Structural Evolution of a Retinal Chromophore in the Photocycle of

Article Cite This: J. Phys. Chem. A XXXX, XXX, XXX−XXX

pubs.acs.org/JPCA

Structural Evolution of a Retinal Chromophore in the Photocycle of Halorhodopsin from Natronobacterium pharaonis Published as part of The Journal of Physical Chemistry virtual special issue “Time-Resolved Vibrational Spectroscopy”. Misao Mizuno,† Ayumi Nakajima,† Hideki Kandori,‡ and Yasuhisa Mizutani*,† †

Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan, and ‡ Department of Frontier Materials, Nagoya Institute of Technology, Showa-ku, Nagoya, Aichi 466-8555, Japan S Supporting Information *

ABSTRACT: We revealed the chloride ion pumping mechanism in halorhodopsin from Natronobacterium pharaonis (pHR) by exploring sequential structural changes in the retinal chromophore during its photocycle using time-resolved resonance Raman (RR) spectroscopy on the nanosecond to millisecond time scales. A series of RR spectra of the retinal chromophore in the unphotolyzed state and of the three intermediates of pHR were obtained. Using singular value decomposition analysis of the CC and C−C stretch bands in the time-resolved RR spectra, we identified the spectra of the K, L, and N intermediates. We focused on structural markers of the RR bands to explore the structure of the retinal chromophore. In the unphotolyzed state, the retinal chromophore is in the planar all-trans, 15-anti geometry. The bound ion affects the polyene chain but does not interact with the protonated Schiff base. In the observed intermediates, the chromophore is in the 13-cis configuration. The chromophore in the K intermediate is distorted due to the photoisomerization of retinal. The hydrogen bond is weak in the unphotolyzed state and in the K intermediate, resulting in exchange of the hydrogen-bond acceptor to a water molecule in the K-to-L transition, relaxation of the polyene chain distortion, and generation of an alternative distortion near the Schiff base. The bound halide ion interacts with the protonated Schiff base through the water molecule bound to the protonated Schiff base. In the L-to-N transition, the hydrogen acceptor of the protonated Schiff base switches from the water molecule to another species, although the strong hydrogen bond of the protonated Schiff base remains. This paper reports the first observation of sequential changes in the RR spectra in the pHR photocycle, provides information on the structural evolution of the retinal chromophore, and proposes a model for chloride ion translocation in pHR.

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INTRODUCTION

A major function of microbial rhodopsins is to pump ions across the cell membrane in a light-driven reaction. Microbial rhodopsins contain all-trans-retinal as a chromophore, which is covalently bound to a lysine residue through a protonated Schiff base linkage (Figure 1). Absorption of a photon induces chromophore isomerization and leads to a cyclic reaction involving a series of intermediates, each of which exhibits a distinct absorption band. The protonated Schiff base in the

retinal chromophore is the most important functional group for ion pumping because it is part of the ion transport pathway. In order to explore the ion pumping mechanism of microbial ion pumps, it is essential to elucidate the sequential structural changes of the retinal chromophore. The absorption band due to each intermediate is distinctly located in the visible region. Using resonance Raman (RR) spectroscopy, the vibrational bands due to the retinal chromophore are resonantly enhanced with visible light excitation. Bacteriorhodopsin (BR) acts as an outward proton pump and is a paradigmatic microbial ion pump. BR has been studied using static and transient RR spectroscopic analysis of both the unphotolyzed state and the intermediates.1−20 Furthermore, vibrational assignments of the

Figure 1. Structure of the retinal chromophore.

Received: December 15, 2017 Revised: February 20, 2018 Published: February 20, 2018

© XXXX American Chemical Society

A

DOI: 10.1021/acs.jpca.7b12332 J. Phys. Chem. A XXXX, XXX, XXX−XXX

Article

The Journal of Physical Chemistry A

evolution of the retinal chromophore of pHR and propose a model for the chloride ion pumping mechanism.

RR bands due to the retinal chromophore have been conducted for static RR spectra of the unphotolyzed state.11,12 These vibrational assignments have aided detailed study of the structural evolution of the retinal chromophore in the photointermediates during the proton pumping process. Halorhodopsin (HR) is a different type of microbial ion pump rhodopsin from BR and pumps a chloride ion from the extracellular side to the cytoplasmic side during a photocyclic reaction. Two chloride ion pumps have been well studied: HR from Halobacterium sarinarum (sHR) and HR from Natronobacterium pharaonis (pHR). Absorption spectroscopy shows that the HR chloride-ion pumping process sequentially involves the intermediates designated K, L, N, and O in the HR photocycle on nanosecond to millisecond time scales.21−23 RR spectroscopic explorations of the structural changes in the retinal chromophore of HR utilize similarities with structural changes in BR. To date, only a few RR spectroscopic studies have been carried out for the structure of sHR24−30 and pHR.31 Fragmentary information on the chromophore structure has been obtained for the L and O intermediates and for the unphotolyzed state, and even less information is available regarding sequential changes in the chromophore structure. Models for the ion pumping mechanism of HR, such as structural changes in the retinal chromophore and protein moiety, have been proposed based on time-resolved and lowtemperature FTIR spectroscopy studies.32−37 Two states have been kinetically distinguished in the L intermediate. The transition between the two L states is accompanied by structural changes in the protein moiety around the retinal chromophore.23,33,35 Recently, crystallographic structures of pHR intermediates have been determined.38 Light-induced structural changes at low-temperature showed that the halide ion moves across the Schiff base of the retinal chromophore in the L1-to-L2 transition. Time-resolved or cryo-trapped measurements have been used to investigate the chromophore structures in the intermediates because similar absorption spectra of the retinal chromophore in the intermediates can be observed using both techniques. However, the RR spectrum of the retinal chromophore in the K intermediate trapped at 77 K is different from the time-resolved RR spectrum at room temperature.17 This discrepancy is likely due to the measurements being conducted under different sample environments, causing structural changes in the protein moiety and influencing the chromophore structure. Consequently, differences in protein motions between ambient and low temperature must be taken into account. A new model of the ion pumping mechanism requires direct evidence for structural changes in the retinal chromophore based on kinetically and spectrally resolved data obtained using physiological conditions. In this study, we measured the time-resolved RR spectra of pHR on the nanosecond to millisecond time scales at room temperature. Specifically, we obtained a series of RR spectra of the pHR retinal chromophore in the unphotolyzed state and of the three intermediates of pHR; based on these data, we discuss the structural evolution of the retinal chromophore. The effects of deuteration on the RR spectra were investigated to explore the structure around the protonated Schiff base, which is a part of the ion transport pathway. Based on the halide ion dependence, we determined the interaction between the retinal chromophore and a pumped ion both in the unphotolyzed state and in the L intermediate. Lastly, we explored the structural

