Distortion and a Strong Hydrogen Bond in the Retinal Chromophore

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Distortion and a Strong Hydrogen Bond in the Retinal Chromophore Enable Sodium Ion Transport by the Sodium Ion Pump KR2 Nao Nishimura, Misao Mizuno, Hideki Kandori, and Yasuhisa Mizutani J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.9b00928 • Publication Date (Web): 04 Apr 2019 Downloaded from http://pubs.acs.org on April 5, 2019

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The Journal of Physical Chemistry

Distortion and a Strong Hydrogen Bond in the Retinal Chromophore Enable Sodium Ion Transport by the Sodium Ion Pump KR2 Nao Nishimura,† Misao Mizuno,† Hideki Kandori,‡ and Yasuhisa Mizutani†,* †

Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan ‡

Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Showa-ku,

Nagoya, Aichi 466-8555, Japan

1 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT

We conducted a comprehensive time-resolved resonance Raman spectroscopic study of the structures of the retinal chromophore during the photocycle of the sodium ion pump Krokinobacter rhodopsin 2 (KR2). We succeeded in determining the structure of the chromophore in the unphotolyzed state and in the K, L, M, and O intermediates, by overcoming the problem that only a small fraction of the M intermediate is accumulated in the KR2 photocycle. The Schiff base in the retinal chromophore forms a strong hydrogen bond in the unphotolyzed state and in the K, L, and O intermediates, and is deprotonated in the M intermediate. Formation of this strong hydrogen bond facilitates deprotonation of the Schiff base, which is necessary for the sodium ion to move past the Schiff base. The polyene chain in the chromophore of KR2 is twisted in all the states of the photocycle: the portion near the Schiff base is largely twisted in the unphotolyzed state and in the K intermediate, whereas the middle portion of the polyene chain becomes largely twisted in the L, M, and O intermediates. During the photocycle, the twisted structure of the polyene chain and the strong hydrogen bond at the Schiff base are advantageous for transient relocation of the Schiff base proton. The obtained RR data clarified the unique structural features of the KR2 chromophore, which are not accessible by other methods.


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INTRODUCTION Krokinobacter rhodopsin 2 (KR2) is a newly discovered light-driven ion pump from the marine flavobacterium Krokinobacter eikastus. Each cycle of KR2 pumps a sodium ion outward using light energy under physiological conditions or pumps a proton under conditions of low sodium ion concentration.1-2 The photocyclic reaction of KR2 during sodium ion pumping involves a series of intermediates: the J, K, L, M, and O intermediates.1, 3-4 These intermediates are similar to those of proton pumps, although their kinetics are different.5-8 Absorption of a photon by the retinal chromophore of KR2 (Figure 1A) yields the vibrationally excited J intermediate immediately following the trans-to-cis isomerization of the retinal, which in turn is followed by the formation of the vibrationally relaxed K intermediate.3 The K intermediate relaxes to the L intermediate and these two states exist in equilibrium for timescales ranging from picoseconds to microseconds.4 Subsequently, a new equilibrium is reached between the L and M intermediates in a few tens of microseconds. The M-to-O transition has a time constant of ~1 ms and is accompanied by the uptake of a sodium ion from the cytoplasmic side. Finally, the O intermediate reverts to its initial state over a timescale of hundreds of milliseconds.1 KR2 has a characteristic amino acid sequence compared to other ion pumps.9 For example, in bacteriorhodopsin (BR), which is the best-studied proton pump, Asp85, Thr89, and Asp96 are found in the C-helix and form the DTD motif. Asp85 is a negatively charged proton acceptor of the protonated Schiff base in the retinal chromophore, whereas Asp96 works as a proton donor from the cytoplasmic side. The three residues located at the corresponding positions in KR2 form the NDQ motif: the two negatively charged carboxylic residues in BR are replaced by the neutral amino acid residues Asn112 and Gln123 in KR2, and Asp116 occupies the position corresponding to Thr89 in BR. The crystallographic structure of KR2 was determined soon after



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the discovery of the protein (Figure 1B).10-11 The proton acceptor from the Schiff base in KR2 is different from that in BR, although the two protein structures are overall similar. In KR2, the Schiff base proton points towards Asp116. The deprotonated Asp116 is assumed to act as a proton acceptor for the Schiff base, even though it is located one helical pitch toward the cytoplasmic side compared to the position of Asp85 in BR (Figure 1C). A solid-state NMR study identified the presence of a hydrogen bond between the Schiff base proton and Asp116.12 Prior to the discovery of KR2,1 it was believed that ion-pump rhodopsins could not act as sodium ion pumps because cations other than protons repulsively interact with the positive charge on the protonated Schiff base in the retinal chromophore during the cation pumping process. As a result, the assumption was that cations other than protons were unable to be transported near the protonated Schiff base as part of the ion transport pathway. Deprotonation of the Schiff base is necessary for the sodium ion to avoid electrostatic repulsion near the chromophore. A better understanding of the structural changes in the retinal chromophore and its surroundings during the ion pumping process is required, although the crystallographic structure did shed some light on the mechanism used to avoid electrostatic repulsion. Under acidic conditions, the protonated side chain of Asp116 flips away from the Schiff base and forms hydrogen bonds with Asn112 and Ser70. This flipping motion of Asp116 was proposed to reduce the positive charge density around the Schiff base and enable transport of a sodium ion near the Schiff base.11 However, only an approximate structure of the deprotonated intermediate was reported and the true chromophore structures of the intermediates in the photocycle remain unknown. Time-resolved spectroscopic approaches are useful for obtaining comprehensive structural information on changes to the retinal chromophore during the photocycle. Transient stimulated


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Raman4 and step-scan FTIR13 studies demonstrated that the chromophore remains in the twisted 13-cis structure in the intermediates. These time-resolved vibrational spectra, however, were composites of multiple spectral contributions because more than one intermediate simultaneously appears due to the transient equilibrium between intermediates in the KR2 photocycle.1,

4

Selective observation of the vibrational spectra of a single intermediate using these methods is therefore challenging. Time-resolved resonance Raman (RR) spectroscopy enables us to observe the vibrational spectrum of the retinal chromophore in intermediates with high sensitivity by virtue of resonance enhancement of the scattering intensity. We recently constructed a time-resolved visible RR spectrometer operating on the nanosecond to millisecond time scales with wide wavelength tunability to selectively detect the spectrum of single intermediates in the photocycle. For example, we measured time-resolved RR spectra of the retinal chromophore in halorhodopsin from Natronobacterium pharaonis (NpHR) and revealed the sequential process of structural changes in the retinal chromophore in the K, L, and N intermediates.14 In this study, we used time-resolved RR spectroscopy to determine the chromophore structure of each intermediate during the KR2 photocycle when acting as a sodium ion pump. We succeeded in selectively obtaining a series of transient RR spectra of the retinal chromophore in the unphotolyzed state and in the K, L, M, and O intermediate states, all with high S/N ratios, allowing us to determine the structural evolution of the chromophore during the photocycle. Two characteristic features of the KR2 chromophore structure during sodium ion pumping were identified: the hydrogen bond at the protonated Schiff base is strong in the protonated species, and the polyene chain is twisted in all states during the photocycle. We discuss the mechanism underlying proton relocation between the Schiff base in the retinal chromophore and a nearby



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residue (required to avoid electrostatic repulsion during sodium ion pumping by KR2). Elucidation of this mechanism is based on a comparison of structural changes in the KR2 and BR chromophores during the photocycle.

