Spectroscopic Study of Proton Transfer Mechanism of Inward Proton

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Spectroscopic Study of Proton Transfer Mechanism of Inward Proton Pump Rhodopsin, Parvularcula oceani Xenorhodopsin Keiichi Inoue, Shinya Tahara, Yoshitaka Kato, Satoshi Takeuchi, Tahei Tahara, and Hideki Kandori J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b01279 • Publication Date (Web): 29 May 2018 Downloaded from http://pubs.acs.org on May 29, 2018

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The Journal of Physical Chemistry

Spectroscopic Study of Proton Transfer Mechanism of Inward Proton Pump Rhodopsin, Parvularcula oceani Xenorhodopsin. Keiichi Inoue†,‡,§,║, Shinya Tahara⊥, Yoshitaka Kato†, Satoshi Takeuchi┴,#, Tahei Tahara*┴,#, and Hideki Kandori*†,‡ †

Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan OptoBioTechnology Research Center, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan § Frontier Research Institute for Material Science, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan ‡

║

PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.

┴

Molecular Spectroscopy Laboratory, RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan. #Ultrafast Spectroscopy Research Team, RIKEN Center for Advanced Photonics (RAP), RIKEN, 2-1 Hirosawa, Wako 3510198, Japan

ABSTRACT: Parvularcula oceani xenorhodopsin (PoXeR) is the first light-driven inward proton pump. To understand the mechanism of inward proton transport, comprehensive transient absorption spectroscopy was conducted. Ultrafast pump-probe spectroscopy revealed that the isomerization time of retinal is 1.2 ps, which is considerably slower than those of other microbial rhodopsins (180–770 fs). Following the production of J, the K intermediate was formed at 4 ps. Proton transfer occurred on a slower timescale. Proton release and uptake were observed on the L/M-to-M and M decay, respectively, by monitoring transient absorption changes of pH-indicating dye, pyranine. While a proton was released from Asp216 into the cytoplasmic medium, no protondonating residue was identified on the extracellular side in mutation experiments. We revealed that a branched retinal isomerization (from 13-cis-15-anti to 13-cis-15-syn and all-trans-15-anti) occurred simultaneously with proton uptake. Furthermore, while the proton release showed a large kinetic isotope effect (KIE), the KIE of proton uptake was negligible. These results suggest that retinal isomerization is the rate-limiting process in proton uptake and that the regulation of pKa of the retinal Schiff base by thermal isomerization enables the uptake from extracellular medium. This proton uptake mechanism differs from that of the outward proton pump with an internal proton donor and is important for understanding how the direction of ion transport by membrane proteins is determined.

INTRODUCTION Proton motive force (pmf) is the most fundamental free energy source for various physiological activities such as ATP synthesis and proton-coupled transport. Microbial rhodopsin is a photoreceptive heptahelical membrane protein widely found in various microorganisms, including eubacteria, archaea, and unicellular eukaryotes 1. All-trans retinal is the chromophore of most of microbial rhodopsin, and photo-isomerization to the 13-cis form induces various types of biological functions. The most abundant microbial rhodopsin is the light-driven outward proton pump. The first outward proton pump rhodopsin, bacteriorhodopsin (BR), was discovered in 1971 in the halophilic archaea Halobacterium salinarum 2. Since then, many types of outward proton pump rhodopsins have been identified in various species 3-8. They are thought to generate pmf in the cell, and the wide distribution of the outward proton pump rhodopsin especially for proteorhodopsins which widely distributed among various marine bacteria suggests their vital role in producing energy in microorganisms9-12. However, a new type of light-driven inward proton pump rhodopsin, PoXeR, was recently identified in the deep-sea marine bacterium Parvularcula oceani 13. PoXeR is a member of the xenorhodopsins (XeRs), a sub-family of microbial rhodopsins

