Signaling-State Formation Mechanism of a BLUF Protein PapB from

Article pubs.acs.org/JPCB

Signaling-State Formation Mechanism of a BLUF Protein PapB from the Purple Bacterium Rhodopseudomonas palustris Studied by Femtosecond Time-Resolved Absorption Spectroscopy Tomotsumi Fujisawa,† Satoshi Takeuchi,†,‡ Shinji Masuda,§ and Tahei Tahara*,†,‡ †

Molecular Spectroscopy Laboratory, RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan Ultrafast Spectroscopy Research Team, RIKEN Center for Advanced Photonics (RAP), RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan § Center for Biological Resources and Informatics, and Earth-Life Science Institute, Tokyo Institute of Technology, Yokohama 226-8501, Japan ‡

S Supporting Information *

ABSTRACT: We studied the signaling-state formation of a BLUF (blue light using FAD) protein, PapB, from the purple bacterium Rhodopseudomonas palustris, using femtosecond timeresolved absorption spectroscopy. Upon photoexcitation of the dark state, FADH• (neutral flavin semiquinone FADH radical) was observed as the intermediate before the formation of the signaling state. The kinetic analysis based on singular value decomposition showed that FADH• mediates the signalingstate formation, showing that PapB is the second example of FADH•-mediated formation of the signaling state after Slr1694 (M. Gauden et al. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 10895− 10900). The mechanism of the signaling-state formation is discussed on the basis of the comparison between femtosecond time-resolved absorption spectra of the dark state and those obtained by exciting the signaling state. FADH• was observed also with excitation of the signaling state, and surprisingly, the kinetics of FADH• was indistinguishable from the case of exciting the dark state. This result suggests that the hydrogen bond environment in the signaling state is realized before the formation of FADH• in the photocycle of PapB.

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INTRODUCTION A bacterial photoresponse begins with absorption of light by a pigment (chromophore) inside a photoreceptor protein.1−3 The chromophore then undergoes a photochemical reaction (i.e., photocycle), which accompanies structural changes of the protein and generates the signaling state to transmit the stimulus of light through allosteric interactions.4 Currently identified bacterial photoreceptors contain chromophores such as retinal, tetrapyrroles, p-coumaric acid, and flavins,5,6 which cover the photosensing triggered by light from blue to far-red. Among them, the flavin-binding photoreceptors, known as LOVs (light−oxygen−voltage domains), cryptochromes, and BLUFs (sensors of blue light using FAD), make up major families of blue light receptors. They have flavin pigments (FAD, flavin adenine dinucleotide; or FMN, flavin mononucleotide) in common but exhibit distinct photocycles which involve different intermolecular chemical processes between the flavins and amino acid residues.4,5,7 BLUFs are one class of blue-light receptors that use FAD and work for the light detection to eventually regulate phototaxis,8,9 photosynthetic gene expression,10 and biofilm formation11,12 of bacteria. The BLUF protein before light absorption is referred © 2014 American Chemical Society

to as the dark state, and photoexcitation of the dark state triggers a photocycle to yield the signaling state which typically shows a ∼10 nm red-shift of the absorption band.10 An early finding was that the red-shift of the absorption is lost by mutating either Gln or Tyr that is highly conserved in all BLUF proteins.13−18 X-ray crystallography revealed that these Gln and Tyr are located near FAD and are capable of forming hydrogen bonds with the chromophore.15,19,20 The present consensus is that the red-shift of the absorption of the signaling state is caused by the rearrangement of the hydrogen bond network that involves Gln, Tyr, and FAD.4,7,21 The signaling state of BLUFs is unique in a sense that the chromophore undergoes only small structural change but can trigger a significant structural change of the protein for signal transduction.22,23 Time-resolved UV−vis and IR spectroscopies have been applied to study the photocycle of BLUF proteins,24−31 and revealed that the signaling-state formation process has a variety. AppA BLUF-domain from the purple bacterium Rhodobacter Received: July 29, 2014 Revised: November 6, 2014 Published: November 19, 2014 14761

dx.doi.org/10.1021/jp5076252 | J. Phys. Chem. B 2014, 118, 14761−14773

The Journal of Physical Chemistry B

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sphaeroides28,29 was first studied by time-resolved UV−vis absorption spectroscopy, and it was shown that the signaling state is formed directly from the first excited singlet state (S1 state) after photoexcitation and that it returns to the dark state in 30 min.28,29 The observed short S1 lifetime (∼500 ps), as well as the low fluorescence quantum yield (∼0.02), was attributed to electron transfer (ET) between FAD and the Tyr, although no reaction intermediate indicating ET was observed in the signaling-state formation process.28,29,32,33 The absence of the transient signal due to the FAD anion radical (FAD•−), which is the intermediate expected with ET, has been rationalized by fast back-ET,29,34 although there is still a controversy about the involvement of ET.35 The formation rate of the signaling state of AppA is insensitive to the H/D isotope exchange, suggesting that proton transfer is not involved.33 Recent studies of other BLUF sensors, BlrB (from Rhodobacter sphaeroides) and BlsA (from Acinetobacter baumannii), have also shown that the signaling states are directly formed from the S1 state.25,31,36 On the other hand, the time-resolved absorption study of Slr1694 (alternative name: SyPixD) from cyanobacterium Synechocystis showed that the neutral flavin semiquinone FADH radical (FADH•) is generated as an intermediate during the signalingstate formation.27 This implies that not merely ET but protoncoupled ET takes place in Slr1694.27,37,38 The photocycle of Slr1694 differs from that of AppA, in terms of a clear appearance of the intermediate that indicates proton transfer in the signaling-state formation. Except for Slr1694, the FADH•mediated signaling-state formation has not been reported so far. With the above-described variety of primary photochemical processes of BLUFs, the mechanism of signaling-state formation has been discussed, and several models were proposed.4 The first model was proposed on the basis of the studies of AppA BLUF domain.18,29 In this model, ET occurs between the Tyr and FAD with photoexciation, which induces rotation of Gln to generate the signaling state.29 Another model incorporating proton transfer was proposed for Slr1694, for which FADH• was observed as an intermediate.27 In this second model, photoexcitation induces proton migration from Tyr to FAD through Gln and generates a pair of FADH• and Tyr•, which bring about rotation of Gln as well as rearrangement of the hydrogen bond in the FAD-binding site.39−44 These two models were proposed with experimental studies, and both propose the rotation of Gln without its chemical change. On the other hand, theoretical studies proposed a different scheme, in which proton transfer occurs from Tyr to FAD through keto−enol tautomerization of the Gln. Thus, this model implies that the signaling state contains the Gln enol tautomer.39−43,45 Unfortunately, no experimental evidence that supports either rotation or tautomerization of Gln has been obtained, and the molecular mechanism of the signaling-state formation of BLUFs is still unclear.19,46−48 In this paper, we report a femtosecond time-resolved absorption study on the signaling-state formation of a BLUF sensor PapB from the purple bacterium Rhodopseudomonas palustris. The biological function of PapB was characterized previously, and it was shown that PapB works as a light-sensing module to regulate the phosphodiesterase activity of the partner protein PapA:12,49 PapB under blue light enhances the enzyme activity of PapA to promote hydrolysis of cyclicdimeric-GMP that is the second messenger for the biofilm formation.12 Figure 1 illustrates the active site of PapB.50 The present time-resolved absorption study shows that PapB undergoes FADH•-mediated signaling-state formation