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EXPERIMENT Sample Preparation. The pHR sample was prepared as described previously39 with some modifications. Briefly, Escherichia coli BL21(DE3) harboring a plasmid encoding pHR with a histidine tag at the C-terminus was grown in 2 × YT medium containing 50 μg/mL ampicillin. At the end of the logarithmic growth phase, 1 mM isopropyl-β-D-thiogalactopyranoside and 10 μM all-trans-retinal were added, and the cells were harvested after 3 h. The gray-colored cells were sonicated and the cell membranes were solubilized with 1% (w/v) ndodecyl-β-D-maltoside (DDM). The solubilized membranes were purified using a Ni-affinity column (GE Healthcare, HisTrap HP) and an ion-exchange chromatography column (GE Healthcare, HiTrap Q HP). Purified samples with an absorption ratio at 280 and 570 nm of less than 1.5 were used for spectroscopic measurements. The samples were suspended in a buffer containing 10 mM MOPS-NaOH (pH 7.0), 150 mM NaCl, 283 mM Na2SO4, and 0.1% DDM. For measurements of halide ion dependence, 150 mM NaBr or 150 mM NaI was used instead of NaCl. To measure the deuteration effect, the H2O buffer was exchanged for D2O buffer or a mixed buffer (H2O:D2O = 1:1). The pD of the D2O buffer was adjusted to 7.0 using the convention that the pH value of a D2O buffer read using a glass electrode is 0.4 pH unit lower than the pH reading of a H2O solution.40 The mixed buffer was prepared by mixing the same volume of H2O and D2O buffers after adjusting their pH or pD. Pump−Probe Time-Resolved RR Measurements. The 475 nm probe pulses were the second harmonic of the output of a nanosecond Ti:sapphire laser pumped by a Q-switched diode-pumped Nd:YLF laser (Photonics Industries, TU-L) operating at 1 kHz. The pulse width was 30 ns. A typical pulse energy was 1.6 μJ at the sample point. The probe pulses were focused downward onto the sample cell at 45° and the 45° backscattered Raman light was collected and focused onto the entrance slit of a prefilter coupled to a spectrograph (HORIBA Jobin Ybon, iHR550) and detected with a CCD camera (Roper Scientific, SPEC-10:400B/LN-SN-U). The Raman shifts was calibrated using the Raman bands of 2-propanol and ethanol and the calibration error was as large as 4 cm−1. We used two types of pump−probe methods for timeresolved measurements. Time-resolved RR spectra were obtained using the same optical setup as for probing the scattered light but with different pumping systems and a series of time-resolved RR spectra in the very wide temporal range from tens of nanoseconds to 10 ms were obtained. Spectra on the nanosecond to early microsecond time scales were obtained with 50 ns resolution by conducting pump−probe measurements using two nanosecond pulses. Pump pulses at 532 nm with 20 ns pulse widths were generated with a Q-switched diode-pumped Nd:YAG laser (Megaopto, LR-SHG) operating at 1 kHz. A typical pulse energy was 65 μJ at the sample point. The pump pulses spatially overlapped on the probe pulse at the focal point on the sample cell. The instrumental response time was 50 ns. We obtained spectra on the late microsecond to millisecond time scales with 100-μs resolution using a dualbeam rapid-flow method.6 The output of a cw DPSS laser (Cobolt, Jive 05−01) was used as a 561 nm pump beam to continuously initiate the photoreaction in the sample. A typical pump power was 100 mW at the sample point. The pump B


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Figure 2. Time-resolved RR spectra of pHR in H2O buffer probed at 475 nm on the −100 ns to 50 μs time scale (A) and on the −160 μs to 4 ms time scale (B). The pump wavelengths were 532 and 561 nm for measurements with a 50 ns resolution (delay time from −100 ns to 50 μs) and 100 μs resolution (delay time from −160 μs to 4 ms), respectively. The spectra of the buffer and the emission background were subtracted. The top traces are the Raman spectra without the pump beam and thus are the spectra of the unphotolyzed state. The other spectra are time-resolved difference spectra obtained by subtracting the spectral contribution of the unphotolyzed state from the Raman spectrum obtained with the pump beam at each delay time.

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RESULTS Time-Resolved RR Spectra. Figure 2 shows time-resolved RR spectra of pHR probed at 475 nm on the nanosecond to millisecond time scales. The top trace in each panel of Figure 2 is the spectrum measured using only probe pulse irradiation and represents the RR spectrum of the unphotolyzed state. The other traces are the time-resolved difference spectra of the photolyzed protein obtained by subtracting the spectral contributions of the unphotolyzed state. Bands were observed at 1008, 1172, 1204, 1212, 1351, 1527, 1579, 1600, and 1634 cm−1 for the unphotolyzed state. In the 50 ns spectra, bands were observed at 974, 1013, 1199, 1533, and 1622 cm−1. These bands decayed as the delay time increased and at delay times greater than 1 μs, bands were instead observed at 1187, 1202, 1550 cm−1. The band at 1643 cm−1 at 200 ns was upshifted to 1651 cm−1 at 2 μs and its intensity increased. The spectral feature of the time-resolved spectra did not change between 2 and 50 μs, whereas at 80 μs, shoulder bands at 1558 and 1648 cm−1 were observed in addition to those observed at 10 μs. All the bands decayed on the millisecond time scale. The strongest bands appearing in the 1500−1600 cm−1 region for all states were assigned to the CC stretching band, which can be used as a marker to identify the intermediate. The three CC stretch bands due to intermediates were identified in Figure 2: the band at 1533 cm−1 from 0 to 300 ns, the band at 1550 cm−1 after 100 ns, and the shoulder band at 1558 cm−1 after 80 μs. The pHR photocycle as a chloride ion pump is described by the scheme:

beam was independently introduced to the sample solution from a direction different from that of the probe pulses and was focused into a line on the sample using a cylindrical lens. The instrumental response time under these conditions was 140 μs. Detailed descriptions of the experimental setup are provided in the Supporting Information. Single-Beam Time-Resolved RR Measurements. A cw DPSS laser (Cobolt, Samba 04−01) with a wavelength of 532 nm was used to photolyze the sample and to probe RR scattering. The sample solution was placed in a glass tube used as a spinning cell and the 90° scattered Raman light was collected and focused onto the entrance slit of a spectrograph (HORIBA Jobin Ybon, iHR320) equipped with a CCD camera (Roper Scientific, PyLoN:400B_eXelon VISAR). The Raman shifts were calibrated using the Raman bands of cyclohexane, toluene and acetone. The calibration error was within 1 cm−1. To obtain a spectrum of the photolyzed sample, we separately measured spectra of the same sample solution using different laser powers. At high laser flux, we detected the spectral contribution of an intermediate accumulated during the residence time in the laser beam in addition to the contribution of the unphotolyzed state. The residence time was determined by the beam focal point size on the sample and the spin speed of the sample cell. The time-resolved spectrum was obtained by subtracting the spectrum obtained at low laser power (1 mW) from that obtained at high power (25 mW). Detailed descriptions of the experimental setup and data acquisition methodologies are provided in the Supporting Information. C


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Figure 3. Overview of the transient RR spectra of pHR in H2O buffer probed at 475 nm. (a) Unphotolyzed state, and (b) K, (c) L, and (d) N intermediates. The spectra of the buffer and the emission background were subtracted.