including homologs of Anabaena sensory rhodopsin 14. The most prominent characteristic of XeRs is their proline residue at the position of Asp212 of BR, which is highly conserved in other rhodopsins. Other XeRs of Nanohaloarchaea were also shown to have inward proton pump function 15. Although the physiological role of inward proton transport by XeRs remains unclear, their presence in various bacteria indicates their importance in the bacterial ecosystem. Studies of the mechanism of inward proton transport with a similar structural architecture to the outward proton pump would provide insight into the factors affecting the ion-transport direction of membrane proteins. Excited PoXeR undergoes a photocyclic reaction involving several photo-intermediates (K, L, L/M, M, and PoXeR13C) (Fig. 1) 13, 16. Low-temperature Fourier transform infrared (FTIR) spectroscopy revealed that retinal isomerizes to the 13cis form via the K intermediate. However, the dynamics of isomerization at room temperature are unclear. Next, the retinal Schiff base (RSB) deprotonates on the M intermediate, and a proton is transferred from RSB to the cytoplasmic side. Another proton then binds to RSB from the extracellular side. FTIR analysis showed that Asp216 on the cytoplasmic side of the seventh helix (helix-C) receives a proton from RSB to form M. Asp216 functions as a proton acceptor, and mutation

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of this residue significantly affects transport efficiency 13. However, it is not clear when the proton is released from Asp216 to the cytoplasmic side in the photocycle. In contrast to the proton release process, the pathway of proton uptake from the extracellular side to RSB is not fully understood. Reprotonation of RSB from the extracellular side occurs with the decay of M to PoXeR13C which was shown to have 13-cis15-syn retinal configuration by FTIR analysis13. The rate of the decay does not depend on the pH of the outer medium, indicating that some residue(s) donate protons to RSB 13. For the outward proton pump, aspartic acid, glutamic acid, and lysine were reported to donate protons to RSB 17-19. Although PoXeR has two acidic residues (Glu3 and Asp119) on the extracellular side of the transmembrane helices, neither are related to proton transport 13. Furthermore, PoXeR contains no lysine residue on the extracellular halves of the transmembrane helices, and any histidine that could transfer a proton is not present on the extracellular side. Therefore, reprotonation of RSB in PoXeR is likely achieved through a different mechanism from outward proton pumps. To reveal the mechanism of inward proton transport, we conducted a comprehensive spectroscopic analysis. First, the ultrafast pump-probe transient absorption measurement was applied for the real-time observation of trans-cis isomerization process of the retinal in PoXeR which is the primary process to trigger the conformational change of the protein to achieve inward proton pump function. Second, we measured the transient absorption change of a pH-indicating dye (pyranine) to reveal the precise time-profile of proton release/uptake processes. Third, the kinetic isotope effect on the photoreaction process was investigated to gain new insights into the kinetics of proton transfer involved in the photocycle of PoXeR. Such wide-encompassing approach makes us possible to understand the molecular mechanism related to the unique inward proton pump function of PoXeR in more detail. MATERIALS AND METHODS Sample Preparation of PoXeR. PoXeR with six histidines at the C-terminus was expressed in the E. coli. C43 (DE3) strain. The protein was purified via Co2+-NTA affinity column chromatography as previously described and solubilized in 0.1% DDM 13. For pyranine measurement, the purified sample was dialyzed against buffer-free solution (100 mM NaCl, 0.05% DDM, neutral pH). The dialyzed sample was diluted to 0.5 O.D. at the λmax, followed by addition of pyranine. The final concentration of pyranine in the sample solution was 50 µM. The pH was adjusted to 7.2 just before analysis. For KIE analysis, the purified sample was reconstituted into a mixture of 1palmitoyl-2-oleoyl-phosphatidyl-ethanolamine (POPE) and 1palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG, sodium salt) lipids (molecular ratio was POPE:POPG = 3:1, PoXeR:Lipid = 1:20) with Bio-Beads (SM-2, Bio-Rad, Hercules, CA, USA). Reconstituted PoXeR was washed 6 times with H2O (20 mM H3PO4 100 mM NaCl, pH 8.0) or D2O buffer (20 mM D3PO4, 100 mM NaCl, pD 8.0). The washed sample was suspended in each buffer and sonicated. The samples suspended in H2O, D2O, and mixture buffer were equilibrated overnight. pH and pD were adjusted by adding NaOH and NaOD, respectively. pD in D2O was

