole
Canadian Journal of Physiology and Pharmacology, NRC Research Press , 2005, 83 (3), pp.243 . The heart of adult rat offspring, born to mothers treated with trace concentrations of lindane (0.5 to 2 ppb) through a beverage and to mothers chronically treated with lindane (CL-T) with the same trace concentration, also through a beverage, during lactation and growth has a round shape and accumulates lindane. The left ventricle (LV) presents a hypertrophied area, atrophied papillary muscles, and unorganized collagen bundles and layers. These observations led us to study the electrical activity of their left ventricle papillary muscles (LVPM) by recording action potential using intracellular microelectrodes. CL-T shortened LVPM action potential duration (APD): 1 ppb shortened the plateau; 2 ppb shortened the plateau and the slow repolarizing phase. In CL-T (2 ppb) and untreated groups, low temperature (22 degrees C) decreased the resting potential and prolonged APD. TEA (tetraethylammonium; 1-2 mmol/L) partially lengthened CL-T (2 ppb lindane) APD. Quinidine (0.2 mmol/L) and E-4031 (10 nmol/L) prolonged CL-T APD, suggesting that the rapid delayed outward K+ current (I-Kr) was increased. Our results indicate the silent effects of chronic exposure to trace concentrations of lindane on the morphological and electrical activity of heart muscle. They demonstrate that chronic lindane treatment of female rats alters the tissue integrity and electrical activity in the LV of their offspring. (10.1139/Y04-132)
DOI : 10.1139/Y04-132
Fourier transform measurement of two-photon excitation spectra: applications to microscopy and optimal control
- Ogilvie Jennifer P.
- Kubarych Kevin J.
- Alexandrou Antigoni
- Joffre Manuel
Optics Letters, Optical Society of America - OSA Publishing , 2005, 30, pp.911 . We report a novel Fourier transform method for measuring two-photon excitation spectra. We demonstrate this method using simple dye molecules and discuss its applications in two-photon fluorescence microscopy and optimal control. This method facilitates an intuitive interpretation of recent control experiments in terms of tuning the nonlinear spectrum of the exciting laser source.
Chiroptical effects in the second harmonic signal of collagens I and IV
- Pena Ana-Maria
- Boulesteix Thierry
- Dartigalongue Thibault
- Schanne-Klein Marie-Claire
Journal of the American Chemical Society, American Chemical Society , 2005, 127 (29), pp.10314 . We performed polarization-resolved surface second harmonic generation (SHG) experiments on thin films of collagen I and IV molecules, as well as conventional CD measurements. We found that collagen IV presents little CD and no SHG optical activity, whereas collagen I exhibits large chiroptical effects involving both one-electron and excitonic coupling mechanisms. We estimated that these chiral components enhance the SHG signal from fibrillar collagen in biological tissues by typically a factor of 2. By comparing the distinct behaviors of collagens I and IV in SHG microscopy and in surface SHG experiments, we concluded that SHG microscopy is a sensitive probe of the micrometer-scale structural organization of collagen in biological tissues. Cop. 2005 American Chemical Society. (10.1021/ja0520969)
DOI : 10.1021/ja0520969
In vivo modulation of morphogenetic movements in Drosophila embryos with femtosecond laser pulses
- Supatto Willy
- Débarre Delphine
- Moulia Bruno B.
- Brouzés Eric
- Martin Jean-Louis
- Farge Emmanuel
- Beaurepaire Emmanuel
Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences , 2005, 102 (4), pp.1047 . The complex biomechanical events associated with embryo development are investigated in vivo, by using femtosecond laser pulse-induced ablation combined with multimodal nonlinear microscopy. We demonstrate controlled intravital ablations preserving local cytoskeleton dynamics and resulting in the modulation of specific morphogenetic movements in nonmutant Drosophila embryos. A quantitative description of complex movements is obtained both in GFP-expressing systems by using whole-embryo two-photon microscopy and in unlabeled nontransgenic embryos by using third harmonic generation microscopy. This methodology provides insight into the issue of mechano-sensitive gene expression by revealing the correlation of in vivo tissue deformation patterns with Twist protein expression in stomodeal cells at gastrulation. (10.1073/pnas.0405316102)
DOI : 10.1073/pnas.0405316102
Velocimetric third-harmonic generation microscopy: micrometer-scale quantification of morphogenetic movements in unstained embryos
- Débarre Delphine
- Supatto Willy
- Farge Emmanuel
- Moulia Bruno B.
