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Transient Resonance Raman Spectroscopy of a Light-Driven Sodium-Ion-Pump Rhodopsin from Indibacter alkaliphilus Kousuke Kajimoto, Takashi Kikukawa, Hiroki Nakashima, Haruki Yamaryo, Yuta Saito, Tomotsumi Fujisawa, Makoto Demura, and Masashi Unno J. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 19 Apr 2017 Downloaded from http://pubs.acs.org on April 20, 2017

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The Journal of Physical Chemistry

Transient Resonance Raman Spectroscopy of a Light-Driven Sodium-Ion-Pump Rhodopsin from Indibacter alkaliphilus Kousuke Kajimoto,† Takashi Kikukawa,‡,|| Hiroki Nakashima,† Haruki Yamaryo,† Yuta Saito,‡ Tomotsumi Fujisawa,† Makoto Demura,‡,|| and Masashi Unno*,† †Department of Chemistry and Applied Chemistry, Graduate School of Science and Engineering, Saga University, Saga 8408502, Japan ‡ Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan || Global Station for Soft Matter, Global Institution for Collaborative Research and Education, Hokkaido University, Sapporo 060-0810, Japan ABSTRACT: Sodium-ion-pump rhodopsin (NaR) is a microbial rhodopsin that transports Na+ during its photocycle. Here we explore the photocycle mechanism of NaR from Indibacter alkaliphilus with transient absorption and transient resonance Raman spectroscopy. The transient absorption data indicate that the photocycle of NaR is K (545 nm) → L (490 nm)/M (420 nm) → O1 (590 nm) → O2 (560 nm) → NaR, where the L and M are formed as equilibrium states. The presence of K, L, M, and O intermediates was confirmed by the resonance Raman spectra with 442 and 532 nm excitation. The main component of the transient resonance Raman spectra was due to L that contains a 13-cis retinal protonated Schiff base. The presence of an enhanced hydrogen outof-plane band as well as its sensitivity to the H/D exchange indicate that the retinal chromophore is distorted near the Schiff base region in L. Moreover, the retinal Schiff base of the L state forms a hydrogen bond that is stronger than that of the dark state. These observations are consistent with a Na+ pumping mechanism that involves a proton transfer from the retinal Schiff base to a key aspartate residue (Asp116 in Krokinobacter eikastus rhodopsin 2) in the L/M states.

INTRODUCTION Microbial rhodopsins are photoactive membrane proteins that contain retinal as a chromophore.1 The light-driven proton pump bacteriorhodopsin (BR) and chloride ion pump halorhodopsin (HR) were found in 1971 and 1977, respectively, from halophilic archaea.2,3 Light-driven proton pumps such as BR were also found in marine prokaryotes and are called proteorhodopsins.4,5 A similar light-driven proton pump was also discovered in eubacteria such as Gloeobacter rhodopsin (GR).6,7 In addition to these light-driven ion-pumps, microbial rhodopsins, such as sensory rhodopsin I and sensory rhodopsin II (SRII), function as photosensors responsible for attractive or repellent phototaxis, respectively.1 All of these microbial rhodopsins contain an all-trans retinal chromophore that binds to a lysine residue in the seventh helix through a protonated retinal Schiff base linkage1 (Figure 1A). Recently, a new group of microbial rhodopsins capable of light-driven Na+ transport was identified8–11 and is desiginated sodium-ion-pump rhodopsins (NaR). The first discovered NaR was Krokinobacter eikastus rhodopsin 2 (KR2), and its crystal structures were reported.12,13 The light-driven proton pumps such as BR and PR contain a proton acceptor (Asp85 in BR) and donor (Asp96 in BR) near the protonated retinal Schiff base (Figures 1B and 1C). The negatively charged proton acceptor Asp85 acts as the counterion of the protonated Schiff base.14 Both Asp85 and Asp212 anchor three water molecules, and one of them forms a hydrogen bond with the protonated Schiff base NH moiety. In NaR, the carboxylic residues are