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Figure 1. Photocycle of PoXeR reported in Ref 13 (upper) and putative H+-transport pathway (lower).

calculated as apparent pH + 0.41. UV-vis Spectra Measurement of Dark- and Light-adapted PoXeR and ASR. UV-visible spectra of dark- and lightadapted (DA and LA) PoXeR were measured with V-650 or V-730 spectrometers (JASCO, Tokyo, Japan) at 4°C. Samples were illuminated for 2 min by a 1-kW tungsten–halogen projector lamp (Master HILUX-HR, Rikagaku, Kawasaki, Japan) through long-pass (λ > 580 nm, R-60, AGC Techno Glass, Shizuoka, Japan) and heat absorbing (Sigma Koki, Tokyo, Japan) filters. After illumination, spectra were measured every minute automatically. Spectra of PoXeR containing all-trans and 13-cis retinal were calculated from the DA and LA spectra, and compatible HPLC data which were measured in parallel by spectroscopy. Three independent measurements were taken and averaged. For ASR, the sample was illuminated for 4 min with identical equipment except for the long-pass filter (λ > 560 nm, O-58, AGC Techno Glass, Japan). HPLC Analysis. The isomeric ratio of PoXeR was obtained by HPLC analysis as reported previously 13 in parallel with UV-vis spectroscopy. Three independent measurements were taken and averaged. Femtosecond Time-resolved Absorption Measurement. Femtosecond time-resolved absorption data were obtained as previously described 20. Briefly, a Ti:sapphire regenerative amplifier system (800 nm, 1.2 mJ, 80 fs, 1 kHz, Legend Elite, Coherent, CA, USA) was used as the light source. The 0.2-mJ portion of the amplifier output was attenuated and focused into a 3-mm-thick calcium fluoride (CaF2) plate to generate a visible white light continuum. This continuum pulse was split into two, which were used as the probe and reference pulses. The remaining 1.0-mJ portion of the amplifier output was used to drive an optical parametric amplifier (TOPAS, Light Conversion, Vilnius, Lithuania), and its signal output was frequency-


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doubled to generate a pump pulse at 573 nm. Polarization of the pump pulse was adjusted to the magic angle (54.7°) with respect to the horizontally polarized probe pulse. The incident energy of the pump pulse was 100 nJ. The pump and probe pulses were focused into a 1-mm-thick flow cell, in which the sample solution was circulated. The circulation speed was adjusted so that the sample solution in the excited volume was replaced with fresh solution between each laser shot. Spectra of the probe pulse after passing through the sample and reference pulse were acquired with a polychromator (500 is/sm, Chromex, NM) equipped with a CCD camera (PIXIS-256E, Princeton Instruments, Trenton, NJ, USA). The group delay dispersion of the continuum probe pulse was examined by the optical Kerr effect measurement 21 on the buffer. The obtained optical Kerr effect signal was used to determine the time zero at each probe wavelength as well as the instrumental response function. The full width at half maximum (FWHM) of the instrumental response function was 100 fs. Transient Absorption Measurements by Nanosecond Laser Flash Photolysis. The transient absorption changes of PoXeR and pyranine were monitored by flash photolysis. The sample was illuminated with a beam of second harmonics of a nanosecond pulsed Nd3+:YAG laser (λ = 532 nm, 3-4 mJ/pulse, INDI40, Spectra-Physics, Santa Clara, CA, USA). The change in absorption after laser excitation was probed by monochromated light from the output of an Xe arc lamp (L9289−01, Hamamatsu Photonics, Japan), and the change in intensity of the probe light passed through the sample was monitored by a photomultiplier tube (R10699, Hamamatsu Photonics, Shizuoka, Japan). The signal from the photomultiplier tube was averaged and stored by a digital-storage-oscilloscope (DPO7104, Tektronix, Beaverton, OR, USA). To remove the contribution of the PoXeR13C photoreaction, 0.6 mL of sample solution was used for each measurement and was replaced with a fresh dark-adapted sample for every photoexcitation. RESULTS Femtosecond Time-resolved Absorption Spectroscopy. To characterize the primary process of PoXeR, we carried out femtosecond time-resolved absorption measurements at an excitation wavelength of 573 nm. The obtained femtosecond time-resolved absorption spectra at selected delay times are shown in Fig. 2A. Immediately after photoexcitation (0.05 ps), the transient spectrum showed a positive band in the 430–530 nm region and negative bands in the 540–630 and >670 nm regions. These bands were assigned to the Sn←S1 absorption (excited state absorption, ESA), S0 bleaching (ground-state bleaching), and S1→S0 stimulated emission (SE), respectively. Both the ESA and SE bands due to the S1 state decay in the early picosecond time region, indicating deactivation of the S1 state on this time scale. Accompanying these decays, a positive band assignable to the photoproduct absorption (PA) appeared at approximately 650 nm. Because the observed PA absorption is nearly identical to the spectrum of the J intermediate in the photocycle of microbial rhodopsins 20, 22-24, this spectral change was attributed to the formation of J of PoXeR which possesses 13-cis retinal generated by trans-cis isomerization. Following these initial spectral changes, -