- Schanne-Klein Marie-Claire
- Beaurepaire Emmanuel
Optics Letters, Optical Society of America - OSA Publishing , 2004, 29, pp.2881-2883 . We demonstrate the association of third-harmonic generation (THG) microscopy and particle image velocimetry (PIV) analysis as a novel functional imaging technique for automated micrometer-scale characterization of morphogenetic movements in developing embryos. Using a combined two-photon-excited fluorescence and THG microscope, we characterize the optical properties of Drosophila embryos and show that sustained THG imaging does not perturb sensitive developmental dynamics. Velocimetric THG imaging provides a quantitative description of the dynamics of internal structures in unstained wild-type and mutant embryos. (10.1364/OL.29.002881)
DOI : 10.1364/OL.29.002881
Second-harmonic microscopy of unstained living cardiac myocytes: measurements of sarcomere length with 20-nm accuracy.
- Boulesteix Thierry
- Beaurepaire Emmanuel
- Sauviat Martin-Pierre
- Schanne-Klein Marie-Claire
Optics Letters, Optical Society of America - OSA Publishing , 2004, 29 (17), pp.2031-3 . We extend second-harmonic generation (SHG) microscopy to the measurement of sarcomere length in unstained living cardiac myocytes with 20-nm accuracy. We quantify individual sarcomere shortening in the presence of saxitoxin and find that it is in agreement with mechanical measurements of atrial tissue contracture. This functional application of SHG microscopy is generally applicable to quantify the physiological effects of drugs on contractile tissue. Our data also suggest that packed myosin heads in sarcomere thick filaments are responsible for the large second-harmonic endogenous signal in muscle tissue. (10.1364/OL.29.002031)
DOI : 10.1364/OL.29.002031
From The Cover: An aminoacyl-tRNA synthetase-like protein encoded by the Escherichia coli yadB gene glutamylates specifically tRNAAsp
- Dubois Daniel
- Blaise Mickaël
- Becker Hubert F.
- Campanacci Valérie
- Keith Gerard
- Cambillau Christian
- Lapointe Jacques
- Kern Daniel
- Becker D.
- Giegé Richard
Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences , 2004, 101 (20), pp.7530-7535 . The product of the Escherichia coli yadB gene is homologous to the N-terminal part of bacterial glutamyl-tRNA synthetases (GluRSs), including the Rossmann fold with the acceptor-binding domain and the stem-contact fold. This GluRS-like protein, which lacks the anticodon-binding domain, does not use tRNA(Glu) as substrate in vitro nor in vivo, but aminoacylates tRNA(Asp) with glutamate. The yadB gene is expressed in wild-type E. coli as an operon with the dksA gene, which encodes a protein involved in the general stress response by means of its action at the translational level. The fate of the glutamylated tRNA(Asp) is not known, but its incapacity to bind elongation factor Tu suggests that it is not involved in ribosomal protein synthesis. Genes homologous to yadB are present only in bacteria, mostly in Proteobacteria. Sequence alignments and phylogenetic analyses show that the YadB proteins form a distinct monophyletic group related to the bacterial and organellar GluRSs (alpha-type GlxRSs superfamily) with ubiquitous function as suggested by the similar functional properties of the YadB homologue from Neisseria meningitidis. (10.1073/pnas.0401634101)
DOI : 10.1073/pnas.0401634101
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
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
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
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
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