Figure 1. Illustration of the PapB active site. The protein structure is modeled in SwissModel server50 using the crystal structure of BlrP1 from Klebsiella pneumoniae (PDB entry: 3GFZ) as the template. The chromophore, FAD, is shown with the atom numbering of the isoalloxazine ring.

as previously reported for Slr1964.27 Furthermore, we report femtosecond time-resolved absorption data of the signaling state that is produced by photoexcitation, which show that FADH• is generated also with excitation of the signaling state. Surprisingly, the FADH• kinetics observed with the dark-state excitation and signaling-state excitation are the same. This implies that the same hydrogen bond structure around the FAD-binding site is realized before the formation of FADH•. The present study indicates that the hydrogen bonds around FAD are rearranged before the proton transfer to generate FADH• in the photocycle of PapB.

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EXPERIMENTAL SECTION Preparation of R. palustris PapB. The nucleotide sequences of R. palustris papB and the corresponding amino acid sequence for PapB (RPA0522) were retrieved from the CyanoBase database at the KAZUSA DNA Research Institute Web site (http://genome.kazusa.or.jp/cyanobase/). Expression plasmid for nontagged PapB was constructed by amplifying the PapB code in PCR using the genomic DNA as the template and the primer for papB: 5′-GGGGGGGCATATGCCGAGCGAGCTGTATCG-3′ (NdeI) and 5′-GGGAATTCTTAGGCGGCGCGGGCTTCTTC-3′ (EcoRI). The italicized sequences lead to restriction sites in the PCR products. The amplified DNA fragment for papB was digested with NdeI and EcoRI, and the product was cloned into a pTYB12 vector (New England Biolabs). The inserted DNA sequence was confirmed by sequencing, and the plasmid was transferred into E. coli BL21(DE3) cells. The protein was expressed after induction with isopropyl 1-thio-D-galactopyranoside (final concentration of 1 mM) at 16 °C. PapB was purified using chitin beads (New England Biolabs) according to the manufacturer’s instructions. Since we found that purified PapB binds different flavin molecules including riboflavin, flavin mononucleotide, and FAD, cell-free extract was incubated with a large molar excess of FAD (Sigma) before purification, which results in replacement of the flavins to FAD.12,51 Purified PapB was dialyzed against 10 mM Tris-HCl (pH 8.0) and 5 mM NaCl. The absorbance of the 14762



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flavin chromophore at 450 nm and a molar extinction coefficient of 11 300 M−1 cm−1 were used to determine the concentration. Femtosecond Time-Resolved Absorption Measurements. The details of the femtosecond time-resolved absorption setup have been reported previously.52 Briefly, the light source was a Ti:sapphire oscillator/regenerative amplifier system (MIRA-900F/Legend Elite, Coherent) that generates 1.1 mJ pulses at 800 nm with a duration of ∼80 fs at 1 kHz.52 About 80% of the 800 nm output was used to pump an optical parametric amplifier (TOPAS, Light Conversion) to generate a 450 nm pump pulse. This pulse was attenuated down to 240 nJ and focused onto the sample for photoexcitation (the beam diameter was ∼150 μm at the sample position). About 1.5% of the residual output was focused on a slowly translating CaF2 crystal to generate white-light continuum (350−750 nm). After dividing the white-light continuum into the probe and reference pulses, the probe pulse was introduced to the excited volume of the sample solution. Then, the probe pulse after passing through the sample and the reference pulse were simultaneously dispersed in a 50 cm polychromator (Chromex, 500 is/sm) and detected by a CCD using different vertical positions. The probe and reference spectra of every five laser shots were read out at 100 Hz from the CCD that was synchronized with the laser system. The reference spectrum was used for the correction of spectral fluctuations of the white-light continuum. The relative polarization of the pump and probe pulses was set at the magic angle (54.7°). To calibrate the chirp of the white-light continuum used for the probe, we recorded the wavelength dependence of OKE (Optical-KerrEffect) signals of the buffer solution. The time-zero dispersion curve was obtained, and the time origin of the time-resolved absorption data was corrected accordingly. With this correction, we achieved an effective time resolution of ∼100 fs. The sample solution of (∼0.3 mM, ∼500 μL) in a Tris buffer (10 mM Tris, 5 mM NaCl, pH 8.0) was circulated through a square capillary cell (2 mm × 2 mm; VitroCom) during the measurements. The sample flow rate was carefully adjusted because the flow rates lower than 30 μL/s substantially accumulate the signaling state (lifetime: ∼6 s) in the excited volume. In other words, we can selectively photoexcite the dark state or the signaling state by controlling the flow rate. Therefore, we set the flow rate at ∼55 μL/s to avoid the accumulation of the signaling state when we measured time-resolved absorption spectra of the dark state. For measuring timeresolved absorption spectra of the signaling state, we set the flow rate at ∼5 μL/s and accumulated the signaling state in the excitation volume. The detailed descriptions about the flow-rate dependence of the signaling state population are given in Supporting Information. For the D2O experiments, the sample solution was prepared by the repeated buffer exchange using centrifugal filter (Amicon Ultra; Merck Millipore). With this procedure, the buffer was replaced by the Tris buffer (10 mM Tris, 5 mM NaCl, pD 8.0) in deuterium oxide (99.9% deuterium; Sigma-Aldrich). The sample solution was left to stand overnight to complete the H/D exchange in the protein before the measurement.