Figure 2. Femtosecond time-resolved absorption spectroscopy of PoXeR. (A) Femtosecond time-resolved absorption spectra of PoXeR observed at selected delay times after photoexcitation at 573 nm. The wavelength region shaded in white is distorted by the scattering of the excitation pulse. (B) Temporal profiles of the femtosecond time-resolved absorption signals at several wavelengths. The colored curves show the experimental data and black solid curves represent the best fits. Note that the horizontal axis is changed from the linear scale to the logarithmic scale after 2 ps. The time region around the time origin is shaded in white because the signal is distorted by the instantaneous response such as coherent artifacts.

the PA band exhibited a ~20-nm blue-shift within 20 ps. Similar blue-shifts have been observed in various rhodopsins such as bacte riorhodopsin 22, 23 and sensory rhodopsins 25, 26, and have been assigned to the conversion from J to K. Thus, the blue-shift observed for PoXeR was also attributable to the J→ K conversion in the photocycle of PoXeR. After 20 ps, no



further spectral change was observed in the time range of the femtosecond absorption measurement (5 s. This indicates that an additional proton was taken up on PoXeR13C, which was released to result in recovery to the initial state. If we assume that one proton is released on L/M → M, the relative amplitude of the rise component in Fig. 3 indicates that 1.17 protons is taken up on the M-decay and 17% of product from the Mis more protonated than in the initial state. Proton Uptake Process of PoXeR Arg71 Mutants. A previous study showed that the rate of M-decay does not depend on external pH, and thus the presence of a residue functioning as the proton donor to RSB on the extracellular side was suggested 13. However, mutants of both acidic residues in the extracellular halves of the transmembrane helices, PoXeR E3Q and


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D119N, showed identical proton pump activities to that of wild-type. Thus, a non-acidic residue

type, R71A and R71Q showed significantly lower activities (Fig.

Figure 5. Observation of branching process from the M intermediate to PoXeR13C and initial state (A) Transient absorption change at 570 nm. (B) Absorption spectra of all-trans (black solid line) and 13-cis (red solid line) forms of PoXeR. Red dashed represents the expected absorption change at 570 nm for 100% conversion from all-trans to 13-cis form. The observed absorption change upon formation of the 13-cis forms (grey dashed line) indicates that the efficiency is 57.2 ± 0.2%.

Figure 4. Hydrogen and deuterium isotope effect on the photoreaction process of PoXeR. (A) Transient absorption change of PoXeR reconstituted in POPE/POPG (molar ratio = 3:1) membrane monitored at 400 (blue) and 570 (green) nm which represent M-accumulation and bleaching of the initial state, respectively, in H2O (dashed lines) and D2O (solid lines). (B) Proton inventory plot of the rates of L-to-L/M (blue filled circle), L/M-to-M (pink opened squares), and M-decay (green filled triangles). Dashed lines are fitting curve based on “Infinite site” multi proton catalysis model 31: k/k0 = (k1/k0)n where k, k1, and k0 are rate in D2O/H2O mixture with fraction of D2O (n), k at n = 1 and 0, respectively.

