Structural Effects on the Ultrafast Photoisomerization of Photoactive

Publication Date (Web): April 16, 2009 ... Fax: +33 144 323 863. ... Subpicosecond transient absorption spectroscopy was used to address the role of t...
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J. Phys. Chem. C 2009, 113, 11605–11613

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Structural Effects on the Ultrafast Photoisomerization of Photoactive Yellow Protein. Transient Absorption Spectroscopy of Two Point Mutants† Pascale Changenet-Barret,*,‡ Pascal Plaza,‡ Monique M. Martin,*,‡ Haik Chosrowjan,*,§ Seiji Taniguchi,§ Noboru Mataga,§ Yasushi Imamoto,| and Mikio Kataoka⊥ Ecole Normale Supe´rieure, De´partement de Chimie, UMR-CNRS 8640 PASTEUR, 24 rue Lhomond, 75231 Paris Cedex 05, France, Institute for Laser Technology (ILT), Utsubo-Hommachi 1-8-4, Nishiku, Osaka 550-0004, Japan, Department of Biophysics, Graduate School of Science, Kyoto UniVersity, Kyoto 606-8502, Japan, and Graduate School of Materials Science, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan ReceiVed: February 13, 2009; ReVised Manuscript ReceiVed: March 26, 2009

Subpicosecond transient absorption spectroscopy was used to address the role of the local environment on the photoisomerization process of the p-coumaric thioester chromophore in photoactive yellow protein (PYP) by studying two point mutants, T50V and E46Q. These mutations introduce alterations of the hydrogen-bond network involving the amino acids of the active site close to the chromophore and the chromophore phenolate group, respectively. Transient-absorption spectra of T50V and E46Q are found to be qualitatively similar to those of the wild-type PYP (WT) and R52Q, suggesting that the earliest steps of the photoinduced processes in all three mutants remain similar to those of the WT. Target analyses of the transient spectra of T50V, E46Q, R52Q, and WT, were successfully performed by using a model based on the one previously published by Larsen et al. (Biophys. J. 2004, 87, 1858), which involves heterogeneous excited-state populations undergoing deactivation along two competitive relaxation pathways. A so-called reactive pathway leads to the sequential formation of the well-characterized cis intermediates, I0 and I1, of the photocycle. The second pathway is non reactive and produces a transient species that restores the initial trans ground-state in 3-6 ps. This transient is tentatively attributed to a distorted vibrationally hot trans ground state. The most prominent effect of mutation is observed for T50V and R52Q which exhibit significantly slower excited-state deactivations, whereas E46Q behaves like the WT protein. This difference is analyzed in terms of a significant decrease, in T50V and R52Q, of the fraction of heterogeneous excited-state population that undergoes isomerization. The quantum yield of isomerization deduced from the target analyses was found to be 0.31 ( 0.08 for WT, 0.22 ( 0.06 for T50V, 0.29 ( 0.08 for E46Q, and 0.19 ( 0.05 for R52Q. The decrease of isomerization yield observed in T50V and R52Q is mainly attributed to the loss of rigidity of the protein active site, induced by these mutations, rather than to the deletion of the positive charge of Arg52 in R52Q. 1. Introduction

SCHEME 1: Schematic Representation of the PYP Active Site in the Ground State

Many experimental and theoretical studies have been devoted to the characterization of the trans-cis isomerization mechanism of the PYP (photoactive yellow protein) chromophore and to the identification of the precursors of the relaxed cis isomer, the so-called I1 state.1-11 PYP is a blue-light photoreceptor thought to be responsible for the photoavoidance response of the purple bacterium Halorhodospira halophila.12 It is a small cytosolic chromoprotein, the chromophore of which is the deprotonated trans-p-hydroxycinnamic acid, covalently linked †

Part of the “Hiroshi Masuhara Festschrift”. * Corresponding authors. Monique M. Martin, Ecole Normale Supe´rieure, De´partement de Chimie, CNRS-ENS-UPMC UMR 8640 PASTEUR, 24 rue Lhomond, 75231 Paris Cedex 05, France. Tel: +33 144 322 412. Fax: +33 144 323 863. E-mail: [email protected]. Pascale ChangenetBarret, Ecole Normale Supe´rieure, De´partement de Chimie, CNRS-ENSUPMC UMR 8640 PASTEUR, 24 rue Lhomond, 75231 Paris Cedex 05, France. Tel: +33 144 322 413. Fax: +33 144 323 863. E-mail: [email protected]. Haik Chosrowjan, Institute for Laser Technology (ILT), Utsubo-Hommachi 1-8-4, Nishiku, Osaka 550-0004, Japan. Tel: +81 664 927 613. Fax: +81 664 925 641. E-mail: [email protected]. ‡ UMR-CNRS 8640 PASTEUR. § Institute for Laser Technology. | Kyoto University. ⊥ Nara Institute of Science and Technology.

via a thioester bond to Cys69 (Scheme 1). Upon blue-light irradiation, PYP undergoes a photocycle ultimately leading to signal transduction.13-15 Like for rhodopsins, the trans-cis isomerization of the chromophore has been identified as the first overall molecular process of the PYP photocycle, with the formation of a relaxed cis isomer (I1) in the nanosecond regime.13-15 The isomerization mechanism involves a photoin-

10.1021/jp901343x CCC: $40.75  2009 American Chemical Society Published on Web 04/16/2009