neutralized (Asn112 and Gln123 in KR2), whereas an aspartate (Asp116 in KR2) is introduced into the corresponding position of Thr89 in BR.9 NaR contains all-trans retinal (~90% for KR2),9 and its Schiff base is protonated and forms a hydrogen bond with the deprotonated Aps116 in the dark state.12,13,15,16 The transient absorption spectroscopy of KR2 revealed the sequential appearance of red-shifted K, blue-shifted L/M, and red-shifted O intermediates.9 Additional early intermediates were reported in recent ultrafast spectroscopic analyses.17,18 Although the structural information for the photocycle intermediates of NaR is limited, structural and functional studies suggested that Asp116 is the H+ acceptor from the retinal Schiff base upon formation of M.12,13,15,16 In the M state, the partially electronegative deprotonated Schiff base may attract the sodium ion, which could lead to a translocation of Na+ across the Schiff base moiety. This mechanism implies that the hydrogen-bonding interaction between the protonated Schiff base and the deprotonated Aps116 is retained in the L state. It is therefore crucial to determine the active site structure of the L state in order to understand the Na+ pumping mechanism in NaR. Thus, in the present study, we applied transient absorption and single-beam transient resonance Raman spectroscopy19 to NaR from Indibacter alkaliphilus (IaNaR).11 Transient absorption spectroscopy in the ultraviolet-visible region is a powerful technique to investigate the kinetics of the photocycle. On the other hand, transient resonance Raman spectroscopy is ideally suited to examining the molecular structures of the intermedi-

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ate states. In particular, the vibrational spectra of specific intermediate states can be preferentially measured by tuning the laser wavelength in the resonance Raman technique. Using both transient absorption and transient resonance Raman spectroscopy, we have characterized the photointermediates of IaNaR. Specifically, the present study demonstrates that the 13-cis retinal chromophore is distorted in the L state. Moreover, the protonated Schiff base moiety forms a strong hydrogen bond, probably with the deprotonated Aps116 residue (Asp115 in IaNaR). These results provide an evidence for a gate structure of NaR during its Na+ pumping process.

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(3.0 mm height x 0.2 mm depth). The sample was pumped through the closed loop system by a non-pulsation pump at 0.30~3.0 mL/min (PUS-3; GL Sciences Inc., Japan). The amount of photointermediate produced by the laser depends upon the photoalteration parameter, F = (3.824 x 10–21) Pεφ/vd.19,22,23 In this equation, P is the laser power (photons/s), ε is the extinction coefficient (M–1 cm–1), φ is the quantum yield for the photochemical reaction, v is the flow velocity (cm/s), and d is the focused beam diameter (cm). A low laser power (0.05 mW) was used as a probe beam (F = 0.26 for λex = 532 nm; F = 0.06 for λex = 441.6 nm) for the measurements of the dark state NaR, while higher laser powers were utilized for the intermediate states (F = 2.6 for λex = 532 nm; F = 1.1 for λex = 441.6 nm). Details of the other parameters are summarized in the Supporting Information. The transient kinetic Raman data were obtained by varying the resident time of a sample within a laser beam (∆t), adjusting the laser power to keep the same photoalteration parameter F.

RESULTS AND DISCUSSION

FIGURE 1. Structures of the all-trans retinal chromophore and the active site of KR2 and BR. (A) The all-trans retinal protonated Schiff base. (B, C) The active site structures of NaR (KR2) and BR based on their crystal structures. ATR stands for all-trans retinal. PDB codes: 3X3C (KR2) and 1C3W (BR).

