Multiscale Transient Absorption Study of the Fluorescent Protein Dreiklang and Two Point Variants Provides Insight into Photoswitching and Nonproductive Reaction Pathways.

Dreiklang is a reversibly photoswitchable fluorescent protein used as a probe in advanced fluorescence imaging. It undergoes a unique and still poorly understood photoswitching mechanism based on the reversible addition of a water molecule to the chromophore. We report the first comprehensive study of the dynamics of this reaction by transient absorption spectroscopy from 100 fs to seconds in the original Dreiklang protein and two point variants. The picture that emerges from our work is that of a competition between photoswitching and nonproductive reaction pathways. We found that photoswitching had a low quantum yield of 0.4%. It involves electron transfer from a tyrosine residue (Tyr203) to the chromophore and is completed in 33 ns. Nonproductive deactivation pathways comprise recombination of a charge transfer intermediate, excited-state proton transfer from the chromophore to a histidine residue (His145), and decay to the ground state via micro-/millisecond-lived intermediates.

Ultrafast dynamics of fully reduced flavin in catalytic structures of thymidylate synthase ThyX.

Thymidylate is a vital DNA precursor synthesized by thymidylate synthases. ThyX is a flavin-dependent thymidylate synthase found in several human pathogens and absent in humans, which makes it a potential target for antimicrobial drugs. This enzyme methylates the 2'-deoxyuridine 5'-monophosphate (dUMP) to 2'-deoxythymidine 5'-monophosphate (dTMP) using a reduced flavin adenine dinucleotide (FADH-) as prosthetic group and (6R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2THF) as a methylene donor. Recently, it was shown that ThyX-catalyzed reaction is a complex process wherein FADH- promotes both methylene transfer and reduction of the transferred methylene into a methyl group. Here, we studied the dynamic and photophysics of FADH- bound to ThyX, in several substrate-binding states (no substrate, in the presence of dUMP or folate or both) by femtosecond transient absorption spectroscopy. This methodology provides valuable information about the ground-state configuration of the isoalloxazine moiety of FADH- and the rigidity of its local environment, through spectra shape and excited-state lifetime parameters. In the absence of substrate, the environment of FADH- in ThyX is only mildly more constrained than that of free FADH- in solution. The addition of dUMP however narrows the distribution of ground-state configurations and increases the constraints on the butterfly bending motion in the excited state. Folate binding results in the selection of new ground-state configurations, presumably located at a greater distance from the conical intersection where excited-state decay occurs. When both substrates are present, the ground-state configuration appears on the contrary rather limited to a geometry close to the conical intersection, which explains the relatively fast excited-state decay (100 ps on the average), even if the environment of the isoalloxazine is densely packed. Hence, although the environment of the flavin is dramatically constrained, FADH- retains a dynamic necessary to shuttle carbon from folate to dUMP. Our study demonstrates the high sensitivity of FADH- photophysics to the constraints exerted by its immediate surroundings.

Ultrafast photoreduction dynamics of a new class of CPD photolyases.

NewPHL is a recently discovered subgroup of ancestral DNA photolyases. Its domain architecture displays pronounced differences from that of canonical photolyases, in particular at the level of the characteristic electron transfer chain, which is limited to merely two tryptophans, instead of the "classical" three or four. Using transient absorption spectroscopy, we show that the dynamics of photoreduction of the oxidized FAD cofactor in the NewPHL begins similarly as that in canonical photolyases, i.e., with a sub-ps primary reduction of the excited FAD cofactor by an adjacent tryptophan, followed by migration of the electron hole towards the second tryptophan in the tens of ps regime. However, the resulting tryptophanyl radical then undergoes an unprecedentedly fast deprotonation in less than 100 ps in the NewPHL. In spite of the stabilization effect of this deprotonation, almost complete charge recombination follows in two phases of ~ 950 ps and ~ 50 ns. Such a rapid recombination of the radical pair implies that the first FAD photoreduction step, i.e., conversion of the fully oxidized to the semi-quinone state, should be rather difficult in vivo. We hence suggest that the flavin chromophore likely switches only between its semi-reduced and fully reduced form in NewPHL under physiological conditions.

