Pascal Plaza

Characterization of two members of the cryptochrome/photolyase family from Ostreococcus tauri provides insights into the origin and evolution of cryptochromes.

Cryptochromes (Crys) are blue light receptors believed to have evolved from the DNA photolyase protein family, implying that light control and light protection share a common ancient origin. In this paper, we report the identification of five genes of the Cry/photolyase family (CPF) in two green algae of the Ostreococcus genus. Phylogenetic analyses were used to confidently assign three of these sequences to cyclobutane pyrimidine dimer (CPD) photolyases, one of them to a DASH-type Cry, and a third CPF gene has high homology with the recently described diatom CPF1 that displays a bifunctional activity. Both purified OtCPF1 and OtCPF2 proteins show non-covalent binding to flavin adenine dinucleotide (FAD), and additionally to 5,10-methenyl-tetrahydrofolate (MTHF) for OtCPF2. Expression analyses revealed that all five CPF members of Ostreococcus tauri are regulated by light. Furthermore, we show that OtCPF1 and OtCPF2 display photolyase activity and that OtCPF1 is able to interact with the CLOCK:BMAL heterodimer, transcription factors regulating circadian clock function in other organisms. Finally, we provide evidence for the involvement of OtCPF1 in the maintenance of the Ostreococcus circadian clock. This work improves our understanding of the evolutionary transition between photolyases and Crys.

Excited-state relaxation dynamics of a PYP chromophore model in solution: influence of the thioester group

Abstract Cis–trans photoisomerization of a photoactive yellow protein chromophore model, the deprotonated trans S -phenyl thio- p -hydroxycinnamate, is studied in aqueous solution by subpicosecond transient absorption and gain spectroscopy. The excited-state deactivation is found to involve the formation, in 1.7 ps, of an intermediate state which decays in 2.8 ps. A persistent bleaching signal is observed at longer times indicating that the excited state not only relaxes to the ground state but also partly forms a stable photoproduct, possibly the cis isomer. This behavior is analogous to that of the native photoactive yellow protein.

Ultrafast intramolecular charge transfer in the merocyanine dye DCM

Abstract Time-resolved transient absorption and gain spectra with subpicosecond laser excitation are reported for weakly polar and highly polar solutions of the merocyanine dye DCM (4-dicyanomethylene-2-methyl-6-( p -dimethylamino-styryl)-4-pyran) in the 380–800 nm spectral range. A temporary isosbestic point is observed in the transient gain spectra during 3 to 18 ps after excitation depending on the solvent. The results are interpreted in terms of the rapid formation of an emissive intramolecular charge-transfer state from the emissive initially excited singlet state. A further evolution of the transient spectra includes a blue-shift of the gain band during a few tens of picoseconds in solvents having ultrashort solvation time whereas a red-shift is found for solvent of slow response. A relaxation mechanism including vibrational cooling and solvation of the charge-transfer state is discussed to explain the observed resolved differential spectra. Other processes such as isomerization or single bond twisting are also discussed.

Spectro-temporal characterization of the photoactivation mechanism of two new oxidized cryptochrome/photolyase photoreceptors.

The photoactivation dynamics of two new flavoproteins (OtCPF1 and OtCPF2) of the cryptochrome photolyase family (CPF), belonging to the green alga Ostreococcus tauri , was studied by broadband UV-vis femtosecond absorption spectroscopy. Upon excitation of the protein chromophoric cofactor, flavin adenine dinucleotide in its oxidized form (FAD(ox)), we observed in both cases the ultrafast photoreduction of FAD(ox): in 390 fs for OtCPF1 and 590 fs for OtCPF2. Although such ultrafast electron transfer has already been reported for other flavoproteins and CPF members, the present result is the first demonstration with full spectral characterization of the mechanism. Analysis of the photoproduct spectra allowed identifying tryptophan as the primary electron donor. This residue is found to be oxidized to its protonated radical cation form (WH(*+)), while FAD(ox) is reduced to FAD(*-). Subsequent kinetics were observed in the picosecond and subnanosecond regime, mostly described by a biexponential partial decay of the photoproduct transient signal (9 and 81 ps for OtCPF1, and 13 and 340 ps for OtCPF2), with reduced spectral changes, while a long-lived photoproduct remains in the nanosecond time scale. We interpret these observations within the model proposed by the groups of Brettel and Vos, which describes the photoreduction of FADH(*) within E. coli CPD photolyase (EcCPD) as a sequential electron transfer along a chain of three tryptophan residues, although in that case the rate limiting step was the primary photoreduction in 30 ps. In the present study, excitation of FAD(ox) permitted to reveal the following steps and spectroscopically assign them to the hole-hopping process along the tryptophan chain, accompanied by partial charge recombination at each step. In addition, structural analysis performed by homology modeling allowed us to propose a tentative structure of the relative orientations of FAD and the conserved tryptophan triad. The results of preliminary transient anisotropy measurements performed on OtCPF2 finally showed good compatibility with the oxidation of the distal tryptophan residue (WH(351)) in 340 ps, hence, with the overall Brettel-Vos mechanism.

Primary events in the photoactive yellow protein chromophore in solution

Abstract The photoinduced cis–trans isomerization of the photoactive yellow protein chromophore, the deprotonated trans-p -coumaric acid, is studied in solution by subpicosecond transient absorption and gain spectroscopy. A nonemissive photoproduct absorbing in the UV region, attributed to the cis isomer, is found to appear in 10 ps at the rate of the excited-state decay, demonstrating that there is no detectable intermediate in the relaxation pathway of the photoactive yellow protein chromophore in solution.