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duced charge redistribution within the chromophore,7,10,16-18 followed by trans to cis isomerization of the ethylenic bond through the flip of the chromophore carbonyl tail in the femtopicosecond regime.7,10,19,20 This evolution leads to the formation of at least one precursor of I1, named I0.1,2,4-8,10,11 Subsequently, a progressive reorganization of the hydrogen bond (HB) network around the chromophore accommodates the cis conformation on the nanosecond time scale and leads to the formation of I1.7,10,20,21 Interestingly, the photoisomerization yield of wild-type PYP (WT) is reported to be about 30%,22-24 whereas that of the isolated chromophore, as modeled by the deprotonated thioester derivative of 4-hydroxycinnamic acid, is negligible in water.25-27 The excited-state deactivation of the free chromophore involves instead the formation of a short-lived transient state, absorbing around 450 nm, from which the molecule returns to the initial trans configuration.8,25,28-33 The formation of this intermediate, proposed to exhibit a larger charge shift and localization than the emissive state, has been correlated to the electron donor-acceptor structure of the molecule.27,32-34 It was therefore proposed that the chromophore isomerization in PYP occurs thanks to the nearby positively charged Arg52, which would prevent the large photoinduced charge shift of the chromophore.32,33,35 QM/MM calculations indeed predicted that the presence of Arg52 above the chromophore phenolate group promotes its photoisomerization.36 We however recently discarded such crucial role of Arg52 on the photoisomerization process in PYP on the basis of subpicosecond transientabsorption measurements carried on the R52Q mutant,11 in which Arg52 was replaced by a neutral glutamine. These experiments revealed that the early excited-state deactivation steps of R52Q remain very similar to those of WT.11 The mutation was nonetheless found to slow down the excited-state deactivation16,37 and the first steps of the photocycle.11 Previous studies carried out on PYP mutants16,17,37-42 already highlighted the substantial role of the local protein environment on the chromophore excited-state deactivation. Alterations of the HB network in the chromophore binding pocket (also called protein nanospace, PNS) have been found to slow down significantly the excited-state decay and was interpreted as mainly arising from a loosening of the PNS.16,37 The present paper is devoted to the study of E46Q and T50V mutants, in order to further clarify the role of the local aminoacids on the deactivation of PYP. In these mutants, Glu46 and Thr50 residues have been replaced by a glutamine and a valine, respectively, leading to alterations of the HB network around the chromophore. The HB coordination of the chromophore phenolate group is directly weakened within E46Q, while the slightly more distant HB between Thr50 and Tyr42 is weakened in T50V. We have probed the photoinduced processes in these mutants by subpicosecond transient absorption spectroscopy. Target analysis of the presently reported differential spectra of E46Q and T50V, as well as of previously published spectra of R52Q and WT,11 was performed with a kinetic model inspired from the one proposed by the group of van Grondelle.5,43 The reaction mechanism involves three heterogeneous excited states and one deactivation pathway in competition with the known isomerization photocycle. The model allows quantifying the impact of mutation on the quantum yield of isomerization of the chromophore. The role of the PNS structure on the chromophore photoinduced reactivity is discussed within the context of recent theoretical studies of WT and R52Q36,44-46 and of crystallographic data on the structures of the mutants.47-50

Changenet-Barret et al. SCHEME 2: Kinetic Model Used for the Target Analysis of WT-PYP and Its Three Mutantsa

a xi are the relative populations of the three (heterogeneous) S1 states. ki are the (total) rate constants of deactivation of each S1 states. The branching ratios φi correspond to the yields of formation of transient I0 from the S1 states. Correspondingly (1 - φi) stands for the yields of formation of transient X from the S1 states. The rate of formation of I1 from I0 is noted kI0. The yield of the I0 f I1 process is assumed to be unity. The decay rate of X noted kX.

2. Materials and Methods 2.1. Materials. E46Q and T50V mutants were produced as previously described.51,52 Samples were prepared in Tris-HCl buffer (10 mM) at pH 8.1. For transient absorption spectroscopy, the sample absorbance was adjusted to about 1 (1 mm optical path). 2.2. Pump-Probe Measurements. Transient absorption and gain experiments were performed by the pump-probe technique. 500-fs pulses at 428 and 570 nm were simultaneously generated by a homemade dye laser system described elsewhere.53 Pump pulses at 428 nm, with energies ranging between 60 and 90 µJ, were focused on a diameter of 2 mm on the sample. The probe was a continuum of white light, generated in a 1-cm water cell with 300-µJ pulses at 570 nm. The continuum was split into a reference beam and a sample beam which crossed the pumped volume on a diameter of about 1 mm. Both continua were sent to a spectrograph (Jobin-Yvon Spex 270M, entrance slit 64 µm) through 600-µm optical fibers and simultaneously analyzed by a CCD camera (128 × 1024, Roper Scientific). The time delay between pump and probe beams was varied with a motorized optical delay line. Pump and probe beams were set at the magic polarization angle to avoid any contribution from rotational diffusion. A 5-ml sample solution was recirculated through a 1-mm cell, at room temperature, to avoid photolysis during the experiment. The time-resolved differential absorbance spectra (∆Α(λ,t) ) A(λ,t) - A0(λ) with A(λ,t) the excited-sample absorbance and A0(λ) the steady-state absorbance) were averaged over 500 shots and corrected from the chirp of the probe beam. The steadystate absorption spectra of the samples were measured with a double-beam UV-visible spectrophotometer (UVmc2, Safas). The spectra of the samples used in the pump-probe experiments were measured before and after each experiment to check that the samples were not degrading. The steady-state fluorescence spectra were measured with diluted samples using a response-corrected spectrofluorimeter (Fluoromax 3, Horiba Jobin Yvon). 2.3. Data Analysis. The technical details of our data analysis procedures are given as Supporting Information (see section 1). In brief, a multiexponential global analysis was first performed by singular value decomposition (SVD)54 in order to obtain decay associated differential spectra (DADS), i.e., spectra of the amplitudes associated to the different time components, and to filter noise out. Target analysis43 was performed on kinetic traces at 18 significant wavelengths with the help of the kinetic model described in Scheme 2. This model is in fact a simplified version of the one initially proposed by Larsen et al.5 The additional