Figure 2. (A) S1 ← S0 absorption of the dark state of PapB (pH = 8.0, in H2O). The spectrum of the 450 nm excitation pulse used for the time-resolved absorption measurement is also shown. (B) Femtosecond time-resolved absorption spectra of PapB (pH = 8.0, in H2O): upper, 0.7−97 ps; lower, 97−473 ps.

spectrum immediately after photoexcitation (the red spectrum in Figure 2B: at 0.7 ps) shows positive absorption signals around 400, 520, and wavelengths longer than 650 nm, which are attributed to the broad Sn ← S1 transition(s). Two negative bands are also observed around ∼450 and ∼550 nm, corresponding to the dark-state bleach and the stimulated emission, respectively. The S1-state absorption gradually decays on a time scale of tens of picoseconds, as most clearly seen as the decreasing absorption in the 650−750 nm region. As the S1 state decays, the time-resolved absorption spectrum develops into a different spectral shape, exhibiting an isosbestic point at 640 nm. This indicates an efficient formation of a reaction intermediate in the formation process of the signaling state. The reaction intermediate shows a broad absorption in the 500−650 nm region, as most clearly seen in the time-resolved spectrum at 97 ps (the blue spectrum in Figure 2B,C). After the complete decay of both the S1 state and the reaction intermediate at 473 ps (the green spectrum in Figure 2B), the time-resolved absorption spectrum exhibits a sharp positive feature around ∼500 nm accompanied by a negative feature due to the dark-state bleach on the shorter wavelength side. This dispersive spectral shape corresponds to a slight red-shift of the S1 ← S0 absorption of the chromophore, which is the typical spectral signature of the signaling-state formation.15,28,53,54

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RESULTS AND DISCUSSION Time-Resolved Absorption Spectra of PapB Dark State. Figure 2 shows femtosecond time-resolved absorption spectra of the dark state of PapB in H2O buffer at pH 8.0, as well as its steady-state absorption. The time-resolved absorption spectra were measured with excitation of the S1 ← S0 transition using the pump pulse at 450 nm. The time-resolved 14763



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represent them by linear combinations of the spectral vectors ui=1,2,3 (vide infra). Then, we can decompose the time-resolved absorption spectra into the spectrum of each chemical species to obtain the associated kinetics. Hereafter, we use the terms of the “S1-state spectrum”, “signaling-state spectrum”, and “reactionintermediate spectrum” for describing the corresponding speciesspecific spectra obtained with this procedure. Note that these spectra are actually the difference between the spectra of the three species and the spectrum of the dark state. The S1-state spectrum and the signaling-state spectrum were determined by choosing proper time points and were constructed using SU3V in eq 2, as follows. Given that the initial spectral change in the transient absorption occurs over tens of picoseconds, the time-resolved spectrum at ∼1 ps just after photoexcitation can be considered the S1-state spectrum

The resultant signaling state does not decay in the delay time range of the present experiment (∼1 ns). The quantum yield of the signaling state was measured separately with a relative actinometric method using ruthenium trisbipyridine, [Ru(bpy)32+], as a reference. We obtained the quantum yield ΦPapB = 52% using the following equation:28 ΦPapB =

430nm ·ΔA 498nm ΔεRu(bpy) 2+ PapB 3

498nm 430nm ΔεPapB ·ΔARu(bpy) 2+

·ΦRu(bpy)32 + =

7500 × 0.0037 × 0.95 4000 × 0.0127

3

= 0.518

where ΦRu(bpy)2+3 is the quantum yield of the triplet-state formation of [Ru(bpy)32+], and Δε (M−1 cm−1) and ΔA represent the change of the molar extinction coefficient and absorbance at the wavelength indicated in the superscript, respectively, after photoirradiation. Spectral Decomposition of Time-Resolved Absorption Data. To quantitatively analyze the time-resolved absorption spectra observed, we performed spectral decomposition of the time-resolved absorption data using SVD (singular value decomposition) and obtained the temporal-profiles of the S1 state, reaction intermediate, and signaling state. In SVD, the time-resolved absorption matrix S is decomposed into the singular-value-weighted orthonormal spectral matrix U and orthonormal kinetic matrix V:55−57 ⎛ w1 ⎞ ⎜ ⎟ w2 ⎟ × VT S=U×⎜ w3 ⎟ ⎜ ⎜ ⎟ ⎝ ⋱⎠ wi : i th singular value, w1 ≥ w2 ≥ w3 ≥ ... S = ( st1 st2 st3 ... stN ), sti : time‐resolved absorption spectrum at ti U = ( u1 u 2 u3 ... uM ), u i : i th orthonormal spectral vector V = ( v1 v2 v3 ... vN ), vi: i th orthonormal kinetic vector

