Publications

2010

• Expression in E. coli and characterization of the catalytic domain of Botrytis cinerea chitin synthase.
  
  • Magellan Hervé
  • Drujon Thierry
  • Thellend Annie
  • Piffeteau Annie
  • Becker Hubert F.
  BMC Research Notes, BioMed Central, 2010, 3 (1), pp.299. BACKGROUND: Chitin synthase 3a (CHS3a) from Botrytis cinerea (Bc) catalyses the multiple transfer of N-acetylglucosamine (GlcNAc) residues to the growing chitin chain. Chitin, a β-1,4 linked GlcNAc homopolymer, is an essential cell wall component of filamentous fungi. Chitin synthase, processive membranous protein, has been recognized as a promising target for new antifungicides. Enzymatic characterizations of chitin synthases have been limited, mainly because purity and amounts of integral enzyme obtained after purification procedures have not been sufficient. FINDINGS: We undertook the preparation of two BcCHS3a fragment proteins, containing only the central domain and devoid of the N-terminal and transmembrane C-terminal regions. The central domain of CHS3a, named SGC (Spsa GntI Core), is conserved in all UDP-glycosyltransferases and it is believed to contain the active site of the enzyme. CHS3a-SGC protein was totally expressed as inclusion bodies in Escherichia coli. We performed recombinant CHS3a-SGC purification in denaturing conditions, followed by a refolding step. Although circular dichroism spectra clearly exhibited secondary structures of renatured CHS3a-SGC, no chitin synthase activity was detected. Nevertheless CHS3a-SGC proteins show specific binding for the substrate UDP-GlcNAc with a dissociation constant similar to the Michaelis constant and a major contribution of the uracil moiety for recognition was confirmed. CONCLUSIONS: Milligram-scale quantities of CHS3a-SGC protein with native-like properties such as specific substrate UDP-GlcNAc binding could be easily obtained. These results are encouraging for subsequent heterologous expression of full-length CHS3a. (10.1186/1756-0500-3-299)
  DOI : 10.1186/1756-0500-3-299
• Direct mid-infrared femtosecond pulse shaping with a calomel acousto-optic programmable dispersive filter
  
  • Maksimenka Raman
  • Nuernberger Patrick
  • Lee Kevin F.
  • Bonvalet Adeline
  • Milkiewicz Jadwiga
  • Barta Cestmir
  • Klima Milos
  • Oksenhendler Thomas
  • Tournois Pierre
  • Kaplan Daniel
  • Joffre Manuel
  Optics Letters, Optical Society of America - OSA Publishing, 2010, 35 (21), pp.3565. Direct amplitude and phase shaping of mid-infrared femtosecond pulses is realized with a calomel-based acousto-optic programmable dispersive filter transparent between 0.4 and 20 mu m. The shaped pulse electric field is fully characterized with high accuracy, using chirped-pulse upconversion and time-encoded arrangement spectral phase interferometry for direct electric field reconstruction techniques. Complex mid-infrared pulse shapes at a center wavelength of 4: 9 mu m are generated with a spectral resolution of 14 cm(-1), which exceeds by a factor of 5 the reported experimental resolutions of calomel-based filters. Cop 2010 Optical Society of America (10.1364/OL.35.003565)
  DOI : 10.1364/OL.35.003565
• Quantum calculation of the second-order hyperpolarizability of chiral molecules in the "one-electron" model
  
  • Hache François
  Journal of Physical Chemistry A, American Chemical Society, 2010, 114 (37), pp.10277-10286. Quantum calculation of the hyperpolarizabilty tensor is carried out for chiral molecules displaying a "one-electron" chirality. Calculation is made possible by introducing a chiral perturbation term in the potential energy surface. We show that a one-electron chiral molecule is intrinsically nonlinear and diplays a nonzero electric chiral hyperpolarizability. Existence of magnetic contributions is discussed, and it is shown that higherorder perturbation terms are necessary to introduce such magnetic effects in the second-order hyperpolarizability. Cop. 2010 American Chemical Society. (10.1021/jp105123m)
  DOI : 10.1021/jp105123m
• Dispersion-based pulse shaping for multiplexed two-photon fluorescence microscopy
  
