Unique Photochemistry Observed in a New Microbial Rhodopsin | The

Letter Cite This: J. Phys. Chem. Lett. 2019, 10, 5117−5121

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Unique Photochemistry Observed in a New Microbial Rhodopsin Chihiro Kataoka,† Keiichi Inoue,†,‡,§,∥ Kota Katayama,†,‡ Oded Béjà,⊥ 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 § The Institute for Solid State Physics, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8581, Japan ∥ PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan ⊥ Faculty of Biology, Technion-Israel Institute of Technology, Haifa 3200003, Israel Downloaded via NOTTINGHAM TRENT UNIV on August 22, 2019 at 14:42:28 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: Light energy is first captured in animal and microbial rhodopsins by ultrafast photoisomerization, whose relaxation accompanies protein structural changes for each function. Here, we report a microbial rhodopsin, marine bacterial TAT rhodopsin, that displays no formation of photointermediates at >10−5 s. Low-temperature ultraviolet− visible and Fourier transform infrared spectroscopy revealed that TAT rhodopsin features all-trans to 13-cis photoisomerization like other microbial rhodopsins, but a planar 13-cis chromophore in the primary K intermediate seems to favor thermal back isomerization to the original state without photocycle completion. The molecular mechanism of the early photoreaction in TAT rhodopsin will be discussed.

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changes for function. This scenario is common for all known microbial rhodopsins without exception.1,5−10 Here, we report an atypical microbial rhodopsin in the photochemical dynamics. Members of the α-proteobacterial SAR11 clade are the most abundant organisms in the world’s oceans.15 Analysis of the genome of SAR11 HIMB11416,17 revealed the existence of two microbial rhodopsins, SAR11 HIMB114 proteorhodopsin (PR) and TAT rhodopsin18 (Figures S1 and S2). It is well-known that the function of microbial rhodopsins can be distinguished by characteristic sequence motifs in their helix 3.19,20 While the archaeal lightdriven proton pump bacteriorhodopsin (BR) has the DTD motif (D85, T89, and D96) in helix 3, most bacterial lightdriven proton pumps, including PRs, contain a DTE motif (Figure 1a).9 SAR11 HIMB114 rhodopsin contains the DTE motif (Figure 1a), suggesting a function similar to that of a light-driven proton pump like PR. On the other hand, the second rhodopsin possesses a unique TAT motif in helix 3 and is thereby named TAT rhodopsin (Figure 1a). Interestingly, the absorption spectrum of TAT rhodopsin at pH 8.0 displays two λmaxs at 400 and 561 nm corresponding to the deprotonated and protonated states, respectively (Figure 1b). While decreasing the pH to 4.0 shifts the equilibrium toward the protonated state, deprotonation was observed with an increase in pH (Figures S3 and S4). The pH titration analysis

hodopsin is a large family of retinal-binding photoactive proteins found in animals and microbes.1 While animal rhodopsins are specialized photosensory G-protein-coupled receptors,1−4 microbial rhodopsins perform various functions, including gene expression, enzymatic reactions, and lightdriven ion conductance such as pumps and channels.1,5−10 While animal and microbial rhodopsins share a common seven-transmembrane α-helical architecture with their N- and C-termini located extracellularly and intracellularly, respectively, they share almost no sequence homology and differ largely in their functions. Due to their rapid and reversible ion conductance, microbial rhodopsins such as channelrhodopsins11 are being widely used in optogenetics to manipulate neuronal activity.12,13 The recent discovery of heliorhodopsins (HeRs) possessing an opposite membrane topology as compared to that of other microbial rhodopsins14 has reestablished their vast diversification. All microbial rhodopsins feature all-trans retinal attached by a Schiff base linkage to the ε-amino group of a lysine side chain in the middle of helix 7. Protonation of the all-trans retinal Schiff base is the common property of microbial rhodopsins and provides a wide range of absorption and color tuning. Upon light absorption, ultrafast photoisomerization takes place from the all-trans to the 13-cis form on a time scale of 10−13 to 10−12 s. Such fast photochemical reaction causes distortion of the retinal chromophore in the primary K intermediate, where light energy is stored through specific chromophore−protein interactions and its relaxation accompanies protein structural © XXXX American Chemical Society

Received: July 7, 2019 Accepted: August 13, 2019

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DOI: 10.1021/acs.jpclett.9b01957 J. Phys. Chem. Lett. 2019, 10, 5117−5121

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The Journal of Physical Chemistry Letters

Figure 1. Rhodopsins from SAR11 HIMB114. (a) Multiple-sequence alignment of characteristic regions between various microbial rhodopsins. The positions corresponding to D85, T89, and D96 in BR are marked with red circles. (b) Absorption spectrum of TAT rhodopsin, which was reconstituted into a lipid membrane (pH 8.0). (c) pH-dependent absorption changes at 403 and 568 nm.