(1)

Here, a time-resolved absorption spectrum sti is expressed by a linear combination of the spectral vector ui, and the timedependent contribution of each ui is represented by the kinetic vector vi. The amplitude of ith singular value, wi, measures the weight of ith spectral and kinetic vectors. In the PapB case, the time-resolved absorption spectra SPapB are well-reproduced by three primary spectral and kinetic vectors (i = 1, 2, 3), and the spectral amplitude of i ≥ 4 is as low as 0.2 mOD, which is comparable to the experimental error. Thus, SPapB is safely represented by SPapB ≅ SU3V = ( w1u1 w2 u 2 w3 u3 ) × ( v1 v2 v3 )T (w1 , w2 , w3) = (0.632, 0.331, 0.130)

(2)

Equation 2 means that the time-resolved absorption spectrum of PapB at any delay time is expressed by the combination of three independent spectral components. However, ui=1,2,3 and vi=1,2,3 do not directly stand for the spectra and kinetics of real chemical species because they are forced to be orthonormal in the mathematical treatment. Thus, we need to determine the spectra of the S1 state, reaction intermediate, and signaling state, and

Figure 3. (A) S1-state spectrum (red) and signaling-state spectrum (green) reconstructed from three primary spectral vectors obtained by the SVD analysis (see text). The spectra of the S1 state and the signaling state are determined so as to reproduce the time-resolved absorption data at 1 and 963 ps, respectively. (B) Time-resolved absorption spectra obtained after the subtraction of the S1-state contribution. FADH• spectrum (blue) is determined so as to reproduce the spectrum at 17 ps. 14764



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which is hardly contaminated by other chemical species. Thus, we use the time point of 1 ps in SU3V as the S1-state spectrum mS1 (Figure 3A, red): S1

m =

U3V s1ps

m St 1‐ subtraction = stPapB − ct · mS1,

ct =

ΔA 700 − 735nm, t ΔA 700 − 735nm,1ps

stPapB : time‐resolved absorption spectrum at t

= ( w1u1 w2 u 2 w3 u3 ) × ( v1,1ps v2,1ps v3,1ps )

T

ΔA 700 − 735nm, t : mean absorbance change over 700−735 nm at t (5)

vi ,1ps: vi component at 1 ps

Figure 3B displays the delay-time dependence of mSt 1‑subtraction. After eliminating the S1-state contribution, the spectrum consists of the dark-state bleach, the absorption of the reaction intermediate, and the absorption of the signaling state. In the first ∼20 ps, the spectral shape over 500−650 nm closely resembles that of the neutral flavin semiquinone FADH radical (FADH•),60 indicating that the reaction intermediate is FADH•. The spectral component of FAD•−,61 which is expected in the case of ET, is not observed during the growth of FADH•. After 40 ps, the signaling-state formation is visible as the growing absorption at ∼500 nm. Meanwhile, FADH• decays on a time scale of hundreds of picoseconds. Because the kinetics of FADH• is different from that of the signaling state, the FADH• spectrum can be readily extracted from the spectra shown in Figure 3B. In fact, because the signaling-state contribution is negligible before ∼20 ps, we consider the spectrum at a time point of 17 ps in Figure 3B as • the FADH• spectrum mFADH :

(3)

The S1-state spectrum exhibits positive Sn ← S1 absorption signals around 400, 520, and over 650 nm with two negative bands around 450 nm (dark-state bleach) and 550 nm (stimulated emission). Similarly, the signaling-state spectrum mSignaling (Figure 3A, green) can be constructed by selecting a long time delay such as 963 ps when the S1 state and reaction intermediate completely vanish to leave only the signaling state: U3V m Signaling = s 963ps

= ( w1u1 w2 u 2 w3 u3 ) × ( v1,963ps v2,963ps v3,963ps )T vi ,963ps: vi component at 963 ps

(4)

The spectral component of the reaction intermediate significantly overlaps with the spectra of the S1 state and signaling state in the time-resolved spectra shown in Figure 2. Therefore, it is necessary to eliminate these other contributions from the raw data to extract the spectrum of the reaction intermediate. It was reported that the amplitude ΔA700−735nm is a good measure of the S1-state contribution when the triplet state is absent in the flavin-containing photoreceptors.27,28,58,59 Thus, we used the mean amplitude of the time-resolved absorption in the 700−735 nm region (ΔA700−735nm) to estimate the S1-state contribution and subtract it from the raw data. The transient spectrum after the subtraction of the S1-state contribution is mSt 1‑subtraction, which is expressed as

S1− subtraction m17ps ≅ m FADH •, U3V m FADH • =s17ps − c17ps·mS1

= ( w1u1 w2 u 2 w3 u3 ) × ( v1,17ps − c17ps·v1,1ps v2,17ps − c17ps·v2,1ps v3,17ps − c17ps·v3,1ps )T

(6)

Note again that, like the S1-state spectrum and the signaling-• state spectrum, the obtained FADH • spectrum m FADH (Figure 3B, blue) is the difference spectrum between the spectra of FADH• and the dark state. Consequently, by combining eqs 2−6, we can decompose the time-resolved absorption data SPapB into the species-specific spectra M and obtain the associated kinetics K, which are given by

SPapB ≅ SU3V = M × KT M = ( mS1 m Signaling m FADH • ) = ( w1u1 w2 u 2 w3 u3 ) × P, m X : molecular spectrum of X KT = ( kS1 kSignaling ⎛ v1,1ps v1,963ps ⎜ P = ⎜ v2,1ps v2,963ps ⎜v ⎝ 3,1ps v3,963ps

−1 T T X X k FADH • ) = P × ( v1 v2 v3 ) , k : kinetics associated with m v1,17ps − c17ps·v1,1ps ⎞ ⎟ v2,17ps − c17ps·v2,1ps ⎟ v3,17ps − c17ps·v3,1ps ⎟⎠