  • Labroille Guillaume
  • Pillai Rajesh
  • Solinas Xavier
  • Boudoux Caroline
  • Olivier Nicolas
  • Beaurepaire Emmanuel
  • Joffre Manuel
  Optics Letters, Optical Society of America - OSA Publishing, 2010, 35 (20), pp.3444. We demonstrate selective two-photon excited fluorescence microscopy with shaped pulses produced with a simple yet efficient scheme based on dispersive optical components. The pulse train from a broadband oscillator is split into two subtrains that are sent through different amounts of glass. Beam recombination results in pulse-shape switching at a rate of 150 MHz. Time-resolved photon counting detection then provides two simultaneous images resulting from selective two-photon excitation, as demonstrated in a live embryo. Although less versatile than programmable pulse-shaping devices, this novel arrangement significantly improves the performance of selective microscopy using broadband shaped pulses while simplifying the experimental setup. Cop. 2010 Optical Society of America. (10.1364/OL.35.003444)
  DOI : 10.1364/OL.35.003444
• A thermodynamic limit of the melting/freezing processes of water under strongly hydrophobic nanoscopic confinement
  
  • Deschamps Johnny
  • Audonnet Fabrice
  • Brodie-Linder Nancy
  • Schoeffel Markus
  • Alba-Simionesco Christiane
  Physical Chemistry Chemical Physics, Royal Society of Chemistry, 2010, 12 (7), pp.1440. We studied liquid water confined within nanopores which present a high level of hydrophobicity thanks to a new method of synthesis. We found that the liquid state persists down to temperatures much lower than in the bulk and in hydrophilic materials of comparable sizes, allowing us to define a thermo- dynamic limit for the melting/crystallization of water (10.1039/b920816j)
  DOI : 10.1039/b920816j
• Beam waist measurement for terahertz time-domain spectroscopy experiments
  
  • Podzorov Alexander
  • Wojdyla Antoine
  • Gallot Guilhem
  Optics Letters, Optical Society of America - OSA Publishing, 2010, 35 (7), pp.901. Classical masking aperture methods are found to be mostly inaccurate to determine the terahertz beam size in terahertz time-domain spectroscopy (TDS) experiments, owing to complex diffraction effects. Here, we present a simple and reliable method for measuring beam waists in terahertz TDS. It is based on the suecessive diffraction by an opaque disk followed by a small circular aperture. Cop. 2010 Optical Society of America. (10.1364/OL.35.000901)
  DOI : 10.1364/OL.35.000901
• Selective probing of a NADPH site controlled light-induced enzymatic catalysis
  
  • Lambry Jean-Christophe
  • Beaumont Edward
  • Tarus Bogdan
  • Blanchard-Desce Mireille
  • Slama-Schwok Anny
  Journal of Molecular Recognition, Wiley, 2010, 23 (4), pp.379. Achieving molecular recognition of NADPH binding sites is a compelling strategy to control many redox biological processes. The NADPH sites recognize the ubiquitous NADPH cofactor via highly conserved binding interactions, despite differences in the regulation of the hydride transfer in redox active proteins. We recently developed a photoactive NADPH substitute, called nanotrigger NT synchronizing the initiation of enzymatic catalysis of the endothelial NO-synthase (eNOS) with a laser pulse. Spatial and temporal control of enzymatic activity by such a designed light-driven activator would benefit from achieving molecular selectivity, i.e. activation of a single NADPH-mediated enzyme. In this work, we probe the ability of NT to discriminate between two NADPH sites with light. The selected NADPH sites belong to dihydrofolate reductase dihydrofolate reductase enzyme (DHFR) and endothelial NO-synthase (eNOS). Ultrafast kinetics showed that NT could not activate DHFR catalysis with a laser pulse in contrast with the observed trigger of eNOS catalysis leading to NO formation. Homology modelling, molecular dynamics simulations showed that NT discriminated between the two NADPH sites by different donor to acceptor distances and by local steric effects hindering light activation of DHFR catalysis. The data suggested that the narrow NADPH site required a tight fit of the nanotrigger at a suitable distance/angle to the electron acceptor for a specific activation of the catalysis. The ability of the nanotrigger to activate eNOS combined with a low reactivity in unfavourable NADPH sites makes NT a highly promising tool for targeting eNOS in endothelial cells with a laser pulse. Cop 2009 John Wiley and Sons, Ltd. (10.1002/jmr.1009)
  DOI : 10.1002/jmr.1009
• Sickling of red blood cells through rapid oxygen exchange in microfluidic drops
  