Table 1. Rate Constants Obtained in H2O and D2O Solution and Their KIEs (mean ± SD). kL / s-1

kL/M / s-1

kM / s-1

in H2O

6050 ± 150

213 ± 6

11.62 ± 0.13

in D2O

710 ± 30

27.0 ± 1.6

6.9 ± 0.2

KIE

8.59 ± 0.05

7.90 ± 0.06

1.69 ± 0.03

can function as the proton donor. Lysine was identified as the proton donor in the outward proton pump of Exiguobacterium sibiricum, and histidine also may be involved in proton transfer 19. However, neither were present on the extracellular side of PoXeR. The possibility of the involvement of BR Arg82 in proton transfer to the extracellular side has been suggested 32. This arginine is highly conserved among most microbial rhodopsins and is homologous to PoXeR Arg71. To investigate the role of Arg71 in reprotonation of RSB, we constructed three mutants, PoXeR R71K, R71A, and R71Q. While R71K showed identical inward proton transport efficiency to wild-

S1A). These results suggest the importance of Arg71 in proton transport by PoXeR. We further investigated the photocycle of the R71Q mutant. If Arg71 is the proton donor, the pH dependence of M-decay should differ from that of wild-type and the rate would be slower at higher pH. Although the decay rate was slower than that of wild-type, it was not pH-dependent (Fig. S1B). Thus, all types of extracellular residues (Asp, Glu, His, and Arg) likely do not function as internal proton donors to achieve proton uptake with a rate independent of external pH. To reveal the mechanism of pH-independent proton uptake of PoXeR, we conducted additional spectroscopic studies. Kinetic Isotope Effect on the Photocycle of PoXeR. The rate of the chemical process related to proton transfer generally slows upon exchange of H2O with D2O 33. Figure 4A shows the absorption change at 400 (probing M accumulation) and 570 nm (L accumulation and initial state bleach) in solvents containing H2O (dashed lines) and D2O (solid lines). While a significant delay in the rise of M accumulation was observed in D2O, the difference in the decay rate of M was not significant between H2O and D2O. The ratios between rate constants in D2O to H2O (referred as kinetic isotope effect, KIE) for the decays of L, L/M, and M were 8.58 ± 0.05, 7.89 ± 0.06, and 1.69 ± 0.04, respectively (Table 1). The smaller KIE of Mdecay suggests mechanistic difference of proton transfer from the other two processes. We will discuss this issue later. Branched Reaction Pathway of M Decay. Figure 5A shows the bleaching of the initial state at 570 nm (pink solid line) by laser excitation. Although most of the bleached absorption recovered in the 100 ms to 1 second time region at the same time as M decay, some of this absorption remained without full recovery to the initial level (black solid line in Fig. 5A) even after 1 s, representing the difference between the initial (PoXeRAT, “AT” represents the protein containing all-trans retinal) and PoXeR13C states. The difference between the initial full bleach by laser excitation obtained by extrapolating



