New Insights into the Ultrafast Photophysics of Oxidized and Reduced

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New Insights into the Ultrafast Photophysics of Oxidized and Reduced FAD in Solution Johanna Brazard,†,‡,§ Anwar Usman,†,‡,§ Fabien Lacombat,†,‡,§ Christian Ley,†,‡,§ Monique M. Martin,†,‡,§ and Pascal Plaza*,†,‡,§ cole Normale Superieure, 24, rue Lhomond, 75005 Paris, France Departement de Chimie, E Universite Pierre et Marie CurieParis 6, 4 Place Jussieu, 75005 Paris, France § CNRS UMR 8640, 75005 Paris, France † ‡

bS Supporting Information ABSTRACT: The ultrafast photophysics of oxidized and reduced flavin adenine dinucleotide (FAD) in aqueous solution was studied by broadband UVvis femtosecond transient absorption spectroscopy. We observed that oxidized FAD (FADox) in solution readily aggregates at submillimolar concentration. Upon excitation of FADox, 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•.

1. INTRODUCTION Flavin adenine dinucleotide (FAD) is an essential photoactive cofactor found in all blue-light activated proteins of the cryptochrome/photolyase family (CPF).13 CPF includes different types of DNA photolyases, cryptochromes, and the so-called cryptochromes DASH. Photolyases are enzymes able to photorepair pyrimidine dimers of UV-damaged DNA.1,4,5 Cryptochromes are signaling photoreceptors,1,610 and cryptochromes DASH are now believed to be specialized photolyases for single-stranded DNA .11,12 FAD can in general be found in three different redox forms: oxidized (FADox, Figure 1, left), semireduced (FADH•), and reduced (FADH, Figure 1, right).13 For each of these redox forms, two acidbase equilibria give rise to three different protonation forms (protonated, neutral, and deprotonated). In solution, the radical species are not stable enough to be isolated and at neutral pH the structures represented in Figure 1 are mainly found. The biological functions of CPF proteins are deeply rooted in the photoinduced reduction or oxidation of FAD. In photolyases, photoexcitation of FADH transfers an electron to the pyrimidine dimer, resulting in DNA repair.1,4,5 In cryptochromes, the excited flavin has been proposed to transfer an electron either to an intra or to an interprotein partner, creating a primary signaling state.3,6,9 On the other hand, it has been shown in vitro that photoexcitation of FADox or FADH• bound to CPF proteins leads to ultrafast reduction of the flavin through electron hopping along a chain of three conserved Trp residues.5 r 2011 American Chemical Society

The important photoactivated role of FAD in the biochemical processes of CPF proteins has prompted considerable interest in deciphering the intrinsic excited-state properties of FAD, in solution. In aqueous solution, oxidized FAD is known to have two distinct conformations: a closed conformation in which the isoalloxazine and adenine moieties are stacked, and an extended (open) one in which the two moieties are separated from each other.14 At pH ranging from 4 to 8, and, at room temperature, 80% of ground-state FADox is in the closed conformation, while 20% is in the open conformation.14,15 Note that in CPF proteins, FAD is in a U-shaped conformation, with the isoalloxazine and adenine moieties lying in close proximity.13 The two conformations in solution have very different photophysical behaviors, as reported by several time-resolved spectroscopic studies.1622 The excited-state lifetime of the open conformation of FAD is about 2.5 ns.17,18,20 The lifetime of the closed conformation is much shorter: the reported values lie between 1 and 20 ps.1622 The excited-state quenching of FADox in the closed conformation has been attributed to an intramolecular electron transfer from the adenine to the electronically excited isoalloxazine.17,18 This reaction is expected to produce an intermediate state: the Received: November 10, 2010 Revised: January 20, 2011 Published: March 25, 2011 3251

dx.doi.org/10.1021/jp110741y | J. Phys. Chem. A 2011, 115, 3251–3262

The Journal of Physical Chemistry A

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Figure 1. Chemical structures of flavin adenine dinucleotide oxidized (FADox, left) and reduced (FADH, right).

radical pair of adenine cation and isoalloxazine anion (noted Ade•þIso•). This intermediate was not observed in several time-resolved spectroscopic experiments (UVvis and mid-IR) performed on FADox at neutral pH,16,19,21 suggesting that the putative intramolecular electron transfer is followed by ultrafast charge recombination. In a recent femtosecond transient absorption and fluorescence study of FADox (at pH 7.5), Kao et al., however, observed two ultrafast time components instead of one. A 4.5 ps component was assigned to the excited-state decay of FADox in the closed conformation, with formation of the radical pair. An additional time component of 3040 ps was interpreted as the lifetime of the radical pair, which decays by charge recombination.20 More recently, Li et al. deduced from femtosecond mid-IR transient absorption spectroscopy that the stacked conformer undergoes intramolecular photoinduced electron transfer from the adenine to the flavin with a time component of 1.1 ps and the geminate recombination with a time component of 9 ps.22 As far as reduced FAD is concerned, several spectroscopic studies reported the photophysical behavior of FADH or FADH2 in aqueous solution.20,2326 These time-resolved studies report a multiexponential decay of the excited state, with a distribution of lifetimes ranging from ps to ns, and most authors invoke the role of the flexibility of the molecule in this decay.20,25,26 It is indeed known that reduced flavins adopt a bent geometry of the isoalloxazine ring.2730 The bending (about the N5N10 axis) motion which interconverts two boat conformers into each other is called the “butterfly” motion. Li et al.,26 in particular, attributed two subns decays of FADH to the presence of two butterfly conformers of the isoalloxazine ring, while Kao et al.20 attributed the multiexponential decay to a deactivation mechanism occurring by butterfly bending motion across conical intersections. In addition, let us note that Enescu et al. considered that biphotonic absorption could lead to the ionization of reduced flavins and concomitant production of solvated electron, but finally rejected the hypothesis on the ground of the linear dependence of their transient absorption spectra on the excitation energy.25

In the present study, we report new results on the excited dynamics of both FADox and FADH in aqueous solution (at pH 8.0), obtained by femtosecond broadband transient-absorption spectroscopy. Concerning oxidized FAD, our goal was to further investigate the formation of the Ade•þIso• radical pair intermediate in the excited-state decay of the stacked conformation of the molecule. As detailed above, previous reports gave quite diverse and diverging conclusions on this issue. We took advantage of the ability of our experimental setup to produce full transient spectra in the visible range to identify the nature of intermediate species. We also checked the role of the solute concentration on the transient absorption spectroscopy of FADox because this technique generally requires concentrated samples and it is known that oxidized FMN (flavin mononucleotide, which lacks the adenine moiety) undergoes dimerization at submillimolar concentrations.3135 It is therefore important to evaluate the relevance of this issue in the case of FADox. Concerning FADH, we report transient absorption spectra over an extended spectral range (down to 340 nm), as compared to previous published works, and discuss them in the context of the above-mentioned interpretations. We, however, concentrated our effort on the role of the photon fluence (number of incident photons per unit area; further abbreviated as fluence) of excitation on the excited-state behavior and, particularly, reinvestigated the hypothesis of multiphotonic ionization of the molecule and concomitant production of hydrated electron.