EXPERIMENTAL SECTION Sample preparation. The KR2 sample was prepared as described previously15 with some modifications. Briefly, Escherichia coli C41(DE3) harboring a plasmid encoding KR2 with a histidine tag at the C-terminus was grown in 2 ×YT medium containing 50 μg/mL ampicillin. In the middle 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 incubation. The redcolored cells were sonicated and the cell membranes were solubilized with 1.5% (w/v) ndodecyl-β-D-maltoside (Annatrace, DDM). The solubilized membranes were purified using a Co2+-affinity column (GE Healthcare, HiTrap TALON crude) and an anion-exchange chromatography column (GE Healthcare, HiTrap Q HP). Purified samples with an absorption ratio at 280 and 526 nm of less than 2.0 were used for spectroscopic measurements. The samples were suspended in a buffer containing 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 300 mM Na2SO4, and 0.1% DDM. The protein concentration was 30 μM. The H2O buffer was exchanged with a D2O buffer as described previously.14 RR measurements. RR scattering of unphotolyzed KR2 was excited using a cw DPSS laser (Cobolt, Samba 04-01) with a wavelength of 532 nm. The sample solution was placed in a 1-cm ϕ glass NMR tube used as a spinning cell. The probe power at the sample point was reduced to 0.25 mW to avoid photolysis of the protein sample. The scattered light was collected and focused onto the entrance slit of a spectrograph (HORIBA Jobin Yvon, iHR320) equipped with a CCD


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camera (Roper Scientific, PyLoN:400B_eXelon VISAR). A long-pass dielectric filter (Asahi Spectra, LV0550) was placed in front of the entrance slit of the spectrograph to eliminate Rayleigh scattering. The Raman shifts were calibrated using the Raman bands of cyclohexane, toluene, and acetone. We adopted a single-beam time-resolved method to measure the transient RR spectra of the short-lived K intermediate and used a nanosecond Q-switched diode-pumped Nd:YAG laser (Megaopto, LR-SHG) to photolyze the sample and probe RR scattering. The wavelength and pulse width of the laser were 532 nm and 20 ns, respectively. The sample solution was circulated in a 1.5-mm ϕ cylindrical glass tube and the scattered light was detected with the same spectrograph and detector described above. The Raman shifts were calibrated using the Raman bands of ethanol, 2-propanol, and acetone. The spectral contribution of the photolyzed sample accumulated during a single laser pulse was obtained by subtracting the contribution of the unphotolyzed sample measured at low laser flux (2.0 μJ) from the spectrum measured at high laser flux (75 μJ). The data acquisition methods were as described previously14, with modifications. The transient RR spectra of the L, M, and O intermediates were measured using a dual-beam rapid-flow method described previously.14 RR spectra of the L intermediate were obtained using the 475 nm beam (the second harmonic of the output of a Ti:sapphire laser; Photonics Industries, TU-L) and the 532 nm beam (the output of the cw DPSS laser) was used to continuously initiate the photoreaction of the sample. The sample solution was circulated in a rectangular-shaped (1×4 mm2) glass cell. Typical probe and pump beam power inputs at the sample point were 1.6 and 100 mW, respectively. The delay time was set to 80 μs by changing the spatial separation between the probe and pump beams. The scattered light was collected and focused onto the



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entrance slit of a Czerny-Turner configured Littrow prism prefilter (Bunkoukeiki, PF-200MP) coupled with a spectrograph (HORIBA Jobin Yvon, iHR550) and detected with a CCD camera (Roper Scientific, SPEC-10:400B/LN-SN-U). The RR spectra of the M and O intermediates were measured using the 405 and 594 nm beams as the probe beams. The former and the latter were the output of a cw diode laser (Innovative Photonic Solutions, Model #I0405SD0050B) and of a cw DPSS laser (Cobolt, Mambo 04-01), respectively. The pump beam was the output of an Ar+ laser (Spectra Physics, BeamLok) with a wavelength of 514.5 nm. Typical probe and pump beam power inputs at the sample point were respectively 4.0 and 100 mW for the M spectra measurements and 2.0 and 100 mW for the O spectra measurements. The sample solution was circulated in a 1.5-mm ϕ cylindrical glass tube. The delay time was set to 100 μs and 1 ms to obtain the M and O spectra, respectively. The scattered light was collected and focused onto the entrance slit of a spectrograph (HORIBA Jobin Yvon, iHR320) equipped with a CCD camera (Roper Scientific, PyLoN:400B_eXelon VISAR). A holographic super Notch filter (Kaiser Optics) was placed in front of the entrance slit of the spectrograph for each probe wavelength. The Raman shifts were calibrated using the Raman bands of ethanol, 2-propanol, and acetone. The observed spectral shape probed at 405 and 594 nm were affected by sensitivity variations at each pixel in the CCD camera because the weak Raman signals were overlapped with a strong background signal due to emission by the protein sample. The sensitivity of the detector was calibrated using the fluorescence spectra of standard samples as described previously,16-17 with modifications. A detailed description is provided in Section 1 of the Supporting Information. The spectrum of the M intermediate was obtained by exciting at 405 nm and additional bands were observed, as shown in Figure S1 in the Supporting Information. These additional bands are


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likely due to the deprotonated chromophore in incorrectly folded KR2. Spectra of the retinal chromophore in the correctly folded protein were obtained by subtracting the spectral contribution of incorrectly folded protein from the raw spectra, as described in Section 1 of the Supporting Information.

RESULTS Figure 2 shows RR spectra of the retinal chromophore in KR2. Trace a is the RR spectrum of the unphotolyzed state and traces b−e represent transient RR spectra of the intermediates obtained by subtracting the spectral contribution of the unphotolyzed state from the photolyzed spectrum measured under each experimental condition. The transient RR spectra of the K, L, M, and O intermediates were observed using the probe wavelengths 532, 475, 405, and 594 nm, respectively, based on the transient absorption spectra reported previously.1 Because the K intermediate is generated with a time constant of 500 fs after photoexcitation3 and decays within several microseconds,1 we used a laser pulse duration of 20 ns to photolyze the sample and obtain the K intermediate spectrum, allowing us to conduct single-color time-resolved measurement. Pump-probe measurements were used to obtain the spectra of the L, M, and O intermediates. To obtain the delay times of the major populations for each intermediate, we measured the temporal behaviors of the absorbance changes at each probe wavelength (Figure S2 in the Supporting Information). In H2O, the largest positive absorbance changes due to the L, M, and O intermediates at 475, 405, and 594 nm appeared at delay times of 80 μs, 100 μs, and 1 ms, respectively, and we measured time-resolved RR spectra at these delay times. The observed bands in Figure 2 were assigned to the respective vibrational modes based on the normal mode analysis of the retinal chromophore in BR reported by Smith and coworkers.18-19



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The bands in the 1619−1656, 1524−1602, 1370−1449, 1272−1356, 1165−1243, 1004−1010, and 800−1000 cm−1 regions are ascribed to the C=N stretching modes at the Schiff base, the ethylenic C=C stretching modes, deformations of the methyl groups, the C−CH/C−NH rocking modes, the skeletal C−C stretching modes, the methyl rocking modes, and the hydrogen-out-ofplane (HOOP) wagging modes, respectively. We also measured the static and transient RR spectra of KR2 in D2O, as shown in Figure S3 in the Supporting Information. Because the largest positive absorbance change at each probe wavelength in D2O was similar to that in H2O (Figure S2 in the Supporting Information), the delay times for measurement of the RR spectra of the L, M, and O intermediates in D2O were set to 80 μs, 100 μs, and 1 ms, respectively. Significant spectral changes were observed for the C=N stretch and the HOOP bands following deuteration. C=C stretch bands. As shown in Figure 2, the strongest band in the ethylenic C=C stretch band region was observed at 1532 cm−1 in trace a, and it appeared at 1531, 1542, 1567, and 1524 cm−1 in traces b−e, respectively. In the L intermediate, a relatively strong shoulder band at 1552 cm−1 was observed on the band at 1542 cm−1 (trace c in Figure 2). This doublet feature is similar to that observed in the RR spectra of the L intermediate of BR.20-21 The observed C=C stretch band consists of two bands: the band with the lower frequency is assigned to in-phase C=C stretching vibrations and the band with the higher frequency is due to out-of-phase vibrations.1819