SVD analysis for the spectral changes in the 1120−1240 (C−C stretch bands) and 1480−1600 cm−1 (CC stretch bands) regions (Figures S5−7 and related text in the Supporting Information). Based on the kinetic model, the time constants of the K-to-L (τ1), forward L-to-N (τ2), and backward N-to-L (τ3) transitions and an apparent decay of the N intermediate (τ4) were calculated to be 680 ± 50 ns, 470 ± 80 μs, 850 ± 340 μs, and 3.1 ms, respectively. These time constants were comparable to those reported by previous absorption studies.21−23 The reconstructed spectra of the C−C and CC stretch bands in the K, L, and N intermediates are shown in Figures S5B and S6B in the Supporting Information. The strongest CC stretch band at 1527 cm−1 in the unphotolyzed state shifted to 1533, 1550, and 1559 cm−1 in K, L, and N, respectively. In the unphotolyzed state and the K and L intermediates, the CC stretch bands were wide and asymmetric. In the N intermediate, an additional band was observed at 1542 cm−1 as a shoulder on the 1559 cm−1 band. It is likely that two bands were included in the observed CC stretch bands for all states and we fitted these CC stretch bands with two Lorentzian functions (Figure S8 in the Supporting Information). The peaks of the band pairs were observed at 1525/1535, 1529/1537, 1542/1552, and 1542/ 1559 cm−1 in the unphotolyzed state and in the K, L, and N intermediates, respectively. The lower frequency bands can be assigned to the in-phase stretching vibrations of the CC bonds in the polyene chain, whereas the higher frequency bands are due to the out-of-phase vibrations.44 The absorption maximum wavelengths were previously reported to be 575, 570, 520, and 520 nm in the unphotolyzed state and in the K, L, and N intermediates, respectively.23 We found that the in-phase CC stretching frequencies exhibited an inverse linear correlation with the absorption maximum wavelengths, which were also observed in the RR spectra of the photocycle of BR.18

hν

HR → K ⇄ L ⇄ N ⇄ O → HR′ → HR.21−23 The band at 1550 cm−1 observed in the present spectra was similar to that reported previously in the L intermediate measured using 514.5 nm excitation31 and thus this band was attributed to the L intermediate. The 1533 cm−1 band was most likely due to the K intermediate, which is the precursor of the L intermediate. The shoulder band at 1558 cm−1 was assigned to the N intermediate and was not attributed to the O intermediate. There is an empirical inverse linear correlation between the CC stretching frequency and the absorption maximum of the retinal chromophore.41−43 A frequency of 1558 cm−1 is too high to be assigned to the O intermediate: the CC stretching frequency in the O intermediate, with the absorption maximum at 600 nm, would be lower than that in the unphotolyzed state with the absorption maximum at 575 nm, as observed in the photocycles of BR6 and sHR.30 In addition, the 475 nm probe light was poorly resonant with the electronic transition of the red-shifted O intermediate. We identified the spectral components of the three intermediates (K, L, and N) in the present time-resolved spectra (Figure 2). The time-resolved spectra in the −100 ns to 50 μs range obtained with the instrument response time of 50 ns were expressed by the sum of the spectra of the K and L intermediates, whereas the spectra in the −160 μs to 4 ms range obtained with the instrument response time of 140 μs were consistent with the sum of the spectra of the L and N intermediates (Figures S3 and S4 and related text in the Supporting Information). Based on the temporal changes of the band intensities, we model the kinetic scheme as τ2

τ4 τ1 HR → K → L→ ←N→, assuming the minimum number of the hν

τ3

intermediates. This scheme is consistent with that proposed by the previous time-resolved absorption studies.21−23 We confirmed the validity of the decomposition by conducting D


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Figure 4. Overview of the transient RR spectra of pHR in D2O buffer probed at 475 nm. (a) Unphotolyzed state, and (b) K, (c) L, and (d) N intermediates. The spectra of the buffer and the emission background were subtracted.

1647 cm−1 in N. The CN bond is present in the Schiff base. The sequential shift of the CN stretch band is attributed to structural changes in the Schiff base. In the time-resolved RR spectra, we also found that the peak frequency of the CN stretch band in the L intermediate gradually upshifted from 1643 cm−1 at 200 ns to 1651 cm−1 at 10 μs, although the neighboring CC stretching frequency remained unchanged. We plotted the peak positions of the C N stretch band against the delay time in Figure S9 in the Supporting Information. The time constant of the frequency upshift was calculated to be 580 ± 50 ns, which is comparable to that for L formation (680 ± 50 ns). This indicates that L formation coincides with the structural change in the Schiff base. Deuteration Effect. A proton bound to the Schiff base can be replaced with a deuteron when a H2O solvent is exchanged with a D2O solvent, whereas all other hydrogen atoms in the chromophore remain unexchanged. Thus, the RR bands due to the vibrational modes of the Schiff base would be affected by deuteration. To further investigate the structures in the Schiff base region, time-resolved RR spectra of pHR in D2O buffer were measured on the nanosecond to millisecond time scales (Figure S10 in the Supporting Information). The RR spectra of the retinal chromophore in the unphotolyzed state and in the K, L, and N intermediates in D2O are shown in Figure 4. A comparison of Figures 3 and 4 shows deuteration effects on the frequencies and widths of the CN stretch bands at 1610− 1650 cm−1, on the frequencies of the C−C stretch bands at 1150−1250 cm−1, and on the frequencies and intensities of the bands at 950−1030 cm−1. Figure 5 shows expanded spectra of the CN stretch bands in H2O (red traces) and D2O (blue traces). Downshifts of the CN stretch bands were observed upon deuteration for all the states. In the unphotolyzed state, the band at 1634 cm−1 in H2O shifted to 1624 cm−1 in D2O. In the K, L, and N

