Fabien Lacombat

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.

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.

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.

New insights into the ultrafast photophysics of oxidized and reduced FAD in solution.

The ultrafast photophysics of oxidized and reduced flavin adenine dinucleotide (FAD) in aqueous solution was studied by broadband UV-vis femtosecond transient absorption spectroscopy. We observed that oxidized FAD (FAD(ox)) in solution readily aggregates at submillimolar concentration. Upon excitation of FAD(ox), three excited-state lifetimes were found and assigned to three different species: the closed (stacked) conformation of the monomer (∼5.4 ps), the open (extended) conformation of the monomer (∼2.8 ns), and the dimer (∼27 ps). In the case of the stacked conformation of the monomer, we show that intramolecular electron transfer from the adenine to the isoalloxazine ring occurs with a time constant of 5.4 ps and is followed by charge recombination on a faster time scale, namely, 390 fs. We additionally demonstrate that deprotonated reduced flavin (FADH(-)) undergoes biphotonic ionization under high excitation fluence and dissociates into a hydrated electron and the neutral semiquinone radical FADH(•).

Primary photodynamics of a biomimetic model of photoactive yellow protein (PYP)

The present work aims at characterizing the photophysical behavior of a first biomimetic cyclodextrin model (CD-PYP1) of the photoactive site of photoactive yellow protein (PYP). The hydrophobic cyclodextrin cavity in which the chromophore self-includes, mimics its local environment within the protein. The photoinduced behavior of deprotonated CD-PYP1 (dp-CD-PYP1) has been probed by femtosecond transient-absorption spectroscopy and compared to those of the free deprotonated chromophore (pCT(-)) and of wild-type PYP. The excited-state deactivation of dp-CD-PYP1 is found to be non-exponential, with slower time components and higher quantum yield of fluorescence than pCT(-). Like in PYP, the non-exponential decay is attributed to ground-state structural heterogeneities of the self-inclusion complexes. A long-lived photoproduct is observed in the transient spectra of dp-CD-PYP1 and identified as the cis isomer. The isomerization quantum yield of dp-CD-PYP1 is estimated to be about 4%, in contrast with the free chromophore in solution which does not photoisomerize at all. This demonstrates the active role of the cyclodextrin environment to promote the photoisomerization of the chromophore, as is thought to be the case for wild-type PYP. The effects of chromophore inclusion in the cyclodextrin on the photoinduced processes are rationalized within the framework of recent theoretical calculations involving two competitive deactivation channels: (i) trans to cis isomerization and (ii) rotation of the phenolate group, leading to trans ground-state recovery. Inclusion is proposed to favor isomerization by hindering the rotation of the phenolate group. Optimizing the structure of this first model in order to better reproduce the primary photoresponse of PYP thus appears very promising.

Photoinduced cation translocation in a calix[4]biscrown: towards a new type of light-driven molecular shuttle.

The photophysics of a ditopic receptor of potassium ion consisting of a 1,3-alternate calix[4]biscrown with a merocyanine dye (DCM) inserted into each crown is reported. Thanks to the large difference between the binding affinity for one and two potassium ions, one can find relative total concentrations of ligand and potassium ion at which the 1:1 complex is most predominant with respect to the free ligand and the 2:1 complex whose amounts are a few percents. Investigation of the 1:1 complex by femtosecond transient absorption spectroscopy provides evidence for the ultrafast movement of a potassium ion through the calix[4]arene tube upon excitation at 400 nm of the dye. Phototranslocation occurs in the picosecond timescale with a non-exponential kinetics without competition with photoejection towards the bulk. The translocation time includes two main short components: 0.83 ps and 10 ps. A smaller-weighted third component of 101 ps might include a competition between phototranslocation and excitation energy transfer as shown by using Försters theory. These findings open the way to new strategies for light-driven molecular shuttles with the aim of information storage and binary logic computing at a nanometric scale.

Femtosecond to Subnanosecond Multistep Calcium Photoejection from a Crown Ether‐Linked Merocyanine

Photoinduced calcium release from the crown ether-linked merocyanine DCM-crown is reexamined by femtosecond transient absorption spectroscopy with sub-100 fs time resolution. Photodisruption of the bond linking the cation to the nitrogen atom shared by the crown and the chromophore is found to take place in 130 fs. Confirming our previous reports, the photoinduced intraligand charge transfer is observed in the picosecond regime but kinetics involving three-components (1 ps, 8 ps and 77 ps), together with a 56 ps stimulated-emission time-resolved red shift, are found in the present study. Both delayed intraligand charge transfer and cation release are assumed to occur in this time range due to repulsion effects between the positively charged nitrogen of the crown ether moiety and the cation. In the subnanosecond regime, a 670 ps time-resolved red shift of the stimulated-emission spectrum of the charge-transfer state, similar to the shift previously observed with Sr(2+), demonstrates the motion of the cation away from the crown to the bulk. A thorough examination of the present data allows us to conclude that calcium ion is photoejected to the bulk in a multistep process.

Ultrafast delocalization of cationic states in poly(N-vinylcarbazole) solid leading to carrier photogeneration

Femtosecond measurements of the transient dichroism and near-IR time-resolved spectra revealed the ultrafast delocalization of the cationic state in poly(N-vinylcarbazole), leading to carrier photogeneration.

Photo-induced cation translocation in a molecular shuttle based on a calix[4]-biscrown including DCM and DMABN chromophores.

We present a new molecular shuttle, consisting of a calixarene core attached to two different photoactive centers, DCM and DMABN. We show that a K(+) ion bound to the DCM-grafted crown is translocated towards the other site of the molecule upon photoexcitation, but not released to the bulk.

Ultrafast Dynamics of a Green Fluorescent Protein Chromophore Analogue: Competition between Excited-State Proton Transfer and Torsional Relaxation

The competition between excited-state proton transfer (ESPT) and torsion plays a central role in the photophysics of fluorescent proteins of the green fluorescent protein (GFP) family and their chromophores. Here, it was investigated in a single GFP chromophore analogue bearing o-hydroxy and p-diethylamino substituents, OHIM. The light-induced dynamics of OHIM was studied by femtosecond transient absorption spectroscopy, at different pH. We found that the photophysics of OHIM is determined by the electron-donating character of the diethylamino group: torsional relaxation dominates when the diethylamino group is neutral, whereas ultrafast ESPT followed by cis/trans isomerization and ground-state reprotonation are observed when the diethylamino group is protonated and therefore inactive as an electron donor.