the single exponential curve corresponding to the M-decay to t = 0 andremaining bleach at a longer time region (solid green and dashed gray lines in Fig. 5A, respectively) was larger than that between the absolute absorption of PoXeRAT and PoXeR13C states at 570 nm (black solid and red dashed lines in Fig. 5B, respectively), which were reported previously 13. This indicates that not all excited PoXeR converted to PoXeR13C, and some excited molecules directly recovered to PoXeRAT. Thus, the photoreaction branches from the M to PoXeRAT and PoXeR13C states. The ratio between the levels of dashed gray and red lines in Fig. 5A (0.57) suggested that while 57% of the excited molecules converted to PoXeR13C and recovered to the PoXeRAT with τ = 91 s 13, 43% directly recovered from M to PoXeRAT (Fig. 6A). DISCUSSION PoXeR is the first natural light-driven inward proton pump identified. However, its proton transport mechanism remained unclear. To gain insight into its photoreaction, we conducted comprehensive time-resolved spectroscopic analysis, and succeeded in obtaining multi-step photoreaction dynamics of PoXeR over wide time region (Fig. 6B) Primary Photoreaction Dynamics of PoXeR. In this study, we investigated the primary process of PoXeR by femtosecond time-resolved absorption spectroscopy. It was revealed that PoXeR undergoes trans-cis photoisomerization of the chromophore from the S1 state (shown as PoXeR* in Fig. 6) to form the J intermediate with a time constant of 1.2 ps. The J intermediate is subsequently converted to the K intermediate in ~4.0 ps. The primary photoreaction scheme of PoXeR is essentially the same as those of other rhodopsins that exhibit the normal outward proton pumps. This indicates that proton pump rhodopsins utilize a common photoreaction scheme in the femto- to pico-second time region, regardless of the direction of the pump, and that the photoreaction dynamics unique for the inward proton pump function may emerge on a later timescale. However, photoisomerization in PoXeR was found to be significantly slower than those of other microbial rhodopsins at physiological pH (e.g. BR: 500 fs 22; outward sodium pump KR2: 180–200 fs 20, 24; sensory rhodopsin I of Salinibacter ruber: 640 fs 25; sensory rhodopsin II of Halobacterium salinarum and Natronomonas pharaonis: 300–400 fs 26 ; xanthorhodopsin: 700 fs 34; thermophilic rhodopsin: 360 fs 35 , and ASR: ~770-870 fs36, 37). To obtain insight into the origin of this slower photoisomerization in PoXeR, we focused on the amino acid arrangement around the chromophore. Figure S2 shows the crystal structure of BR (1M0L) 38 as well as the expected structure of PoXeR based on the crystal structure of the ASR D217E mutant (4TL3) 39, which is the phylogenetically closest to PoXeR among rhodopsins whose structure is determined. In this “PoXeR” structure, 23 residues were located within 5 Å of the chromophore, and 9 were different from those in BR. We consider that this large difference in the amino acid arrangement made the chromophore environment in PoXeR different from that in BR, resulting in with differences observed in the photoisomerization rates. Remarkably, however, the photoisomerization in PoXeR was still slower than that in ASR, although ASR and PoXeR possess similar amino acid arrangements around the chromophore. In fact, only two residues near the chromophore of ASR, Val136 and Gln109, were replaced with Cys135 and Asp108 in PoX-