(7)

of each species was obtained with SVD in the same way (see Supporting Information). The temporal traces in D2O are also shown in Figure 5. They clearly show the significant H/D isotope effect. Kinetics of the Signaling-State Formation of PapB. The temporal profiles of the S1 state, FADH•, and signaling state shown in Figure 5 reveal the following important features of the signaling-state formation of PapB. First, the decay of the S1 state is not single exponential. It includes fast and slow decay components, suggesting structural inhomogeneity in the FADbinding site. Second, the fast S1 decay proceeds in parallel with the rise of FADH•, and then the signaling state grows as

Figure 4 shows a typical example of the spectral decomposition based on eq 7. The time-resolved absorption spectrum observed at 73 ps (black) contains the contributions from the S1 state, FADH•, and the signaling state which are significantly overlapped. Clearly, the spectrum is well-reproduced by the linear •combination of the three species-specific spectra, mS1, mFADH , and mSignaling. The associated temporal profiles, kS1, •

kFADH , and kSignaling, obtained with this analysis, are shown in Figure 5. Time-resolved absorption spectra of PapB were also measured in a Tris D2O buffer, and the temporal profile 14765



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Figure 4. Typical example of the spectral decomposition of timeresolved absorption spectra. The experimental time-resolved absorption spectrum at 73 ps (black) is reconstructed by the contributions of the S1 state (red), FADH• (blue), and signaling state (green). The reconstructed spectrum is shown in yellow.

FADH• decays. This indicates that the S1 state showing the shorter lifetime is relevant to the FADH• formation and that the signaling state is formed via FADH•. On the basis of these results, the reaction scheme shown in Scheme 1 is derived for Scheme 1 Figure 5. Experimental temporal profiles of the S1 state (red), FADH• (blue), and signaling state (green). The closed and open circles are the data measured in H2O and D2O, respectively. Lines are the best fits based on Scheme 1.

signaling state, while the other (b %) goes back to the original dark state after photoexcitation. Because of the delayed rise of the signaling state, we excluded the possibility that the S1 state directly produces the signaling state. The kinetics of each species based in Scheme 1 is given as follows:

PapB. This scheme assumes two different states in the dark state: one state (a %) produces FADH• and then relaxes to the

[S1] = A × [a exp( −(k1 + kIC)t ) − b exp( −k4t )], [FADH•] = B × [exp(− (k1 + kIC)t ) − exp(− (k 2 + k 3)t )], ⎡ 1 − exp( −(k + k )t ) 1 − exp( −(k 2 + k 3)t ) ⎤ 1 IC ⎥, [Signaling] = C × ⎢ − k1 + kIC k 2 + k3 ⎣ ⎦ where A = [S1]0 , B =

ak1[S1]0 ak1k 2[S1]0 ,C= , and b = 1 − a (k 2 + k 3) − (k1 + kIC) (k 2 + k 3) − (k1 + kIC)

The best fits based on eq 8 are shown and compared with the experimental temporal profiles obtained by SVD in Figure 5. This fitting employs seven parameters of k1 + kIC, k2 + k3, k4, a, A, B, and C. As shown in this figure, the best fits agree very well with the experimental kinetic data. The S1 decay is reproduced by two time constants of 29 ± 4 ps (64%) and 114 ± 18 ps (36%). The faster S1 decay leads to formation of FADH•, and the resultant FADH• decays with a time constant of 119 ± 10 ps to produce the signaling state with the yield of k2/(k2 + k3). The slower S1 decay corresponds to the direct relaxation to the original dark state. Table 1 lists the parameters obtained by the fitting (a and b, relative populations of the two dark states;

(8)

τa = (k1 + kIC)−1 and τb = (k4)−1, lifetimes of the S1 state; τFADH• = (k3)−1, lifetime of FADH•.) The decay of the S1 state and that of FADH• exhibit a significant H/D kinetic isotope effect (KIE). The KIE values are 1.2, 1.4, and 1.7 for τa, τb, and τFADH•, respectively. As described in the Introduction, two different photoreaction processes have been observed in the signaling-state formation of BLUFs.25,27−29 One is the signaling-state formation directly from the S1 state (S1 → signaling), which was observed for AppA, BlrB, and BlsA.25,28,29,31 This type of signaling-state formation proceeds in a H/D isotope independent manner.25,33 The other is the signaling-state formation through FADH• 14766



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The nonreactive decay shows KIE of 1.4, implying that proton transfer is involved. Time-Resolved Absorption Measurement of PapB Signaling State. Although the kinetics of the signaling-state formation from the dark state has been clarified, it is not clear what kind of structural change is induced in the FAD-binding site during the process. To address this issue, we photoexcite the signaling state and measured the time-resolved absorption spectra, thinking that comparison of the time-resolved absorption spectra between the signaling state and the dark state may provide information about the difference in their FAD-binding site. Previously, time-resolved absorption spectra of the signaling state of AppA and Trp91Phe mutant of Slr1694 (Slr1694-W91F) were measured to examine the photoreactions60,62 and the photoreversibility60 of the signaling states. In those cases, the lifetimes of the signaling states are very long (AppA, ∼0.5 h; Slr1694-W91F, ∼4 min), so that the sufficient signaling-state population can be generated by continuous bluelight illumination using LED. For PapB, however, the lifetime of the signaling state is as short as ∼6 s,12 and hence we cannot generate the sufficient population by the same method. Instead, we found that we can convert almost all the dark state of PapB to the signaling state by reducing the sample flow rate in the present experiments: The slowly flowing sample is repeatedly photoexcited by laser pulses while it passes through the excited volume, and the time-resolved absorption measurements can be performed practically for the signaling state accumulated by multiple photoexcitation. Figure 7 shows the time-resolved absorption spectra of PapB measured with the flow rate as slow as ∼5 μL/s. The time-resolved spectrum immediately after photoexcitation