Transient Absorption Spectroscopy of Two Point Mutants

Figure 1. Normalized steady-state absorption and fluorescence spectra of (a) WT, (b) R52Q, (c) E46Q, and (d) T50V, in 10 mM Tris-HCl buffer solution at pH 8.1. The fluorescence data were recorded upon excitation at 440 nm.

use of a minimal set of spectral constraints (see the Supporting Information, section 1) allowed optimizing the various parameters of the model, i.e., the different rates and branching ratios (see the footnote of Scheme 2) and obtaining species associated spectra (SAS), i.e., spectra of the generalized extinction coefficients (including transient absorption and stimulated emission) attached to each transient. Previous pump-probe spectroscopy studies carried out on the isolated PYP chromophore in water30,32,55 and on WT5 showed the formation of radical-electron pair due to two-photon ionization of the chromophore. This nonlinear laser-induced reaction, which bears no relationship with the sun-driven onephoton photocycle of PYP, was found to occur to a small extent in the PYP mutants. This process was included in our kinetic model as a separate excitation channel instantly leading to a long-lived species (see the Supporting Information, section 2). 3. Results 3.1. Steady-State Spectra. The normalized steady-state absorption and fluorescence spectra of E46Q and T50V are displayed in Figure 1, together with those of WT and R52Q for comparison. The absorption and emission spectra of WT peak at 446 and 494 nm, respectively. As previously observed,37,52 the steady-state spectra of E46Q and T50V are comparatively abs flu abs ) 460 nm, λmax ) 501 nm for E46Q, λmax ) red-shifted: λmax flu 456 nm, λmax ) 497 nm for T50V. In these two mutants, the HB network of the PYP active site was shown to be altered.47-49 The large red shift of E46Q absorption spectrum is due to the weakening of the HB between the chromophore phenolate and residue 46 in which the hydroxyl group has been replaced by an amide group.47,48,56 This substitution likely induces an increased electron delocalization on the chromophore responsible of the red shift of its absorption spectrum.38,47,48,56 In T50V, the red shift of the spectra is smaller since residue 50 does not form direct HB with the chromophore. Crystallographic data however indicate that replacing Thr50 by Val induces a weakening of the HB between Glu46 and Tyr42,49 leading to a loosening of the HB between the chromophore phenolate group and Tyr42.38 In contrast, suppressing the positively charged Arg52 barely abs ) 446 nm for R52Q) affects the steady-state absorption (λmax flu ) 497 nm) spectra of PYP.11,37,52 Recent and emission (λmax calculations indeed showed that Arg52 causes essentially the same stabilization of the ground and excited states of the chromophore, in the Franck-Condon geometry.44

J. Phys. Chem. C, Vol. 113, No. 27, 2009 11607 3.2. Time-Resolved Transient Absorbance Spectra. The absorption difference spectra of E46Q and T50V in 10 mM TrisHCl buffer at pH 8.1 are given in Figure 2, along with their corresponding steady-state absorption and fluorescence spectra (upper frame). After excitation, the transient spectra display three bands: the excited-state absorption (ESA) band at 380 nm and the ground-state bleaching (GSB) and stimulated-emission (SE) bands in the regions of steady-state absorption and fluorescence spectra, respectively. In about 10 ps, the ESA band disappears and the SE band is replaced by a small transient absorption band located around 510 nm for E46Q and 500 nm for T50V. In the subnanosecond regime, this absorption band turns into a blue-shifted transient absorption band centered at 490 and 480 nm, respectively for E46Q and T50V. Note that the spectro-temporal behavior of the differential spectra of E46Q and T50V is qualitatively similar to those of WT and R52Q we previously measured.11 The two consecutive transient absorption bands of the mutants in the 480-510 nm region seem in particular to be close spectral analogs of intermediate I0 (500 nm)1,5 and its successor I1 (480 nm)1,5,57 of WT identified as cis isomers of the chromophore.7,10,20,58 The slight bathochromic shift of the absorption maxima of these intermediates in E46Q, as compared to the native photoreceptor, could be explained by the weakening of HB between the chromophore and residue 46. X-ray spectroscopy and UV pump mid-infrared probe measurements indeed showed that this HB is lost in I1 for E46Q whereas it is reinforced in the native protein.24,47 Global kinetic analysis of the complete data set of T50V and E46Q required the sum of four exponential components and a plateau. The lifetimes are 1.4, 3.4, 26, and 461 ps for T50V and 0.9, 3.5, 27, and 676 ps for E46Q. Figure 3 illustrates the five decay associated differential spectra (including the plateau), numbered DADS1 to DADS5 with increasing lifetime. For comparison, the DADS previously obtained for WT and R52Q11 are also shown. One sees that the spectra of the three mutants are qualitatively similar to those of WT. The relative amplitude of DADS1 (black line) is, however, significantly reduced for T50V and R52Q. As already stressed for R52Q,11 the smaller amplitude of this short component stems from the significant slowing down of the overall excited-state decay of these two mutants, with respect to WT (0.8, 2.6, 16, and 406 ps), as was already known from their fluorescence decays.16,37 For all compounds, DADS1 to DADS3 illustrate the loss of ESA (below 400 nm) and SE (above 480 nm) with the corresponding lifetimes. DADS2 and DADS3 exhibit an additional GSB contribution (between 400 and 480 nm) whereas no such contribution appears on DADS1. The presence of the negative component in the spectral region located around 480 nm is observed instead and is assigned to the rise of the first transient, we call X. The presence of such a transient was also reported in previous pump-probe measurements carried out on WT.5 DADS4 corresponds to the decay of the intermediate I0. DADS5 is assigned to intermediate I1, which contributes to the ∆A spectra in the nanosecond time scale. 3.3. Target Analyses. 3.3.1. Kinetic Model. The transient spectroscopy of T50V and E46Q is qualitatively similar to that of WT and R52Q, both in terms of time evolution and spectral signatures. We interpret this similarity as an indication that the early photoinduced processes of the four systems are essentially the same, hence that the photocycle of the three mutants leads to the cis isomer through the same intermediates as in WT. The qualitative analysis of the present transient spectra of T50V and E46Q, as well as of the previously reported spectra of WT and