  • Abbyad Paul
  • Tharaux Pierre-Louis
  • Martin Jean-Louis
  • Baroud Charles N.
  • Alexandrou Antigoni
  Lab on a Chip, Royal Society of Chemistry, 2010, 10 (19), pp.2505. We have developed a microfluidic approach to study the sickling of red blood cells associated with sickle cell anemia by rapidly varying the oxygen partial pressure within flowing microdroplets. By using the perfluorinated carrier oil as a sink or source of oxygen, the oxygen level within the water droplets quickly equilibrates through exchange with the surrounding oil. This provides control over the oxygen partial pressure within an aqueous drop ranging from 1 kPa to ambient partial pressure, i.e. 21 kPa. The dynamics of the oxygen exchange is characterized through fluorescence lifetime measurements of a ruthenium compound dissolved in the aqueous phase. The gas exchange is shown to occur primarily during and directly after droplet formation, in 0.1 to 0.5 s depending on the droplet diameter and speed. The controlled deoxygenation is used to trigger the polymerization of hemoglobin within sickle red blood cells, encapsulated in drops. This process is observed using polarization microscopy, which yields a robust criterion to detect polymerization based on transmitted light intensity through crossed polarizers. Cop. 2010 The Royal Society of Chemistry. (10.1039/c004390g)
  DOI : 10.1039/c004390g
• QUANTUM YIELD MEASUREMENTS OF SHORT-LIVED PHOTOACTIVATION INTERMEDIATES IN DNA PHOTOLYASE : TOWARD A DETAILED UNDERSTANDING OF THE TRIPLE TRYPTOPHAN ELECTRON TRANSFER CHAIN
  
  • Byrdin M.
  • Lukacs Andras
  • Thiagarajan V.
  • Apm Eker
  • Brettel K.
  • Vos Marten H.
  Journal of Physical Chemistry A, American Chemical Society, 2010, 114 (9), pp.3207-3214. The light-dependent DNA repair enzyme photolyase contains a unique evolutionary conserved triple tryptophan electron transfer chain (W382−W359−W306 in photolyase from E. coli) that bridges the ∼15 Å distance between the buried flavin adenine dinucleotide (FAD) cofactor and the surface of the protein. Upon excitation of the semireduced flavin (FADH°), electron transfer through the chain leads to formation of fully reduced flavin (FADH−; required for DNA repair) and oxidation of the most remote tryptophan residue W306, followed by its deprotonation. The thus-formed tryptophanyl radical W306°+ is reduced either by an extrinsic reductant or by reverse electron transfer from FADH−. Altogether the kinetics of these charge transfer reactions span 10 orders of magnitude, from a few picoseconds to tens of milliseconds. We investigated electron transfer processes in the picosecond−nanosecond time window bridging the time domains covered by ultrafast pump−probe and "classical" continuous probe techniques. Using a recent dedicated setup, we directly show that virtually no absorption change between 300 ps and 10 ns occurs in wild-type photolyase, implying that no charge recombination takes place in this time window. In contrast, W306F mutant photolyase showed a partial absorption recovery with a time constant of 0.85 ns. In wild-type photolyase, the quantum yield of FADH− W306°+ was found at 19 ± 4%, in reference to the established quantum yield of the long-lived excited state of [Ru(bpy)3]2+. With this yield, the optical spectrum of the excited state of FADH° can be constructed from ultrafast spectroscopic data; this spectrum is dominated by excited state absorption extending from below 450 to 850 nm. The new experimental results, taken together with previous data, allow us to propose a detailed kinetic and energetic scheme of the electron transfer chain. Copyright © 2009 American Chemical Society (10.1021/jp9093589)
  DOI : 10.1021/jp9093589
• Nonlinear beam shaper for femtosecond laser pulses, from Gaussian to flat-top profile
  