Table 1 PapB dark state Parameters from the Kinetic Analysis a 0.64 (0.49 in D2O) b=1−a 0.36 (0.51 in D2O) S1-State Lifetime 29 ± 4 ps τa = (k1 + kIC)−1 (36 ± 6 ps in D2O) 114 ± 18 ps τb = (k4)−1 (157 ± 14 ps in D2O) FADH• Lifetime 119 ± 10 ps τFADH• = (k2 + k3)−1 (197 ± 15 ps in D2O)

PapB signaling state 0.65 (0.47) 0.35 (0.53) 24 ± 4 ps (28 ± 5 ps) 136 ± 12 ps (146 ± 19 ps) 135 ± 10 ps (181 ± 20 ps)

(i.e., S1 → FADH• → signaling). This shows a significant H/D kinetic isotope effect.27 So far, the second type of the signalingstate formation has been reported only for Slr1694 (from cyanobacteria Synechocystis).27 In the present study, the formation of FADH• was clearly observed, and the kinetic H/D isotope effect on the signaling-state formation was also clarified by the analysis based on SVD. Therefore, it is concluded that PapB belongs to the second type in which proton-coupled electron transfer to FAD generates FADH• as the intermediate in the signaling-state formation. The most probable donor of electron and proton is Tyr (Tyr10 in PapB) that is located near FAD and is conserved in BLUFs.27,42,62 It may be worth mentioning that although it was claimed that the spectral component attributable to FAD•− was recognized as the precursor of FADH• in a previous study of Slr1694,27 the signal assignable to FAD•− was not directly observed in the present timeresolved absorption measurements of PapB (vide infra). Figure 6 illustrates the photocycle of PapB with the kinetics of the signaling-state formation clarified in this study.

Figure 6. Photocycle of PapB. The lifetime of PapB signaling state (∼6 s) is estimated from the half time (∼4 s) reported in ref 12. τa and τb are the lifetimes of the S1 state.

Photoexcitation of the dark state generates the S1 state which exhibits a biexponential decay. The S1 state having a lifetime of 29 ± 4 ps generates FADH• through proton-coupled electron transfer, and the signaling state (lifetime: ∼6 s)12 is then formed from FADH• with back-transfer of the proton and electron. On the other hand, the S1 state having a lifetime of 114 ± 18 ps directly relaxes to the initial dark state without generating any photoproducts. The amplitude of this nonreactive decay is 36% of the total amplitude of the S1 decay (in H2O, Table 1). These two different S1 decays highly likely arise from the structural inhomogeneity in the FAD-binding site.

Figure 7. Femtosecond time-resolved absorption spectra of PapB obtained by excitation of the signaling state (pH = 8.0, in H2O, flow rate ∼5 μL/s): upper, 0.7−78 ps; lower, 78−693 ps. 14767



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(the red spectrum: at 0.7 ps) exhibits spectral features which are characterized by the stimulated emission at ∼550 nm, Sn ← S1 transition around 400, 520, and wavelengths longer than 650 nm, as well as the ground-state bleach around ∼450 nm. Then, the absorption in the 550−650 nm region increases on the time scale of tens of picoseconds as most clearly recognized for the spectrum at 78 ps (the blue spectrum), which is attributable to the formation of FADH•. Until this delay time, the spectral evolution is similar to that observed with the darkstate excitation. However, a marked difference is observed after the decay of the FADH• signal; that is, any signals indicating photoproducts are hardly observed except for small offset-like signals of ∼0.2 mOD (the green spectrum: at 693 ps). This is in a sharp contrast to the time-resolved spectra observed with the dark-state excitation in which the formation of the photoproduct (the signaling state) is clearly observed. This difference reveals that, with reducing flow rate of the sample, the accumulated signaling state is predominantly photoexcited instead of the dark state, and photoexcitation of the signaling state ends up with generation of the same signaling state, leaving no spectral difference after the transients (S1 and FADH•) vanish. It is also noted that the time-resolved spectra immediately after photoexcitaion exhibit a significantly different spectral feature around 500 nm, as compared to those observed with excitation of the dark state (Figure 2). This difference is explainable as the difference in the S0 absorption of the initial state because the S0 absorption of the signaling state is red-shifted by ∼10 nm compared to that of the dark state (vide infra). The time-resolved data of the signaling state taken with the slow flow rate was analyzed with SVD in a way similar to the case of the dark state, and we obtained the three spectra attributable to the S1 state, FADH•, and the long-lived component as shown in Figure 8. As shown for a typical case at

Figure 9. Comparison of the species-specific spectra obtained with dark-state excitation and signaling-state excitation. Upper: the S1-state spectrum from the signaling state (red, obtained at ∼5 μL/s flow rate) versus that from the dark state (black, obtained at ∼55 μL/s flow rate). The gray shadow is the difference between the two S1-state spectra. Lower: FADH• spectrum from the singling state (blue) and that from the dark state (black). The gray shadow is the difference between the two FADH• spectra.