The spectrum of the M intermediate (trace d in Figure 2) shows a shoulder band at 1544 cm−1

near the 1567 cm−1 band. The frequency of the shoulder band (1544 cm−1) is similar to that of the strongest band observed at 1542 cm−1 in the spectrum of the L intermediate. Moreover, the intensity of the 1544 cm−1 band increased relative to the intensity of the 1567 cm−1 band under longer probing wavelengths conditions (Figure S4 in the Supporting Information). The shoulder


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band in the spectrum of the M intermediate is due to the C=C stretch band of the coexisting L intermediate at 100 μs and can be resonantly enhanced with 405 nm Raman excitation. The C=C stretching frequencies in the intermediates were close to the frequencies observed in the recently reported transient RR spectra of another sodium ion pump from Indibacter alkaliphilus.22 It should be noted that the in-phase C=C stretching frequency showed an inverse correlation with the absorption maximum wavelength for each KR2 intermediate. The absorption maximum wavelengths of KR2 are red-shifted in the K and O intermediates compared to the maximum wavelength in the unphotolyzed state,1 whereas they are blue-shifted in the L intermediate and greatly blue-shifted in the M intermediate. The C=C stretch band at 1531 cm−1 of the K intermediate was broad compared to that of the 1532 cm−1 band of the unphotolyzed state. This suggests that the band at 1531 cm-1 involves two or more components. We conducted a band fitting analysis using two Lorentzian functions for the C=C stretch band as shown in Figure S5 in the Supporting Information. As shown in trace b in Figure S5, it was found that the band can be described as a sum of two Lorentzian bands at 1527 and 1538 cm−1. The former and the latter are assigned to an in-phase and out-of-phase stretching modes, respectively.18-19 The in-phase C=C stretching frequency of the K intermediate was determined to be 1527 cm−1. The 5-cm−1 lower in-phase C=C stretch frequency of the K intermediate than the unphotolyzed state is consistent with the red-shifted absorption peak of the K intermediate. Similarly to the K intermediate, the strongest C=C stretch band was observed as a doublet feature in the L intermediate and showed a asymmetric band shape in the M and O intermediates. Based on the band fitting analysis, the inphase stretching frequencies were determined to be 1540, 1569, and 1520 cm−1 in the L, M, and O intermediates, respectively. The frequency of the strongest in-phase C=C stretch band exhibits



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an inverse linear correlation with the maximum wavelength of a distinct absorption band of each intermediate, as reported for transient RR spectra of the BR intermediates.23 C−C stretch bands. Skeletal C−C stretch bands at 1165, 1171, 1187, and 1199 cm−1 were observed in the spectrum of the unphotolyzed state (trace a in Figure 2). The spectral features were similar to that of BR568 containing an all-trans chromophore,18 indicating that the retinal is in the all-trans configuration in the unphotolyzed state of KR2. In comparison, the intensity of the 1165 cm−1 band decreased in the intermediates and the intensities of the band at 1185−1187 cm−1 increased (traces b−e in Figure 2). These bands were observed in both BR54819 and the photointermediates of BR containing a 13-cis chromophore.21,

23-28

The present observations

suggest that the chromophore adopts the 13-cis form in all KR2 intermediates, consistent with a recent step-scan FTIR study of KR2 showing that the chromophore is in the 13-cis form in the intermediates.13 In the O intermediate, the retinal in KR2 adopts the 13-cis configuration, in contrast to the all-trans configuration found in the O intermediate of BR.29 We did not observe significant differences in the C−C stretch bands upon H/D exchange, except for a slight decrease in band intensity at 1187 cm−1 in the unphotolyzed state. The C−C stretching modes are coupled with the C−H and N−H rocking modes. The coupling between the C14−C15 stretching and the N−H rocking modes can be used to determine the configuration of the C15=N bond.20, 24, 30 If the C15=N bond is in the 15-syn configuration, a large coupling results in a pronounced deuteration shift of the C14−C15 stretch band, from 1167 to 1208 cm−1, as observed in BR548. In contrast, if the C15=N bond is in the 15-anti configuration, a weak rock-stretch coupling makes the C14−C15 stretching frequency insensitive to deuteration, analogous to that observed for BR568. The present observations indicate that the C15=N bond in the chromophore


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of KR2 remains in the 15-anti configuration in the unphotolyzed state and in all intermediates of the photocycle. C=N stretch bands. To further investigate the structural evolution at the Schiff base region of the chromophore during the photocycle, we focused on spectral changes in the C=N stretch band at the Schiff base. Figure 3 shows expanded views of the RR spectra of the C=N stretch band region for each intermediate. In H2O (darker colored traces for each intermediate), the band at 1640 cm−1 in the unphotolyzed state downshifted to 1635 cm−1 in the K intermediate and upshifted to 1656 cm−1 in the L intermediate, whereas two bands appeared (at 1619 and 1656 cm−1) in the spectra of the M intermediate. The relative intensity of the 1619 cm−1 band to the 1656 cm−1 band increased under shorter probing wavelengths conditions (Figure S4 in the Supporting Information). Thus, the band at 1619 cm−1 is attributed to the M intermediate and the band at 1656 cm−1 is due to the L intermediate coexisting at 100 μs. In the O intermediate, the C=N stretch band upshifted to 1642 cm−1. We examined deuteration effects on the C=N stretch band in each intermediate. The lighter colored traces in Figure 3 are the spectra measured in D2O. In the unphotolyzed state, the band at 1640 cm−1 in H2O shifted to 1618 cm−1 in D2O (trace a in Figure 3). In the K, L, and O intermediates, the bands at 1635, 1656, and 1642 cm−1 shifted to 1615, 1621, and 1615 cm−1 following deuteration, respectively, whereas the band at 1619 cm−1 did not shift in the M intermediate (traces b−e in Figure 3). In the protonated Schiff base, the C=N stretching mode is strongly coupled with the N−H rocking mode. Coupling between these two modes increases the observed C=N stretching frequency compared to the intrinsic C=N stretching frequency. Deuteration of the Schiff base gives rise to decoupling between the two modes, resulting in a downshift of the C=N stretch band. If the Schiff base is deprotonated, no frequency shift is



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observed. The present observations show that the Schiff base is protonated in the unphotolyzed state and in the K, L, and O intermediates, and is deprotonated in the M intermediate. The deuteration shifts for the C=N stretch band of the protonated Schiff base in the unphotolyzed state and in the K, L, and O intermediates were 22, 20, 35, and 27 cm−1, respectively. In the unphotolyzed state, the observed frequency and amplitude of the deuteration shift for the C=N stretch band were comparable to those of another sodium ion pump22 and of proton pumps,18,

31-34

and larger than those of chloride ion pumps.14,

35-37

The observed

frequencies and amplitudes of the deuteration shifts of the intermediates were larger than those of other ion pumps.14, 21, 23-26, 31, 37 The stronger the hydrogen bond in the protonated Schiff base, the higher the frequency of the C=N stretch band and the larger the deuteration shift.38 The observed large deuteration shifts for KR2 indicate that the hydrogen bond is very strong at the protonated Schiff base in all the protonated intermediates. The strong hydrogen bond is highly likely required for sodium ion pumping. Notably, the amplitude of the deuteration shift in the L intermediate is the largest of all the intermediates, indicating that a strong hydrogen bond is formed and lowers the barrier height for transfer of the Schiff base proton. We compared the width of the C=N stretch band in H2O with that in D2O as shown in Figure S6 in the Supporting Information. If the proton acceptor of the Schiff base is a water molecule, resonance vibrational energy transfer results in broadening of the C=N stretch band only in H2O because of the coupling between the C=N stretching mode at the protonated Schiff base and the HOH bending mode of the nearby water molecule.39 The bandwidth was insensitive to deuteration of the Schiff base for the unphotolyzed state and the L and O intermediates, indicating the absence of the resonance energy transfer. In the spectra of the K intermediate, the 1615 cm−1 band in D2O looked broader than the 1635 cm−1 band in H2O. The broadening in D2O