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Figure 2. Transient absorption spectra of E46Q (left) and T50V (right) in 10 mM Tris-HCl buffer solution at pH 8.1, for various pump-probe delays after subpicosecond excitation at 428 nm (the scattered pump light has been masked).

Figure 3. Decay associated differential spectra (DADS) of the five time components obtained from the multiexponential global fit of the transient absorption spectra of WT-PYP, R52Q, E46Q, and T50V. DADS are numbered with increasing lifetime of the components.

R52Q,11 is consistent with the formation of the three distinct transients: X, I0, and I1. We performed a target analysis of our time-resolved differential absorption spectra including those of WT and R52Q with the kinetic model shown in Scheme 2. This model is a slightly simplified version of the inhomogeneous model developed for WT in the group of van Grondelle.5,43 The reaction scheme involves two competitive routes. One so-called reactive pathway leads to the formation of the photocycle intermediates I0 then I1, thus to isomerization, whereas the second, nonreactive, pathway produces a short-lived transient (X) returning back to the initial trans ground-state. In our model state X cannot be reached from I0. Note that inserting a third intermediate (I0‡) between I0 and I1, as proposed by the group of Cusanovich,1,2 was not necessary to satisfactorily fit our experimental data. One might argue that the three decay components could be an artifact resulting from hypothetical continuous spectral shifts

or shape changes during relaxation, which would indeed not be properly captured by the global analysis. For WT and its mutants, neither the fluorescence spectra59-62 nor the stimulated emission exhibit significant dynamical Stokes shift within the time resolution of our apparatus.5,11,29 The shift or reshaping of the other absorption bands cannot be fully excluded but, as discussed in a former letter,11 we rather believe the triexponential decays of the SE signal to arise from the existence of groundstate conformational heterogeneities leading, upon excitation, to excited-state heterogeneities. This hypothesis is consistent with NMR studies of WT in solution, which show that the protein is found to have many different conformations.63 On the basis of temperature effects on the fluorescence dynamics, Mataga et al. in fact initially proposed the existence of a distribution of activation barriers in the excited-state deactivation of PYP and its mutants.37,64 In the simplest picture, the three exponential decays of SE are thus assumed to come from three independent excited-state populations, like in the van Grondelle model.5,43 They are believed to correspond to different protein conformations and to have different lifetimes and isomerization yields. Interestingly, recent crystallographic data indeed emphasize the existence of two populations of PYP molecules, one leading to the photocycle and another one being nonreactive.65 In our model, we assumed that the three excited states have very similar spectra. The photoproducts, X, I0 and I1, formed from each of these states are also supposed to be indistinguishable: their spectra and decay kinetic parameters are identical, whatever the initial excited-state they are produced from. 3.3.2. Kinetics Parameters and Photoisomerization Yields. Figure 4 displays the target analysis results of the T50V differential absorption data, using the model of Scheme 2. The figure gives the fits at selected wavelengths (on the left side) and the species associated spectra (SAS, on the right side) of S1, X, I0, and I1. Similar analyses were also performed for E46Q, as well as for WT and R52Q (see the Supporting Information, section 3). They lead to the same qualitative results. A good description of the time-resolved differential spectra of all studied proteins is thus obtained with the same kinetic model. The optimized rate constants and branching ratios corresponding to Scheme 2 are gathered in Table 1 for each system.

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Figure 4. Left: ∆A(t) kinetics at selected wavelengths of mutant T50V, in 10 mM Tris-HCl buffer solution at pH 8.1, after excitation at 428 nm. The fits corresponding to the target analysis with the model of Scheme 2 are represented by the solid lines. Right: Species associated spectra (SAS) of species S1, X, I0, and I1, obtained by target analysis of the differential absorption spectra of T50V. The initial S0 absorption spectrum is recalled for comparison.

TABLE 1: Parameters of the Kinetic Model of Scheme 2, As Optimized by Target Analysis of the Differential Absorption Spectra of WT and Its R52Q, E46Q, and T50V Mutantsa xexc, xexc2

relative populations

branching ratios

rate constants (ps-1)