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eR, respectively, indicating that either or both of these residues affect the photoisomerization rate. In contrast to the photoisomerization rate, the time scale for the J→K conversion in PoXeR is comparable to those observed in BR (~3 ps) and SRs (~2.5 ps for sensory rhodopsin I of Salinibacter ruber and 4–5 ps for SRI and II of Halobacterium salinarum and Natronomonas pharaonis) 22, 25, 26. The J intermediate of BR has been assigned to the vibrationally hot K intermediate, and the J→K conversion corresponds to the vibrational cooling process of the chromophore.40 It was recently indicated that this process is also accompanied with the distortion of the chromophore41. The similarity between the time constants for J →K conversion in BR and PoXeR suggests that similar relaxation processes occur in PoXeR. Proton Transfer Processes in PoXeR. In a previous study, we showed that the sequential reaction was composed of the K → L → L/M → M → PoXeR13C → PoXeRAT processes (Fig. 1) . The transient absorption change in pyranine indicated that a proton was released to the cytoplasmic side and taken up from the extracellular side during the decays of L/M and M, respectively (Figs. 3 and 6A). FTIR analysis of cytoplasmic proton transfer revealed that Asp216 received a proton from RSB 13, which was released into the extracellular medium upon L/M → M. We observed significant difference of KIE between the M-accumulation (the decay of L and L/M) and Mdecay (Table 1). KIEs with various H2O/D2O compositions (proton inventory plot) are shown in Fig. 4B. The plots of rates of L → L/M (kL) and L/M → M (kL/M) showed curved lines. The curved inventory plots were well-reproduced by the “Infinite site” proton catalysis model31, indicating that multiple proton transfer between several amino acid residues and/or internal water molecules is related to these proton release processes31, 33. In contrast, because the KIE of M-decay (kM) was much smaller, the proton inventory plot did not show significant curvature and appeared to be well-fitted to a liner regression curve. Thus, it is difficult to conclude whether multiple proton transfer is involved in the proton uptake process. The curved proton inventory plot suggests that the multiple proton transfer process is related to both L → L/M and L/M → M (Fig. 4B). The X-ray crystallographic structure of the inward proton pumping ASR D217E mutant (PDB code: 4TL3) showed a long-distance hydrogen bonding network from the main-chain carbonyl of retinal-binding Lys210 to water502, Ser214, water503, and Glu217 39. Glu217 of the ASR D217E mutant also functioned as a proton acceptor as PoXeR Asp216, long-distance hydrogen-bonding network between Glu217, and retinal Schiff-base region, indicating multiple proton transfer among amino acid residues and water502 and 503; this is supported by the curved proton inventory plots of proton transfer to the cytoplasmic side (Fig. 4B). Recently, xenorhodopsin from Nanosalina (NsXeR) was reported to have an inward proton pump function 15. The cytoplasmic hydrogen bonding network in X-ray crystallographic NsXeR differs from that of ASR D217E and is composed of the mainchain carbonyl of retinal-binding Lys213 to water2, His48, and Asp220 (homologous to PoXeR Asp216) 15. Therefore, each XeR evolved different types of hydrogen bonding networks for long-distant proton transfer to the cytoplasmic acceptor. Although the proton release pathway from Asp216 to the extracellular milieu remains unclear, mutation of PoXeR Glu35 located on the cytoplasmic side of helix B significantly diminished proton transport efficiency 13, and the homologous