EXPERIMENTAL SECTION The expression and purification of IaNaR were conducted as described in the Supporting Information. All measurements of the transient absorption spectroscopy were performed at 25°C. The details of the transient absorption apparatus and the procedure for data analysis were reported previously.20,21 The calculation of Pi spectra shown in Figure 3 requires the P0 (dark state) spectrum, whose determination was described in the Supporting Information. The resonance Raman spectra were obtained as described earlier with some modifications.20 Briefly, the spectrometer system was composed of a heliumcadmium laser (IK5651R-G; Kimmon Electric, Ltd., Japan) or a semiconductor solid-state green laser (Ventus532-1500; Laser Quantum, U.K.), a 0.5 m single spectrograph (Spex 500M; HORIBA Jobin Yvon, NJ), and a liquid nitrogen-cooled UVcoated CCD detector (Spec-10:400B; Roper Scientific Inc., NJ). A 90° scattering geometry was employed, a Triax190 spectrometer (HORIBA Jobin Yvon, NJ) was used to remove the excitation light, and the first order of the dispersed light by the 500M spectrometer was imaged on the detector. An entrance slit width of 0.1 mm corresponded to a spectral resolu-

Figure 2A shows time traces for the absorbance changes of NaR at the three diagnostic wavelengths, 410, 520, and 600 nm. The depletion signal at 520 nm reflects the amount of bleach due to the formation of intermediate states. The signal at 410 nm primarily corresponds to the formation and decay of L and M, while the signal at 600 nm is mainly due to the formation and decay of K and O. Figure 2B displays the flashinduced light-minus-dark difference spectra between 330 and 740 nm. Figure S1 in the Supporting Information displays the data in three separate time ranges. These results are consistent with a previous transient absorption study on KR2.9 We also confirmed that the photocycle kinetics of IaNaR in DDM detergent is very close to that in lipid as reported11 (see Figure S2 in the Supporting Information). The data observed at all wavelengths from 330 to 740 nm were fitted simultaneously with a multiexponential equation, and five exponential terms were required to obtain satisfactory fits. These data were further analyzed as described previously.20,24 In this analysis, τi represents the decay time constant of the ith component that is expressed as Pi, and P0 corresponds to the initial dark state NaR.

tion of ∼4 cm–1. All spectra were taken at room temperature (∼25°C), and all Raman spectra were calibrated by using neat fenchone as a standard. The transient resonance Raman spectra were obtained by a single-beam rapid-flow technique.19 The NaR samples were dissolved in 50 mM Tris-HCl, pH 8.0, 0.4 M NaCl, and 0.05% n-dodecyl-β-D-maltoside (DDM). ~3 mL of a 50 µM solution was placed in a recirculating flow system consisting of a reservoir connected to a quartz flow cell


(1)

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The Journal of Physical Chemistry 1.3 ms) shows two absorption maxima, indicating that P3 consists of at least two physically defined intermediates that attain equilibrium at a rate much faster than the rate of decay. The absorption maximum of the short wavelength minor component is ~490 nm, and thus was ascribed to L. If we compare the 490 nm components between P2 and P3, the absorption spectrum of P2 seems to contain a minor band around 420 nm, suggesting the presence of M.9 As reported for KR2, this observation indicates the co-existence of L and M as equilibrium states. The absorption spectrum for the main component of P3 is centered at 590 nm and can be related to O. A small shoulder of P2 that can be discerned near 600 nm is probably due to O. P4 (τ4 = 3.3 ms) also corresponds to a red-shifted intermediate with an absorption maximum at 560 nm. Although the structural origin of the spectral difference is not certain, we denote the 590 and 560 nm components as O1 and O2, respectively. P5 has the longest lifetime of all intermediates (τ5 = 65 ms), and its absorption spectrum is quite similar to that of the initial dark state NaR (P0). The structure of this species is probably similar to that of NaR, and this slowest process may be attributed to the protein relaxation to the initial state.