2. MATERIALS AND METHODS 2.1. Sample Preparation. Flavin adenine dinucleotide disodium salt hydrate (FAD, HPLC grade) was purchased from Sigma-Aldrich and used without further purification. FADox was prepared by simply dissolving those compounds in an aqueous solution at pH 8.0 containing Tris-HCl 100 mmol L1, NaCl 100 mmol L1, glycerol (10% vol/vol). This buffer was chosen to be identical to the one we previously used for studying the photoactivation reaction of oxidized CPF proteins,36 thereby allowing direct comparison of the two sets of experiments (FADox in 3252

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The Journal of Physical Chemistry A solution and bound to CPF protein). Note that the presence of glycerol is however shown, in section 3.1.2, not to play any role on the observed ultrafast processes observed and discussed in the present paper. A series of FADox samples with different concentrations was prepared by diluting a concentrated solution (∼2.5 mmol L1). FADH was prepared in an aqueous solution at pH 8.0 by photoreduction of the oxidized molecules in the presence of a reductant, ethylenediaminetetraacetic acid (EDTA; purchased from Sigma-Aldrich) as in ref 25, after 40 min of argon bubbling to remove oxygen. The solution was irradiated at 450 nm for 30 min, under an argon atmosphere. The buffer was made of TrisHCl (50 mmol L1) and EDTA (20 mmol L1) and was set at pH 8.0 by adding HCl (1.0 mol L1). 2.2. Steady-State Spectroscopy and Concentration Effect. UVvis absorption spectra were recorded from 300 to 700 nm with a double-beam UV spectrophotometer: either UV-mc2 (Safas) or Cary 300 (Varian). Fluorescence spectra were measured with a fully corrected Fluoromax-3 (Jobin-Yvon) spectrofluorometer. All experiments were carried out in a cell thermostated at 5 °C by a temperature-controlled bath (Minichiller CC, Huber). The concentration of FADox was determined by fitting the absorption spectrum of a dilute sample (50 μmol L1; leading to negligible aggregation; see section 3.1.1) with a known molar absorption coefficient spectrum of FADox, reported by Song et al.37 The concentrations of all other samples (ranging from 9 μmol L1 to 2.27 mmol L1) were deduced by applying appropriate dilution factors. Samples with concentrations below 100 μmol L1 were contained in 1 cm fused-silica cuvettes, while samples with concentrations higher than 100 μmol L1 were contained in 1 mm fused-silica cuvettes. 2.3. Time-Resolved Absorption Spectroscopy. Femtosecond transient absorption spectra were obtained by the broadband pumpprobe technique. The source was an amplified Ti: Sapphire laser system (Tsunami þ Spitfire, Spectra-Physics) providing 50 fs pulses at 775 nm and 1 kHz. Two different excitation wavelengths, 470 and 387.5 nm, were used to selectively excite the oxidized and reduced FAD, respectively. These excitation wavelengths lied on the red edge of the lowest absorption transition for both oxidized and reduced flavins. To produce the pulses (∼50 fs) at 470 nm, most of the 775 nm beam was used to run a noncollinear optical parametric amplifier (NOPA, 387.5-nm pumped, Clark MXR). To generate the 387.5 nm beam, most of the 775 nm beam was frequencydoubled in a BBO crystal. Pulses having an energy ranging from 0.10 to 0.33 μJ were focused on a section of about 5 103 μm2 of the sample. Care was taken to check that the excitation fluence lay in the linear regime. The continuum probe (extending from 340 to 750 nm) was generated by focusing a few μJ (per pulse) of the 775 nm beam on a moving CaF2 window. The probe was then split into two parts: a reference and a sample beam. The sample beam was focused onto the 1 mm fused-silica cell sample and crossed the pump beam at an angle of ∼5°. The polarizations of the pump and probe beams were set at the magic angle (54.7°). The pumpprobe delay was adjusted by a motorized optical delay line. The reference and the probe beams were sent onto the entrance slit of a spectrograph (Acton SP306i; spectral resolution ∼2 nm). The spectra were recorded at 333 Hz on a CCD matrix detector (Roper Scientific, 130 1340 pixels) and accumulated over 3000 pump shots. The sample solutions were cooled down

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to 5 °C and moved up and down (20 mm s1) to prevent photolysis (the steady-state absorption of the sample was continuously monitored during the experiments). The differential absorption spectra (ΔA) were corrected from the chirp of the probe beam (due to group velocity dispersion in the crossed materials). 2.4. Data Analysis. The full transient absorption data (all wavelengths and delay times) were globally fitted to a sum of four exponential functions, convoluted by a Gaussian function representing the instrument response function. The procedure used dimensional reduction and noise filtering by singular value decomposition (SVD).38 The number of retained singular value at the stage of analysis of the truncated data matrix (where times below 200 fs were removed to get rid of a cross-phase modulation artifact during pumpprobe overlap) varied from four to eight, which was enough to ensure that no information loss could alter the subsequent global fitting procedure (see technical details in ref 39). The mean amplitude of the residue (2D rms) after fitting was of the order of 0.2 mOD. The decay-associated differential spectrum (DADS), that is, spectrum of pre-exponential factors, of each time component was then calculated over the entire experimental spectral range following methods published in ref 40.