  • Mercier Brigitte
  • Rousseau Jean-Philippe
  • Jullien A.
  • Antonucci Laura
  Optics Communications, Elsevier, 2010, 283 (14), pp.2900. We present a straightforward method to transform a spatially Gaussian femtosecond laser beam into a flattop shaped beam. The proposed technique takes advantage of a nonlinear phase induced in positive Kerr medium followed by a simple optical system. The variation of the refractive index with the laser intensity creates a phase plate which induces changes in the beam profile after propagation; flat-top and doughnut profiles are observed. The shaping conditions are computed numerically and confirmed experimentally. The method does not introduce energy losses. The device is very simple, self-regulated, flexible and does not need a manufactured phase plate or precise alignment. This method can be useful for light-matter interaction and laser machining. Cop 2010 Elsevier B.V. All rights reserved. (10.1016/j.optcom.2010.04.004)
  DOI : 10.1016/j.optcom.2010.04.004
• Quantum yield measurements of short-lived photoactivation intermediates in DNA photolyase: Toward a detailed understanding of the triple tryptophan electron transfer chain
  
  • Byrdin Martin
  • Lukacs Andras
  • Thiagarajan Viruthachalam
  • Eker André P.M.
  • Brettel Klaus
  • Vos Marten H.
  Journal of Physical Chemistry A, American Chemical Society, 2010, 114 (9), pp.3207-3214. The light-dependent DNA repair enzyme photolyase contains a unique evolutionary conserved triple tryptophan electron transfer chain (W382−W359−W306 in photolyase from E. coli) that bridges the 15 Å distance between the buried flavin adenine dinucleotide (FAD) cofactor and the surface of the protein. Upon excitation of the semireduced flavin (FADH°), electron transfer through the chain leads to formation of fully reduced flavin (FADH−; required for DNA repair) and oxidation of the most remote tryptophan residue W306, followed by its deprotonation. The thus-formed tryptophanyl radical W306°+ is reduced either by an extrinsic reductant or by reverse electron transfer from FADH−. Altogether the kinetics of these charge transfer reactions span 10 orders of magnitude, from a few picoseconds to tens of milliseconds. We investigated electron transfer processes in the picosecond−nanosecond time window bridging the time domains covered by ultrafast pump−probe and "classical" continuous probe techniques. Using a recent dedicated setup, we directly show that virtually no absorption change between 300 ps and 10 ns occurs in wild-type photolyase, implying that no charge recombination takes place in this time window. In contrast, W306F mutant photolyase showed a partial absorption recovery with a time constant of 0.85 ns. In wild-type photolyase, the quantum yield of FADH− W306°+ was found at 19 ± 4%, in reference to the established quantum yield of the long-lived excited state of [Ru(bpy)3]2+. With this yield, the optical spectrum of the excited state of FADH° can be constructed from ultrafast spectroscopic data; this spectrum is dominated by excited state absorption extending from below 450 to 850 nm. The new experimental results, taken together with previous data, allow us to propose a detailed kinetic and energetic scheme of the electron transfer chain. (10.1021/jp9093589)
  DOI : 10.1021/jp9093589
• Multiply excited vibration of carbon monoxide in the primary docking site of hemoglobin following photolysis from the heme
  
  • Nuernberger Patrick
  • Lee Kevin F.
  • Bonvalet Adeline
  • Vos Marten H.
  • Joffre Manuel
  Journal of Physical Chemistry Letters, American Chemical Society, 2010, 1 (14), pp.2077. We investigate ultrafast vibrational ligand dynamics in carboxyhemoglobin using chirped pulse upconversion and demonstrate the formation of vibrationally multiply excited carbon monoxide trapped in the primary docking site of hemoglobin after photolysis. The bleach signal due to ligand dissociation and the incipient docking-site absorption signal are about 200 cm-1 apart and differ by more than an order of magnitude in absorbance. In conventional approaches, these signals are monitored individually. Our method allows simultaneous observation of these signals with both high spectral resolution and high sensitivity. The large amount of vibrationally hot CO in the docking site as observed under Soret band excitation of the heme is discussed in the context of excess energy provided by the pump photon and is shown to be in quantitative agreement with predictions based on changes in the CO equilibrium distance upon instantaneous dissociation. Cop 2010 American Chemical Society. (10.1021/jz1006324)
  DOI : 10.1021/jz1006324
• Polarization-resolved Second Harmonic microscopy in anisotropic thick tissues
  