Ultrafast Oxidation of a Tyrosine by Proton-Coupled Electron Transfer Promotes Light Activation of an Animal-like Cryptochrome.

The animal-like cryptochrome of Chlamydomonas reinhardtii (CraCRY) is a recently discovered photoreceptor that controls the transcriptional profile and sexual life cycle of this alga by both blue and red light. CraCRY has the uncommon feature of efficient formation and longevity of the semireduced neutral form of its FAD cofactor upon blue light illumination. Tyrosine Y373 plays a crucial role by elongating , as fourth member, the electron transfer (ET) chain found in most other cryptochromes and DNA photolyases, which comprises a conserved tryptophan triad. Here, we report the full mechanism of light-induced FADH• formation in CraCRY using transient absorption spectroscopy from hundreds of femtoseconds to seconds. Electron transfer starts from ultrafast reduction of excited FAD to FAD•- by the proximal tryptophan (0.4 ps) and is followed by delocalized migration of the produced WH•+ radical along the tryptophan triad (∼4 and ∼50 ps). Oxidation of Y373 by coupled ET to WH•+ and deprotonation then proceeds in ∼800 ps, without any significant kinetic isotope effect, nor a pH effect between pH 6.5 and 9.0. The FAD•-/Y373• pair is formed with high quantum yield (∼60%); its intrinsic decay by recombination is slow (∼50 ms), favoring reduction of Y373• by extrinsic agents and protonation of FAD•- to form the long-lived, red-light absorbing FADH• species. Possible mechanisms of tyrosine oxidation by ultrafast proton-coupled ET in CraCRY, a process about 40 times faster than the archetypal tyrosine-Z oxidation in photosystem II, are discussed in detail.

Ultrafast photoinduced flavin dynamics in the unusual active site of the tRNA methyltransferase TrmFO.

Flavoproteins often stabilize their flavin coenzyme by stacking interactions involving the isoalloxazine moiety of the flavin and an aromatic residue from the apoprotein. The bacterial FAD and folate-dependent tRNA methyltransferase TrmFO has the unique property of stabilizing its FAD coenzyme by an unusual H-bond-assisted π-π stacking interaction, involving a conserved tyrosine (Y346 in Bacillus subtilis TrmFO, BsTrmFO), the isoalloxazine of FAD and the backbone of a catalytic cysteine (C53). Here, the interaction between FAD and Y346 has been investigated by measuring the photoinduced flavin dynamics of BsTrmFO in the wild-type (WT) protein, C53A and several Y346 mutants by ultrafast transient absorption spectroscopy. In C53A, the excited FAD very rapidly (0.43 ps) abstracts an electron from Y346, yielding the FAD˙-/Y346OH˙+ radical pair, while relaxation of the local environment (1.3 ps) of the excited flavin produces a slight Stokes shift of its stimulated emission band. The radical pair then decays via charge recombination, mostly in 3-4 ps, without any deprotonation of the Y346OH˙+ radical. Presumably, the H-bond between Y346 and the amide group of C53 increases the pKa of Y346OH˙+ and slows down its deprotonation. The dynamics of WT BsTrmFO shows additional slow decay components (43 and 700 ps), absent in the C53A mutant, assigned to excited FADox populations not undergoing fast photoreduction. Their presence is likely due to a more flexible structure of the WT protein, favored by the presence of C53. Interestingly, mutations of Y346 canceling its electron donating character lead to multiple slower quenching channels in the ps-ns regime. These channels are proposed to be due to electron abstraction either (i) from the adenine moiety of FAD, a distribution of the isoalloxazine-adenine distance in the absence of Y346 explaining the multiexponential decay, or (ii) from the W286 residue, possibly accounting for one of the decays. This work supports the idea that H-bond-assisted π-π stacking controls TrmFO's active site dynamics, required for competent orientation of the reactive centers during catalysis.

Delocalized hole transport coupled to sub-ns tryptophanyl deprotonation promotes photoreduction of class II photolyases.