3. RESULTS In the following subsections, we first report our study of oxidized flavin adenine dinucleotide (FADox) in aqueous solution by broadband femtosecond transient absorption spectroscopy. The main scopes are (i) looking for a spectroscopic proof of intramolecular electron transfer, from the adenine to the isoalloxazine, in the closed conformation of the molecule and (ii) evaluating the impact of the solute concentration on the excited-state dynamics. This work has been paralleled by a similar study of FMNox, the main results of which are provided as Supporting Information (see sections 14). We subsequently present an investigation of the role of the excitation fluence on the photophysics of reduced FAD in its deprotonated form (FADH). 3.1. Oxidized FAD: FADox. 3.1.1. Concentration Effect on the Steady-State Absorption Spectrum. Figure 2A shows the absorp-

tion spectra of FADox at different concentrations, ranging from 9 μmol L1 to 2.27 mmol L1, expressed as effective extinction coefficient (εeff = absorbance divided by total solute concentration and optical path). The characteristic spectral features of FADox in solution24 are seen on all these spectra: two broad bands centered at 450 and 377 nm. It is remarkable that the amplitude of these spectra decreases as the concentration increases, without major changes of the shape. This hypochromism phenomenon, well-known for molecular aggregates or biopolymers,41 may in the case of FADox be due to some screening of the chromophores arranged in stacked aggregates. Those aggregates would likely be quite loose in order to explain the very small variation of the spectral shape. It is indeed known that FMNox undergoes dimerization at submillimolar concentrations.3135 We therefore analyzed our data within the simplest aggregation model, the formation of a dimer (D) out of two monomers (M): MþM / D 3253

ð1Þ



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combination of monomer and dimer molar coefficients, as follows: εeff ¼

Figure 2. Effective extinction coefficient spectra of FADox at different concentration (red, 9 μmol L1; green, 152 μmol L1; blue, 1.14 mmol L1; black, 2.27 mmol L1) are represented in (A). The effective extinction coefficient of FADox as a function of the concentration C at the two absorption maxima (377 nm: square, 450 nm: circle) and their fits by the dimer formation model (lines) are displayed in (B). The extinction coefficient spectrum of FADox monomer (line) and FADox dimer (dots) are shown in (C).

The corresponding thermodynamic equilibrium constant Kdim, as well as dimer and monomer concentrations as a function of the total solute concentration C, read ½D C° ½M2

ð2Þ

pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 8 Kdim C þ 1 1 4 Kdim

ð3Þ

C ½M 2

ð4Þ

Kdim ¼

½M ¼ and

½D ¼

where C° is the standard concentration equal to 1 mol L1. A global fit of all the εeff spectra was applied, assuming that each spectrum could be described by a linear

εM ½M þ εD ½D C

ð5Þ

The procedure yielded satisfactory fits of the data (see εeff vs C at the two absorption maxima in Figure 2B) with a dimerization constant of Kdim(FADox, 5 °C) = 640 ( 40 (the half width of a 95% confidence interval is obtained by multiplying the error by ∼2). It also provided the monomer and dimer molar absorption coefficient spectra displayed in Figure 2C. One clearly sees that the absorption spectrum of the dimer is less than twice as intense as that of the monomer but retains its major spectral features. According to this estimation, the fraction of dimer in a concentrated solution of FADox normally used for transient absorption spectroscopy may not be negligible. For instance at 0.5 mmol L1 (optical density at 450 nm of the order of 0.5 for 1 mm optical path), one would obtain [D]/C = 0.15 and [M]/C = 0.7. This means that 30% of the FADox molecules would be in the form of dimers (or higher order aggregates which have not been considered in the present simplified model). The role of this aggregated population on the transient absorption spectra will be examined in sections 3.1.2 and 3.1.3. As shown in section 1 of Supporting Information, our global analysis of the absorption spectra of FMNox as a function of concentration confirms previously published results, namely that FMNox in aqueous solution readily forms dimers. We found a dimerization constant of Kdim(FMNox, 5 °C) = 470 ( 10, in relatively good agreement with the value published by Lukasiewicz et al. (314 at 3 °C).35 3.1.2. Transient Absorption Spectroscopy: “Dilute” Sample. In this section, we report transient absorption spectra of a so-called “dilute” solution of FADox. The concentration was 0.23 mmol L1, which yielded an optical density of 0.25 at 450 nm for 1 mm optical path. According to the above dimerization model, the monomer fraction ([M]/C) of this solution is 81%. Figure 3 presents an overview of transient spectra of FADox after excitation at 470 nm, for pumpprobe delays ranging from 225 fs to 1.4 ns. Times below 225 fs are not shown because the corresponding spectra are too contaminated by a cross-phase modulation artifact generated during pumpprobe overlap. At a delay of 225 fs (Figure 3A), one observes three positive ΔA bands dominated by transient absorption contributions: a large one starting below 410 nm, a small one around 508 nm and a broad structure extending beyond 628 nm. Ground-state bleaching however dominates between 408 nm and (with extrapolation) 493 nm, where it gives rise to a net negative band. Stimulated emission is in turn dominant between 540 and 628 nm, where it produces another net negative peak. The temporal evolution of the spectra shows three phases: • Phase 1 (Figure 3A): The blue and red positive bands, together with the bleaching band, do not change much between 225 fs and 3 ps and then start decaying significantly. Most significantly, the stimulated-emission band continuously shifts to the red by 8 nm between 225 fs and 5 ps, while the amplitude of the positive band at 508 nm increases and slightly blue shifts. • Phase 2 (Figure 3B): Between 5 and 25 ps, all bands decay and exhibit subtle shape changes, with in particular a small blue shift of the positive band around 508 nm and a decrease 3254



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Figure 4. DADS attached to each of the four time components found in the global analysis of the transient absorption spectra of the “dilute” (A, [FADox] = 0.23 mmol L1) and “concentrated” (B, [FADox] = 0.63 mmol L1) solutions of FADox, recorded under excitation at 470 nm.

Figure 3. Transient absorption spectra of FADox ([FADox] = 0.23 mmol L1) in Tris buffer after excitation at 470 nm. The evolution of the spectra between 225 fs and 5 ps is displayed in (A), between 5 and 25 ps in (B), and between 25 and 1400 ps in (C). The steady-state absorption and fluorescence spectra of FADox are recalled in gray lines in (C).