WT

xexc ) 0.179 ( 0.006 xexc2 ) 0.003

x1 ) 0.50 ( 0.03 x2 ) 0.42 ( 0.03 x3 ) 0.08 ( 0.03

φ1 ) 0.58 ( 0.07 φ2 ) 0.03 ( 0.06 φ3 ) 0.03 ( 0.08

R52Q

xexc ) 0.244 ( 0.006 xexc2 ) 0.016

x1 ) 0.29 ( 0.02 x2 ) 0.44 ( 0.01 x3 ) 0.27 ( 0.03

φ1 ) 0.63 ( 0.08 φ2 ) 0.01 ( 0.03 φ3 ) 0.01 ( 0.03

E46Q

xexc ) 0.204 ( 0.007 xexc2 ) 0.003

x1 ) 0.39 ( 0.02 x2 ) 0.48 ( 0.02 x3 ) 0.13 ( 0.04

φ1 ) 0.55 ( 0.09 φ2 ) 0.13 ( 0.05 φ3 ) 0.13 ( 0.05

T50V

xexc ) 0.28 ( 0.01 xexc2 ) 0.01

x1 ) 0.31 ( 0.02 x2 ) 0.48 ( 0.02 x3 ) 0.21 ( 0.04

φ1 ) 0.60 ( 0.08 φ2 ) 0.05 ( 0.02 φ3 ) 0.05 ( 0.03

k1 ) 1.5 ( 0.1 k2 ) 0.35 ( 0.03 k3 ) 0.066 ( 0.005 kx ) 0.31 ( 0.03 kI0 ) 0.0027 ( 0.0001 k1 ) 1.3 ( 0.1 k2 ) 0.21 ( 0.01 k3 ) 0.038 ( 0.001 kx ) 0.27 ( 0.02 kI0 ) 0.0019 ( 0.0002 k1 ) 1.4 ( 0.2 k2 ) 0.33 ( 0.02 k3 ) 0.042 ( 0.003 kx ) 0.33 ( 0.03 kI0 ) 0.0012 ( 0.0001 k1 ) 1.7 ( 0.2 k2 ) 0.31 ( 0.01 k3 ) 0.033 ( 0.001 kx ) 0.18 ( 0.01 kI0 ) 0.0017 ( 0.0002

PYP

a The table provides the fraction xexc of molecules excited by one-photon absorption, the fraction xexc2 of molecules undergoing two-photon ionization and the fraction xi of (one-photon) excited population found in each of the three S1 states immediately after excitation (xi ) xi(0) with the notations given as Supporting Information). ki is the (total) decay rate of each S1 state and φi is the corresponding formation yield of I0. The errors are given assuming the general constraints detailed as Supporting Information. Note that the yield of the I0 f I1 process was fixed to 1.

Since the formation yield of I1 from I0 is assumed to be unity (φI0fI1 ) 1, see details of the fitting constraints in the Supporting Information, section 1), the average formation yield of I1 from 3 S1 is thus calculated as 〈φI1〉 ) ∑i)1 xiφi from the parameters of Table 1. One finds the following: 0.31 ( 0.08 for WT, 0.22 ( 0.06 for T50V, 0.29 ( 0.08 for E46Q, and 0.19 ( 0.05 for R52Q (values collected in Table 2). Note that they are in good agreement with other values reported by several groups: 0.35-0.28 ( 0.05 for WT,22-24 0.31-0.20 ( 0.1 for E46Q,24,66 and 0.20 ( 0.08 for R52Q.67 Table 1 also shows that the excitedstate population having the fastest decay rate has the higher yield of I0 and I1 formation, in agreement with the previous

3 TABLE 2: Average Excited-State Lifetime (〈τS1〉 ) ∑i)1 xiki-1, Calculated from Table 1), Average Formation Yield of 3 I1 (〈OI1〉 ) ∑i)1 xiOi)

PYP

WT

R52Q

E46Q

T50V

〈τS1〉 〈φI1〉

2.7 ( 0.8 ps 0.31 ( 0.08

9 ( 1 ps 0.19 ( 0.05

5 ( 1 ps 0.29 ( 0.08

8 ( 2 ps 0.22 ( 0.06

report by Larsen et al. on WT.5 The other fractions of the excited-state population almost do not form intermediate I0, essentially following the competitive path toward X. Our results show that mutation decreases the fraction (x1) of excited population undergoing isomerization.

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4. Discussion In the following sections, we analyze the effects of mutation on the competitive pathways of the model (Scheme 2) we used to describe our data. The nature of the transient X is also discussed. 4.1. Effect of PNS Rigidity on the Isomerization Process. As shown in Table 2, the largest effects of mutation are observed for T50V and R52Q, for which the average excited-state lifetime 3 xiki-1) is increased by a factor of 3 as compared (〈τS1〉 ) ∑i)1 to WT. On the contrary, the excited-state decay of E46Q exhibits almost the same dynamics as the natural photoreceptor. Table 1 shows that the significant slowing down of the mean excitedstate decay of T50V and R52Q mainly arises from a change of the sample heterogeneity. In WT, only one of the three excited state, the one exhibiting the fastest decay, has the correct configuration to undergo isomerization. The same is true for T50V and R52Q but the fraction of this short-lived reactive population is reduced. The consequence is a globally slower excited-state decay than in WT and a significantly smaller isomerization yield (〈φI1〉 ) 0.31 for WT, 0.19 for R52Q and 0.22 for T50V). Our study thus suggests that T50V and R52Q alter the distribution of protein conformations in such a way that the particular structures needed to allow the reactive pathway and trigger the photocycle are less abundant. Mataga et al. proposed that isomerization requires a tight protein nanospace and that the slowing down of excited-state decay observed for T50V is due to a loosening of the immediate surroundings of the chromophore.37 The crystallographic structure of T50V indeed shows the loss of HB between residue 50 and both Glu46 and Tyr42,49 leading to a looser nanospace. Mataga et al. proposed that the slower excited-state deactivation of R52Q could also be sought in the loss of rigidity of the chromophore environment induced by the absence of the positively charged Arg52.37 The recent studies of the X-ray crystallographic structure of R52Q indeed brought evidence for a loosening of the PNS (protein nanospace) of this mutant.50 The absence of the guanidino group in R52Q opens a cavity occupied by two water molecules sitting near the chromophore. Gln52 is linked to Tyr98 through those two water molecules. Furthermore, the two HB linking Arg52 to Thr50 and Tyr98 are lost.50 It is interesting to compare our interpretation to a previous report on femtosecond transient absorption studies of two other mutants of PYP, E46A and Y42F, in which the HB of the chromophore with Glu46 and Tyr42 is respectively deleted.42 This work showed a significant slowing down of the excitedstate decay kinetics as compared to WT which was interpreted as a possible “consequence of allowing rotational motions of the phenolic ring that decrease the coupling between the vibronic states of the thioester bond and those of the π-electron system of the aromatic portion of the chromophore”. We believe this explanation is in fact fully compatible with our view of a mutation-induced decrease of the active site rigidity. Finally, the weak impact of mutation on the photoisomerization yield we observe for E46Q (Table 2) can be explained by the limited alteration of the active site rigidity induced by mutation in this case. This likely reduces the population of the nonisomerizing excited state (Table 1: x2 + x3 ) 0.6 for E46Q, 0.5 for WT and 0.7 for the other mutants). The mutation induces a weakening of the HB between the chromophore phenolate group and residue 46, the short HB with Tyr42 being maintained.47