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residue (Glu36) of the ASR D217E mutant was suggested to

relay

a

proton

from

Figure 6. The summary of photo reaction dynamics of PoXeR. (A)Photo-reaction scheme of PoXeR suggested in this study. (B) Combined 2D representation of transient absorption change (upper) and transient absorption change at specific wavelengths from femtosecondpicosecond region to microsecond-millisecond and second region. To make it easy to see, the signal intensity of three different time regions (femtosecond-picosecond, microsecond-millisecond, and second region) were adjusted by multiplying the factors shown in Table S2.

Glu217 to the cytoplasmic side 39. The proton inventory plot of the proton release process to the cytoplasmic milieu (L/M → M) (Fig. 4B) suggests that multiple proton transfer is involved in proton release from PoXeR Asp216 via Glu35.

Complicated hydrogen bonding networks involving several amino-acid residues and water molecules exist in the crystallographic structures of both ASR D217E and NsXeR 15, 39. If multiple proton transfer occurs during the extracellular proton uptake process with the large hydrogen bonding network as in



cytoplasmic proton release, the decay of M should also show a large KIE and curved proton inventory plot. However, the KIE of M decay was small (1.69) and the curved proton inventory plot was not obvious. The much smaller KIE suggests that another rate-limiting process occurs before proton uptake. A 15-anti-to-syn isomerization was observed during the M → PoXeR13C process in a previous study 13. Precise comparison between the transient absorption change and absolute absorption spectra of PoXeR and PoXeR13C revealed direct recovery of M → PoXeR (13-cis-to-trans isomerization) competed with the formation of PoXeR13C. Both types of isomerization orient the lone-pair of deprotonated Schiff base from the cytoplasmic side towards the extracellular side. This is expected to make the Schiff base more easily protonated (by elevating its pKa). Thus, we consider that the isomerization of retinal is the rate-limiting step of proton uptake from the extracellular side and explains the negligible KIE and pH-independency of the proton uptake rate for PoXeR wild-type and mutants of the putative proton donor (Asp3, Arg72, and Asp119). Branched Reaction of Thermal Isomerization of Retinal in PoXeR. Finally, the branched photocycle of PoXeR on M decay will be discussed. While branched photocycles have been observed for many types of channelrhodopsins 42, 43, light-driven ion pump rhodopsins generally show unbranched photocycles. To our knowledge, the branching reaction of PoXeR from 13-cis-15-anti to all-trans-15-anti and 13-cis-15syn with comparable rates (43% and 57%, respectively) is the first example among pump rhodopsins. Interestingly, transient absorption change of pyranine suggested that excess proton is taken up after the M-decay. Since 57% of the M directly recovers to the PoXeRAT, only long-lived PoXeR13C is related to the binding of excess proton. According to the branching ratio to PoXeR13C (43%) and the amount of excess proton (equivalent to 17% of product from the total M), 41% of PoXeR13C is more protonated than PoXeRAT. In contrast to the comparable blanching ration of PoXeR, in the case of ASR, which is also a member of XeR family and evolutionally close to PoXeR, we reported that the M exclusively decayed to ASR with a 13-cis15-syn configuration (ASR13C) in a previous study 44. Because the significant difference between PoXeR and ASR may have been caused by the different methods (transient absorption spectroscopy at room temperature and low-temperature UVvisible absorption spectroscopy) used for analysis, the branching rate of the M-decay of ASR was determined using the same method as that used for PoXeR. The ratio between fullbleaching of ASR by laser excitation estimated by extrapolating the single exponential fitting line corresponding to the Mdecay (Fig. S3A, green line) to t = 0 and remaining bleaching after ASR13C formation (Fig. S3A, red dashed line) was very close to that between the absorbance of ASRAT (ASR having all-trans-15-anti retinal) (Fig. S3B, black solid line) and ASR13C (Fig. S3B red dashed line) at 570 nm. This result suggests that 91.7 ± 6.0% of the photo-excited ASR was converted to ASR13C, and the branching was more biased than that of PoXeR. In contrast, recently reported NsXeR shows no significant branching reaction 15. Thus, the degrees of branching are diverse among different XeRs, and may be regulated by the different amino acid residues around retinal (e. g. PoXeR Asp108 is replaced by glutamine and glutamic acid in ASR and NsXeR, respectively, and NsXeR contains a tryptophan at

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the position of PoXeR Arg71, which is one of the most conserved arginine residues in many microbial rhodopsins including ASR). Although PoXeR is well-expressed in mammalian cells, the recovery from PoXeR13C to PoXeRAT is very slow (91 s) 13. Thus, the construction of PoXeR with no branching to PoXeR13C by mutation is important for developing a more ideal optogenetic tool for controlling the inner-pH of synaptic vesicles, lysosomes, and vacuoles, among other structures. CONCLUSIONS The inward proton pump mechanism of PoXeR is studied by comprehensive spectroscopic measurement. Femtosecond time-resolved absorption measurement revealed that the retinal isomerization process of PoXeR is significantly slower compared with those of other microbial rhodopsins. Subsequently, proton release and uptake which were observed by the transient absorption change of pH-indicating pyranine dye occur on the processes of L/M-to-M and M-decay, respectively. While proton is released from Asp216 to cytoplasmic milieu, deprotonated retinal Schiff base is suggested to directly uptake proton from extracellular bulk solvent upon the configuration change from 13-cis-15-anti to 13-cis-15-syn and all-trans-15anti which proceeds without showing significant KIE. This result suggests that proton uptake process of inward proton pump is highly different from that of outward proton pump which has specific proton donor, and configuration change of retinal plays a critical role for the former.

ASSOCIATED CONTENT Supporting Information. Amplitudes of four kinetic components obtained by fitting analysis for the temporal traces of femtosecond time-resolved absorption spectroscopy, magnification factors for temporal traces (Fig. 6B) in each time region at each wavelength, transport activity and transient absorption change of PoXeR Arg71 mutants, amino-acid residues around retinal of BR and modeled PoXeR, and calculation of the conversion efficiency from the M to ASR13C. This information is available free of charge via the Internet at http://pubs.acs.org

AUTHOR INFORMATION Corresponding Authors *[email protected] and [email protected]

ACKNOWLEDGMENT This work was supported by JSPS KAKENHI Grant Number 26708001, 26620005, 17H03007 to K.I (26708001, 26620005, 17H03007), JP25104005 to T. T., and 25104009, 15H02391to H.K

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Spectroscopic Study of Proton Transfer Mechanism of Inward Proton

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