of the intensity ratio of the bleaching band to the stimulatedemission band. • Phase 3 (Figure 3C): All bands decay in a seemingly proportional way between 25 ps and 1.4 ns. Two slightly nontrivial temporary isosbestic points (ΔA small but nonzero) are, however, seen at 533 nm and around 660 nm, demonstrating that the decay is in fact not strictly proportional at all wavelengths. Global analysis of the full data set was performed with best results by using four exponential components. The lifetimes of the exponentials were found to be τ1 = 1.0 ( 0.1 ps, τ2 = 5.5 ( 0.3 ps, τ3 = 31 ( 3 ps, and τ4 = 3.0 ( 0.3 ns. The corresponding DADS (pre-exponential factor spectra) are given in Figure 4A. DADS1, attached with the lifetime of 1.0 ps, is closely related to the continuous band shift observed during Phase 1 (see above). The band shifting is in fact only crudely described by this single global exponential but, as we will not be interested into its details, it can be said that the 1.0 ps component essentially captures the existence of the phenomenon. We mostly ascribe it to solvation dynamics,42 that is, the relaxation of solvent molecules around FADox in the excited state, which has previously been reported in the same time scale by other time-resolved spectroscopic studies of FADox in aqueous solution.18,20 The lifetime of 1.0 ps is rather close to the longest component of solvation dynamics in water (880 fs).43 It is also close to the shortest component of solvation dynamics in glycerol (the buffer used contains 10% glycerol), but the complete solvation dynamics in glycerol is considerably longer (average value of 196 ps),44 and does not correspond to any observed band shift. We may therefore conclude that FADox is preferentially solvated by water molecules in the buffer used in this study. In addition, DADS1 might as well include some contributions of structural relaxation along low-frequency modes of the isoalloxazine ring, as has been recently suggested for riboflavin.45 One finally notes

that DADS1 is positive in the bleaching domain (around 450 nm). The other three DADS are positive in the transient absorption domains and negative in the bleaching and stimulated-emission domains. These spectral features sign the decay of excited-state species with concomitant ground-state recovery. DADS3 (attached to the lifetime of 31 ps) and DADS4 (3.0 ns) have similar shapes but substantially differ from DADS2 (5.5 ps); see normalized spectra in Supporting Information, section 5. The bleaching band of DADS2 extends more toward the red than those of DADS3 and DADS4, and its stimulated-emission band is relatively less intense (upon normalization at 358 nm). This agrees well with the observation of minor shape changes of the transient absorption spectra between 5 and 25 ps (Phase 2, see description above). DADS3 and DADS4 in fact also differ slightly from each other, DADS4 being in particular relatively more intense than DADS3 between 600 and 750 nm. At 655 nm, DADS3 = 0 and DADS4 > 0, which justifies the existence of a nontrivial isosbestic point at this wavelength between 25 ps and 1.4 ns (Phase 3). 3.1.3. Transient Absorption Spectroscopy: “Concentrated” Sample. The transient absorption spectra of a 2.7 times more concentrated solution of FADox (0.63 mmol L1) where the monomer fraction ([M]/C) is only 65%, show essentially the same features as those described above for the “dilute” solution (see spectra in Supporting Information, section 6). The best global fit of the time-resolved spectra was obtained with the four following lifetimes: τ1 = 0.9 ( 0.1 ps, τ2 = 5.2 ( 0.3 ps, τ3 = 22 ( 2 ps, and τ4 = 2.6 ( 0.2 ns. Those lifetimes are very close to the ones obtained for the “dilute” solution, and can in fact be considered identical within confidence intervals. The corresponding DADS, shown in Figure 4B, are also very similar to those of the “dilute” solution, and can be identified one-to-one to them. The major difference between the two experiments lies in the relative amplitudes of their DADS. In Figure 4A, one notes that DADS3 (31 ps) and DADS4 (3.0 ns) of the “dilute” solution of FADox have the same amplitude, whereas in Figure 4B it is seen that, for the “concentrated” solution, the amplitude of DADS3 (22 ps) is larger than that of DADS4 (2.6 ns). 3.2. Reduced and Deprotonated FAD: FADH. The transient absorption spectra of FADH under femtosecond irradiation at 3255



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Figure 6. DADS attached to each of the four time components found in the global analysis of the transient absorption spectra of FADH at low fluence (A, j = 0.05) and at high fluence (B, j = 0.2), recorded under excitation at 387.5 nm.

Figure 5. Transient absorption spectra of FADH at low fluence (j = 0.05) in Tris buffer at pH 8.0 after excitation at 387.5 nm. The time evolution of the spectra between 220 fs and 1.2 ps is displayed in (A), between 1.2 and 20 ps in (B), and between 20 and 1400 ps in (C). The steady-state absorption and fluorescence (reproduced from Kao et al.20) spectra of FADH are recalled in gray lines in (C).

387.5 nm were recorded with two different excitation fluences (F: number of incident photons per cm2). Fluences are here expressed as a dimensionless so-called reduced fluence j, defined as the ratio of the fluence F to the saturation fluence Fs, itself defined as the inverse of the ground-state absorption cross section σ (cm2) at the excitation wavelength46 j¼

F ¼ Fσ ¼ 3:82 1021 Fε Fs

ð6Þ

where ε is the molar extinction coefficient (at the excitation wavelength). The reduced fluence is a convenient way of measuring of how close the fluence of an ultrashort excitation pulse approaches saturation of the absorption transition (j , 1 means low saturation; j g 1 means high saturation).46 For FADH in aqueous solution at 387.5 nm, ε ∼ 3300 L mol1 cm1,37 giving Fs = 7.9 1016 cm2. 3.2.1. Transient Absorption Spectroscopy Under “Low” Excitation Fluence. The experiment was first recorded with an excitation energy of 0.10 μJ per pulse focused on a surface of 5 103 μm2. This corresponds to a reduced fluence of j = 0.05, situated well below the saturation threshold (j = 1). Figure 5 shows transient absorption spectra of FADH for pumpprobe delay ranging from 220 fs to 1.4 ns. The transient absorption spectra present two positive bands, around 390 and 520 nm, dominated by transient absorption contributions. They could correspond to two separate excited-state absorption transitions but the hole between them, situated at about 440 nm, matches in fact rather well the expected position of stimulated emission. This can be verified by looking at the steady-state fluorescence spectrum of FADH, peaking at 450 nm, reported by Kao et al.20 and reproduced in gray line in the lower frame of Figure 5. The resulting picture is therefore that the ΔA spectra may be constituted of either one very broad or two separate excited-state