  • Gusachenko Ivan
  • Latour Gaël
  • Schanne-Klein Marie-Claire
  Optics Express, Optical Society of America - OSA Publishing, 2010, 18 (18), pp.19339-19352. We thoroughly analyze the linear propagation effects that affect polarization-resolved Second Harmonic Generation imaging of thick anisotropic tissues such as collagenous tissues. We develop a theoretical model that fully accounts for birefringence and diattenuation along the excitation propagation, and polarization scrambling upon scattering of the harmonic signal. We obtain an excellent agreement with polarization-resolved SHG images at increasing depth within a rat-tail tendon for both polarizations of the forward SHG signal. Most notably, we observe interference fringes due to birefringence in the SHG depth profile when excited at π/4 angle from the tendon axis. We also measure artifactual decrease of ρ = χxxx/χxyy with depth due to diattenuation of the excitation. We therefore derive a method that proves reliable to determine both ρ and the tendon birefringence and diattenuation. © 2010 Optical Society of America (10.1364/OE.18.019339)
  DOI : 10.1364/OE.18.019339
• Nitric oxide binds to the proximal heme coordination site of the ferrocytochrome c/cardiolipin complex: Formation mechanism and dynamics
  
  • Silkstone G.
  • Kapetanaki Sofia M.
  • Husu I.
  • Vos Marten H.
  • Wilson M.T.
  Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2010, 285 (26), pp.19785. Mammalian mitochondrial cytochrome c interacts with cardiolipin to form a complex (cyt. c/CL) important in apoptosis. Here we show that this interaction leads to structural changes in ferrocytochrome c that leads to an open coordinate site on the central iron, resulting from the dissociation of the intrinsic methionine residue, where NO can rapidly bind (k = 1.2 x 10(7) m(-1) s(-1)). Accompanying NO binding, the proximal histidine dissociates leaving the heme pentacoordinate, in contrast to the hexacoordinate nitrosyl adducts of native ferrocytochrome c or of the protein in which the coordinating methionine is removed by chemical modification or mutation. We present the results of stopped-flow and photolysis experiments that show that following initial NO binding to the heme, there ensues an unusually complex set of kinetic steps. The spectral changes associated with these kinetic transitions, together with their dependence on NO concentration, have been determined and lead us to conclude that NO binding to cyt. c/CL takes place via an overall scheme comparable to that described for cytochrome c' and guanylate cyclase, the final product being one in which NO resides on the proximal side of the heme. In addition, novel features not observed before in other heme proteins forming pentacoordinate nitrosyl species, include a high yield of NO escape after dissociation, rapid (<1 ms) dissociation of proximal histidine upon NO binding and its very fast binding (60 ps) after NO dissociation, and the formation of a hexacoordinate intermediate. These features all point at a remarkable mobility of the proximal heme environment induced by cardiolipin. (10.1074/jbc.M109.067736)
  DOI : 10.1074/jbc.M109.067736
• High Up-Conversion Efficiency of YVO4:Yb,Er Nanoparticles in Water down to the Single-Particle Level
  
  • Mialon Genevieve
  • Tuerkcan Silvan
  • Dantelle Geraldine
  • Collins Daniel P.
  • Hadjipanayi Maria
  • Taylor Robert A.
  • Gacoin Thierry
  • Alexandrou Antigoni
  • Boilot Jean-Pierre
  Journal of Physical Chemistry C, American Chemical Society, 2010, 114 (51), pp.22449. We report up-conversion emission from an aqueous solution of YVO4:Yb3+,Er3+ nanocrystals synthesized by an original method that produces nanoparticles with excellent crystallinity and no porosity. We show that these YVO4:Yb3+,Er3+ nanocrystals are not very sensitive to nonradiative relaxations, leading to a high green-to-red emission ratio of 6.3. Using a comparison with YVO4:Eu3+ particles, we determined the quantum yield of the up-conversion emission of the aqueous YVO4:Yb3+,Er3+ dispersion to be 0.09 +/- 0.04% for an excitation intensity of only 0.55 kW.cm(-2) at 970 nm. Furthermore, single YVO4:Yb,Er particles with an estimated size down to 10 nm can be detected using a wide-field microscope under a 970 nm, 8 kW.cm(-2) excitation. Because of their unexpectedly high up-conversion emission without intermittency, their water dispersibility, and their photostability, YVO4:Yb3+,Er3+ nanoparticles are highly appropriate both for single-biomolecule and for in vivo imaging. (10.1021/jp107900z)
  DOI : 10.1021/jp107900z
• Imaging by second harmonic generation: the example of pulmonary and renal fibrosis. [Imagerie par génération de seconde harmonique: l'exemple des fibroses pulmonaires et rénales.]
  