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observed in the spectrum of the K intermediate is not due to the resonance energy transfer, because the DOD bending frequency of approximately 1240 cm−1 does not match the C=N stretching frequency of 1615 cm−1. The broadening in D2O is because the C=C stretch band appearing around 1600 cm−1 overlaps the C=N stretch band in D2O. Accordingly, the present observation suggests that a water molecule is not the hydrogen bond acceptor of the protonated Schiff base in the K intermediate. In addition, band shape of the higher frequency side of the C=N stretch band in D2O was similar to that in H2O as shown in trace b in Figure S6 in the Supporting Information, implying that the broadening of the C=N stretch band did not occur in the K intermediate. In the M intermediate, the Schiff base is deprotonated, resulting in loss of the coupling between the C=N stretching mode and the bending mode of the water molecule. Thus, the width of the C=N stretch band did not change. The present observation suggests that for KR2, a water molecule is not the acceptor of the Schiff base proton during the whole photocycle; rather, Asp116 is the most probable candidate, as proposed by previous studies.10-11 In the unphotolyzed state, this result is consistent with the crystallographic structure showing that no water molecule locates in the direction of the hydrogen bond from the Schiff base proton. In the intermediates, the present results are in contrast to observations of other ion pumps, such as BR23, 40

and NpHR,14 in which the proton acceptor is a water molecule. The strong hydrogen bond

between the protonated Schiff base and Asp116 is characteristic of sodium ion pumping by KR2, including in the photointermediates. HOOP bands. The HOOP bands are resonantly enhanced when the polyene chain is twisted. Thus, the appearance of HOOP bands suggests distortion of the retinal chromophore. Figure 4 shows expansions of the RR spectra of the HOOP bands region. The darker colored traces were measured in H2O buffer and intense HOOP bands were observed in both the intermediates and



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unphotolyzed state. This is contrast to the RR spectra of BR, in which very weak HOOP bands are observed in the unphotolyzed state.18 The present observation indicates that the polyene chain in the retinal chromophore is largely twisted throughout the photocycle and is characteristic of the spectra of KR2. Based on normal mode analyses of all-trans-retinal,41 13-cis-retinal,42 and the retinal chromophore in the unphotolyzed state and in intermediates of BR,18-19,

23, 29

vibrational assignments for the observed HOOP bands of KR2 were conducted and are summarized in Table 1. We compared the RR spectra measured in D2O (the lighter colored traces in Figure 4) to those measured in H2O. Significant deuteration effects on the HOOP bands were observed in the spectra of the unphotolyzed state and of the K intermediate, whereas the HOOP bands in the L, M, and O intermediates were insensitive to deuteration of the Schiff base. Spectral contamination was closely examined for traces in Figure 4. Details of the examination are described in Section 1 of the Supporting Information. In the unphotolyzed state (trace a in Figure 4), spectral changes were clearly observed for the strong bands at 1006 and 877 cm−1 following deuteration. The 1006 cm−1 band comprises contributions from the C15H=NH in-phase (Au mode in the 15-anti configuration with an approximately C2h local symmetry) wagging mode, as observed in the RR spectrum of the L intermediate of BR.20, 23, 43 The 877 cm−1 band is attributed to the C14H HOOP mode. In addition, other HOOP bands at 971, 961, 900, 845, 829, and 822 cm−1 were observed and are due to C7H=C8H (Au mode in the local trans configuration), C11H=C12H (Au in the trans), C10H, C7H=C8H (Bg), C11H=C12H (Bg), and Lys wagging modes, respectively. The appearance of a number of HOOP bands suggests that the twist is distributed throughout the polyene chain in the unphotolyzed state. The intensities of the bands due to C15H=NH and C14H wagging were strong


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compared to that in the unphotolyzed states of other ion pumps,14, 20, 24, 34, 37, 44-46 indicating that the polyene chain near the Schiff base is considerably twisted in KR2. The determined structure is consistent with that observed by solid-state NMR: namely, that the Schiff base region is twisted.12 In the K intermediate, deuteration effects were apparent for the bands at 1004 and 778 cm−1 (trace b in Figure 4), with the former being assigned to the C15H=NH (Au) wagging mode and the latter to the C14H wagging mode characteristic of the 13-cis configuration.19 Other HOOP bands were observed at 970 and 956 cm−1 and are due to the Au modes of C7H=C8H and C11H=C12H wagging.19 The appearance of HOOP bands in the K intermediate spectra indicates that the polyene chain is twisted not only near the protonated Schiff base, but also in the middle region of the retinal chromophore after photoisomerization. In the L intermediate (trace c in Figure 4), little deuteration effect was observed, except for the band at 981 cm−1 in D2O which is due to the N−D rocking mode at the Schiff base whose N−H rocking frequency is about 1350 cm−1. The observed bands at 1008, 951, 893, 859, and 799 cm−1 are assigned to the methyl rocking mode and the C11H=C12H (Au), C10H, C7H=C8H (Bg), and C14H wagging modes, respectively. The K-to-L transition causes an increase in intensity of the C11H=C12H wag band, indicating that the C11=C12 bond is largely twisted, while a decrease in intensity of the C15H=NH wag band suggests that distortion near the Schiff base is reduced. In the M intermediate (trace d in Figure 4), the HOOP bands were not affected by H/D exchange because the Schiff base is deprotonated. The 1010 cm−1 band is due to the methyl rocking mode. The bands observed at 953 and 892 cm−1 can be assigned to the C11H=C12H (Au) and C10H wagging modes, respectively. These observations suggest that the polyene chain is twisted at the bonds at the C10, C11, and C12 positions. The appearance of the strong C11H=C12H



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wag band was similar to that observed in the spectrum of the L intermediate and indicates that the polyene chain retains a similar structure in the L and M intermediates, consistent with the experimental finding that these two intermediates are in equilibrium in the KR2 photocycle.1 In the O intermediate (trace e in Figure 4), the frequencies of the HOOP modes changed little upon deuteration. The bands at 1006, 954, 945, 892, and 805 cm−1 were attributed to the methyl rocking mode and the C11H=C12H (Au), C7H=C8H (Au), C10H, and C14H wagging modes, respectively. These bands suggest that the middle of the chromophore is twisted whereas the polyene chain near the Schiff base is less twisted. The present RR study shows that the polyene chain in the retinal chromophore of KR2 remains twisted in all the species in the photocycle. The vibrational assignments of the observed HOOP bands allowed us to identify the twisted positions for each species: the polyene chain is largely twisted near the Schiff base, especially in the unphotolyzed state and in the K intermediate, while it is twisted at the middle of the chain in the L, M, and O intermediates. Our findings regarding polyene chain distortion are supported by recent stimulated Raman3 and FTIR12,

14

studies

showing that several HOOP bands appear as transient signals following photoisomerization. Furthermore, our results regarding the persistence of the twisted chromophore in the photointermediates are consistent with previous spectroscopic studies of KR2. However, there are differences between the present RR and previous spectroscopic results. First, only our present RR spectra show intense HOOP bands in the unphotolyzed state. These intense bands demonstrate that the chromophore is twisted in the unphotolyzed state. In contrast, previous reports4, 13, 15 showed unclear HOOP bands in the unphotolyzed state. This discrepancy is due to differences in obtaining the spectra of the unphotolyzed state. The spectra reported previously were represented as difference spectra between the unphotolyzed state and the intermediates, and