absorption bands (positive) superimposed on the stimulatedemission band (negative). Ground-state bleaching likely contributes to the falling blue wing of the 390 nm ΔA band. The temporal evolution of our transient spectra shows three phases: • Phase 1 (Figure 5A): Between 220 fs and 1.2 ps, the amplitude of the 390 nm band slightly decreases, while the blue side of the 520 nm band remains nearly constant. A clear narrowing of the 520 nm band is however visible on its red side. The dip at 440 nm concomitantly deepens and slightly shifts toward the blue. • Phase 2 (Figure 5B): Between 1.2 and 20 ps, one notes a strong decay of all the bands, during which the maximum of the 520 nm band becomes rounder and its red side continues, and completes, its narrowing. • Phase 3 (Figure 5C): The main bands disappear completely in less than 100 ps. This decay is quasi-proportional. At 100 ps a weak, very broad, positive band, however, remains. It extends for wavelengths higher than 400 nm and shows an apparent maximum around 700 nm. This spectrum decays then very slowly and is still seen with only slightly reduced amplitude at 1.4 ns. The best global fit of the data was obtained by using four exponential components with the following lifetimes: τ1 = 1.0 ( 0.2 ps, τ2 = 5.5 ( 0.6 ps, τ3 = 32 ( 1 ps, and τ4 = 5 ( 2 ns. Figure 6A shows the corresponding DADS. DADS1, associated to the lifetime of 1.0 ps, captures the band narrowing observed during the early phase of the dynamics (see description above). Its “oscillatory” shape with two alternating negative and positive regions, suggest that DADS1 could in fact be dominated by the blue shift, and narrowing, of two excitedstate absorption contributions. Because, as noted above, the longest component of solvation dynamics in water is 880 fs,43 solvation dynamics of water is likely to contribute to this ultrafast dynamic step but structural relaxation of the excited molecule could as well be involved. DADS2 and DADS3 have comparable shapes with two positive maxima; they convey the decay of the two main transient absorption bands. DADS2 is, however, broader on the red side of the two maxima and its long-wavelength maximum is significantly red-shifted as compared to that of DADS3. These differences mean that the transient absorption bands go on 3256



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defined shape. Another striking difference is that DADS1, associated with the lifetime of 0.5 ps, is here negative for wavelengths below 623 nm, while it is positive at “low” fluence in this spectral range. This corresponds to the above-mentioned rise of transient absorption signal in the far red region during the first picosecond of the excited-state dynamics.

4. DISCUSSION In the following subsections, we first discuss the photodynamics of FADox focusing on the role of the dimers and on the hypothesis of intramolecular charge transfer in the closed (stacked) conformation of the molecule. The excited-state behavior of FADH is then discussed, with special interest on the photoionization of the molecule under high irradiation fluence. 4.1. Photodynamics of FADox. 4.1.1. Effect of Aggregation on the Photoinduced Dynamics of FADox. It is clear from section

Figure 7. Transient absorption spectra of FADH at high fluence (j = 0.2) in Tris buffer at pH 8.0 after excitation at 387.5 nm. The time evolution of the spectra between 250 fs and 1.4 ps is displayed in (A), between 1.4 and 20 ps in (B), and between 20 and 1400 ps in (C). The steady-state absorption and fluorescence (reproduced from Kao et al.20) spectra of FADH are recalled in gray lines in (C).

narrowing on their red sides while decaying, with a time constant of 5.5 ps. The final decaying phase, without narrowing, takes place with a time constant of 32 ps. It is interesting to note that the continuous narrowing phenomenon reported during the 1 ps phase continues during the 5.5 ps phase, which is much longer than solvation dynamics in water. It is therefore likely that some process other than solvation dynamics contributes to the phenomenon. Finally, DADS4, associated to the lifetime of 5 ns, shows a very broad band extending at least down to 700 nm, which strongly evokes the spectral signature of hydrated electron, the maximum of which lies at 720 nm.4749 3.2.2. Transient Absorption Spectroscopy under “High” Excitation Fluence. Because the spectrum of the long-lived species (DADS4) found in the preceding transient absorption measurement of FADH with j = 0.05 was quite weak, we carried out a new experiment with a four times higher fluence (j = 0.2). The transient spectra are very similar to the case of “low” excitation fluence and evolve in the same way, as shown in Figure 7. The amplitude of the spectrum left after decay of the two main transient absorption bands is, however, much larger. The presence of this broad long-lived spectrum is in fact clear beyond about 680 nm at the very start of the dynamics. One even notices that the transient absorption signal in this far red region significantly grows during the first picosecond. A global fit of the data with a sum of four exponentials provided the following lifetimes: τ1 = 0.5 ( 0.1 ps, τ2 = 4.7 ( 0.2 ps, τ3 = 28 ( 1 ps, and τ4 = 5 ( 1 ns. Those lifetimes are very close to the ones obtained for the “low” excitation fluence and may be considered essentially identical. The corresponding DADS are shown in Figure 6B. The most obvious difference with the “low” fluence case lies in the amplitude of DADS4, associated with the lifetime of 5 ns, which is comparatively larger at “high” fluence and has much better

Results that the photoinduced dynamics of FADox at “low” and “high” concentrations essentially share the same decay time constants and shapes of decay-associated difference spectra. The main difference lies in the relative amplitude of the τ3 = 2231 ps decay, associated with the differential spectrum DADS3. Our experiments thus show that the amplitude of DADS3 increases with the fraction of FADox dimers. As a matter of fact, according to the model of section 3.1.1, the fraction of dimers (expressed as 2[D]/C) increases from 19% in solutions of “low” concentration in FADox to 35% in solutions of “high” concentration. We therefore propose to assign the lifetime of 2231 ps to the deactivation of the excited dimers. While DADS1 (associated to the lifetime of τ1 = 0.91 ps), which corresponds to the early relaxation dynamics (solvation and structure), may contain mixed contributions of both the monomers and the dimers, the dynamic steps of τ2 = 5.25.5 ps (DADS2) and τ4 = 2.63.0 ns (DADS4) can be attributed to the monomers. In agreement with previous reports,1622 the 5.25.5 ps component is assigned to the excited-state decay of the closed (stacked) conformation of the monomer, while the 2.63.0 ns component is the deactivation of its open (extended) conformation. Because DADS3 has a closer resemblance to DADS4 than to DADS2, and because τ3 is significantly longer than τ2, it may be tentatively proposed that the structure of FADox within the dimers (or higher order aggregates not included in the simplified model of section 3.1.1) would rather be extended than stacked. It may further be hypothesized that the key parameter controlling the excited-state lifetime of each configuration of the molecule is the spatial separation between the isoalloxazine and adenine moieties: short distance in the closed conformation (fast decay), large distance in the open conformation (long decay), and intermediate in the dimer (intermediate decay). It may additionally be noted that the excited-state lifetime of FMNox, which lacks the adenine moiety, is even longer (∼4.7 ns;17,50 see also global analysis in Supporting Information, section 4) than that of the open conformation of FADox. This points to a still active quenching role of the adenine within the open conformation of FADox, possibly because the distance between the isoalloxazine and the adenine is not large enough to cancel any interaction. 4.1.2. Intramolecular Electron Transfer in the Stacked Conformation of FADox. Kao et al. proposed that intramolecular electron transfer from the adenine to the isoalloxazine occurs in 3257