Changenet-Barret et al. 4.2. Effect of Local Charge on the Isomerization Process. On the margin of its incidence on the PNS rigidity, the presence of the positively charged Arg52 is thought to be one of the crucial factors controlling the isomerization pathway of PYP. QM/MM simulations carried on WT36 show that the presence of Arg52 markedly alters the topology of the potential energy surfaces of the chromophore at the perp geometry (90° torsion of the ethylenic bond) as compared to vacuum. The presence of this positive charge just above the negatively charged phenol ring of the chromophore would induce a drastic decrease of the S1-S0 energy gap at the perp configuration of the chromophore, thereby allowing the formation of the cis isomer. A theoretical model involving a single point charge near the phenolate group of an anionic analog of the PYP chromophore, showed that the charge drives the isomerization about the ethylenic bond.68 Other calculations carried on WT indeed indicated that presence of Arg52, placed sideways with respect to the chromophore, would only induce a moderate stabilization of the S1 state at the perp geometry.45 In a preceding letter,11 we excluded a crucial role of the positive charge borne by Arg52 by experimentally comparing the transient absorption decays of R52Q and WT. We showed that deleting the positive charge in R52Q does not prevent the formation of spectral analogs of the cis intermediates I0 and I1 (see also the Supporting Information, section 3). In a recent theoretical work on R52Q,46 Groenhof et al. claimed that the first photoproduct of this mutant is not a cis ground-state as in WT but a carbonyl-flipped trans ground state, formed with a yield of 20%, through the rotation of the phenolate. This transient would be the first ground-state intermediate triggering the photocycle of this mutant. The remaining 80% of the excited population is proposed to return to the initial ground-state through a conical intersection linking the S1 and S0 surfaces.46 Interestingly, the above calculated yield of formation of the carbonyl-flipped trans ground-state of R52Q closely matches the experimental yield of formation of I1 (pR) reported by Takeshita et al.67 for R52Q as well as our own I0/I1 average yield for this same mutant (see 〈φI1〉 in Table 2). The comparison with our work must however be handled with care because the calculations of Groenhof et al.46 were based on a single configuration of the protein while our analysis concludes to the existence of three configurations having very different excitedstate properties, and I0/I1 yield, in agreement with the previous report of Larsen et al. on WT.5 We retain the fact that the absorption spectra of I0 and I1 we found for WT, R52Q, as well as T50V and E46Q, are strikingly similar, which suggests a similar nature of these intermediates in all cases. On the other hand, our analysis shows that the fraction of short-lived reactive excited state (x1, Table 1) is similarly reduced for R52Q and T50V (as compared to WT), which may be taken as an indication that the positive charge of Arg52 has a moderate impact on the excited-state deactivation. 4.3. Effect of Mutation on the Formation of the Relaxed cis-Isomer I1. Femtosecond visible-pump-mid-infrared probe measurements showed that, upon excitation, a charge translocation occurs in the chromophore, from the phenolate group to the ethylenic bond, leading to a weakening of the HB with Glu46 and Tyr42. The HB network around the phenolate is subsequently restored during the formation of the ground-state I0, while the HB involving the cystein group gets cleaved.7,10,24 The rate of formation of I1 from the cis isomer I0, corresponding to arelaxationofboththechromophoreandtheproteinenvironment,7,10,20,21 was also found to be sensitive to mutation.1,2,41 The reaction from I0 to I1 was shown to involve a strengthening of the HB