FIGURE 2. Transient flash-induced absorption changes of NaR from Indibacter alkaliphilus. Measurements were performed at 25ºC in a solution of 50 mM Tris-HCl, pH 8.0, 0.4 M NaCl, 0.05% DDM. (A) The kinetic traces were monitored at 410, 520, and 600 nm. The solid lines (black) show the multiexponential fit to the data. (B) The transient absorption spectra at 0.01, 0.05, 0.2, 0.7, 1.3, 2.5, 5.0, 10 ms after illumination.

FIGURE 3. Spectra of intermediates obtained by global fitting analysis of the transient absorption data of NaR from I. alkaliphilus. The estimated decay time constants for P1, P2, P3, P4, and P5 are τ1 = 39 µs, τ2 = 0.40 ms, τ3 = 1.3 ms, τ4 = 3.3 ms, and τ5 = 65 ms, respectively.

Figures 3 and S3 in the Supporting Information show the results of this analysis. P1 (τ1 = 39 µs) exhibits an absorption maximum at 545 nm and is assigned to red-shifted K. A recent femtosecond time-resolved absorption study on KR2 observed K on the red side of the absorption band of the dark state.17 The absorption maximum of P2 is 490 nm, and this species is mainly assigned to L (τ2 = 0.40 ms). The spectrum of P3 (τ2 =

FIGURE 4. Resonance Raman spectra of NaR and selected microbial rhodopsins under the dark state. (a) BR (light-adapted state) in the purple membrane of Halobacterium salinarum, 532 nm excitation. (b) HR from Natronomonas pharaonis, 532 nm excitation. (c) GR, 441.6 nm excitation. (d) NaR from I. alkaliphilus, 441.6 nm excitation. (e) SRII from N. pharaonis, 441.6 nm excitation. Trace e is adapted from ref 20.

Figure 4 shows the resonance Raman spectrum of NaR with 441.6 nm excitation in comparison with those of BR, HR, GR, and SRII. The spectra for BR and HR were measured with 532 nm excitation, while 441.6 nm excitation was used for GR and SRII. The resonance Raman spectra for BR, HR, and GR are



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consistent with those reported previously.25–27 The spectrum of IaNaR is consistent with the stimulated Raman spectrum of KR2,18 and the overall spectral features for NaR are similar to those for the other microbial rhodopsins. The most prominent feature for NaR is the ethylenic stretch νC=C observed at 1534 cm–1. As illustrated in Figure S4 in the Supporting Information, an approximately linear correlation exists between the absorption maximum (λmax) of retinal compounds and the ethylenic stretching frequency νC=C.20,28 In NaR, the 1534 cm–1 frequency of νC=C correlates well with the measured λmax of 525 nm. The Raman band at 1644 cm–1 is assigned to the C=N stretching (νC=N) mode of a Schiff base linkage to the protein. This assignment as well as the protonation state of the Schiff base is demonstrated by the sensitivity of the νC=N band to deuteration. A comparison of the spectra (black and blue traces) in Figure 5 (a) indicates that the H/D exchange downshifts the νC=N band at 1644 cm–1 by 22 cm–1. For retinals, the C–C skeletal stretching modes in the “fingerprint” region (1100– 1300 cm–1) are sensitive indicators of the chromophore geometry.19,22 The spectra of NaR are characterized by bands at 1201 and 1173/1166 cm–1, indicating an all-trans configuration. The mode at 1201 cm–1 is assigned to the C14–C15 stretch, and the C12–C13 stretch and C10–C11 stretch are the bands at 1167 and 1173 cm–1, respectively.22,29