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59 ps (at pH 7.4) in the closed conformation of FADox and that charge recombination follows in 3040 ps.20 Considering this interpretation, the intermediate lifetime (τ3 = 2231 ps) found in the present study might thus stem from charge recombination in the closed conformation of FADox rather than from the excited dimers dynamics. Within this hypothesis, DADS3 (Figure 4A,B) should be constituted of a sum of the differential absorption spectra, formed during the preceding step (τ2 = 5.25.5 ps) and decaying with τ3 = 2231 ps. However, the actual shape of DADS3, contains a clear stimulated-emission contribution and is very similar to DADS4 (doubtlessly associated to the decay of an excited species: FADox in its open conformation). On the other hand, the reconstructed transient absorption spectrum expected for the radical photoproducts formation (see spectrum in Supporting Information, section 7) does not match with DADS3. We thus do not retain the hypothesis of DADS3 being the signature of charge recombination and confirm our interpretation in terms of the deactivation of excited dimers. The question of intramolecular electron transfer followed by charge recombination is however central to the question of the fast decay of the closed conformation of FADox.17,18 Li et al. recently proposed charge separation to occur in 1.1 ps and geminate recombination in 9 ps.22 Our present measurements provide roughly comparable time constants (τ1 = 0.91 ps and τ2 = 5.25.5 ps), but the same interpretation does not apply. Just as for the preceding discussion, DADS2 contains a clear stimulated-emission contribution and its shape does not match the expected difference spectrum of the adenine cation and isoalloxazine anion radicals (let us note it DSrad) alone. An alternative proposal is to consider that the same mechanism applies but in an “inverted kinetic” regime, that is, with the rate of charge recombination (krec) being faster than the rate of charge separation (ksep). This point of view agrees well with previous works which failed to identify any intermediate in the excited-state relaxation of FADox in its closed conformation.19,21 Within such a scheme, the amplitudes (DADS) of the fast (recombination) and slow (separation) components read: DADSfast ¼

DSrad ksep ksep krec

DADSslow ¼ DSes

DSrad ksep ksep krec

ð7Þ

ð8Þ

where DSes is the difference spectrum associated with the precursor excited state. Because krec > ksep, DADSfast bears the spectral signature of the products (DSrad), with a negative sign and vanishing amplitude as krec increases. DADSslow is a sum of the spectral signature of the excited-state precursor (DSes) and that of the products (DSrad), with a positive sign (and vanishing amplitude as krec increases). At this point, it is interesting to note that DADS2 and DADS4, respectively, associated to the decay of the closed and open conformations of FADox, are not simply proportional as one would grossly expect if the only difference between the two cases were the lifetime of the isoalloxazine excited state. If one normalizes DADS2 and DADS4 at the maximum of the bleaching (see spectra in Supporting Information, section 5), it appears that DADS2 is less negative than DADS4 in the stimulatedemission band where the expected photoproducts of the intramolecular charge separation absorb.

Figure 8. Decomposition of DADS2, associated at the lifetime of 5.5 ps (“dilute” solution of FADox) into a sum of two contributions: (i) the transient absorption spectrum of FADox* in its open conformation (i.e., DADS4), and (ii) the expected differential spectrum of the photoproducts obtained after electron transfer from the adenine to the isoalloxazine. This spectrum was reconstructed by summing the absorption spectra of the radical anion of the riboflavin51 and the radical cation of the adenine52 and subtracting the absorption spectrum of FADox in solution.15 Coefficient R, expressing the ratio of the (RF•- þ Ade•þ) contribution to the FADox* contribution, was found to be 0.079.

The picture therefore emerges where DADS2 could play the role of DADSslow with a finite and positive contribution DSrad. We checked this hypothesis by trying to fit DADS2 by a sum of DSes and DSrad, affected with positive coefficients. DADS4, associated to the excited open conformation FADox, was used as a substitute for DSes, assuming that the intrinsic signature of the excited state of the isoalloxazine ring does not depend much on the closed/open state of the flavin. To convert DADS4 in molar extinction coefficient, the concentration of the excited state was estimated by compensating the bleaching in DADS4 by adding a fraction of the ground-state spectrum of FADox in solution.15 The concentration of the excited open conformation was thereby estimated to 2 μmol L1. DSrad was reconstructed by summing the absorption spectra of the radical anion of the riboflavin (RF•)51 and the radical cation of the adenine (Ade•þ),52 subtracting the absorption spectrum of FADox (see spectrum in Supporting Information, section 7).15 Our best fit (R2 = 0.98), obtained with a ratio of the (RF• þ Ade•þ) contribution to the FADox* contribution (coefficient R) equal to 0.079, is shown in Figure 8. We take the relative good quality of the fit as a strong argument in favor of an “inverse kinetic” scheme where intramolecular charge transfer occurs with a rate constant ksep of ∼1.9 1011 s1 (corresponding to the mean value of 5.2 and 5.5 ps) and is followed by charge recombination on a faster time scale. This latter process is, however, slow enough to leave a measurable trace of it within DADS2. We made an estimation of the rate constant of charge recombination by equating coefficient R (0.079) to expression ksep/(ksep krec) (corresponding term in eq 8). It follows that krec is of the order of 2.6 1012 s1. This value in turn corresponds to a time constant of 390 fs, which would most likely have blended into the shortest lifetime we observed (0.91 ps; DADS1), which we initially assigned to solvation dynamics in water and structural relaxation of the excited molecule. This latter idea agrees in fact well with the marked difference of shape found between the respective DADS1 of FADox (Figure 4) and FMNox (Supporting Information, section 4). DADS1(FMNox), which approximates the ultrafast dynamics of the isoalloxazine ring alone, does not exhibit a large positive peak 3258



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Figure 9. Loglog plots of the transient absorption signal of FADH, for a pumpprobe delay of 100 ps, as a function of excitation energy (Ep), at selected wavelengths: 360 (blue square), 520 (green circle), and 680 nm (red triangle); at low fluence (A, j = 0.05) and high fluence (B, j = 0.2). The slope of the linear fits performed on those data is given as coefficient “a”.

