Changenet-Barret, P (Changenet-Barret, Pascale); Plaza, P (Plaza, Pascal); Martin, MM (Martin, Monique M.); Chosrowjan, H (Chosrowjan, Haik); Taniguchi, S (Taniguchi, Seiji); Mataga, N (Mataga, Noboru); Imamoto, Y (Imamoto, Yasushi); Kataoka, M (Kataoka, Mikio) Role of arginine 52 on the primary photoinduced events in the PYP photocycle CHEMICAL PHYSICS LETTERS, 434 (4-6): 320-325 FEB 5 2007 PHOTOACTIVE YELLOW PROTEIN; EXCITED-STATE DYNAMICS; FEMTOSECOND-PICOSECOND FLUORESCENCE; SITE-DIRECTED MUTAGENESIS; ECTOTHIORHODOSPIRA-HALOPHILA; PRIMARY PHOTOREACTION; CHROMOPHORE MODEL; SPECTROSCOPY; NANOSPACES; EVOLUTION We investigated the earliest steps of the photocycle of photoactive yellow protein (PYP) and its R52Q mutant in aqueous solution, by subpicosecond transient absorption spectroscopy. Our aim was to address the role of the positively charged Arg52 residue. We found that the relaxation mechanism of R52Q is similar to that of wt-PYP and discarded that Arg52 plays a key role in the chromophore photoreactivity. The excited-state decay of R52Q is however slower, confirming previous fluorescence up-conversion data. The experiments also reveal a slower stabilization of the cis isomer product. The loosening of the protein pocket and structural heterogeneities are discussed. Plaza, P (Plaza, Pascal); Mahet, M (Mahet, Mathilde); Martin, MM (Martin, Monique M.); Checcucci, G (Checcucci, Giovanni); Lenci, F (Lenci, Francesco) Target analysis of primary photoprocesses involved in the oxyblepharismin-binding protein JOURNAL OF PHYSICAL CHEMISTRY B, 111 (4): 690-696 FEB 1 2007 UNICELLULAR EUKARYOTE BLEPHARISMA; UP PHOTOPHOBIC RESPONSE; ENHANCED RAMAN-SPECTROSCOPY; PHOTOACTIVE YELLOW PROTEIN; HUMAN SERUM-ALBUMIN; PHOTOSENSORY TRANSDUCTION; ELECTRON-TRANSFER; ACTION SPECTRA; BACTERIAL PHOTOSYNTHESIS; PHOTORECEPTOR PIGMENT Target analysis is performed on previously published transient absorption spectra of the 200-kDa oxyblepharismin-binding protein (OBIP) thought to tric?gger the photophobic response of the ciliate Blepharisma japonicum. The OBIP sample is considered as heterogeneous and made of two distinct classes of chromophore-protein complexes. A so-called nonreactive class is seen to be comparable to free oxyblepharismin in organic solution. Another, reactive, class is shown to undergo a fast picosecond photocycle involving the formation in 4 ps of an intermediate state noted Y-1. The spectrum associated to Y-1 bears striking similarities with that of the oxyblepharismin radical cation. This element favors the hypothesis that an excited-state intermolecular electron-transfer could be the primary step of the sensory transduction chain of B. japonicum. Proton release is also considered as a possible secondary step. These possibilities support the idea that reactive OBIP functions like an electron or proton pump. We alternatively propose a new hypothesis stating that the fast photocycle of reactive OBIP actually does not generate any photoproduct or protein change of conformation but is involved in another biological function. It would act as a kind of solar screen, providing additional protection to the light-adapted form of B. japonicum in case of excessive illumination. Espagne, A (Espagne, Agathe); Changenet-Barret, P (Changenet-Barret, Pascale); Baudin, JB (Baudin, Jean-Bernard); Plaza, P (Plaza, Pascal); Martin, MM (Martin, Monique M.) Photoinduced charge shift as the driving force for the excited-state relaxation of analogues of the Photoactive Yellow Protein chromophore in solution JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY A-CHEMISTRY, 185 (2-3): 245-252 JAN 25 2007 4-hydroxycinnamic acid; PYP; photoisomerization; solvent effects; ultrafast spectroscopy ISOMERIZATION DYNAMICS; PHOTON-ABSORPTION; PRIMARY EVENTS; PHOTOISOMERIZATION; MODEL; PYP; CONSTANTS; LACTONES; VISION Transient absorption spectroscopy with subpicosecond laser excitation is used to probe the primary photoinduced processes in two ester analogues (linear and cyclic) of the Photoactive Yellow Protein (PYP) chromophore in solution. The PYP chromophore is the thioester derivative of the deprotonated trans-4-hydroxycinnamic acid. The results found for the ester analogues are compared to those previously obtained for the deprotonated trans-4-hydroxycinnamic acid and its amide and thioester derivatives. Special attention is paid to the role of the electron donor-acceptor character of the chromophore substituents and of the molecular flexibility on the excited-state relaxation pathway and kinetics. Solvent viscosity and polarity effects on the