Transient Absorption Spectroscopy of Two Point Mutants linking the chromophore with Glu46, likely because of the shortening of the distance between the phenolate group and Glu46.7 In the present measurements the formation of I1 from I0 is found to occur in the subnanosecond regime. The corresponding time constant of 370 ps (kI0-1, see Table 1) found for WT is much shorter than the values found in the literature, which spread between 0.8 and 3 ns.1,2,5,7 The discrepancy might be related to the existence of intermediate I0‡ sitting between I0 and I1, proposed by the group of Cusanovich,1,2 and that we did not detect. According to these authors I0‡ rises in 220 ps and decays in 3 ns. If I0‡ actually exists, our I0 f I1 process could be viewed as a mean approximation of the successive I0 f I0‡ and I0‡ f I1 steps. In contrast, the characteristic time of formation of I1 in E46Q is the same as the previously reported ones, lying between 700 and 800 ps.24,41 As a general trend, it appears here that the rate of formation of I1 from I0 is slowed down upon mutation in the three cases we studied. 4.4. Nature of the Transient State X in the Competitive Path. Given that our kinetic model is close to the one previously developed by the group of van Grondelle for WT,5,43 it is interesting to compare our transient X (Scheme 2) to its “GSI” counterpart. The formation of GSI occurs in competition with that of I0 and was described as failed attempts of the chromophore at initiating the photocycle.5 On the basis of recent target analysis of visible-pump-mid-infrared probe measurements with a kinetic model including the formation of GSI, it was proposed that this transient could be a distorted groundstate with a conformation close to the cis, where the HB between the chromophore carbonyl group and Cys69 would be maintained (the formation of I0 requires the breaking of this HB).24 On the other hand, it is worth recalling that WT transient spectra at short times bear strong similarities with that of the isolated chromophore in solution, as modeled by the deprotonated thioester derivative of 4-hydroxycinnamic acid.25-27 Although this analog does not lead to the formation of a stable cis isomer in water,25-27 its deactivation is known to involve the formation of an intermediate absorbing around 450 nm (between the bleaching and the SE bands) displaying spectral similitude with the transient X of WT.8,25,28-33 The nature of this transient has not been fully clarified yet but its formation was however shown to be directly related to the electron donor-acceptor character of the chromophore.27,32-34 Several proposals have been made concerning its ground-state or excited-state nature and it was discussed if it is a TICT-like state or if it involves the torsion of the ethylenic bond.27,32-34 Several recent theoretical studies concluded that the excitedstate deactivation of deprotonated PYP chromophore analogues, in vacuum, would involve the rotation of the phenolate group as a competitive pathway to the torsion around the ethylenic bond.36,44,45,68 The attribution of X to a phenolate-twisted S1 intermediate however faces several difficulties for WT or its mutants. In the first place, the occurrence of a “phenolate” route within WT is notobservedinvisible-pump-mid-infraredprobemeasurements.7,10,24 On the other hand, the spectrum of such a twisted transient is expected to differ considerably from that of the initial trans ground state, because of the π decoupling of the phenolate group from the rest of the chromophore. This is inconsistent with the spectrum of X, which strikingly resembles that of the initial trans ground state, with a red shift of about 10 nm (Figure 4). The similarity of the electronic spectra of X and the initial ground state, both in shape and in intensity, suggests that X would rather be a vibrationally hot ground state, with a geometry close to trans, situated on the way back to the initial trans ground

J. Phys. Chem. C, Vol. 113, No. 27, 2009 11611 state. Our kinetic analysis indicates that its formation involves rate constants in the subpicosecond regime (ki(1 - φi), see Table 1). It is known from the photoinduced processes in stilbene or rhodopsins that cis to trans isomerization may indeed proceed very quickly, in a few hundred femtoseconds, and generate hot ground-state photoproducts.69-71 As far as WT is concerned, the formation of a vibrationally hot trans ground-state was in fact previously proposed by Heyne et al.10 in order to interpret their transient IR spectra of the protein. This hot ground-state may be recognized in the QM/MM simulations carried out by Groenhof et al. on WT.36 After passing through a conical intersection at a twisted geometry the system may end up in the trans conformation, with excess vibrational energy and probably slightly distorted geometry. The decay of X in about 3-6 ps might thus be explained by a combination of conformational relaxation and cooling of the chromophore in the ground state. As matter of fact the relaxation of X is seen to be quite sensitive to the immediate surroundings of the chromophore since kX is reduced by a factor 2 in R52Q and T50V, in which the PNS is loosened. 5. Conclusion In the present work, we address the active role of the protein environment on the photoisomerization process of PYP chromophore by studying two point mutants, T50V and E46Q, by ultrafast transient absorption spectroscopy. In these mutants, alterations of the hydrogen-bond network of the protein active site near the chromophore are achieved. Transient spectroscopy of these mutants suggests that the earliest steps of the photocycle remain similar to those of WT and of its R52Q mutant we previously reported.11 Target analysis with a kinetic model inspired from the one developed for WT by the group of van Grondelle5,24,43 was found to satisfactorily describe the differential absorption spectra in all cases. This model involves the deactivation of a set of three independent excited states (heterogeneous distribution of protein conformation) along two competitive relaxation pathways. One pathway leads to the known photocycle with the sequential formation of the I0 and I1 cis intermediates, whereas the second pathway produces a transient state X returning back to the initial trans ground-state in 3-6 ps. The absorption spectrum of this short-lived transient is found to be similar in shape and absorption coefficient to the initial trans ground state, with an additional 10-nm red shift. This similarity leads us to propose its attribution to a slightly distorted vibrationally hot trans ground state. The most prominent effects of mutation on the PYP photocycle are observed for T50V and R52Q, for which a significantly slowing down of the excited-state deactivation is observed. On the contrary, E46Q exhibits almost the same excited-state dynamics as WT, in excellent agreement with previous timeresolved fluorescence measurements.37 Target analysis shows that mutation effects mainly stem from the distribution of initial excited states. In WT and E46Q half of the exited-state population exhibits a subpicosecond decay with high isomerization yield. In T50V and R52Q most of the excited-state population is localized in less reactive excited states, exhibiting slow decay and a low isomerization yield. We attribute these effects to a loss of the rigidity of the hydrogen bond network in the close surrounding of the chromophore, induced by the mutation of the residues T50 and R52. The slight decrease of the isomerization yield of R52Q, as compared to T50V, is thought to arise from the deletion of the positively charged Arg52 residue near the chromophore, in addition to the alteration of the hydrogen bond network of the active site.