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thus the intensities of the HOOP bands of the unphotolyzed state were likely cancelled out with the transient signals. Second, in the previous time-resolved FTIR study, the authors observed peaks at ~990 and ~950 cm−1 for all the intermediates, with the former peak being sensitive and the latter insensitive to deuteration. The band at ~990 cm−1 led the authors to propose that the distortion near the Schiff base is retained in all the intermediates,13 contrary to our RR result that the polyene chain near the Schiff base is less twisted in the L, M, and O intermediates. This difference is likely due to several intermediates coexisting in equilibrium during the KR2 photocycle and thus the multiple spectral contributions cannot be distinguished in the FTIR spectra. The band at ~990 cm−1 may originate from a vibrational mode of the protein moiety. Comparison of the polyene chain structures of KR2 and BR. The obtained information on the distortion of the retinal chromophore enables us to discuss the sodium ion pumping mechanism. Here, we summarize the insights into the chromophore structure resulting from the present RR study and discuss the evolution of this structure based on the observed RR spectra of all the photointermediates in the photocycle. Figure 5 depicts the structural evolution of the KR2 chromophore in comparison with the BR chromophore. The sodium ion pumping mechanism was elucidated by noting changes in the polyene chain distortion of the retinal chromophore. As reported previously, the hydrogen bond acceptor from the protonated Schiff base in the unphotolyzed state is located at different positions in the KR2 and BR proteins (Figure 1B). We thus postulate that the polyene chain structure can be a determining factor for the direction of proton transfer. Here, we compare the polyene chain structures in KR2 and BR, based on the RR spectra of the unphotolyzed state and of the L intermediate. In the unphotolyzed state, the polyene chain is largely twisted near the Schiff base in KR2, as shown by the intense HOOP bands due to C14H and C15H=NH wagging (Figure 4A). In contrast,



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in the RR spectrum of the unphotolyzed state of BR (Figure S7A in the Supporting Information), the HOOP bands at 958 and 881 cm−1 are moderate in intensity and are assigned to C11H=C12H and C14H wagging, respectively. The deuteration effect for the band at 1007 cm−1 was insignificant, indicating that this band is not attributed to the C15H=NH wagging mode but solely to the methyl rocking mode. These differences between the KR2 and BR spectra suggest that the polyene chain near the Schiff base is less twisted in BR compared to KR2 (Figure 5A and E). The distortion of the polyene chain near the Schiff base in KR2 would keep the Schiff base pointed toward the hydrogen-bond partner of the Schiff base, namely, Asp116 (Figure 5A). The L intermediate appears prior to formation of the deprotonated M intermediate. The M intermediate plays a key role in the ion pumping mechanism. Comparison of the RR spectra of KR2 and BR showed different spectral features for the HOOP bands. For KR2, appearance of the strong HOOP band at 951 cm−1 (trace c in Figure 4) indicated large distortion of the polyene chain at the C11=C12 bond in the middle portion of the chromophore. In addition, because the observed HOOP bands were insensitive to deuteration of the Schiff base, the polyene chain close to the Schiff base is more planar compared to that in the unphotolyzed state of KR2. Formation of the hydrogen bond between the Schiff base and Asp116 in the L intermediate of KR2 requires the middle part of the chromophore to be largely twisted to compensate for the planar structure near the Schiff base. In the spectrum of BR (Figure S7B in the Supporting Information), a significant deuteration effect was detected for the strong band at 1006 cm−1, and the intensities of the other HOOP bands were very weak, suggesting that the polyene chain is largely twisted near the Schiff base in BR. This distortion of the BR chromophore is probably due to the unrelaxed structure of the chromophore following the isomerization of retinal. We found that the twisted position in the retinal chromophore of the L intermediate is different between KR2 and BR and is


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likely due to the hydrogen-bond partner being located at different positions in the two proteins (Figure 5B and F). This difference in the twisted positions in the polyene chain prior to deprotonation of the Schiff base can determine the direction of proton transfer from the Schiff base.

DISCUSSION In this study, we observed the transient RR spectra of all the photointermediates in the photocycle of KR2. The resulting insights into changes in the chromophore structure enable us to discuss the mechanism by which sodium ion is pumped out of a cell. Prior to this study, the RR spectrum of the M intermediate had not been reported due to low transient accumulation of the intermediate in the photocycle, as revealed by transient absorption measurements.1 The successful observation of the RR spectrum of the M intermediate reported here provides an elusive missing link in the structural evolution in the photocycle of KR2. The proton of the Schiff base in the retinal chromophore is key to the sodium ion pumping mechanism because the proton is electrostatically repulsive to the sodium ion being transported in the protein. In proton pumping, electrostatic repulsion between the proton of the Schiff base and the transported proton is avoided by the act of proton pumping itself. In contrast, sodium ion pumping requires a mechanism to avoid repulsion between the proton of the Schiff base and the transported sodium ion, without release of the proton from the protein. It was previously proposed that the proton of the Schiff base is transiently relocated in Asp116 (which locates close to the Schiff base of the chromophore) and is transferred back to the Schiff base after a sodium ion moves past the chromophore.11



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The crystallographic structure of the unphotolyzed state of KR2 shows a distinct difference in the relative position between the Schiff base and its proton acceptor from that of BR (Figure 1B). The hydrogen-bond partner of the Schiff base in KR2 is Asp116, which is proposed to be a proton acceptor in the M-like state under acidic conditions.11 In BR, the protonated Schiff base is hydrogen-bonded to a water molecule in the hydrogen-bond network that includes Asp85 as a proton acceptor from the Schiff base.47 The 116th position in KR2 is shifted from the 85th position in BR by one helical pitch. The N−H bond of the Schiff base in KR2 points in a different direction from that in BR. Next, we discuss the mechanism of proton transfer from the Schiff base based on the structural evolution of the retinal chromophore in the KR2 photocycle. Structure characteristics of the KR2 chromophore. The observed spectra revealed unique structural features of KR2 chromophore. The polyene chain in the retinal chromophore remains twisted in all the species in the photocycle, although the position of the distortion changes during the photocycle. The polyene chain near the Schiff base is largely twisted in the unphotolyzed state and in the K intermediate, whereas it relaxes and becomes planar and instead the middle portion of the polyene chain becomes largely twisted in the L, M, and O intermediates. The Schiff base of the KR2 chromophore is protonated in all species except the M intermediate. The hydrogen bond of the protonated Schiff base of KR2 is stronger than those of other ion pumps, based on the amplitude of the deuteration shift of the C=N stretch band.14, 21, 23-26, 31, 37 These structural features provide critical clues to the mechanism by which KR2 avoids electrostatic repulsion between the positive charges of the transported sodium ion and the proton in the Schiff base when the sodium ion moves past the retinal chromophore. Previous crystallographic study under acidic conditions proposed that the Schiff base proton is relocated to Asp116 to remove electrostatic repulsion between the Schiff base and the transported sodium ion.11 In this study, we


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claim that the distorted polyene chain and the strong hydrogen bond in the L intermediate (Figure 5B) is preferable to the proton relocation from the Schiff base to Asp116. After the sodium ion is translocated to the extracellular side of the chromophore (Figure 5C), Asp116 restores the proton to the Schiff base (Figure 5D). The structural features revealed by the present study are associated with the mechanism of proton relocation between the Schiff base and Asp116 to allow ion pumping by KR2. First, we found that the hydrogen bond of the protonated Schiff base in the unphotolyzed state and in the K, L, and O intermediates of KR2 is very strong. We note that in the L intermediate (the precursor of the deprotonated M intermediate), the hydrogen bond of the protonated Schiff base is strong and thus can lower the barrier height for proton transfer from the Schiff base to Asp116. The formation of a strong hydrogen bond at the protonated Schiff base prior to deprotonation of the Schiff base is a common feature in KR2 as shown here and in proton pumps, such as in BR (which shows a large deuteration shift of the C=N stretch band).21, 23 Thus, the strong hydrogen bond of the protonated Schiff base in KR2 helps avoid electrostatic repulsion by accelerating the removal of the proton from the chromophore. Second, a number of HOOP bands were observed for all species in the photocycle, indicating that the polyene chain is twisted in both the unphotolyzed state and in the intermediates. In contrast, the polyene chain in the unphotolyzed state is planar for ion pumps reported to date. Then, the polyene chain is twisted in the primary intermediate following isomerization and relaxes and becomes planar in the subsequent intermediates. Therefore, distortion of the retinal chromophore in all states is characteristic of KR2. Polyene chain distortion of the retinal chromophore in KR2 is essential to direct the N−H bond to Asp116 for proton relocation.