kinetics are also analyzed. Two hypothetical relaxation pathways involving a one-bond flip mechanism are proposed to explain the observation of a transient species in the course of the excited-state relaxation of the analogues bearing the Stronger electron-acceptor substituents. In the first one, the intermediate is described as a perp ground state, whereas the second one involves a twisted excited state where the conformation of the ethylenic bond deviates from 90 degrees. In both cases, the relaxation of the transient state may lead or not to the cis isomer. Mahet, M (Mahet, M.); Plaza, P (Plaza, P.); Martin, MM (Martin, M. M.); Checcucci, G (Checcucci, G.); Lenci, F (Lenci, F.) Primary photoprocesses in oxyblepharismin interacting with its native protein partner JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY A-CHEMISTRY, 185 (2-3): 345-353 JAN 25 2007 Blepharisma japonicum; oxyblepharismin; oxyblepharismin binding protein (OBIP); human serum albumin (HSA); photoinduced electron transfer; ultrafast transient absorption spectroscopy UP PHOTOPHOBIC RESPONSE; UNICELLULAR EUKARYOTE BLEPHARISMA; LASER FLASH-PHOTOLYSIS; AQUEOUS-SOLUTION; ACTION SPECTRA; PHOTORECEPTOR PIGMENTS; TRIPLET-STATE; HYPERICIN; JAPONICUM; FLUORESCENCE The primary photoprocesses in the photoreceptor for the step-up photophobic response of the light-adapted cells of Blepharisma japonicum (OBIP, OxyBlepharismin bInding Protein) have been studied by Ultrafast UV-vis transient spectroscopy. The results are ratc?ionalized in terms of heterogeneity of the OBIP sample. Two independent classes of chromoprotein are proposed: a "reactive" species, which presents a specific 680-nm band decaying in 4 and 56 ps and a "non-reactive" one, which behaves like the free chromophore (OxyBP) in solution. A bimolecular photooxidation of OxyBP in the presence of 1,4-benzoquinone was performed to record the absorption spectrum of the OxyBP radical cation. Comparison with reactive OBIP Suggests that an electron transfer could be involved in the primary photoprocesses, of OBIP and possibly trigger the sensory transduction chain of B. japonicum. In addition, the specificity of the chromophore-protein interaction was investigated by studying the artificial complex that OxyBP forms with human serum albumin (HSA). OxyBP-HSA happens to be spectroscopically much closer to free OxyBP than to OBIP. This highlights the specific nature of the interaction between OxyBP and its native protein partner and further supports the proposal that OBIP is the actual photoreceptor for the photophobic response of B. japonicum. Espagne, A (Espagne, Agathe); Paik, DH (Paik, Daniel H.); Changenet-Barret, P (Changenet-Barret, Pascale); Martin, MM (Martin, Monique M.); Zewail, AH (Zewail, Ahmed H.) Ultrafast photoisomerization of photoactive yellow protein chromophore analogues in solution: Influence of the protonation state CHEMPHYSCHEM, 7 (8): 1717-1726 AUG 11 2006 femtochemistry; isomerization; photochemistry; solvent effects; time-resolved spectroscopy P-COUMARIC ACID; INITIAL PHOTOINDUCED DYNAMICS; FREQUENCY-DEPENDENT FRICTION; GREEN FLUORESCENT PROTEIN; POTENTIAL-ENERGY SURFACE; PRIMARY EVENTS; ECTOTHIORHODOSPIRA-HALOPHILA; CONICAL INTERSECTION; SOLVATION DYNAMICS; PHOTOCHEMICAL ISOMERIZATION We investigate solvent viscosity and polarity effects on the photo-isomerization of the protonated and deprotonated forms of two analogues of the photoreactive yellow protein (PYP) chromophore. These are trans-p-hydroxybenzlidene acetone and trans-p-hydroxyphenyl cinnamatc?e, studied in solutions of different polarity and viscosity at room temperature, by means of femtosecond fluorescence up-conversion. The fluorescence lifetimes of the protonated forms are found to be barely sensitive to solvent viscosity and to increase with increasing solvent polarity. In contrast, the fluorescence of the deprotonated forms are significantly slowed down in viscous media and accelerated in polar solvents. These results elucidate the dramatic influence of the protonation state of the PYP chromophore analogues on their photoinduced dynamics. The viscosity and polarity effects are, respectively, interpreted in terms of different isomerization coordinates and charge redistribution in S-1. A trans-to-cis isomerization mechanism involving mainly the ethylenic double-bond torsion and/or solvation is proposed for the anionic forms, whereas "concerted" intramolecular motions are proposed for the neutral forms. Espagne, A; Changenet-Barret, P; Plaza, P; Martin, MM Solvent effect on the excited-state dynamics of analogues