Class II photolyases utilize for the photoreduction of their flavin cofactor (FAD) a completely different tryptophan triad than most other photolyases and cryptochromes. To counter sped-up back electron transfer, they evolved an unusually fast deprotonation of the distal tryptophanyl radical cation (WH˙+) that is produced after excitation of the flavin. We studied the primary aspects of oxidized FAD photoreduction by ultrafast transient absorption spectroscopy, using the class II photolyase from Methanosarcina mazei. With a time constant of 9.2 ps, the initial reduction step of the excited flavin by the proximal W381 tryptophan proceeds almost twentyfold slower than in other photolyases carrying oxidized FAD, most likely because of the larger distance between the flavin and the proximal tryptophan. The thus formed W381H˙+ radical is tracked by transient anisotropy measurements to migrate in 29 ps with delocalization over several members of the tryptophan triad. This 29 ps phase also includes the decay of a small fraction of excited flavin, reacting on a slower timescale, and partial recombination of the FAD˙-/WH˙+ radical pair. A final kinetic phase in 230 ps is assigned to the deprotonation of W388H˙+ that occurs in competition with partial charge recombination. Interestingly, we show by comparison with the Y345F mutant that this last phase additionally involves oxidation of the Y345 phenolic group by W388H˙+, producing a small amount of neutral tyrosyl radical (YO˙). The rate of this electron transfer step is about six orders of magnitude faster than the corresponding oxidation of Y345 by the deprotonated W388˙ radical. Unlike conventional photolyases, where the electron hole accumulates on the distal tryptophan before the much slower tryptophanyl deprotonation, our data show that delocalized hole transport is concomitantly concluded by ultrafast deprotonation of W388H˙+.

Ultrafast flavin photoreduction in an oxidized animal (6-4) photolyase through an unconventional tryptophan tetrad.

Photolyases are flavoenzymes repairing UV-induced lesions in DNA, which may be activated by a photoreduction of their FAD cofactor. In most photolyases, this photoreduction proceeds by electron transfer along a chain of three tryptophan (Trp) residues, connecting the flavin to the protein surface. Much less studied, animal (6-4) photolyases (repairing pyrimidine-pyrimidone (6-4) photoproducts) are particularly interesting as they were recently shown to have a longer electron transfer chain, counting four Trp residues. Using femtosecond polarized transient absorption spectroscopy, we performed a detailed analysis of the photoactivation reaction in the (6-4) photolyase of Xenopus laevis with oxidized FAD. We showed that the excited flavin is very quickly reduced (∼0.5 ps) by a nearby tryptophan residue, yielding FAD˙- and WH˙+ radicals. Subsequent kinetic steps in the picosecond regime were assigned to the migration of the positive charge along the Trp tetrad, in competition with charge recombination. We propose that the positive charge is actually delocalized over various Trp residues during most of the dynamics and that charge recombination essentially occurs through the proximal tryptophanyl radical. Oxidation of the fourth tryptophan is thought to be reached about as fast as that of the third one (∼40 ps), based on a comparison with a mutant protein lacking the distal Trp, implying ultrafast electron transfer between these two residues. This unusual mechanism sheds light on the rich diversity of electron transfer pathways found in various photolyases, and evolution-related cryptochromes alike.

Photoinduced Chromophore Hydration in the Fluorescent Protein Dreiklang Is Triggered by Ultrafast Excited-State Proton Transfer Coupled to a Low-Frequency Vibration.

Because of growing applications in advanced fluorescence imaging, the mechanisms and dynamics of photoinduced reactions in reversibly photoswitchable fluorescent proteins are currently attracting much interest. We report the first time-resolved study of the photoswitching of Dreiklang, so far the only fluorescent protein to undergo reversible photoinduced chromophore hydration. Using broadband femtosecond transient absorption spectroscopy, we show that the reaction is triggered by an ultrafast deprotonation of the chromophore phenol group in the excited state in 100 fs. This primary step is accompanied by coherent oscillations that we assign to its coupling with a low-frequency mode, possibly a deformation of the chromophore hydrogen bond network. A ground-state intermediate is formed in the picosecond-nanosecond regime that we tentatively assign to the deprotonated water adduct. We suggest that proton ejection from the phenol group leads to a charge transfer from the phenol to the imidazolinone ring, which triggers imidazolinone protonation by nearby Glu222 and catalyzes the addition of the water molecule.

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.

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.

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.

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 Förster's 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.

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.

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.

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.

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.