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Proton transport and proton relocation. The Schiff base is protonated in the unphotolyzed state in ion-pumping rhodopsins. Sodium ion pumps require transient deprotonation of the Schiff base in the photocycle, even though they do not pump protons. It is interesting to correlate the relative position of the proton-accepting residue with respect to the Schiff base with the pumping function. In KR2, Asp116 receives a proton from the Schiff base during the L-to-M transition (Figure 5B). In the M intermediate, Asp116 flips the positive charge away from the Schiff base, causing the sodium ion to transport across the chromophore to the extracellular side of the cell (Figure 5C), then the proton is restored from Asp116 to the Schiff base during the M-to-O transition (Figure 5D). Thus, Asp116 functions as a transient reservoir for an unfavorable proton, permitting sodium ion transport. The distortion of the middle portion of the chromophore, as well as the planar structure at the Schiff base, leads to a strong hydrogen bond between the protonated Schiff base and Asp116. The twisted structure of the chromophore and the strong hydrogen bond of the Schiff base observed for KR2 are advantageous for proton relocation from the Schiff base to Asp116 and vice versa. In BR, in contrast, the proton is transported through the space surrounded by the transmembrane helices, from the cytoplasmic side to the extracellular side. Asp85 receives a proton from the Schiff base during the L-to-M transition (Figure 5F and G). Another proton comes from the cytoplasmic side, then the Schiff base is reprotonated in the N intermediate (Figure 5H). Thus, Asp85 functions as a mediator of proton relay. As discussed above, the purpose of proton release from the Schiff base is different between BR and KR2. In BR, this release is part of the proton transport mechanism whereas in KR2 it is the avoidance of electrostatic repulsion. Asp85 in BR and Asp116 in KR have different roles to achieve the respective purpose as discussed by Kato et al.48 We propose that the positions of the Asp residues are optimized for their roles. The transfer of a proton in the forward direction is


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required for proton pumping whereas lateral transfer of a proton is preferable for transient storage of the proton during sodium ion pumping because the proton is not pumped in either direction. Asp116 locates at the optimal position for lateral transfer in KR2. The distortion of the polyene chain and the strong hydrogen bond of the protonated Schiff base are essential for proton relocation at the given positions of Asp116 and the Schiff base.

CONCLUSION We report RR spectra of the retinal chromophore for the unphotolyzed state and all the photointermediates in the photocycle of the light-driven sodium ion pump KR2. The observed spectra revealed unique structural features of the chromophore. The Schiff base proton in the retinal chromophore forms a strong hydrogen bond in the unphotolyzed state and in all the photointermediates except the M intermediate, in which the Schiff base is deprotonated. The polyene chain is twisted throughout the photocycle: near the Schiff base it is largely twisted in the unphotolyzed state, whereas it relaxes to be more planar and the middle portion of the polyene chain becomes highly twisted in the intermediates. We propose that these unique structural features are essential to proton relocation between the Schiff base and Asp116 to avoid electrostatic repulsion between the positive charges on the Schiff base and the pumped sodium ion in the key step in sodium ion pumping by KR2.



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Figure 1. (A) Structure of the retinal chromophore. (B) Comparison of the crystallographic structure around the retinal chromophore between KR2 (left, PDB ID 3X3C) and BR (right, PDB ID 1C3W). Red arrows represent the proposed direction of proton transfer from the Schiff base.


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Figure 2. RR spectra of the retinal chromophore in KR2. (a) RR spectrum of the unphotolyzed state probed at 532 nm. (b) Transient RR spectrum of the K intermediate probed at 532 nm using the single-beam time-resolved method (delay time < 20 ns). (c) Transient RR spectrum of the L intermediate probed at 475 nm after photoexcitation by 532 nm pump beam irradiation (delay time 80 μs). (d) Transient RR spectrum of the M intermediate probed at 405 nm after photoexcitation by 514.5 nm pump beam irradiation (delay time 100 μs). (e) Transient RR spectrum of the O intermediate probed at 594 nm after photoexcitation by 514.5 nm pump beam irradiation (delay time 1 ms). Spectral contributions of the buffer and emission background were subtracted from each spectrum. The spectra were normalized to the intensity of the band at 1199 cm−1 for the unphotolyzed state and that of the band at 1185−1187 cm−1 for each intermediate. The asterisks represent the band due to the sulfate ion added as an intensity standard.



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Figure 3. Expanded view of the RR spectra of the C=N stretch band region, 1590−1690 cm−1. (a) Unphotolyzed state, and (b) K, (c) L, (d) M, and (e) O intermediates. Darker and lighter colored traces represent the spectra measured in H2O and D2O buffer, respectively, and were normalized to the intensity of the C=N stretch band in each state. Spectral contributions of the buffer and emission background were subtracted from each spectrum.


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Figure 4. Expanded view of the RR spectra of the HOOP bands region, 760−1060 cm−1. (a) Unphotolyzed state, and (b) K, (c) L, (d) M, and (e) O intermediates. Upper dark and lower light colored traces represent the spectra measured in H2O and D2O buffer, respectively. Spectral contributions of the buffer and emission background were subtracted from each spectrum. The asterisks represent the band due to the sulfate ion added as an intensity standard.



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Figure 5. Comparison of the chromophore structure in the photocycle of KR2 (A−D) and BR (E−H). Schematic drawing of the retinal chromophore in the unphotolyzed state (A, E), the L intermediate (B, F), the M intermediate (C, G), and later intermediates (D: the O intermediate for KR2, H: the N intermediate for BR). Purple lines show the twisted positions of the polyene chain, identified based on the RR spectra. Red and orange arrows represent the motions of the proton and the sodium ion, respectively. The present study revealed the structural evolution of the KR2


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chromophore as follows. The retinal chromophore is in the all-trans, 15-anti configuration and is protonated in the unphotolyzed state. Upon photoexcitation, the chromophore is isomerized to the 13-cis configuration in the photointermediates and the 13-cis, 15-anti form remains throughout the cycle in the K, L, M, and O intermediates. The Schiff base is deprotonated during the L-to-M transition and reprotonated during the M-to-O transition. The O intermediate of KR2 showed combined features of the N and O intermediates of BR. The protonated Schiff base forms a strong hydrogen bond to Asp116 in the unphotolyzed state and all protonated intermediates. The polyene chain retains its twist in all species during the photocycle to maintain the direction of the Schiff base proton towards Asp116.