of the photoactive yellow protein chromophore JOURNAL OF PHYSICAL CHEMISTRY A, 110 (10): 3393-3404 MAR 16 2006 P-COUMARIC ACID; LINKED 4-HYDROXYCINNAMYL CHROMOPHORE; INTRAMOLECULAR CHARGE-TRANSFER; HYDROGEN-BONDED COMPLEXES; PUSH-PULL POLYENES; ECTOTHIORHODOSPIRA-HALOPHILA; POLAR-SOLVENTS; PRIMARY EVENTS; ISOMERIZATION DYNAMICS; SOLVATION DYNAMICS We previously reported that two analogues of the Photoactive Yellow Protein chromophore, trans-phydroxycirmarnic acid (pCA(2-)) and its amide derivative (pCM(-)) in their deprotonated forms, undergo a trans-cis photoisomerization whereas the thioester derivative, trans-p-hydroxythiophenyl cinnamate (pCT(-)), does not. pCT- is also the only one to exhibit a short-lived intermediate on its excited-state deactivation pathway. We here further stress the existence of two different relaxation mechanisms for these molecules and examine the reaction coordinates involved. We looked at the effect of the solvent properties (viscosity, polarity, solvationc? dynamics) on their excited-state relaxation dynamics, probed by ultrafast transient absorption spectroscopy. Sensitivity to the solvent properties is found to be larger for pCT- than for pCA2- and pCM-. This difference is considered to reveal that either the relaxation pathway or the reaction coordinate is different for these two classes of analogues. It is also found to be correlated to the electron donor-acceptor character of the molecule. We attribute the excited-state deactivation of analogues bearing a weaker acceptor group, pCA(2-) and pCM(-), to a stilbene-like photoisomerization mechanism with the concerted rotation of the ethylenic bond and one adjacent single bond. For pCT-, which contains a stronger acceptor group, we consider a photoisomerization mechanism mainly involving the single torsion of the ethylenic bond. The excited-state deactivation of pCT(-) would lead to the formation of a ground-state intermediate at the "perp" geometry, which would return to the initial trans conformation without net isomerization. Pieroni, O; Plaza, P; Mahet, M; Angelini, N; Checcucci, G; Malatesta, M; Martin, MM; Lenci, F Circular dichroism of the photoreceptor pigment oxyblepharismin PHOTOCHEMISTRY AND PHOTOBIOLOGY, 81 (6): 1343-1346 NOV-DEC 2005 BLEPHARISMA-JAPONICUM; HYPERICIN; PROTEIN; ENANTIOMERS; BARRIER Circular dichroism (CD) was used to study the structure of oxyblepharismin (OxyBP), the photoreceptor chromophore for the photophobic response of the blue form of Blepharisma japonicum. Both the chromophore associated to its native protein and the free chromophore in ethanol solution were investigated. CD spectra in the far-UV range indicate that OxyBP induces a slight increase in the a-helix content of the protein matrix. CD spectra in the near-UV and visible region of the spectrum show that OxyBP adopts a chiral conformation with a preferential geometry not only when associated to its protein matrix, but also when isolated and dissolved in ethanol. This experimental result is related to the existence of ac? high-energy interconversion barrier between two enantiomeric structures of the molecule and discussed on the basis of an asymmetric biosynthesis of its precursor, blepharismin. Plaza, P; Mahet, M; Martin, MM; Angelini, N; Malatesta, M; Checcucci, G; Lenci, F Spectroscopic study of the chromophore-protein association and primary photoinduced events in the photoreceptor of Blepharisma japonicum PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES, 4 (9): 754-761 2005 UP PHOTOPHOBIC RESPONSE; UNICELLULAR ORGANISM; STENTOR-COERULEUS; FLASH-PHOTOLYSIS; CILIATE STENTOR; ACTION SPECTRA; FLUORESCENCE; PIGMENT; TRANSDUCTION; HYPERICIN Blepharisma japonicum is a ciliated protozoan exhibiting a strong step-up photophobic response upon illumination. The photoreceptor chromophores responsible for this response have been identified to be hypericin-like chromophores ( blepharismin and oxyblepharismin), complexed to a 200 kDa non-water-soluble protein. The present work opens up new perspectives on the primary phototransduction steps of B. japonicum's light perception through a joined approach by steady-state fluorescence spectroscopy, time-resolved fluorescence anisotropy and sub-picosecond transient absorption spectroscopy. The free chromophore of the light-adapted form of the cell ( oxyblepharismin) was studied in various solvents and its spectroscopic properties, as well as its primary excited-state reactivity, compared with those of the corresponding pigment - protein complex, extracted by phosphate-concentration-step chromatography