showed pKa values in a reconstituted membrane and a detergent of 8.4 (Figure 1c) and 7.3 (Figure S4), respectively, suggesting that TAT rhodopsin features an equilibrium between its protonated and deprotonated states at physiological pH. Notably and like other microbial rhodopsins, both the protonated and deprotonated states of TAT rhodopsins harbored an all-trans form bound to the opsin protein moiety (Figure S5). TAT rhodopsin reconstituted into liposomes at physiological pH (8.0) was photoexcited by a nanosecond laser pulse at 532 nm, and transient absorption was measured with a time resolution of 40 μs (Figure 2). Surprisingly, no transient absorption changes were observed 38 μs, 50 ms, or 100 ms after the laser excitation (Figure 2). If photoisomerization is blocked by use of 11-cis or all-trans locked retinal analogues, no photochemical reaction takes place for animal21,22 or microbial23 rhodopsins, respectively. However, there have been no reports of native rhodopsins exhibiting no intermediate formation at microsecond and slower time scales. This observation can be interpreted in the following alternative ways. One possibility is an absence of photoisomerization, as is the case for artificial pigments with locked retinal.21−23 Another possibility is that photoisomerization takes place, but the primary intermediate reverts to the original state without a further photocycle. To gain insight into this unusual behavior and to assess the possibility of an absence of photoisomerization, we performed low-temperature ultraviolet−visible analyses of TAT rhodopsin. Figure 3 clearly shows the formation of the red-shifted K intermediate at 77 K, suggesting that the existence of photoisomerization followed by the thermal reisomerization of the K intermediate to the resting state.

Figure 2. Photoreaction dynamics of TAT rhodopsin. Transient absorption spectra of TAT rhodopsin in a lipid membrane (pH 8.0). The black dotted line represents the transient absorption spectrum of SAR11 PR.

To determine the structural changes that take place during photoisomerization of TAT rhodopsin, we conducted lightinduced difference Fourier transform infrared (FTIR) spectroscopy. Figure 4a shows the K-minus-TAT difference FTIR spectra in H2O (black line) and D2O (red line) at 77 K. The low-frequency shift of the CC stretch from 1542 cm−1 in TAT rhodopsin to 1526 cm−1 upon formation of the K intermediate is consistent with the spectral red-shift in visible 5118

DOI: 10.1021/acs.jpclett.9b01957 J. Phys. Chem. Lett. 2019, 10, 5117−5121

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Figure 3. Light-induced difference ultraviolet−visible spectra of TAT rhodopsin reconstituted into lipid membranes at 77 K. The sample was prepared at pH 6.0. The hydrated film with H2O was illuminated at 547 ± 10 nm. The light-minus-dark difference spectrum was measured.

Figure 5. Spectral comparison in the HOOP region of light-induced difference FTIR spectra of various microbial rhodopsins at 77 K. See the text for details.

Crystal structures of many microbial rhodopsins reported that each protein accommodates an all-trans retinal in its planar form.30 Upon light activation, the all-trans to 13-cis photoisomerization causes chromophore distortion of the 13cis form,31,32 which drives subsequent protein structural changes.33,34 A partial distortion of the 13-cis chromophore in the protonated K intermediate of TAT rhodopsin might not have sufficient driving force for protein structural changes, and consequently, the K intermediate returns to the resting state. There are essentially two reaction pathways after the K intermediate: (i) relaxation of the 13-cis state by driving protein structural changes, leading to the subsequent intermediates such as L and M, and (ii) thermal 13-cis to alltrans isomerization, leading to the recovery to the initial state. Researchers have never considered pathway ii, because nature uses pathway i exclusively. This indicates that thermal reaction of pathway i is much faster than that of pathway ii in microbial rhodopsins, so that pathway ii is negligible. This suggests that the activation barrier for thermal 13-cis to all-trans isomerization (ii) is much higher than the relaxation of the 13-cis state (i) in general microbial rhodopsins. In the case of TAT rhodopsin, however, the activation barrier for thermal 13-cis to all-trans isomerization (ii) is much lower than the relaxation of the 13-cis state (i), and thus, TAT rhodopsin uses pathway ii exclusively. The unique reaction is achieved by the specific chromophore−protein interaction in TAT rhodopsin. Although little is known about the interaction at present, it is possibly correlated to the low pKa of the Schiff base (Figure 1, Figure S3, and Figure S4), which is unique to TAT rhodopsin. The planarity of the retinal chromophore in the K intermediate (Figure 5) is also unique to TAT rhodopsin. According to the previous computational study, planarization of the retinal chromophore causes a high thermal double bond isomerization and blue-shifted absorption.35 The experimental results presented here suggest efficient thermal 13-cis to all-

Figure 4. Light-induced difference FTIR spectra of TAT rhodopsin reconstituted into lipid membranes at 77 K. The sample was prepared at pH 6.0. The hydrated film with either H2O (black line) or D2O (red line) was illuminated at 547 ± 10 nm. The light-minus-dark difference spectra were measured.