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Table 1. Assignment of the observed HOOP bands

state unphotolyzed state

K intermediate

L intermediate

M intermediate

O intermediate

frequency / cm−1 in H2O in D2O 1006 1011 990 971 971 961 961 900 900 877 880 845 850 829 829 822 nd 1004 1009 988 970 969 956 956 778 796 1008 1008 981 951 952 893 893 859 862 799 800 1010 1011 953 953 892 891 1006 1007 954 954 945 944 892 893 805 803

description methyl rock C15H C7H=C8H Au C11H=C12H Au C10H C14H C7H=C8H Bg C11H=C12H Bg Lys wag methyl rock C15H C7H=C8H Au C11H=C12H Au C14H methyl rock N−D rock C11H=C12H Au C10H C7H=C8H Bg C14H methyl rock C11H=C12H Au C10H methyl rock C11H=C12H Au C7H=C8H Au C10H C14H


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ACKNOWLEDGMENT The authors thank Dr. Rei Abe-Yoshizumi and Dr. Shota Ito at Nagoya Institute of Technology for providing the BR samples for RR measurements. This work was supported by JSPS KAKENHI Grant Number JP16K13933 to MM, MEXT KAKENHI Grant Number JP25104006 to YM, and JP25104009 to HK.

Supporting Information. Details of data corrections, transient absorption spectra of KR2, resonance Raman spectra of the KR2 chromophore in D2O, probe wavelength dependence of spectral features of the C=C and C=N stretch bands, band fitting analysis of the C=C stretch band, deuteration effects on the widths of the C=N stretch bands, and HOOP bands in the resonance Raman spectra of BR (PDF) This information is available free of charge via the Internet at http://pubs.acs.org

AUTHOR INFORMATION Corresponding Author * To whom correspondence should be addressed. E-mail address: [email protected], Phone: +81-6-6850-5776 Notes The authors declare no competing financial interest.



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REFFERENCES (1) Inoue, K.; Ono, H.; Abe-Yoshizumi, R.; Yoshizawa, S.; Ito, H.; Kogure, K.; Kandori, H. A Light-Driven Sodium Ion Pump in Marine Bacteria. Nat. Commun. 2013, 4, 1678. (2) Kandori, H.; Inoue, K.; Tsunoda, S. P. Light-Driven Sodium-Pumping Rhodopsin: A New Concept of Active Transport. Chem. Rev. 2018, 118, 10646-10658. (3) Tahara, S.; Takeuchi, S.; Abe-Yoshizumi, R.; Inoue, K.; Ohtani, H.; Kandori, H.; Tahara, T. Ultrafast Photoreaction Dynamics of a Light-Driven Sodium-Ion-Pumping Retinal Protein from Krokinobacter eikastus Revealed by Femtosecond Time-Resolved Absorption Spectroscopy. J. Phys. Chem. Lett. 2015, 6, 4481-4486. (4) Hontani, Y.; Inoue, K.; Kloz, M.; Kato, Y.; Kandori, H.; Kennis, J. T. M. The Photochemistry of Sodium Ion Pump Rhodopsin Observed by Watermarked Femto- to Submillisecond Stimulated Raman Spectroscopy. Phys. Chem. Chem. Phys. 2016, 18, 2472924736. (5) Mathies, R. A.; Lin, S. W.; Ames, J. B.; Pollard, W. T. From Femtosecond to Biology: Mechanism of Bacteriorhodopsin's Light-Driven Proton Pump. Annu. Rev. Biophys. Biophys. Chem. 1991, 20, 491-518. (6) Oesterhelt, D.; Tittor, J.; Bamberg, E. A Unifying Concept for Ion Translocation by Retinal Proteins. J. Bioener. Biomembr. 1992, 24, 181-191. (7) Váró, G. Analogies between Halorhodopsin and Bacteriorhodopsin. Biochim. Biophys. Acta 2000, 1460, 220-229. (8) Ernst, O. P.; Lodowski, D. T.; Elstner, M.; Hegemann, P.; Brown, L. S.; Kandori, H. Microbial and Animal Rhodopsins: Structures, Functions, and Molecular Mechanisms. Chem. Rev. 2014, 114, 126-163. (9) Inoue, K.; Konno, M.; Abe-Yoshizumi, R.; Kandori, H. The Role of the NDQ Motif in Sodium-Pumping Rhodopsins. Angew. Chem., Int. Ed. 2015, 54, 11536-11539. (10) Gushchin, I.; Shevchenko, V.; Polovinkin, V.; Kovalev, K.; Alekseev, A.; Round, E.; Borshchevskiy, V.; Balandin, T.; Popov, A.; Gensch, T., et al. Crystal Structure of a LightDriven Sodium Pump. Nat. Struct. Mol. Biol. 2015, 22, 390-395. (11) Kato, H. E.; Inoue, K.; Abe-Yoshizumi, R.; Kato, Y.; Ono, H.; Konno, M.; Hososhima, S.; Ishizuka, T.; Hoque, M. R.; Kunitomo, H., et al. Structural Basis for Na+ Transport Mechanism by a Light-Driven Na+ Pump. Nature 2015, 521, 48-53. (12) Shigeta, A.; Ito, S.; Inoue, K.; Okitsu, T.; Wada, A.; Kandori, H.; Kawamura, I. SolidState Nuclear Magnetic Resonance Structural Study of the Retinal-Binding Pocket in Sodium Ion Pump Rhodopsin. Biochemistry 2017, 56, 543-550. (13) Chen, H.-F.; Inoue, K.; Ono, H.; Abe-Yoshizumi, R.; Wada, A.; Kandori, H. TimeResolved FTIR Study of Light-Driven Sodium Pump Rhodopsins. Phys. Chem. Chem. Phys. 2018, 20, 17694-17704. (14) Mizuno, M.; Nakajima, A.; Kandori, H.; Mizutani, Y. Structural Evolution of a Retinal Chromophore in the Photocycle of Halorhodopsin from Natronobacterium pharaonis. J. Phys. Chem. A 2018, 122, 2411-2423. (15) Ono, H.; Inoue, K.; Abe-Yoshizumi, R.; Kandori, H. FTIR Spectroscopy of a LightDriven Compatible Sodium Ion-Proton Pumping Rhodopsin at 77 K. J. Phys. Chem. B 2014, 118, 4784-4792.