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towards the extracellular side and forms a hydrogen bond with a water molecule that is anchored by hydrogen bonds through the Schiff base counterions Asp212 and Asp85 (Figure 1C). In contrast, the orientation of the Schiff base in KR2 is different from that in BR, and the NH moiety forms a hydrogen bond with Asp116. This structural difference accounts for the significant enhancement of the HOOP bands for NaR. Next we measured the transient resonance Raman spectra of photointermediate states by a single-beam rapid flow technique, where the single beam initiates the photoreaction of the sample and excites the Raman spectrum.19,22 Spectra of different intermediates were obtained by increasing the incident laser power and/or changing the flow rate. Since the spectra taken with high photoalteration contain signals from both intermediates and the unphotolyzed states, it is necessary to subtract a low-photoalteration spectrum. In the present case, we used the photoalteration parameter F = 1.1 (λex = 441.6 nm) or 2.6 (λex = 532 nm). Figure S6 in the Supporting Information presents a typical result of the transient resonance Raman experiments on NaR. Trace b is obtained under a low power condition, which gives the spectrum for the dark state as mentioned above. The high power spectrum (trace a) exhibits new bands at ~1180 and 954 cm–1 as well as a shoulder near 1555 cm–1, indicating the formation of intermediate states. Subtraction of the low power spectrum from the high power one to fully subtract the 878 cm–1 intensity due to the dark state gives a difference spectrum (trace c). In addition, the positive or negative 1644 and 1534 cm–1 intensities were minimized in the difference spectrum.

FIGURE 5. Effects of the H/D exchange on the resonance Raman spectra of NaR from I. alkaliphilus with 532 nm excitation. (a) Dark state, (b) ∆t = 0.56 ms, and (c) ∆t = 5.4 ms. The black and blue traces refer to the measurements in H2O and D2O solutions, respectively.

We also observed relatively strong low frequency bands (972, 963, 901, 878 cm–1), which are assigned to hydrogenout-of-plane (HOOP) wagging vibrations. It has been shown that enhanced Raman intensities for HOOP modes are caused by torsional deformations of the retinal polyene chain.30 Figure 1 and Figure S5 in the Supporting Information display the active site structures for NaR12 and BR.31 Although, in both cases, the retinal chromophore adopts an all-trans configuration, its out-of-plane distortions are clearly different. Table S1 in the Supporting Information compares the selected dihedral angles of the retinal chromophore found in X-ray crystallographic and solution NMR structures for some microbial rhodopsins. The dihedral angle τ(C14–C15–N–Cε) for KR2 was 125~142°,12,13 whereas the values for BR,31 PR,32,33 and SRII34 were –143° ~ –172°. In BR, the protonated Schiff base points

FIGURE 6. Transient resonance Raman spectra of NaR from I. alkaliphilus with 532 nm (a–d) and 441.6 nm (e–h) excitation in the three spectral regions. (a) Dark state NaR, (b) a resident time of a sample within a laser beam ∆t = 0.56 ms, (c) ∆t = 2.2 ms, (d) ∆t = 5.4 ms, (e) ∆t = 0.44 ms, (f) ∆t = 1.0 ms, (g) ∆t = 2.0 ms, (h) ∆t = 4.4 ms.

Figure 6 displays the transient resonance Raman spectra of NaR with 532 nm (traces b–d) and 441.6 nm (traces e–h) exci-