The moderately strong C−C stretching bands at 1172, 1204, and 1212 cm−1 were identified in the unphotolyzed state. In the intermediates, the intensities of the bands at 1172 and 1212 cm−1 decreased and new bands at 1185, 1189, and 1186 cm−1 appeared in the K, L, and N intermediates, respectively, which were not observed in the unphotolyzed state. The bands at 1188−1189 cm−1 were observed in the intermediates of BR in the 13-cis configuration.13,18 Based on the population change of each intermediate as calculated by SVD analysis, we obtained RR spectra of the retinal chromophore in addition to the spectra of the C−C and CC stretch bands. Figure 3 shows the transient RR spectra of pHR in the unphotolyzed state and of the three photointermediates; K, L, and N. Changes in the chromophore structure of each intermediate in the pHR photocycle were thus investigated based on the RR spectra of the chromophore. It should be noted that these transient RR spectra represent the first observations of the K and N intermediates in pHR. Here, we focused on spectral changes in the hydrogen-out-of-plane (hoop) wag and the CN stretch bands. An intense hoop band was observed at 973 cm−1 only in the K intermediate in Figure 3. In the BR spectra, the band was observed at 959 cm−1.11 A frequency of the corresponding mode was calculated to be 968 cm−1 for the hoop vibration at the C11 and C12 positions in normal-mode analysis.11,12 The positions of the carbon atoms in the retinal chromophore mentioned hereafter are described in Figure 1. The similarity in the frequency suggested that the 973 cm−1 band observed for the K intermediate of pHR was attributed to the C11−C12 hoop vibration. A strong hoop band at 970 cm−1 was reported for the time-resolved FTIR spectrum of the K intermediate of sHR45 and the time-resolved spectrum of the K intermediate of BR.18 For the CN stretching mode, we found that the band at 1634 cm−1 in the unphotolyzed state downshifted to 1622 cm−1 in K, upshifted to 1652 cm−1 in L, and slightly downshifted to E


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Figure 5. Expanded spectra of the CN stretch band region, 1580− 1700 cm−1. (a) Unphotolyzed state, and (b) K, (c) L, and (d) N intermediates. Red and blue traces represent the spectra measured in H2O and D2O buffer, respectively, and were normalized by the intensity of the CN stretch band in each state.

Figure 6. Expanded RR spectra of the CN stretch band region, 1580−1700 cm−1, of the L intermediate using single-beam timeresolved measurements in (a) H2O buffer, (b) D2O buffer, and (c) mixed buffer (H2O/D2O = 1:1). The wavelength of both the photolyzed and probe beams was 532 nm. The spectral contributions of the buffer and unphotolyzed state were subtracted. Gray curves are the best-fit of the sum of Lorentzian functions, including the wing of the neighboring CC stretch bands. Red and blue curves show the fitting results of the Lorentzian curves representing the CN(H) and CN(D) stretch band, respectively. The fitting parameters of the peak positions and the bandwidths are given in the figure.

intermediates, the bands at 1622, 1652, and 1647 cm−1 shifted to 1613, 1621, and 1622 cm−1, respectively, following deuteration. The amplitudes of the deuteration shifts were 10, 9, 31, and 25 cm−1 in the unphotolyzed state, and the K, L, and N intermediates, respectively. The observed frequencies and amplitudes of the deuteration shift in the unphotolyzed state and the L intermediate were similar to those observed in previous studies of pHR31 and sHR.25−28 The bandwidths in H2O and D2O were 17/15, 13/13, 21/16, and 12/14 cm−1 in the unphotolyzed state and the K, L, and N intermediates, respectively. In the L intermediate the CN stretch band in H2O was broadened compared to that in D2O. Broadening of the CN stretch band in H2O was previously reported for the RR spectra of the BR chromophore, and this band broadening arose from resonance vibrational energy transfer between the CN stretching mode in the Schiff base and the HOH bending mode of a nearby water molecule with similar vibrational frequencies.46 To obtain direct spectroscopic evidence of the resonance vibrational energy transfer at the Schiff base in the L intermediate, we measured the RR spectra of the L intermediate in H2O, D2O, and a 1:1 H2O/D2O mixture using a single-beam time-resolved method (Figure S11 in the Supporting Information). Expanded spectra of the CN stretch region of the L intermediate are shown in Figure 6. We fitted the observed CN stretch bands using Lorentzian functions. The red and blue curves show the band shapes of the CN stretch band in the Schiff base bound to a proton [C N(H)] and to a deuteron [CN(D)], respectively. The peak position of the CN(H) band was 1646.9 cm−1 in H2O and that of the CN(D) band was 1618.4 cm−1 in D2O. The peak positions in the mixed buffer were 1618.4 and 1647.3 cm−1 for the CN(H) and CN(D) bands, respectively, identical to the positions of the corresponding bands in H2O and D2O. The width of the CN(H) band in H2O was 21.1 cm−1, that of the CN(D) band in D2O was 13.6 cm−1, and in the mixed buffer

the CN(H) and CN(D) bandwidths were 19.1 and 15.1 cm−1, respectively. The CN(H) band in H2O was the widest. In the mixed buffer, the CN(H) band was narrower than that in H2O and the CN(D) band was wider than that in D2O. These findings provide strong experimental evidence that the resonance vibrational energy transfer occurs in the L intermediate of pHR. We observed intensity changes in the C−C stretch bands in the fingerprint region from 1150 to 1250 cm−1 for the unphotolyzed state and the L intermediate (Figure S12A in the Supporting Information). In the unphotolyzed state, the intensity of a lower side shoulder of the 1174 cm−1 band in H2O decreased and a higher side shoulder of the 1204 cm−1 band increased its intensity upon deuteration. In the L intermediate, an intensity decrease of the 1189 cm−1 band and increase of the 1191 cm−1 band were observed. We observed no significant differences in the spectra of the K and N intermediates obtained in H2O or D2O. In D2O, new bands appeared at 969, 965, 986, and 986 cm−1 in the RR spectra of the unphotolyzed state and the K, L, and N intermediates, respectively (Figure S12B in the Supporting Information). These new bands were attributed to the N−D rocking mode in the Schiff base. The corresponding N−H rock band in H2O appeared at 1351 cm−1 in the unphotolyzed state but could not be identified in the photointermediates. In addition, in the L intermediate, the shoulder on the band at 1011 cm−1 in H2O shifted to 975 cm−1 in D2O (traces c in Figure S12B in the Supporting Information). A similar downshift was observed in the RR spectra of the L intermediate of BR, indicating that the polyene chain is distorted in the L intermediate.10,18,19 F


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Figure 7. Halide ion dependence of the RR spectra of (a) the unphotolyzed state and (b) the L intermediate of pHR. Red, light blue, and dark yellow traces show the spectra obtained in buffer containing Cl−, Br−, and I−, respectively. The inset shows an expanded view of the band at 1010 cm−1 of the L intermediate, with intensity normalization of the peaks. The probe wavelength was 532 nm. The spectral contributions of the buffer and the unphotolyzed state were subtracted. The asterisks represent the positions of the band due to sulfate ion added as a standard to allow subtraction of the spectral contributions of the buffer. Peak frequencies labeled without numbers in spectra obtained in the presence of Br− and I− are identical with the frequencies of the corresponding bands in spectra obtained in the presence of Cl−. Bands whose frequencies are underlined show significant halide ion dependence.