the signaling state exhibits a red shift (∼10 nm) of the groundstate bleach around ∼450 nm, and so does the FADH• spectrum. (Note that these spectra are the difference spectra between each species and the initial ground state.) The gray filled spectra in Figure 9 depict the difference between the spectra obtained with the signaling-state excitation and the dark-state excitation. They are close to the signaling-state spectrum shown in Figure 4. This further ensures that the timeresolved spectra obtained with the slow flow rate is essentially the time-resolved spectra of the signaling state. Figure 10 shows the temporal profiles of the S1 state and FADH• obtained by the SVD analysis of the time-resolved spectra of the signaling state of PapB in H2O (closed circle). The S1 state exhibits a biexponential decay as in the case of the dark-state excitation. The faster decay of the S1 state matches the rise of FADH•, indicating that FADH• is generated from the S1 state having the shorter lifetime. Because no final photoproduct is observed after the decay of FADH•, it is safely concluded that the FADH•, as well as the S1 state that has the longer lifetime, relaxes to the original signaling state of PapB. A possible origin of the small offset-like long-lived component is the sample degradation. The lifetimes of the S1 state and FADH• were obtained from the fitting using biexponential functions (black lines in Figure 10), and the lifetimes obtained are listed in Table 1. In Figure 10, the temporal profiles in D2O (open circle) are also shown for comparison. The slower kinetics of the S1 state and FADH• indicates the H/D kinetic isotope effect on the decay of these transients, and their lifetimes in D2O are also listed in Table 1. Time-resolved absorption spectra of the signaling state clarified that the S1 state of the signaling state generates FADH• as in the case of photoexcitation of the dark state, and that FADH• decays back into the original signaling state without yielding any photoproducts. In other words, in PapB, FADH• is

Figure 8. Typical example of the spectral decomposition of timeresolved absorption spectra obtained by excitation of the signaling state of PapB. The experimental time-resolved absorption spectrum at 73 ps (black) is reconstructed by the contributions of the S1 state (red), FADH• (blue), and signaling state (green). The reconstructed spectrum is shown in yellow.

73 ps, time-resolved spectra at all the delay time can be nicely reproduced by linear combination of these three spectra. In Figure 9, the S1-state spectrum (red) and FADH• spectrum (blue) obtained with excitation of the signaling state are compared with corresponding spectra obtained with the darkstate excitation (black). The S1-state spectrum obtained with 14768



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determined to be ∼4.5 Å in the crystal.22,47,63,64 Therefore, it is expected that the kinetics of FADH• sharply changes with the change of the hydrogen bond configuration among the Tyr, Gln, and FAD. Figure 12 (lower) compares the temporal profiles of FADH• obtained by the dark-state excitation (black) and the signaling-

Figure 10. Temporal profiles of the S1 state and FADH• after photoexcitation of the signaling state (● in H2O; ○ in D2O). The double exponential fitting curves are shown by black lines.

produced by exciting either the dark state or the signaling state, and FADH• relaxes to the signaling state in both cases. The photochemical process observed with photoexcitation of the signaling state is illustrated in Figure 11, being combined with the photocycle of the dark state to provide a unified view.

Figure 12. Comparison of the temporal profiles of the S1 state (upper, red) and FADH• (lower, blue) obtained with the singling-state excitation (at ∼5 μL/s flow rate) and those obtained with the darkstate excitation (black, at ∼55 μL/s flow rate).

state excitation (blue) of PapB. The most striking finding is that the kinetics of the FADH• is indistinguishable, within the experimental error. This indicates that the rate of the protoncoupled electron transfer from Tyr to FAD through Gln does not noticeably change upon the formation of the signaling state in PapB. Although the observation of FADH• was reported with photoexcitation of the signaling states of AppA and Trp91Phe mutant of Slr1694,60,62 the identical FADH• kinetics is observed for the first time in this study by a direct comparison between the photochemical processes of the dark state and the signaling state of PapB. Furthermore, the biexponential kinetics of the S1 decay is also the same between the darkstate excitation (black) and the signaling-state excitation (red), as shown in Figure 12 (upper). This suggests that the inhomogeneity that renders the S1 decay biexponential is not perturbed, either, upon formation of the signaling state. The same trends were also observed in D2O (see Supporting Information). In a previous work of PapB,49 it was reported that the signaling-state formation causes a ∼10 cm−1 downshift of the C4O stretch frequency of FAD. This ∼10 cm−1 downshift is commonly observed with the formation of the signaling state in BLUFs, and it indicates formation of a stronger hydrogen bond between the FAD carbonyl and the nearby Gln which is preserved in BLUFs. The present study shows that the kinetics of FADH• is the same regardless of the dark state or the signaling state being excited for PapB. This means that the signaling-state formation of PapB is a process that changes the hydrogen bond at the C4O of FAD but does not perturb the proton transfer process among the Tyr, Gln, and FAD.

Figure 11. Unified photochemical processes of PapB. The time constant of each process is depicted. In particular, τa and τb are the lifetimes of the S1 state generated by the dark-state excitation, and τa′ and τb′ are the lifetimes of the S1 state generated by the signaling-state excitation. The lifetime of the signaling state (∼6 s) is estimated from the half time (∼4 s) reported in ref 12.

Mechanism of the Signaling-State Formation of PapB. Because it is considered that FADH• is formed via protoncoupled electron transfer through the hydrogen bonding network, the kinetics of FADH• should significantly change with the change of hydrogen bonds involved. Since FADH• was observed as the intermediate for the signaling-state formation of Slr1694, it has been argued that the nearest Tyr (Tyr8 in Slr1694) is the donor of electron and proton, and the proton is transferred through the hydrogen bonds involving Gln (Gln50 in Slr1694). This is because the Tyr cannot directly form a hydrogen bond with FAD due to a long distance, as the distance between the Tyr hydroxyl and N5 atom of FAD is 14769



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Figure 13. Mechanisms of the signaling-state formation of BLUFs based on Gln flip (A),27 and Gln keto−enol tautomerism (B).40,41 The mechanism proposed for PapB is illustrated in part C: ET, electron transfer; PT, proton transfer; HB, hydrogen bond.

in the hydrogen bond network should affect the kinetics of FADH•. However, the present study clearly shows that the kinetics is the same. In the second model, it is claimed that the enol tautomer of the Gln is formed in the signaling state.40,41,43,45 Theoretical studies indicated that this Gln tautomerization can be realized regardless of the rotation of Gln being involved or not (Figure 13B).41,43 If the enol tautomer of Gln is formed without its rotation, the proton transfer distance among Tyr, Gln, and FAD may not be significantly different between the dark state and the signaling state. Nevertheless, the proton affinity of the enol form should be different from that of the keto form, so that the proton transfer rate should change between the dark-state excitation and the signaling-state excitation. Thus, the second model cannot rationalize the identical FADH• kinetics observed with excitation of the dark state and the signaling states, either. Consequently, an alternative mechanism is necessary to rationalize the FADH• kinetics observed for PapB in this study. The femtosecond time-resolved absorption data have shown that whichever state (the dark state or the signaling state) is photoexcited, the same FADH• kinetics is observed for PapB.