Repair of the (6–4) Photoproduct by DNA Photolyase Requires Two Photons†

It takes two (photons) to tango: Single-turnover flash experiments showed that the flavoenzyme (6-4) photolyase uses a successive two-photon mechanism to repair the UV-induced T(6-4)T lesion in DNA (see picture). The intermediate (X) formed by the first photoreaction is likely to be the oxetane-bridged dimer T(ox)T. The enzyme could stabilize the normally short-lived T(ox)T, allowing repair to be completed by the second photoreaction.

DYNAMICS OF THE FIRST EXCITED STATE OF THE DECATUNGSTATE ANION STUDIED BY SUBPICOSECOND LASER SPECTROSCOPY

Abstract The primary events in decatungstate photolysis are investigated by subpicosecond spectroscopy. Steady-state and picosecond fluorescence experiments are also reported. The absorption peak at 776 nm of the transient state called X, involved in photocatalytic reactions with organic substrates on the nanosecond time scale, rises in a few ps after subtle changes of an initial band, extending from the visible to the near-infrared (NIR). The decay in the NIR edge occurs in 25 ps in acetonitrile and 15 ps in water. X is interpreted as a delocalized ligand-to-metal charge transfer (LMCT) excited state, originating from a localized excited state absorbing in the NIR. Re-excitation of X could contribute to the 580 nm fluorescence observed under picosecond excitation.

Real-Time Monitoring of Chromophore Isomerization and Deprotonation during the Photoactivation of the Fluorescent Protein Dronpa

Dronpa is a photochromic green fluorescent protein (GFP) homologue used as a probe in super-resolution microscopy. It is known that the photochromic reaction involves cis/trans isomerization of the chromophore and protonation/deprotonation of its phenol group, but the sequence in time of the two steps and their characteristic time scales are still the subject of much debate. We report here a comprehensive UV-visible transient absorption spectroscopy study of the photoactivation mechanism of Dronpa, covering all relevant time scales from ∼100 fs to milliseconds. The Dronpa-2 variant was also studied and showed the same behavior. By carefully controlling the excitation energy to avoid multiphoton processes, we could measure both the spectrum and the anisotropy of the first photoactivation intermediate. We show that the observed few nanometer blue-shift of this intermediate is characteristic for a neutral cis chromophore, and that its anisotropy of ∼0.2 is in good agreement with the reorientation of the transition dipole moment expected upon isomerization. These data constitute the first clear evidence that trans → cis isomerization of the chromophore precedes its deprotonation and occurs on the picosecond time scale, concomitantly to the excited-state decay. We found the deprotonation step to follow in ∼10 μs and lead directly from the neutral cis intermediate to the final state.

Excited state relaxation paths in 9,9′-bianthryl and 9-carbazolyl-anthracene: a sub-ps transient absorption study

Abstract Transient absorption spectra with sub-picosecond resolution are reported for 9,9′-bianthryl (BA) and 9-carbazolyl-anthracene (C9A) in ethanol, n -butanol and diethylether, from 320 to 800 nm. Ground-state bleaching and excited-state absorption (ESA) are seen in the UV region. In alcohols, the ESA decay (≈20 ps in ethanol and ≈70 ps in butanol) matches the rise time of the well-known charge-transfer (CT) band in the visible, with a time constant comparable to the average solvation time. An additional change is observed for BA in the fluorescence region (400–480 nm), characterized by the disappearance of a structured gain contribution in less than 1 ps. This is also observed in diethylether, although the subsequent evolution is different. This ultrafast process, which is attributed to the unhindered torsional relaxation of BA out of the Franck–Condon geometry, is less clear for C9A. However, for both compounds in alcohols, the CT state formation is a second step and is more than one order of magnitude slower. In diethylether, unhindered and diffusive conformational changes are invoked. The data support a two-coordinate (internal twisting and solvent motion) relaxation model for BA, the relative importance of these coordinates being strongly solvent dependent. A similar model is proposed for C9A.

Primary photoprocesses involved in the sensory protein for the photophobic response of Blepharisma japonicum.

We present new femtosecond transient-absorption and picosecond fluorescence experiments performed on OBIP, the oxyblepharismin-binding protein believed to trigger the photophobic response of the ciliate Blepharisma japonicum. The formerly identified heterogeneity of the sample is confirmed and rationalized in terms of two independent populations, called rOBIP and nrOBIP. The rOBIP population undergoes a fast photocycle restoring the initial ground state in less than 500 ps. Intermolecular electron transfer followed by electron recombination is identified as the excited-state decay route. The experimental results support the coexistence of the oxyblepharismin (OxyBP) radical cation signature with a stimulated-emission signal at all times of the evolution of the transient-absorption spectra. This observation is interpreted by an equilibrium being reached between the locally excited state and a charge-transfer state on the ground of a theory developed by Mataga and co-workers to explain the fluorescence quenching of aromatic hydrogen-bonded donor-acceptor pairs in nonpolar solvents. OxyBP is supposed to bind to an as yet unknown electron acceptor by a hydrogen-bond (HB) and the coordinate along which forward and backward electron transfer proceed is assumed to be the shift of the HB proton. The observed kinetic isotope effect supports this interpretation. Protein relaxation is finally proposed to accompany the whole process and give rise to the highly multiexponential observed dynamics. As previously reported, the fast photocycle of rOBIP can be interpreted as an efficient sunscreen mechanism that protects Blepharisma japonicum from continuous irradiation. The nrOBIP population, the transient-absorption of which strongly reminds that of free OxyBP in solution, might be proposed to actually trigger the photophobic response of the organism through excited-state deprotonation of the chromophore occurring in the nanosecond regime. Additional femtosecond transient-absorption spectra of OxyBP and peri-deprotonated OxyBP are also reported and used as a comparison basis to interpret the results on OBIP.