Table 1. Halide Ion Dependence on Deuteration Effects of the CN Stretch Band of the Retinal Chromophore in pHR in H2O state unphotolyzed state

L intermediate

a

binding ion −

Cl Br− I− Cl− Br− I−

Frequency/cm

−1

ΔH/D

in D2O Width/cm

1631.8 1631.3 1631.1 1646.9 1645.8 1644.0

12.4 11.9 12.5 22.5 22.2 19.4

−1

Frequency/cm 1621.6 1621.2 1620.5 1618.2 1617.6 1617.6

−1

Width/cm

−1

12.3 12.2 12.1 13.0 12.3 12.8

Shift/cm−1 10.2 10.1 10.6 28.7 28.2 26.4

Standard deviations of the fitting results were within ±0.3 cm−1 and ±1.0 cm−1 for frequencies and bandwidths, respectively.

Halide Ion Dependence. We examined a halide ion dependence on the transient RR spectra of the unphotolyzed state and the L intermediate using single-beam time-resolved measurements. Figure 7a shows the RR spectra of the unphotolyzed state in the presence of Cl−, Br−, and I− ions. We obtained the CC and CN stretching frequencies by fitting the observed spectra in 1510−1700 cm−1 using five Lorentzian functions. The strong in-phase CC stretch band showed a frequency shift, from 1524.3 cm−1 following Cl− binding to 1523.5 and 1522.7 cm−1 following Br− and I− binding, respectively (standard deviation of the fitting results was ±0.1 cm−1). The CN stretching frequencies were 1631.8, 1631.3, and 1631.1 cm−1 following Cl−, Br−, and I− binding, respectively (standard deviation of the fitting results was ±0.3 cm−1). The CN stretching frequencies were the same among the anion bound forms within the experimental error. No significant spectral differences were observed for the

other bands. This observation was consistent with previous RR results for pHR31 and sHR29 but was inconsistent with previous FTIR results.47 As shown in Figure 7b, spectral differences in five bands of the L intermediate were observed upon exchange of the halide ion. The lower side shoulder of the band at 1010 cm−1 and the band at 1239 cm−1 increased in intensity in the order Cl−, Br−, and I−. The bands at 1444, 1548, and 1647 cm−1 observed in the spectrum obtained in the presence of Cl− shifted to 1442, 1547, and 1646 cm−1 upon replacement with Br−, and 1438, 1546, and 1644 cm −1 following replacement with I−, respectively. Based on vibrational assignments of the RR bands of the BR chromophore,11,12 the strongest bands at 1548−1546 cm−1 are assigned to the CC stretching mode of the polyene chain, the bands at 1647−1644 cm−1 are assigned to the CN stretching mode of the Schiff base, and the shoulder of the band at 1010 cm−1 and the band at 1239 cm−1 G


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The Journal of Physical Chemistry A are attributed to the C15−H wagging and lysine rocking modes, respectively. The bands at 1444−1438 cm−1 include spectral contributions from the deformations of two methyl groups bound to the polyene chain. The observed difference in the CN stretching frequency is consistent with a previous RR study.31 We also observed deuteration effects on bands affected by the binding of halide ions (Figure S13 in the Supporting Information). Although no deuteration effects on the CC stretch bands at 1525−1523 cm−1 in the unphotolyzed state or on the 1548−1546 cm−1 band in the L intermediate were observed, the shoulder band at 1010 cm−1 and the bands at 1239, 1444−1438, and 1647−1644 cm−1 shifted to 975, 1246, 1451, and 1618 cm−1, respectively, in the L intermediate. We examined the band shapes of the CN stretching mode in detail. Table 1 shows the fitting results of the frequencies and widths of the CN stretch band obtained using a Lorentzian function. In the unphotolyzed state, the CN(H) and C N(D) stretching frequencies were 1631.1−1631.8 and 1620.5− 1621.6 cm−1, respectively. The amplitudes of the deuteration shifts were 10.1−10.6 cm−1. The bandwidths for both the C N(H) and CN(D) stretching modes were 11.9−12.5 and 12.1−12.3 cm−1, respectively. These values were independent of the bound ions. In the L intermediate, the CN(H) stretching frequencies significantly changed, from 1646.9 cm−1 in the presence of Cl− to 1645.8 cm−1 following replacement with Br− and to 1644.0 cm−1 upon replacement with I−, while the CN(D) stretching frequencies at 1618.2−1617.6 cm−1 were essentially unchanged. The amplitudes of the deuteration shifts changed by 28.7, 28.2, and 26.4 cm−1 in the order Cl−, Br−, and I−. In addition, the widths of the CN(H) stretch bands in H2O were 22.5, 22.2, and 19.4 cm−1 following Cl−, Br−, and I− binding, respectively, while those of the CN(D) stretch bands in D2O were 12.3−13.0 cm−1 and were independent of the bound ion. These observations suggest that the interaction between the protonated Schiff base and the halide ion in the L intermediate is different from that in the unphotolyzed state.

observed for the amide bands and the carboxylic CO stretch band due to an aspartate involved in the hydrogen-bond network close to the Schiff base, while the vibrational bands of the retinal chromophore did not change.34,35 Light-induced changes in a low-temperature HR crystallographic structure demonstrated that the halide ion moves from the extracellular side of the protein to the cytoplasmic side across the Schiff base in the L1-to-L2 transition.38 Time-resolved absorption spectroscopy, in contrast, reported two kinetic components exhibiting identical absorption spectra for the L intermediate,23 indicating that the electronic transitions of the retinal chromophore are spectrally indistinguishable between the L1 to L2 intermediates. We conducted SVD analysis of the spectral changes in the CC and C−C stretch bands. These band shapes are predominantly influenced by the structure of the polyene chain in the retinal chromophore. The present observation of a single L intermediate in the RR spectra indicates that structural changes occur solely in the retinal chromophore and that the structure of the polyene chain remains unchanged between the L1 and L2 intermediates. Additionally, we observed an upshift of the CN stretch band due to the L intermediate, from 1643 to 1651 cm−1 with a time constant of 580 ns, while the CC stretching frequency did not change (Figure 2 and Figure S9 in the Supporting Information). This time constant was close to that of the K-toL transition (680 ns). This shift in the CN stretching frequency reflects structural change in the Schiff base region, and thus formation of the L intermediate is accompanied by structural change in the protonated Schiff base. Structural Changes in the Polyene Chain of the Retinal Chromophore. Spectral changes in the C−C stretch bands in the RR spectra of the unphotolyzed state and intermediates provide structural information on the C13C14− C15N configuration. Our present time-resolved RR study showed that the spectral features of each state of pHR (Figure 3 and Figure S12A in the Supporting Information) were similar to those of the corresponding state of BR. We tentatively assigned the observed RR bands of pHR according to the vibrational assignments of BR.11,12 Here, we characterize the structural changes in the polyene chain of the retinal chromophore during the pHR photocycle. The frequency ordering and differences in the localized C10− C11 and C14−C15 stretch bands can be markers of the C13C14 configuration. In the case of BR as a model protein, it was found that two isomers, called BR568 and BR548, are present in the unphotolyzed state. The C13C14 configuration of the retinal chromophore in BR568 is all-trans, while that of BR548 is in the 13-cis configuration. The C10−C11 and C14−C15 stretch bands appear at 1169 and 1201 cm−1 for the trans form and at 1183 and 1167 cm−1 for the cis form, respectively.11,12 In the RR spectrum of BR548, the intensity of the band at 1169 cm−1 decreased and the intensity of the band at 1183 cm−1 increased compared to BR568.12 The K, L, and N intermediates of BR all contain the chromophore in the 13-cis configuration and the intensities of the bands around 1185 cm − 1 increased.5,8,10,13,18,19 For the pHR photocycle, in the present RR spectrum of the unphotolyzed state of pHR (trace a in Figure 3), no apparent band was observed at 1185 cm−1, indicating that most of the unphotolyzed state is in the all-trans form. In the K, L, and N intermediates (traces b−d in Figure 3), the appearance of new bands at 1185−1189 cm−1 indicates that the retinal chromophore in pHR is also in the 13-cis configuration. The 13-cis configuration in the L intermediate