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tations for a resident time of the sample within a laser beam ∆t from 0.44 ms to 5.4 ms. The figure also shows the spectrum of dark state NaR with 532 nm as a reference (trace a). The spectra in a wide range (1750–900 cm–1) are also illustrated in Figure S7 in the Supporting Information. The transient resonance Raman spectra contain contributions from all species present within the resident time ∆t. The spectra with 532 nm excitation exhibit an ethylenic νC=C band near 1550 cm–1, while the νC=N band is observed at ~1650 cm–1. The spectra are also characterized by bands at 1201, 1190, 1010, 970, and 955 cm– 1 . These features are also seen in the spectra with 441.6 nm excitation. The main spectral component in the transient resonance Raman spectra can be ascribed to the 490 nm species of P2 and P3, which we assign to L for the following reasons. As we described above, the present transient absorption studies indicate that the lifetimes of P2 (τ2 = 0.40 ms) and P3 (τ2 = 1.3 ms) correspond to the resident time of the transient resonance experiments (∆t = 0.44~5.4 ms). As seen in Figure 3, the probe wavelengths of 441.6 and 532 nm are suited for the resonance enhancement of L. It is, therefore, reasonable to assign this main component to L. Indeed, as we will describe later, a global fitting analysis estimates the νC=C frequency as 1547 cm–1, which correlates well with the estimated absorption maximum of 490 nm (Figure S4 in the Supporting Information). The resonance Raman spectrum of L in H2O exhibits a band at 1655 cm–1, which is assigned to νC=N. The shift of the νC=N band to 1623 cm–1 in D2O is good evidence that L has a protonated Schiff base (Figure 5). A closer inspection of the time evolution of the spectra with 532 nm excitation indicates the presence of an early intermediate at ∆t = 0.56 ms, which shows the νC=C and νC=N bands at 1527 and 1643 cm–1, respectively. We assign this early intermediate to K, whose lifetime is τ1 = 39 µs. This assignment is consistent with the νC=C/λmax correlation (Figure S4). The presence of K is also evident in the methyl (950–1050 cm–1) and fingerprint regions (1150–1250 cm–1). As illustrated in Figures 6 and S7, the methyl rocking band for K is observed at 1008 cm–1, which is 2 cm–1 downshifted from the corresponding band for L. The C–C stretching region of the spectrum for K is slightly different from that for L; K shows bands at 1187 and 1200 cm–1, while the corresponding bands for L are 1190 and 1201 cm–1, respectively. The νC=N band of K exhibits a downshift to 1607 cm–1 upon deuteration, confirming the protonated Schiff base in K (Figure 5 (b)). The C=C stretching band of the transient resonance Raman spectra with 441.6 nm excitation reveals an additional shoulder near 1566 cm–1, which is ascribed to M. As mentioned above, the transient absorption spectroscopy estimated the λmax for M at around 420 nm, which correlates well with the νC=C frequency of ~1566 cm–1 (Figure S4). In addition, the absorption spectrum of M seems to have no absorption at 532 nm (Figure 2), implying that M is out of resonance. It is, therefore, reasonable to assign the 1566 cm–1 band only observed with 441.6 nm excitation to M. In addition to K, L, and M intermediates, a minor component is observed in the spectra for ∆t = 4.4 or 5.4 ms. This component exhibits a band at 1175 cm–1 in the C–C stretching region. Furthermore, it seems that there is a C=C stretching band near 1520 cm–1 in the transient resonance Raman spectra with 532 nm excitation. Although the presence of the latter band is not clear, we ascribe this minor component to O. This assignment

is supported by the finding that the resonance Raman spectra of O is analogous to that for BR, which exhibits an intense Raman band at ~1170 cm–1 in the fingerprint region.35

FIGURE 7. A global fitting analysis of the transient resonance Raman spectra of NaR from I. alkaliphilus in the C=C stretching and C=N stretching regions with 532 nm (a–c) and 441.6 nm (d– g) excitation. The transient resonance Raman spectra were globally fitted with a sum of Gaussian band shapes as described in the text.

In order to confirm the above interpretation, we performed a global fitting analysis of the transient resonance Raman spectra in the C=C and C=N stretching regions (Figure 7). The spectra with 532 and 441.6 nm excitation were globally fitted with a sum of four spectral components that correspond to K, L, M, and O. As illustrated in Figure S8 in the Supporting Information, each spectral component was represented by a sum of Gaussian band shapes. Since the relative intensities among different vibrational modes may vary if an excitation wavelength is changed, the relative intensities were allowed to be different between the data for 532 and 441.6 nm excitation. As displayed in Figures 7 and S8, a main spectral component L exhibits a doublet at 1556/1543 cm–1 in the C=C stretching region, while the C=N stretching band is seen at 1656 cm–1. The transient resonance Raman spectra with 532 nm excitation (traces a–c) can be fitted with two additional components that are related to K and O. The spectrum for K is characterized by the νC=C and νC=N bands at 1527 and 1643 cm–1, respectively. On the other hand, the C=C stretching band for O is estimated at 1522 cm–1, although it was difficult to determine its value accurately. Figures 7 and S8 show that the transient resonance Raman spectra with 441.6 nm excitation require an additional