So far, two major models have been proposed for the structural change that occurs around the FAD binding site with signaling-state formation of BLUFs.27,29,40,41,43,45,62 In the first model, the proton coupled electron transfer occurs between Tyr and FAD to form FADH•. (Note that transient formation of FADH• is suggested also for the BLUFs for which FADH• is not directly observed in time-resolved measurements.34,60) Then, the resultant FADH• induces the flip of Gln, which remains in the signaling state even after FADH• vanishes (Figure 13A).27,62 This model can explain the ∼10 cm−1 downshift of the C4O stretch frequency of FAD because the flip of Gln generates a stronger hydrogen bond between the C4O carbonyl of FAD and the amino group of the Gln residue. However, this model contradicts the result of the present study. It is because, if the flip of Gln is induced by the formation of FADH• in the photochemical process of the dark state, it means that FADH• is generated before the Gln flip. Then, the kinetics of FADH• observed with the darkstate excitation should be different from that observed with the signaling-state excitation, because the Gln is already flipped in the ground state in the signaling state so that the difference 14770



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and surprisingly, the FADH• kinetics is indistinguishable from that observed with the dark-state excitation. This result suggests that the hydrogen bond rearrangement is completed before the proton transfer to generate FADH• in PapB, and questions the previous models that propose that FADH• induces the change of the hydrogen bond structure around the chromophore. Presumably, the hydrogen bond rearrangement occurs along with the electron transfer prior to the proton transfer and completes before FADH• is formed.

This indicates that the hydrogen bonds among the Tyr, Gln, and FAD become indistinguishable when FADH• is formed after exciting either the dark state or the signaling state, because the proton transfer rate should be sensitive to the hydrogen bond network. In other words, the femtosecond time-resolved absorption data suggest that the rearrangement of the hydrogen bonds among Tyr, Gln, and FAD completes before the formation of FADH•, and hence, the FADH• kinetics becomes identical. It may be worth mentioning that a recent time-resolved IR study of an AppA mutant suggests that photoexcitation changes the hydrogen bond structure around the FAD-biding site within 100 fs in the S1 state.46 This indicates that the rearrangement of the hydrogen bonds around FAD can be completed in a very short time as indicated in the present study, although the hydrogen-bond rearrangement does not necessarily occur in the S1 state in the case of PapB. In fact, the time-resolved absorption data of the dark state of PapB do not show any spectral evolution that would indicate the hydrogen-bond rearrangement occurring in the S1 state. Therefore, we think that the hydrogen bond rearrangement around the FAD-binding site likely occurs along with the electron transfer from Tyr to FAD before the proton transfer, although the product of the electron transfer, FAD•−, is not observable in the photochemical process of PapB due to its short lifetime. It is well-known that if the lifetime of an intermediate is much shorter than the time constant of its production, the intermediate is not noticeably populated. Thus, although FAD•− was not directly detected in the present femtosecond time-resolved absorption study, we think that electron transfer occurs and plays an important role in the signaling-state formation of PapB. Figure 13C illustrates the scheme that we suggest on the basis of the present study of PapB. In this scheme, the change of the hydrogen bond structure is induced by FAD•− that is generated by electron transfer, and two strong hydrogen bonds are formed bridging Tyr•+, Gln, and FAD•−. Then, a proton migrates from Tyr•+ to FAD•− with the aid of the strong hydrogen bonds, generating and vanishing FADH•, which finally leaves strong hydrogen bonds around the chromophore. The strong hydrogen bond formed between the Gln and the C4O carbonyl of FAD gives rise to the ∼10 cm−1 downshift of the C4O stretch frequency, which is commonly observed in the signaling states of BLUFs.54,65,66 For the strong hydrogen bond between Tyr and Gln, it is worth mentioning that an unusually low O−H streching frequency (2900−2400 cm−1) of the Tyr was recently reported for AppA and PAC (from Euglena gracilis).21 We note that this scheme differs from previously proposed mechanisms27,40 in terms that the hydrogen bond rearrangement occurs before the formation of FADH•, not after it. The present work questions the importance of FADH• to induce the change of the hydrogen bond network around the chromophore.

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ASSOCIATED CONTENT

S Supporting Information *

Time-resolved absorption data of PapB dark state and the signaling state in D2O, and the evaluation for the population of the signaling state in the excited volume. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +81-48-467-7928. Fax: +81-48-467-4539. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by KAKENHI, Grant-in-Aid for Scientific Research (A) (No. 25248009), Grant-in-Aid for Scientific Research (B) (No. 25282229), and Grant-in-Aid for Scientific Research on Innovative Areas (Nos. 25117508, 25104005, and 25117508) from The Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. T.F. was supported by RIKEN Special Postdoctoral Researchers (SPDR) Program.

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REFERENCES

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CONCLUSION The photocycle of PapB from the purple bacterium Rhodopseudomonas palustris and the signaling-state formation mechanism were examined by femtosecond time-resolved absorption spectroscopy. It was shown that FADH• is formed in the photocycle of PapB, and the kinetic analysis showed that the signaling state is formed via FADH•. This signaling-state formation of PapB is similar to that of Slr1694,27 and actually it is the second example of FADH•-mediated signaling-state formation after Slr1694. Besides, it was found that photoexcitation of the signaling state of PapB also produces FADH•, 14771



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