  • Schanne-Klein Marie-Claire
  Annales de Pathologie, Elsevier Masson, 2010, 30 (5 Suppl 1), pp.56.
• Density of states and vibrational modes of PDMS studied by terahertz time-domain spectroscopy
  
  • Podzorov Alexander
  • Gallot Guilhem
  Chemical Physics Letters, Elsevier, 2010, 495 (40969), pp.46. Using time-domain spectroscopy (TDS), we performed high-precision spectroscopic measurements of polydimethylsiloxane (PDMS) in the terahertz domain. We investigated the influence of crosslinking on the terahertz absorption and refractive index, and modeled the data with vibrational density of states and coupling between photons and vibrational modes of the polymer molecules. We also investigated the influence of the temperature on PDMS, and observed the glass transition temperature, as well as the cold crystallization peak and the melting zone. Cop. 2010 Elsevier B.V. All rights reserved. (10.1016/j.cplett.2010.06.050)
  DOI : 10.1016/j.cplett.2010.06.050
• Expression and purification of untagged and histidine-tagged folate-dependent tRNA:m5U54 methyltransferase from Bacillus subtilis
  
  • Hamdane Djemel
  • Skouloubris Stéphane
  • Myllykallio Hannu
  • Golinelli-Pimpaneau Béatrice
  Protein Expression and Purification, Elsevier, 2010, 73 (1), pp.83-89. Folate-dependent tRNA m5U methyltransferase TrmFO is a flavoprotein that catalyzes the C5-methylation of uridine at position 54 in the T-Psi-C loop of tRNA in several bacteria. Here we report the cloning and optimization of expression in Escherichia coli BL21 (DE3) of untagged, N-terminus, C-terminus (His)6-tagged TrmFO from Bacillus subtilis. Tagged and untagged TrmFO were purified to homogeneity by metal affinity or ion exchange and heparin affinity, respectively, followed by size-exclusion chromatography. The tag did not significantly alter the expression level, flavin content, activity and secondary structure of the protein. Cop. 2010 Elsevier Inc. All rights reserved. (10.1016/j.pep.2010.04.013)
  DOI : 10.1016/j.pep.2010.04.013
• Picosecond primary structural transition of the heme is retarded after nitric oxide binding to heme proteins
  
  • Kruglik Sergei G.
  • Yoo Byung-Kuk
  • Franzen S.
  • Vos Marten H.
  • Martin Jean-Louis
  • Négrerie Michel
  Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences, 2010, 107 (31), pp.13678. We investigated the ultrafast structural transitions of the heme induced by nitric oxide (NO) binding for several heme proteins by subpicosecond time-resolved resonance Raman and femtosecond transient absorption spectroscopy. We probed the heme iron motion by the evolution of the iron-histidine Raman band intensity after NO photolysis. Unexpectedly, we found that the heme response and iron motion do not follow the kinetics of NO rebinding. Whereas NO dissociation induces quasi-instantaneous iron motion and heme doming (< 0.6 ps), the reverse process results in a much slower picosecond movement of the iron toward the planar heme configuration after NO binding. The time constant for this primary domed-to-planar heme transition varies among proteins (∼30 ps for myoglobin and its H64V mutant, ∼15 ps for hemoglobin, ∼7 ps for dehaloperoxidase, and ∼6 ps for cytochrome c) and depends upon constraints exerted by the protein structure on the heme cofactor. This observed phenomenon constitutes the primary structural transition in heme proteins induced by NO binding. (10.1073/pnas.0912938107)
  DOI : 10.1073/pnas.0912938107
• Heme-heme and heme-ligand interactions in the di-heme oxygen-reducing site of cytochrome bd from Escherichia coli revealed by nanosecond absorption spectroscopy
  