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spectral component at 1566 cm–1, which is due to M. Notably, the figure indicates that the decay of M is faster than that of L. The faster decay of M should contain important information, but its implications are beyond the scope of the current study, and will be discussed elsewhere. Table 1. Vibrational Frequency (cm-1) and Absorption Maximum (nm) of IaNaR and its Photointermediatesa νC=C

νC=N

∆ν(H/D)

λmax

ASSOCIATED CONTENT Supporting Information Transient absorption spectra, plots of the ethylenic stretching frequency and the absorption maximum, transient resonance Raman spectra, crystal structures of BR and KR2, a list of selected dihedral angles of the retinal chromophore for KR2, BR, PR, and SRII, and a supplemental experimental procedure. (PDF) This information is available free of charge via the Internet at http://pubs.acs.org

H2O

D2O

NaR

1534

1644

1622

–22

525

K

1527

1643

1607

–36

540

AUTHOR INFORMATION

L

1556,1543

1655

1623

–32

490

Corresponding Author

M

1566

nd

nd

nd

~420

* E-mail: [email protected]

O

1522

nd

nd

nd

590,560

ACKNOWLEDGMENT

a

∆ν(H/D) is the D2O-induced shift of νC=N. nd = not determined. The present transient absorption and resonance Raman studies provide spectroscopic evidence of the photocycle that involves K, L/M, O1, and O2 intermediates for IaNaR. The main component observed in the transient resonance Raman spectra is L, and its spectrum indicates a 13-cis-retinylidene chromophore with a protonated Schiff base linkage. The protonated Schiff base C=N stretching frequency and its frequency shift in D2O provide important information about interactions at the Schiff base group.19,36 The frequency shift of the νC=N band upon deuteration of the Schiff base nitrogen depends on the strength of its hydrogen bond with the environment. The correlation between the deuteration shift and the hydrogen bonding strength results from the coupling of the C=N stretching mode with the N–H rocking mode. Deuteration of the nitrogen reduces this coupling as a result of the decreased N–D rocking frequency. A decreased level of hydrogen-bonding interaction at the NH moiety will lower the N–H rocking frequency, causing a lower Schiff base stretch and a reduced deuterationinduced shift.19 As shown in Table 1 and Figure 5, the D2Oinduced shift of νC=N for L is –32 cm–1, which is significantly larger than the corresponding shift of –22 cm–1 for the dark state NaR. This result implies that the Schiff base group experiences a stronger hydrogen bonding environment in L. Another interesting feature of the resonance Raman spectrum of L is the observation of the HOOP band at 955 cm–1 (Figure 6). In BR, a similar band is observed for K at 957 cm–1,37 whereas the L state did not show the corresponding band.38 Thus the observation of the HOOP band in L is a characteristic feature for NaR. As displayed in Figure 5, the HOOP band at 955 cm– 1 is sensitive to the H/D exchange, indicating that the distortions of the retinal chromophore in L are localized in the Schiff base region. On the basis of these observations, it is reasonable to conclude that the 13-cis retinal chromophore is distorted to form a stronger hydrogen bond with the deprotonated Aps116 (Asp115 in IaNaR) residue in the L state of NaR. This active site structure of L is consistent with a proposed Na+-pumping mechanism that involves a proton transfer from the retinal Schiff base to Asp116 in the L/M states.

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We thank Prof. Kwang-Hwan Jung for providing the pKA001 plasmid. This work was supported by JSPS KAKENHI Grants 26410017 and 17K05756 (M.U.), 16K17859 (T.F.), and 26440042 (T.K.).

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