around 450 nm. Such a peak is, however, seen in both DADS1(FADox) and DSrad (with minus sign). We further attempted to fit DADS1(FADox) by a sum of DADS1(FMNox) and DSrad. The result (not shown) is of imperfect quality (R2 = 0.85) but qualitatively explains the differences between DADS1(FADox) and DADS1(FMNox). Moreover, it provides a negative coefficient for the DSrad contribution, just as expected from the “inverse kinetic” scheme presented above (see eq 7 with krec > ksep). Along the arguments developed above for the monomer of FADox in its closed form, it may finally be asked if electron transfer followed by fast charge recombination could explain the lifetime of the dimer (2231 ps). Because DADS3 (dimers) is very similar to DADS4 (open form), it may be concluded that any contribution from a charge-separated intermediate is nearly negligible. In turn, this would mean that, if charge separation occurred in 2231 ps within dimers, charge recombination would be very much faster. However, because no actual proof is available to us, we prefer not to conclude on this issue. 4.2. Photodynamics of FADH. 4.2.1. Excited-State Decay and “Butterfly” Motion. As mentioned in the Introduction, the multimodal excited-state decay of FADH in aqueous solution has been related several times to the bent geometry and flexibility of the molecule along the so-called “butterfly” motion (dihedral N5N10 bending). Enescu et al. invoked a distribution of rapidly interconverting structures,25 Li et al. assigned two picosecond decays to two butterfly conformers,26 and Kao et al. proposed a deactivation mechanism occurring by butterfly bending motion across conical intersections.20 Although the present results have been analyzed in terms of a discrete sum of global exponential decays, the emerging picture is that of a continuous evolution of the transient spectra, with progressive narrowing of the bands on their red side as they decay. We have shown that this narrowing starts within the time window during which solvation dynamics of water is expected (first ps or so) but readily extends in a longer, and unrelated, time scale (τ3 = 2832 ps). We therefore favor the hypothesis that the continuous spectral evolution reveals the relaxation of the molecule along some internal degree of freedom, which can indeed be reasonably identified with the butterfly bending motion. On the other hand, the fast deactivation of the excited state (completed in less than 200 ps) points toward an efficient quenching mechanism, somehow found along the excited-state

relaxation pathway. Because no clear deactivation intermediate was isolated from our measurements, we finally conclude that our transient absorption spectra are qualitatively compatible with the interpretation of Kao et al. involving the crossing of conical intersections found along the butterfly bending coordinate.20 4.2.2. Biphotonic Ionization of the Molecule. Biphotonic ionization of the FADH molecule has been considered by Enescu et al.,25 as an explanation for the far red tail (say beyond 700 nm) of the transient absorption spectra, but the hypothesis was finally rejected because the authors found that the amplitude of this band was directly proportional to the excitation fluence. This previous conclusion contrasts with our present findings which show that the relative amplitude of DADS4, associated to the long-lived species, as compared to the other time components, increases with the excitation fluence. This nonlinear behavior was confirmed by recording the variations of the transient absorption spectra as a function of the excitation fluence. Due to technical constraints, this test was performed for relatively small variations about the two different working fluences reported in section 3.2 (j = 0.05 and j = 0.2). Figure 9 displays loglog plots of the transient absorption signal as a function of excitation energy (excitation surface being held constant) recorded at several characteristic wavelengths (360, 520, and 680 nm) for a pumpprobe delay of 100 ps. At this pump probe delay most of the excited-state population has already decayed. For the “low” fluence excitation (j = 0.05), the 100 ps spectrum is however still dominated by the excited state. For “high” fluence (j = 0.2), the 100 ps spectrum is conversely dominated by the long-lived species. Moreover, the short wavelengths (360 and 520 nm) contain more contributions from the excited state than the far red wavelength (680 nm), which is more characteristic of the long-lived species. Linear fits of the loglog plots were performed, and the corresponding slopes (coefficient “a”) are indicated in Figure 9. It appears that loglog slope is larger than unity even at “low” excitation fluence, especially at 700 nm. The slope, however, clearly increases at “high” excitation fluence and gets close to 2 in the red part of the spectrum. The straightforward interpretation of this behavior is that the excited-state population essentially depends linearly on the excitation fluence while the concentration of long-lived species absorbing in the red wavelengths depends quadratically on it. 3259



Figure 10. Comparison of the transient spectrum associated to the longlived species (DADS4; blue) obtained under “high” excitation fluence of FADH to a reconstructed differential spectrum (DSioniz; black) made of the sum of the absorption spectra of FADH•51 and the hydrated electron,47,48 to which the absorption spectrum of FADH37 is subtracted.

As considered by Enescu et al.,25 one plausible explanation of the existence of the long-lived species is the biphotonic ionization of FADH, leading to the formation of a neutral radical, FADH•, and hydrated electron. To verify this hypothesis, the transient spectrum of the long-lived species (DADS4) recorded under high-fluence excitation was compared to a reconstructed differential spectrum (DSioniz) made of the sum of the absorption spectra of FADH• and the hydrated electron to which the absorption spectra of FADH is subtracted, all of those being properly expressed in the same molar extinction coefficient units. Since the absorption spectrum of FADH• has only been reported in complexes with flavoproteins,37,53,54 we chose to use the one of the riboflavin semiquinone radical (RFH•) published by Land et al.51 The absorption spectrum of hydrated electron was taken from Jou et al.48 and adjusted to the maximum value given by Michael et al.47 Figure 10 shows DADS4 and DSioniz properly scaled to minimize its differences with DADS4. The relative good quality of the match (R2 = 0.995) finally confirms the hypothesis of photoinduced ionization of FADH. It is worth mentioning that the above-mentioned scaling allowed us quantifying the concentration of long-lived hydrated electron for both the “low” and “high” fluence conditions. We found 0.39 ( 0.01 μmol L1 and 5.09 ( 0.02 μmol L1, respectively. The ratio of the two concentrations is 13, while the ratio of the corresponding fluences is 4. Given the uncertainties in comparing the absolute amplitudes of two distinct experiments (imperfect reproducibility of the pump-prove overlap), this result is in relatively good agreement with a quadratic dependence (factor 16 expected) of the hydrated electron concentration on the excitation fluence, thereby confirming that the ionization process is biphotonic. The speculation that FADH• might deprotonate within the observation window of our experiment was further checked by reconstructing the differential spectrum associated with the pair made of one FAD• radical and one hydrated electron. The match with DADS4 was poor (data not shown) and the hypothesis was rejected. As far as the hydrated electron is concerned, it is interesting to recall that a rise of transient absorption signal was observed beyond 680 nm during the first picosecond of the dynamics, in the measurement under “high” excitation fluence. This phenomenon may well correspond to the formation time of the relaxed hydrated electron. It is indeed known that its absorption spectrum shifts from near IR to far red in 0.4 ps.55