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Acknowledgment. H.C. thanks ENS for an invited scientist position at the Chemistry Department of Ecole Normale Supe´rieure in Paris and ILT for financial support. P.C.B., P.P., and M.M.M. thank the French ANR for financial support. P.C.B. also thanks Irene Burghardt for fruitful discussions on the theoretical papers cited in this manuscript. Supporting Information Available: Technical details of our data analysis and additional fitting results. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Ujj, L.; Devanathan, S.; Meyer, T. E.; Cusanovich, M. A.; Tollin, G.; Atkinson, G. H. Biophys. J. 1998, 75, 406. (2) Devanathan, S.; Pacheco, A.; Ujj, L.; Cusanovich, M. A.; Tollin, G.; Lin, S.; Woodbury, N. Biophys. J. 1999, 77, 1017. (3) Imamoto, Y.; Kataoka, M.; Tokunaga, F.; Asahi, T.; Masuhara, H. Biochemistry 2001, 40, 6047. (4) Gensch, T.; Gradinaru, C. C.; van Stokkum, I. H. M.; Hendriks, J.; Hellingwerf, K. J.; van Grondelle, R. Chem. Phys. Lett. 2002, 356, 347. (5) Larsen, D. S.; van Stokkum, I. H. M.; Vengris, M.; van der Horst, M. A.; de Weerd, F. L.; Hellingwerf, K. J.; van Grondelle, R. Biophys. J. 2004, 87, 1858. (6) Kort, R.; Hellingwerf, K. J.; Ravelli, R. B. G. J. Biol. Chem. 2004, 279, 26417. (7) Groot, M.-L.; van Wilderen, L. J. G. W.; Larsen, D. S.; van der Horst, M. A.; van Stokkum, I. H. M.; Hellingwerf, K. J.; van Grondelle, R. Biochemistry 2003, 42, 10054. (8) Changenet-Barret, P.; Espagne, A.; Plaza, P.; Hellingwerf, K. J.; Martin, M. M. New J. Chem. 2005, 4, 527. (9) Larsen, D. S.; van Grondelle, R. ChemPhysChem 2005, 6, 828. (10) Heyne, K.; Mohammed, O. F.; Usman, A.; Dreyer, J.; Nibbering, E. T. J.; Cusanovich, M. A. J. Am. Chem. Soc. 2005, 127, 18100. (11) Changenet-Barret, P.; Plaza, P.; Martin, M. M.; Chosrowjan, H.; Taniguchi, S.; Mataga, N.; Imamoto, Y.; Kataoka, M. Chem. Phys. Lett. 2007, 434, 320. (12) Sprenger, W. W.; Hoff, W. D.; Armitage, J. P.; Hellingwerf, K. J. J. Bacteriol. 1993, 175, 3096. (13) Cusanovich, M. A.; Meyer, T. E. Biochem. 2003, 42, 4759. (14) Hellingwerf, K. J.; Hendriks, J.; Gensch, T. J. Phys. Chem. A 2003, 107, 1082. (15) Hendriks, J.; Hellingwerf, K. J. Photoactive Yellow Protein, the prototype xanthopsin In CRC handbook of organic photochemistry and photobiology, 2nd ed.; Horspool, W., Lenci, F. , Eds.; CRC Press: Boca Raton, FL, 2003; p 123. (16) Chosrowjan, H.; Mataga, N.; Shibata, Y.; Imamoto, Y.; Tokunaga, F. J. Phys. Chem. B 1998, 102, 7695. (17) Mataga, N.; Chosrowjan, H.; Taniguchi, S.; Hamada, N.; Tokunaga, F.; Imamoto, Y.; Kataoka, M. Phys. Chem. Chem. Phys. 2003, 5, 2454. (18) Premvardhan, L. L.; van der Horst, M. A.; Hellingwerf, K. J.; van Grondelle, R. Biophys. J. 2003, 848, 3226. (19) Genick, U. K.; Soltis, S. M.; Kuhn, P.; Canestrelli, I. L.; Getzoff, E. D. Nature (London) 1998, 392, 206. (20) Ren, Z.; Perman, B.; Srajer, V.; Teng, T.-Y.; Pradervand, C.; Bourgeois, D.; Schotte, F.; Ursby, T.; Kort, R.; Wulff, M.; Moffat, K. Biochemistry 2001, 40, 13788. (21) Ihee, H.; Rajagopal, S.; Srajer, V.; Pahl, R.; Anderson, S.; Schmidt, M.; Schotte, F.; Anfinrud, P. A.; Wulff, M.; Moffat, K. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 7145. (22) van Brederode, M. E.; Gensh, T.; Hoff, W. D.; Hellingwerf, K. J.; Braslavsky, S. E. Biophys. J. 1995, 68, 1101. (23) Gensch, T.; Hellingwerf, K. J.; Braslavsky, S. E.; Schaffner, K. J. Phys. Chem. A 1998, 102, 5398. (24) van Wilderen, L. J. G. W.; van der Horst, M. A.; van Stokkum, I. H. M.; Hellingwerf, K. J.; van Grondelle, R.; Groot, M.-L. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 15050. (25) Changenet-Barret, P.; Espagne, A.; Katsonis, N.; Charier, S.; Baudin, J.-B.; Jullien, L.; Plaza, P.; Martin, M. M. Chem. Phys. Lett. 2002, 365, 285. (26) Usman, A.; Mohammed, O. F.; Heyne, K.; Dreyer, J.; Nibbering, E. T. J. Chem. Phys. Lett. 2005, 401, 157. (27) Espagne, A.; Paik, D. H.; Changenet-Barret, P.; Plaza, P.; Martin, M. M.; Zewail, A. H. Photochem. Photobiol. Sci. 2007, 6, 780. (28) Larsen, D. S.; Vengris, M.; Van Stokkum, I. H. M.; van der Horst, M. A.; Cordfunke, R. A.; Hellingwerf, K. J.; van Grondelle, R. Chem. Phys. Lett. 2003, 369, 563. (29) Changenet-Barret, P.; Espagne, A.; Charier, S.; Baudin, J.-B.; Jullien, L.; Plaza, P.; Hellingwerf, K. J.; Martin, M. M. Photochem. Photobiol. Sci. 2004, 3, 823.

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