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DISCUSSION Kinetics. In the time-resolved RR spectra of the retinal chromophore in pHR (Figure 2), we found three distinct CC stretch bands attributed to different intermediates. The CC stretching frequency in the retinal chromophore linearly correlates with its absorption maximum wavelength;41−43 consequently, this band is a marker that can be used to determine the intermediate states. Based on temporal intensity changes in the CC stretch bands, SVD analysis was conducted for spectral changes in the CC stretch bands in addition to spectral changes in the C−C stretch bands (Figures S5 and S6 in the Supporting Information). We observed the kinetics of the three intermediates K, L, and N. The K-to-L transition was unidirectional and its time constant was 680 ns, comparable to the 400 ns time constant obtained previously by time-resolved absorption measurements.23 The L-to-N transition was in equilibrium, and the time constants of the forward and backward transitions were 470 and 850 μs, respectively. Our present time-resolved RR data identified a single L intermediate, whereas previous studies identified two putative L intermediates, L1 and L2, in the HR photocycle.23,33 These earlier studies suggested that the two L intermediates differ due to structural changes in the protein moiety. In low-temperature and time-resolved FTIR spectroscopy, spectral changes were H


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indicates that the polyene chain at the C11C12 position is distorted. Polyene chain distortion was also observed for BR after photoisomerization.17,18 The photoisomerized retinal chromophore holds the distortion in the polyene chain in the initial step of the photocycle. In the L intermediate (traces c in Figure 3 and Figure S12B in the Supporting Information), the hoop band at the 973 cm−1 band was diminished and instead the hoop band was observed as a shoulder band at 1010 cm−1, which was also observed in the BR photocycle.10,18,19 The present observation suggests that coupling between two wagging vibrations of the C15−H and N−H bonds contributed to the bands at 1011 cm−1 in H2O. The C15−H wag bands in D2O shifted to the 975 cm−1 band and the N−D wag bands could not be identified. The band at 1014 cm−1 observed in D2O was the intrinsic band due to the rocking modes of two methyl groups. The observation of the C15−H wag band indicates that the chromophore is distorted at the C15 position in the L intermediate. In the L intermediate, the N−H group of the Schiff base presumably points to the cytoplasmic side, thereby distorting the polyene at the C15 position. In the N intermediate (traces d in Figure 3 and Figure S12B in the Supporting Information), hoop bands were not observed, indicating that the polyene chain is relaxed to a planar geometry, consistent with the crystallographic structure reported previously.38 Structural Changes around the Protonated Schiff Base. In the protonated Schiff base, the CN stretching mode is strongly coupled with the N−H rocking mode. Thus, the CN stretch band is sensitive to structural changes in the hydrogen bond at the protonated Schiff base, which is part of the ion-pumping pathway. In the RR spectra of the retinal chromophore, the CN stretch band is a key marker with respect to ion translocation. Spectral changes in the CN stretch band provide direct information on changes in the structure around the protonated Schiff base in the chromophore. Coupling between the CN stretching and N−H rocking modes increases the observed CN stretching frequency compared to the pure (uncoupled) CN stretching frequency. In D2O, the N−D rocking frequency shifts to about 980 cm−1, the CN stretching mode is decoupled, and downshift of the CN stretch band is observed as a deuteration effect. If the Schiff base is deprotonated, no frequency shift is observed. Downshift of the CN stretch band was observed for all states upon deuteration, as shown in Figure 5, indicating that the Schiff base in the chromophore is protonated in the unphotolyzed state and in the K, L, and N intermediates. When the strong hydrogen bond in the protonated Schiff base is formed, the N−H bending frequency upshifts, and thus the observed CN stretching frequency in the Schiff base also upshifts. It has been reported that the stronger the H-bond, the higher the CN stretching frequency and the larger the deuteration shift.50 In the unphotolyzed state of pHR, the C N stretching frequency in H2O and the amplitude of the deuteration shift were 1634 and 10 cm−1, respectively. For proton pumps, the CN stretch band appeared at 1640−1657 cm−1 and the amplitude of the deuteration shift was over 20 cm−1.51−53 Our present observations suggest that the strength of the hydrogen bond at the protonated Schiff base is weaker for pHR acting as a chloride ion pump compared to its strength in proton pumps. In the K intermediate, the low CN stretching frequency and the small deuteration shift indicate significant weakening of the hydrogen bond upon photo-