  • Rappaport Fabrice
  • Zhang Jie
  • Vos Marten H.
  • Gennis Robert B.
  • Borisov Vitaliy B.
  Biochimica biophysica acta (BBA) - Bioenergetics, Elsevier, 2010, 1797 (9), pp.1657-1664. Cytochrome bd is a terminal quinol:O(2) oxidoreductase of respiratory chains of many bacteria. It contains three hemes, b(558), b(595), and d. The role of heme b(595) remains obscure. A CO photolysis/recombination study of the membranes of Escherichia coli containing either wild type cytochrome bd or inactive E445A mutant was performed using nanosecond absorption spectroscopy. We compared photoinduced changes of heme d-CO complex in one-electron-reduced, two-electron-reduced, and fully reduced states of cytochromes bd. The line shape of spectra of photodissociation of one-electron-reduced and two-electron-reduced enzymes is strikingly different from that of the fully reduced enzyme. The difference demonstrates that in the fully reduced enzyme photolysis of CO from heme d perturbs ferrous heme b(595) causing loss of an absorption band centered at 435 nm, thus supporting interactions between heme b(595) and heme d in the di-heme oxygen-reducing site, in agreement with previous works. Photolyzed CO recombines with the fully reduced enzyme monoexponentially with tau similar to 12 mu s, whereas recombination of CO with one-electron-reduced cytochrome bd shows three kinetic phases, with tau similar to 14 ns, 14 mu s, and 280 mu s. The spectra of the absorption changes associated with these components are different in line shape. The 14 ns phase, absent in the fully reduced enzyme, reflects geminate recombination of CO with part of heme d. The 14-mu s component reflects bimolecular recombination of CO with heme d and electron backflow from heme d to hemes b in similar to 4% of the enzyme population. The final, 280-mu s component, reflects return of the electron from hemes b to heme d and bimolecular recombination of CO in that population. The fact that even in the two-electron-reduced enzyme, a nanosecond geminate recombination is observed, suggests that namely the redox state of heme b(595), and not that of heme b(558), controls the pathway(s) by which CO migrates between heme d and the medium. Cop 2010 Elsevier B.V. All rights reserved. (10.1016/j.bbabio.2010.05.010)
  DOI : 10.1016/j.bbabio.2010.05.010
• D1 protein variants in Photosystem II from Thermosynechococcus elongatus studied by low temperature optical spectroscopy
  
  • Hughes Joseph L.
  • Cox Nicholas
  • Rutherford A.William
  • Krausz Elmars
  • Lai Thanh-Lan
  • Boussac Alain
  • Sugiura Miwa
  Biochimica et Biophysica Acta (BBA) - Reviews on Bioenergetics, Elsevier, 2010, 1797 (1), pp.11-19. In Photosystem II (PSII) from Thermosynechococcus elongatus, high-light intensity growth conditions induce the preferential expression of the psbA3 gene over the psbA1 gene. These genes encode for the D1 protein variants labeled D1:3 and D1:1, respectively. We have compared steady state absorption and photo-induced difference spectra at < 10 K of PSII containing either D1:1 or D1:3. The following differences were observed. (i) The pheophytin Qx band was red-shifted in D1:3 (547.3 nm) compared to D1:1 (544.3 nm). (ii) The electrochromism on the PheoD1 Qx band induced by QA− (the C550 shift) was more asymmetric in D1:3. (iii) The two variants differed in their responses to excitation with far red (704 nm) light. When green light was used there was little difference between the two variants. With far red light the stable (t1/2 > 50 ms) QA− yield was ∼ 95% in D1:3, and ∼ 60% in D1:1, relative to green light excitation. (iv) For the D1:1 variant, the quantum efficiency of photo-induced oxidation of side-pathway donors was lower. These effects can be correlated with amino acid changes between the two D1 variants. The effects on the pheophytin Qx band can be attributed to the hydrogen bond from Glu130 in D1:3 to the 131-keto of PheoD1, which is absent for Gln130 in D1:1. The reduced yield with red light in the D1:1 variant could be associated with either the Glu130Gln change, and/or the four changes near the binding site of PD1, in particular Ser153Ala. Photo-induced QA− formation with far red light is assigned to the direct optical excitation of a weakly absorbing charge transfer state of the reaction centre. We suggest that this state is blue-shifted in the D1:1 variant. A reduced efficiency for the oxidation of side-pathway donors in the D1:1 variant could be explained by a variation in the location and/or redox potential of P+. (10.1016/j.bbabio.2009.07.007)
  DOI : 10.1016/j.bbabio.2009.07.007