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(16) Iwata, K.; Hamaguchi, H.; Tasumi, M. Sensitivity Calibration of Multichannel Raman Spectrometers Using the Least-Squares-Fitted Fluorescence Spectrum of Quinine. Appl. Spectrosc. 1988, 42, 12-14. (17) Higashino, A.; Mizuno, M.; Mizutani, Y. Chromophore Structure of Photochromic Fluorescent Protein Dronpa: Acid-Base Equilibrium of Two Cis Configurations. J. Phys. Chem. B 2016, 120, 3353-3359. (18) Smith, S. O.; Braiman, M. S.; Myers, A. B.; Pardoen, J. A.; Courtin, J. M. L.; Winkel, C.; Lugtenburg, J.; Mathies, R. A. Vibrational Analysis of the All-Trans-Retinal Chromophore in Light-Adapted Bacteriorhodopsin. J. Am. Chem. Soc. 1987, 109, 3108-3125. (19) Smith, S. O.; Pardoen, J. A.; Lugtenburg, J.; Mathies, R. A. Vibrational Analysis of the 13-Cis-Retinal Chromophore in Dark-Adapted Bacteriorhodopsin. J. Phys. Chem. 1987, 91, 804819. (20) Stockburger, M.; Alshuth, T.; Oesterhelt, D.; Gärtner, W. In Spectroscopy of Biological Systems, Clark, R. J. H.; Hester, R. E., Eds. John Wiley & Sons: Chichester, 1986; pp 483-535. (21) Fodor, S. P. A.; Pollard, W. T.; Gebhard, R.; van den Berg, E. M. M.; Lugtenburg, J.; Mathies, R. A. Bacteriorhodopsin's L550 Intermediate Contains a C14-C15 S-Trans-Retinal Chromophore. Proc. Natl. Acad. Sci. U. S. A. 1988, 85, 2156-2160. (22) Kajimoto, K.; Kikukawa, T.; Nakashima, H.; Yamaryo, H.; Saito, Y.; Fujisawa, T.; Demura, M.; Unno, M. Transient Resonance Raman Spectroscopy of a Light-Driven SodiumIon-Pump Rhodopsin from Indibacter alkaliphilus. J. Phys. Chem. B 2017, 121, 4431-4437. (23) Lohrmann, R.; Stockburger, M. Time-Resolved Resonance Raman Studies of Bacteriorhodopsin and Its Intermediates K590 and L550: Biological Implications. J. Raman Spectrosc. 1992, 23, 575-583. (24) Smith, S. O.; Lugtenburg, J.; Mathies, R. A. Determination of Retinal Chromophore Structure in Bacteriorhodopsin with Resonance Raman Spectroscopy. J. Membrane Biol. 1985, 85, 95-109. (25) Doig, S. J.; Reid, P. J.; Mathies, R. A. Picosecond Time-Resolved Resonance Raman Spectroscopy of Bacteriorhodopsin's J, K, and KL Intermediates. J. Phys. Chem. 1991, 95, 63726379. (26) Ames, J. B.; Fodor, S. P. A.; Gebhard, R.; Rapp, J.; van den Berg, E. M. M.; Lugtenburg, J.; Mathies, R. A. Bacteriorhodopsin's M412 Intermediate Contains a 13-Cis,14-S-Trans,15-AntiRetinal Schiff Base Chromophore. Biochemistry 1989, 28, 3681-3687. (27) Fodor, S. P. A.; Ames, J. B.; Gebhard, R.; van den Berg, E. M. M.; Stoeckenius, W.; Lugtenburg, J.; Mathies, R. A. Chromophore Structure in Bacteriorhodopsin's N Intermediate: Implications for the Proton-Pumping Mechanism. Biochemistry 1988, 27, 7097-7101. (28) Terner, J.; Hsieh, C.-L.; Burns, A. R.; El-Sayed, M. A. Time-Resolved Resonance Raman Spectroscopy of Intermediates of Bacteriorhodopsin: The bK590 Intermediate. Proc. Natl. Acad. Sci. U. S. A. 1979, 76, 3046-3050. (29) Smith, S. O.; Pardoen, J. A.; Mulder, P. P. J.; Curry, B.; Lugtenburg, J.; Mathies, R. Chromophore Structure in Bacteriorhodopsin's O640 Photointermediate. Biochemistry 1983, 22, 6141-6148. (30) Smith, S. O.; Myers, A. B.; Pardoen, J. A.; Winkel, C.; Mulder, P. P. J.; Lugtenburg, J.; Mathies, R. Determination of Retinal Schiff Base Configuration in Bacteriorhodopsin. Proc. Natl. Acad. Sci. U. S. A. 1984, 81, 2055-2059.



Page 36 of 38

(31) Gellini, C.; Lüttenberg, B.; Sydor, J.; Engelhard, M.; Hildebrandt, P. Resonance Raman Spectroscopy of Sensory Rhodopsin II from Natronobacterium pharaonis. FEBS Lett. 2000, 472, 263-266. (32) Furutani, Y.; Bezerra, A. G., Jr.; Waschuk, S.; Sumii, M.; Brown, L. S.; Kandori, H. FTIR Spectroscopy of the K Photointermediate of Neurospora Rhodopsin: Structural Changes of the Retinal, Protein, and Water Molecules after Photoisomerization. Biochemistry 2004, 43, 9636-9646. (33) Kralj, J. M.; Spudich, E. N.; Spudich, J. L.; Rothschild, K. J. Raman Spectroscopy Reveals Direct Chromophore Interactions in the Leu/Gln105 Spectral Tuning Switch of Proteorhodopsins. J. Phys. Chem. B 2008, 112, 11770-11776. (34) Miranda, M. R.; Choi, A. R.; Shi, L.; Bezerra, A. G., Jr.; Jung, K. H.; Brown, L. S. The Photocycle and Proton Translocation Pathway in a Cyanobacterial Ion-Pumping Rhodopsin. Biophys. J. 2009, 96, 1471-1481. (35) Alshuth, T.; Stockburger, M.; Hegemann, P.; Oesterhelt, D. Structure of the Retinal Chromophore in Halorhodopsin. FEBS Lett. 1985, 179, 55-59. (36) Maeda, A.; Ogurusu, T.; Yoshizawa, T.; Kitagawa, T. Resonance Raman Study on Binding of Chloride to the Chromophore of Halorhodopsin. Biochemistry 1985, 24, 2517-2521. (37) Gerscher, S.; Mylrajan, M.; Hildebrandt, P.; Baron, M.-H.; Müller, R.; Engelhard, M. Chromohore-Anion Interaction in Halorhodopsin from Natronobacterium pharaonis Probed by Time-Resolved Resonance Raman Spectroscopy. Biochemistry 1997, 36, 11012-11020. (38) Baasov, T.; Friedman, N.; Sheves, M. Factors Affecting the C=N Stretching in Protonated Retinal Schiff Base: A Model Study for Bacteriorhodspsin and Visual Pigments. Biochemistry 1987, 26, 3210-3217. (39) Hildebrandt, P.; Stockburger, M. Role of Water in Bacteriorhodopsin's Chromophore: Resonance Raman Study. Biochemistry 1984, 23, 5539-5548. (40) Althaus, T.; Eisfeld, W.; Lohrmann, R.; Stockburger, M. Application of Raman Spectroscopy to Retinal Proteins. Israel J. Chem. 1995, 35, 227-251. (41) Curry, B.; Broek, A.; Lugtenburg, J.; Mathies, R. Vibrational Analysis of All-TransRetinal. J. Am. Chem. Soc. 1982, 1982, 5247-5286. (42) Curry, B.; Palings, I.; Broek, A.; Pardoen, J. A.; Mulder, P. P. J.; Lugtenburg, J.; Mathies, R. Vibrational Analysis of 13-Cis-Retinal. J. Phys. Chem. 1984, 88, 688-702. (43) Alshuth, T.; Stockburger, M. Time-Resolved Resonance Raman Studies on the Photochemical Cycle of Bacteriorhodopsin. Photochem. Photobiol. 1986, 43, 55-66. (44) Krebs, R. A.; Dunmire, D.; Partha, R.; Braiman, M. S. Resonance Raman Characterization of Proteorhodopsin's Chromophore Environment. J. Phys. Chem. B 2003, 107, 7877-7883. (45) Saint Clair, E. C.; Ogren, J. I.; Mamaev, S.; Russano, D.; Kralj, J. M.; Rothschild, K. J. Near-IR Resonance Raman Spectroscopy of Archaerhodopsin 3: Effects of Transmembrane Potential. J. Phys. Chem. B 2012, 116, 14592-601. (46) Niho, A.; Yoshizawa, S.; Tsukamoto, T.; Kurihara, M.; Tahara, S.; Nakajima, Y.; Mizuno, M.; Kuramochi, H.; Tahara, T.; Mizutani, Y., et al. Demonstration of a Light-Driven SO42Transporter and Its Spectroscopic Characteristics. J. Am. Chem. Soc. 2017, 139, 4376-4389. (47) Luecke, H.; Schobert, B.; Richter, H.-T.; Cartailler, J.-P.; Lanyi, J. K. Structure of Bacteriorhodopsin at 1.55 Å Resolution J. Mol. Biol. 1999, 281, 899-911. (48) Kato, H. E.; Inoue, K.; Kandori, H.; Nureki, O. The Light-Driven Sodium Ion Pump: A New Player in Rhodopsin Research. BioEssays 2016, 38, 1274-1282.


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Distortion and a Strong Hydrogen Bond in the Retinal Chromophore

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