range (Figure 4). The peak pair at 1202 (−)/1196 (+) cm−1 is also characteristic of the difference spectra upon all-trans to 13cis photoisomerization.24 Thus, the difference FTIR spectra of the protonated form of TAT rhodopsin look similar to the typical spectra of microbial rhodopsins at 77 K. Next, we studied why the K intermediate of TAT rhodopsin does not proceed with the formation of subsequent L and M intermediates. The difference FTIR spectra showed hydrogenout-of-plane (HOOP) vibrations in the 1000−800 cm−1 region. The difference spectra show characteristic peaks at 1006 (+)/990 (−)/881 (+) cm−1, among which the D2Ospecific negative peak at 990 cm−1 can be ascribed to the inplane N−D bending vibration of the Schiff base. In comparison to the CC and C−C stretches, positive HOOP bands appeared to be small for TAT rhodopsin. The HOOP vibrations of TAT rhodopsin are much smaller than those of the light-driven proton pump (BR25 and PR26), chloride pump [halorhodopsin (HR)],27 sodium pump (KR2),28 and light sensor [pharaonis sensory rhodopsin II (pSRII)]29 (Figure 5 and Figure S6). As the appearance of HOOP bands is the result of chromophore distortion, these results suggest that the K intermediate of the protonated form of TAT rhodopsin harbors a planar 13-cis chromophore. 5119

DOI: 10.1021/acs.jpclett.9b01957 J. Phys. Chem. Lett. 2019, 10, 5117−5121

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trans isomerization for TAT rhodopsin and red-shifted absorption of the K intermediate (Figure 3) similar to those of other rhodopsins. At present, we have no clear answer for the apparent variance, while the red-shift in the planar K intermediate may be caused by an expansion of the double bond length rather than double bond twisting. Such a change in bond length alteration is possibly induced by the electrostatics of the unique protein cavity of TAT rhodopsin, especially around the Schiff base region. Now, the function of TAT rhodopsin is intriguing. Light converts TAT rhodopsin into the K intermediate, which returns to the original state with a photocycle duration of 10−5 s. This time scale may be too short to consider any functional processes, where light energy is fully converted into thermal energy without propagating further photoreaction. One possibility is that the photoreaction time scale is sufficient for the function of TAT rhodopsin. Sheves and co-workers reported the presence of slow dynamics in bacteriorhodopsin with the all-trans locked retinal analogues, though the photoreaction is complete within the time scale.36,37 Another possibility is the lack of a functional role for the visible absorbing form of TAT rhodopsin, whereas the deprotonated form of TAT rhodopsin may be important for function. This idea is supported by the low pKa of the protonated Schiff base of TAT rhodopsin. We do not know the function of TAT rhodopsin at present, and further studies of TAT rhodopsin will lead to a better understanding of the unique photochemistry of this rhodopsin. In summary, TAT rhodopsin displays an equilibrium between its protonated and unprotonated forms under physiological conditions. Upon illumination of green light, transient absorption was completely silent at microsecond and slower time scales, which was never observed for known microbial rhodopsins. Low-temperature ultraviolet−visible spectroscopy confirmed formation of the K intermediate, as well as other microbial rhodopsins. Low-temperature FTIR spectroscopy further revealed all-trans to 13-cis photoisomerization taking place in TAT rhodopsin. A planar retinal chromophore in the K intermediate was also observed for TAT rhodopsin, which may cause a high reaction barrier for the relaxation to the subsequent intermediates and lead to the recovery to the initial state without photocycle completion. Further chemical and biological studies will lead to a better understanding of this unique rhodopsin protein.

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Letter

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Keiichi Inoue: 0000-0002-6898-4347 Hideki Kandori: 0000-0002-4922-1344 Author Contributions

C.K. and K.I. prepared samples for spectroscopic measurements. C.K. and K.I. conducted pH titration, HPLC analysis, and flash photolysis measurements. C.K. and K.K. conducted FTIR measurements. O.B. performed bioinformatics analysis. H.K. wrote the paper and directed all of the research. All authors discussed and commented on the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology to K.I. (17H03007) and H.K. (18H03986), by CREST, Japan Science and Technology Agency, to H.K. (JPMJCR1753), and by PRESTO, Japan Science and Technology Agency, to K.I. (JPMJPR15P2).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpclett.9b01957. Experimental procedure, multiple-sequence alignment of selected microbial rhodopsins (Figure S1), phylogenetic tree of microbial rhodopsins (Figure S2), pH titration of SAR11 HIMB114 TAT in a lipid membrane (Figure S3), pH titration of SAR11 HIMB114 TAT in a detergent (Figure S4), HPLC analysis of the darkadapted TAT rhodopsin at pH 8.0 and 4.0 (Figure S5), and light-induced FTIR difference spectra of various microbial rhodopsins at 77 K in the 1780−820 cm−1 region (Figure S6) (PDF) 5120

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