was observed in previous RR spectra of pHR31 and sHR,27,28 and the crystallographic structure of the N intermediate likewise showed that the retinal chromophore is in the 13-cis configuration.38 Our present observations are thus consistent with these previous reports. The C14−C15 stretching frequency is sensitive to the C15N configuration 7,8 because the vibrational modes of the neighboring C14−C15 and N−H bonds can be coupled, and coupling between the C14−C15 stretching and N−H rocking modes is different for the trans (15-anti) and cis (15-syn) configurations in the C15N bond. In BR548, in which the C15N bond is in the 15-syn configuration, a large coupling results in a large upshift of the C14−C15 stretching frequency upon deuteration, from 1167 to 1208 cm−1. In contrast, in the 15-anti configuration for BR568, a weak rock-stretch coupling makes the C14−C15 stretching frequency insensitive to deuteration. Accordingly, the deuteration effect on the C14− C15 stretching frequency is a good marker of the C15N configuration. In the unphotolyzed state of pHR, the C14−C15 stretch band at 1204 cm−1 did not shift upon deuteration (traces a in Figure S12A in the Supporting Information), showing that the C15N configuration is in the 15-anti configuration. However, we also found the intensity decrease of the weak shoulder band at 1168 cm−1 as well as the intensity increase of the 1214 cm−1 band upon deuteration in the same traces. Indeed, it has been reported that about 20% of the chromophore adopts the 13-cis configuration21 and thus the observed intensity changes in the unphotolyzed state result from the spectral contribution of the isomer in the 13-cis, 15-syn configuration. On the other hand, in the K intermediate (traces b in Figure S12A in the Supporting Information), no significant shifts of the C−C stretch bands were observed. In the L intermediate (traces c in Figure S12A in the Supporting Information), we observed the C−C stretch band at 1189 cm−1 and a small upshift of this band upon deuteration. In the N intermediate (traces d in Figure S12A in the Supporting Information), no shift following deuteration was detected in the C−C stretch bands. In the photointermediates, the absence of large deuteration shifts indicates that the C15N bond is in the 13-cis, 15-anti configuration. In the N intermediate, the reported crystallographic structure for the Schiff base configuration is the 15-anti geometry.38 Structural information on the polyene chain is provided by the intensities of the hoop bands. The RR bands of the retinal chromophore are enhanced due to the A-term mechanism which depends on the vibrational overlap integrals between two resonant electronic states; consequently, only normal modes with large shifts in equilibrium geometry along the vibrational coordinates upon electronic excitation yield strong RR intensities.48 In the case of hoop bands, distortion of the polyene chain determines the band intensity. Thus, the presence of hoop bands suggests that the polyene chain is distorted. Distorted positions can be estimated by assignments for the observed bands using vibrational assignments of the RR bands of BR.11,12 In the unphotolyzed state (traces a in Figure 3 and Figure S12B in the Supporting Information), no intense hoop bands were identified, indicating that the polyene chain is in a planar geometry, consistent with the crystallographic structure.49 In the K intermediate (traces b in Figure 3 and Figure S12B in the Supporting Information), we observed an intense hoop band at 973 cm−1 which can be assigned to the hoop vibration at the C11C12 position. The presence of the 973 cm−1 hoop band I


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hydrogen-bond strength between the protonated Schiff base and the water molecule. In the unphotolyzed state and in the K and N intermediates, no broadening of the CN stretch band in the H2O buffer was observed, indicating that the resonance energy transfer did not occur. This lack of energy transfer is due to the water molecule either not being attached to the Schiff base proton or not being situated favorably to the hydrogen bond from the protonated Schiff base in the retinal chromophore. In the unphotolyzed state, the crystallographic structure showed that a water molecule and a chloride ion are located next to each other facing the N−H group of the protonated Schiff base. The distance from the nitrogen atom of the Schiff base to the oxygen atom of the closest water molecule is 3.5 Å,49 which is longer than the corresponding hydrogen-bond distance of 2.9 Å in BR.54 In the unphotolyzed state, the hydrogen-bond at the protonated Schiff base is weak, making the resonance energy transfer between the Schiff base and the water molecule less favorable. In the K intermediate, the hydrogen bond acceptor changes to a chloride ion, as previously proposed in a lowtemperature FTIR study.35 In the N intermediate, based on the crystallographic structure, a halide ion is trapped at the cytoplasmic side facing the N−H group of the Schiff base.38 Exchange of the hydrogen-bond acceptor attenuates the resonance energy transfer between the Schiff base and the water molecule, although the strong hydrogen bond remains at the protonated Schiff base in the L-to-N transition. Interaction between the Chloride Ion and Retinal Chromophore. The effects of halide ion on the RR spectra of the unphotolyzed state and the L intermediate were examined to understand the interaction between chloride ion and the retinal chromophore. In the unphotolyzed state, the CC stretching frequencies were 1524.3, 1523.5, and 1522.7 cm−1 following Cl−, Br−, and I− binding, respectively, as shown in traces a in Figure 7. The difference among the observed CC stretching frequencies was 1.6 cm−1, which exceeded the standard deviation of ±0.1 cm−1. The absorption maximum wavelengths of the chromophore were 577, 578, and 580 nm upon the binding of Cl−, Br−, and I−, respectively, and thus the CC stretching frequencies linearly correlate with the absorption maximum. However, the CN stretch bands appeared at 1631.8, 1631.3, and 1631.1 cm−1, which were within the standard deviation of ±0.3 cm−1. These data show that the bound halide ion perturbs π-electron delocalization but does not influence the Schiff base moiety, consistent with RR results reported previously.31 Although the crystallographic structure showed that a chloride ion binding site is located 3.4 Å from the Schiff base proton,49 the halide ion does not interact with the Schiff base. Spectral differences were found in the L intermediate for five bands, as shown in traces b in Figure 7. The frequency shift of the CC stretch bands at 1548−1546 cm−1 originates from changes in the π-conjugation system in the polyene chain. The frequency shift of the CN stretch bands at 1647−1644 cm−1 arises from the structural change in the protonated Schiff base. Intensity changes in both the shoulder band at 1010 cm−1 and the band at 1239 cm−1, due to the C15−H wagging and lysine rocking modes, respectively, reflect changes in the interaction between the bound halide ion and the retinal chromophore, giving rise to the A-term RR enhancement factor. Relative intensity changes in the bands at 1444−1438 cm−1 due to the deformation of two methyl groups result from changes in the interaction between the chromophore and bound ion.

isomerization. In the L and N intermediates, high CN stretching frequencies and large deuteration shifts were observed. The strength of the hydrogen bond increased in the K-to-L transition and changed little in the L-to-N transition. A previous low-temperature FTIR study of pHR observed that the N−D stretching frequency of the protonated Schiff base is lower than the frequency of the unphotolyzed state.35 The N− D stretching frequency is a direct probe of the hydrogen bond strength, and thus the lowering of the N−D stretching frequency indicated formation of a strong hydrogen bond in the K intermediate. The opposite conclusions drawn from our RR and previous FTIR studies are probably due to different protein motions at ambient and low temperatures; the retinal chromophore at 77 K is likely to be trapped in the unrelaxed protein structure after photoisomerization, although the structure at room temperature is relaxed on the nanosecond time scales. The bandwidth of the CN stretching mode helps us to identify an acceptor of the hydrogen bond at the protonated Schiff base. The CN stretching frequency of the protonated Schiff base is close to the HOH bending frequency of the H2O molecule. When a water molecule is directly attached to the proton in the Schiff base, the energy transfer would shorten the dephasing time of the CN stretching mode and the bandwidth would broaden in H2O. In the present case of pHR, band broadening in H2O was observed only for the L intermediate. Broadening of the CN stretch band in H2O was also found for the L intermediate of BR.46 The observed broadening of the CN stretch band may provide spectroscopic evidence for the resonance vibrational energy transfer to the water molecule directly attached to the Schiff base proton. In the present study we obtained the spectra shown in Figure 6 of the L intermediate in H2O, D2O, and a 1:1 H2O/D2O mixed buffer. The CN(H) and CN(D) stretching frequencies at 1647 and 1618 cm−1, respectively, are close to the bending frequency of HOH at 1640 cm−1, and they deviate from the HOD bending frequency at 1440 cm−1 and the DOD bending frequency at 1205 cm−1. Thus, the vibrational frequency of the CN stretching mode in the protonated Schiff base can be resonant only with the HOH bending mode in the water molecule. The mixed buffer comprises 25%, 50%, and 25% H2O, HOD, and D2O, respectively. The 25% H2O isomer may be responsible for broadening the CN(H) stretch band due to the resonance vibrational energy transfer, resulting in the observed narrower CN(H) band in the mixed buffer compared with that in the H2O buffer. This observation provides evidence that the changes in bandwidth upon H/D exchange arise from the resonance vibrational energy transfer between the CN stretching and HOH bending modes. In addition, the CN(H) stretching frequency did not change in the pure H2O and mixed buffers, indicating that the structure of the protonated Schiff base does not change upon H/D exchange and strongly supporting the view that the observed band broadening is due to the resonance energy transfer. In low-temperature FTIR spectroscopy35 of the L1 state, the hydrogen bond at the Schiff base is strengthened. The hydrogen bond acceptor is not a halide ion but could be a water molecule. We concluded that the Schiff base proton is directly hydrogen-bonded to a water molecule in the L intermediate. In addition, the observed upshift of the CN stretching frequency upon L formation (Figure 2 and Figure S9 in the Supporting Information) is attributed to the increase in J