on a hydroxyapatite column. Fluorescence anisotropy together with SDS PAGE electrophoresis results confirm that oxyblepharismin is non-covalently bound to the apoprotein and show that, in the excited state, it is free to rotate in all directions within the binding site where it experiences a large local viscosity. Time-resolved anisotropy measurements on aromatic amino acids confirm that the molecular weight of the protein is of the order of 200 kDa. Although showing very similar steady-state spectra, frc?ee oxyblepharismin and its protein complex have noticeably different excited-state behaviours. In particular, the protein complex exhibits a pronounced short-lived absorption feature in the 640 - 750 nm range, decaying biexponentially in 4 ps and 56 ps. Those decays, also observed in other spectral regions, are not found in the corresponding kinetics of the isolated pigment in solution. This early behaviour of the protein complex might be the signature of the primary phototransduction process, possibly involving an electron transfer from the pigment to a neighbouring protein acceptor residue as it had been suggested in previous studies. Murali, S; Changenet-Barret, P; Ley, C; Plaza, P; Rettig, W; Martin, MM; Lapouyade, R Photophysical properties of pyrrolobenzenes with different linking and substitution pattern: The transition between charge transfer states with large (MICT) and small (TICT) resonance interaction CHEMICAL PHYSICS LETTERS, 411 (1-3): 192-197 AUG 5 2005 Keywords Plus: EXCITED-STATE; DUAL FLUORESCENCE; N-PHENYLPYRROLES; MOLECULAR-CONFORMATION; SOLVENT INTERACTION; DERIVATIVES; MODEL; PYRROLOBENZONITRILE; 1-PHENYLPYRROLE; SPECTRA Abstract: Pyrrolobenzenes, with different linking and substitution patterns, 2'-(4-cyanophenyl)-methylpyrrole (MP2-BN) and 2'-(2,5-cyanophenyl)-methylpyrrole (MP2-B25CN), are investigated by steady-state and time-resolved UV-Vis spectroscopy and compared to the parent compound N-pyrrolobenzonitrile (PBN). Both the electron donor-acceptor linking sites and the strength of the electron acceptor moiety are found to influence the emission characteristics of these compounds. The large radiative rate constant of MP2-BN indicates an allowed emission due to mesomeric interaction between the donor and acceptor moieties (MICT), whereas in the case of PBN and MP2-B25CN, the reduced radiative rate constant indicates a forbidden emission from a twisted intramolecular charge transfer (TICT) state. Plaza, P; Mahet, M; Tchaikovskaya, ON; Martin, MM Excitation energy effect on the early photophysics of hypericin in solution CHEMICAL PHYSICS LETTERS, 408 (1-3): 96-100 JUN 7 2005 H-ATOM-TRANSFER; INTRAMOLECULAR PROTON-TRANSFER; FLUORESCENCE UP-CONVERSION; ST-JOHNS-WORT; GROUND-STATE; HYPOCRELLIN; DEPROTONATION; EQUILIBRIA; SPECTRA; AGENT Picosecond transient absorption spectra of hypericin in solution have been recorded over the 350-850 nm spectral range under excitation fluences, respectively, 3/4 and four times the saturation fluence. At low fluence only a weak sub-100 ps decay component is detected, superimposed to the main nanosecond decay of the excited state. At high fluence a sub-10 ps rising component, comparable to that of earlier reports, is observed. This novel observation is discussed within the context of the state-of-the-art theory of hypericin photophysics, involving intramolecular excited-state H atom transfer. A cooling process in the S, state after biphotonic absorption is proposed as an alternative explanation. Changenet-Barret, P; Espagne, A; Plaza, P; Hellingwerf, KJ; Martin, MM Investigations of the primary events in a bacterial photoreceptor for photomotility: photoactive yellow protein (PYP) NEW JOURNAL OF CHEMISTRY, 29 (4): 527-534 2005 EXCITED-STATE DYNAMICS; P-COUMARIC ACID; LINKED 4-HYDROXYCINNAMYL CHROMOPHORE; FEMTOSECOND-PICOSECOND FLUORESCENCE; HALOPHILIC PHOTOTROPHIC BACTERIUM; PUSH-PULL POLYENES; ECTOTHIORHODOSPIRA-HALOPHILA; PHOTON-ABSORPTION; PHOTOCYCLE INTERMEDIATE; INFRARED-SPECTROSCOPY PYP, the Photoactive Yellow Protein, is a small water-soluble protein extracted from the cytosol of the halophilic purple bacterium Halorhodospira halophila. PYP is thought to mediate the phototactic response of the bacterium against blue light. Its chromophore is the deprotonated trans-p-hydroxycinnamic acid covalently linked, via a thioester bond, to the unique cysteine residue of the protein. Upon blue-light irradiation, PYP undergoes a photocycle. As for rhodopsins, the trans to cis isomerization of the chromophore was shown to be the first overall step of this photocycle.