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It may finally be wondered why the lifetime of the FADH•/ hydrated electron pair is as short as ∼5 ns (with a large error due to the limited observation window of our experiment) since the survival time of hydrated or otherwise solvated electron may be considerably longer.56,57 Diffusion-controlled recombination can be ruled out because the very low concentration of photoproduced neutral semiquinone (μmol L1 range; see section 4.2.2) sets the corresponding rate in the microsecond range (concentration times diffusion constant of the order of 1010 L mol1 s1). On the other hand, it is doubtful that geminate recombination could be as slow as 5 ns, a time scale during which the hydrated electron and FADH• can diffuse away from each other. The recent review by Chen and Bradforth58 states that incomplete tens-to-hundreds picosecond time scale geminate recombination is general to all femtosecond electron photodetachment measurements in water. Fast scavenging by the hydronium cations (k = 2.3 1010 L mol1 s1)59 can be excluded because of their too low concentration at pH 8.0 (1 μmol L1). It is finally known that, in the presence of high solute concentrations, solvated electrons can be scavenged at a rate that depends on the nature of the solute. The exact nature of the hypothetical scavenger involved in the present experiment is not clear, but might be found among the buffer constituents.

5. CONCLUDING REMARKS We showed that FADox in solution readily undergoes aggregation, approximated here by dimerization, at the concentrations we used for transient absorption spectroscopy. By varying the solute concentration we demonstrated that the excited-state decay of FADox actually depends on the dimer fraction. Three excited-state lifetimes were found and assigned to three different species: the closed conformation of the monomer (5.25.5 ps), the open conformation of the monomer (2.63.0 ns), and the dimer (2231 ps). One interesting result is that the spectrum describing the fast decay of the FADox monomer in closed conformation contains remnants of the signature of a charge separated species corresponding to the intramolecular electron transfer from the adenine moiety to the isoalloxazine moiety. Such observation supports the previously considered hypothesis17,18,22 that the deactivation of excited FADox (monomer) in closed conformation is due to intramolecular electron transfer. We however demonstrate that charge recombination follows with a somewhat faster rate constant, but not fast enough to remain completely undetected. We deduce from the analysis of the difference spectrum associated to the 5.25.5 ps decay that the rate constant of the recombination process is of the order of 2.6 1012 s1, while charge separation only proceeds at a rate of ∼1.9 1011 s1. As the excited-state lifetime of the dimer is rather close to that of the open monomer, it may be speculated that the structure of the FADox molecule within the dimer would rather be open than closed and that its quenching would also involve intramolecular charge transfer. The longer rate of charge transfer would be related to the larger distance between the isoalloxazine and adenine rings in the dimer species. Indeed FMNox which lacks the adenine shows longer decay. As far as the excited-state dynamics FADH in aqueous solution is concerned, we showed that the transient spectra continuously evolve (narrow) as they decay. Moreover, this phenomenon extends in a time scale (2832 ps) that is unrelated to solvation dynamics of water, which may contribute to the spectral shifts at a shorter time scale. We favor the hypothesis that 3260


The Journal of Physical Chemistry A this phenomenon reveals the relaxation of the molecule along some internal degree of freedom, along which excited-state deactivation is finally found. This view is compatible with the interpretation of Kao et al. involving the crossing of conical intersections through butterfly bending motion.20 We finally demonstrated that the FADH molecule undergoes biphotonic ionization at the excitation fluences required for femtosecond transient absorption spectroscopy, leading to the formation of both the FADH• semiquinone radical and the hydrated electron.

’ ASSOCIATED CONTENT

bS

Supporting Information. (1) Effective extinction coefficient spectra of FMNox; (2) Transient absorption spectra of a “dilute” solution of FMNox; (3) Transient absorption spectra of a “concentrated” solution of FMNox; (4) Global analysis of the transient absorption data of FMNox; (5) Normalized DADS of a “dilute” solution of FADox; (6) Transient absorption spectra of a “concentrated” solution of FADox; (7) Reconstructed transient spectra of radicals after charge separation in FADox. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected].

’ ACKNOWLEDGMENT This work was supported by the ANR (French National Agency for Research) through the “Femtomotile” Project (ANR-05-BLAN-0188-01). ’ REFERENCES (1) Sancar, A. Chem. Rev. 2003, 103, 2203. (2) M€uller, M.; Carell, T. Curr. Opin. Struct. Biol. 2009, 19, 277. (3) Schleicher, E.; Bittl, R.; Weber, S. FEBS J. 2009, 276, 4290. (4) Weber, S. Biochim. Biophys. Acta Bioenerg. 2005, 1707, 1. (5) Brettel, K.; Byrdin, M. Curr. Opin. Struct. Biol. 2010, 20, 693. (6) Cashmore, A. R. Cell 2003, 114, 537. (7) Lin, C. T.; Shalitin, D. Annu. Rev. Plant Biol. 2003, 54, 469. (8) Partch, C. L.; Sancar, A. Photochem. Photobiol. 2005, 81, 1291. (9) Lin, C. T.; Todo, T. Gen. Biol. 2005, 6, 220. (10) Ritz, T.; Ahmad, M.; Mouritsen, H.; Wiltschko, R.; Wiltschko, W. J. R. Soc. Interface 2010, 7, S135. (11) Selby, C. P.; Sancar, A. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 17696. (12) Pokorny, R.; Klar, T.; Hennecke, U.; Carell, T.; Batschauer, A.; Essen, L. O. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 21023. (13) Massey, V. Biochem. Soc. Trans. 2000, 28, 283. (14) Barrio, J. R.; Tolman, G. L.; Leonard, N. J.; Spencer, R. D.; Weber, G. Proc. Natl. Acad. Sci. U.S.A. 1973, 70, 941. (15) Islam, S. D. M.; Susdorf, T.; Penzkofer, A.; Hegemann, P. Chem. Phys. 2003, 295, 137. (16) Stanley, R. J.; MacFarlane, A. W., IV. J. Phys. Chem. A 2000, 104, 6899. (17) van den Berg, P. A. W.; Feenstra, K. A.; Mark, A. E.; Berendsen, H. J. C.; Visser, A. J. Phys. Chem. B 2002, 106, 8858. (18) Chosrowjan, H.; Taniguchi, S.; Mataga, N.; Tanaka, F.; Visser, A. Chem. Phys. Lett. 2003, 378, 354. (19) Kondo, M.; Nappa, J.; Ronayne, K. L.; Stelling, A. L.; Tonge, P. J.; Meech, S. R. J. Phys. Chem. B 2006, 110, 20107.

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New Insights into the Ultrafast Photophysics of Oxidized and Reduced

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