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The Journal of Physical Chemistry A In the L intermediate, of the five halide ion dependent bands, the shoulder band at 1010 cm−1 and the bands at 1239, 1444, and 1647 cm−1 in the RR spectrum of the Cl− bound form showed significant deuteration effects (Figure S13 in the Supporting Information). The C15−H wag band at ∼1000 cm−1, the lysine rock band at 1239 cm−1, and the CN stretch band at 1647 cm−1 are coupled with the vibration of the N−H group in the Schiff base. These observations suggest that the halide ion is bound close to the Schiff base in the L intermediate. In addition, we observed a deuteration effect on the band at 1444 cm−1 attributed to the deformation of the two methyl groups in the cytoplasmic side of the chromophore. It is therefore likely that the bound halide ion interacts with the methyl groups located on the cytoplasmic side. In this step, the chloride ion would move across the chromophore to the cytoplasmic side. No deuteration effect was observed on the CC stretch band due to little coupling between the CC stretching mode and the vibration of the N−H group in the protonated Schiff base. The CN(H) stretching frequency and the amplitude of the deuteration shift arise from splitting of the degenerate energy levels between the CN stretching and N−H rocking modes. The pure CN stretching frequency coincides with the C N(D) frequency in D2O due to the lack of coupling with the N−D rocking mode. In the present data, the CN(D) stretch bands had similar frequencies, and thus the pure CN stretching frequency would not change upon exchange of the halide ion. Therefore, changes in the N−H rocking frequency would be solely responsible for the halide dependence of the CN(H) stretching frequencies and deuteration shifts. The halide ion could interact with the vibration of the N−H bond in the Schiff base. Broadening of the CN stretch band in H2O shortens the vibrational lifetime of this mode due to the resonance vibrational energy transfer with the water molecule.46 The Schiff base proton is directly hydrogen-bonded to the water molecule, judging from broadening of the CN stretch band. We also found that the bound halide ion affects the vibrational lifetime of the CN(H) stretching mode in H2O. The charge density of the bound halide ion decreases in the order Cl−, Br−, and I−. All observations pertaining to halide dependence strongly suggested that the halide ion is located very close to the Schiff base. In a low-temperature FTIR study, the lack of halide ion dependence of the water bands suggested that the water molecule attached the Schiff base does not hydrate the halide ion.35 On the other hand, the halide ion dependence of spectral changes in the RR bands, coupled with the vibrational mode in the Schiff base and the widths of the CN(H) stretch bands, indicates that the bound halide ion interacts with both the Schiff base and the water molecule. A recent time-resolved FTIR study revealed that the dangling bond of the water molecule disappeared in the L intermediate.37 According to our time-resolved RR results and recent time-resolved FTIR measurements, we conclude that the protonated Schiff base is hydrogen-bonded to the water molecule that hydrates the pumped halide ion in the L intermediate.

protonated Schiff base. The halide ion bound to the protein affects only the π-conjugated system of the polyene chain and does not interact with the Schiff base structure. Upon K formation, the retinal chromophore changes to the 13-cis, 15anti geometry and photoisomerization accompanies the large distortion at the C7C8 position of the polyene chain. The weak hydrogen bond at the protonated Schiff base can promote exchange of the hydrogen-bond acceptor in the subsequent step. In the L intermediate, the chromophore is in the 13-cis, 15-anti configuration with distortion of the C15 position near the Schiff base. The N−H group in the protonated Schiff base points to the cytoplasmic side. The hydrogen bond acceptor replaces the water molecule, accompanied by an increase in hydrogen-bond strength. The strength of the hydrogen-bond between the protonated Schiff base and the water molecule increases with accompanying L formation. In this step, the halide ion moves across the Schiff base and is hydrated by the water molecule hydrogen-bonded to the Schiff base. In the N intermediate, the chromophore is in a planar 13-cis, 15-anti geometry. The hydrogen bond acceptor at the protonated Schiff base switches from the water molecule to another species, although the hydrogen bond of the protonated Schiff base remains strong.

CONCLUSION Sequential changes in RR spectra obtained during the pHR photocycle provided information on structural changes in the retinal chromophore in pHR. In the unphotolyzed state, the retinal chromophore is dominantly in the all-trans, 15-anti form with a planar geometry. A weak hydrogen bond is formed at the

(1) Terner, J.; Hsieh, C.-L.; Burns, A. R.; El-Sayed, M. A. TimeResolved Resonance Raman Spectroscopy of Intermediates of Bacteriorhodopsin: The bK590 Intermediate. Proc. Natl. Acad. Sci. U. S. A. 1979, 76, 3046−3050. (2) Braiman, M.; Mathies, R. Resonance Raman Evidence for an AllTrans to 13-Cis Isomerization in the Proton-Pumping Cycle of Bacteriorhodopsin. Biochemistry 1980, 19, 5421−5428.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpca.7b12332. Details of time-resolved RR measurements, simple spectral decomposition of the time-resolved RR spectra of pHR, SVD analysis of the time-resolved RR spectra of pHR, CC stretching frequencies, shift of the CN stretching frequency in the L intermediate, time-resolved RR spectra of the pHR chromophore in D2O buffer, single-beam time-resolved RR spectra of the L intermediate, deuteration effects on the RR spectra of the intermediates, and deuteration effects on the RR spectra of pHR bound to Cl−, Br−, and I− (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail address: [email protected], Phone: +81-66850-5776. ORCID

Hideki Kandori: 0000-0002-4922-1344 Yasuhisa Mizutani: 0000-0002-3754-5720 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by JSPS KAKENHI Grant Number JP16K13933 to MM and MEXT KAKENHI Grant Numbers JP23750015 to MM and JP25104006 to YM.

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Structural Evolution of a Retinal Chromophore in the Photocycle of

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