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Biophysical Chemistry, Biomolecules, and Biomaterials; Surfactants and Membranes

Production of a Light-gated Proton Channel by Replacing the Retinal Chromophore with Its Synthetic Vinylene Derivative Riho Takayama, Akimasa Kaneko, Takashi Okitsu, Satoshi P. Tsunoda, Kazumi Shimono, Misao Mizuno, Keiichi Kojima, Takashi Tsukamoto, Hideki Kandori, Yasuhisa Mizutani, Akimori Wada, and Yuki Sudo J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.8b00879 • Publication Date (Web): 11 May 2018 Downloaded from http://pubs.acs.org on May 12, 2018

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The Journal of Physical Chemistry Letters

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Production of a Light-gated Proton Channel by

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Replacing the Retinal Chromophore with Its

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Synthetic Vinylene Derivative

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Riho Takayama†,┴, Akimasa Kaneko†,┴, Takashi Okitsu‡,┴, Satoshi P. Tsunoda&,%, Kazumi

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Shimono^, Misao Mizuno#, Keiichi Kojima†,§, Takashi Tsukamoto†,§, Hideki Kandori&,

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Yasuhisa Mizutani#, Akimori Wada‡ and Yuki Sudo†,§,*

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†

Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan

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‡

Laboratory of Organic Chemistry for Life Science, Kobe Pharmaceutical University, Kobe 658-

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8558, Japan

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&

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%

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0012, Japan

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^

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#

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Japan

Department of Frontier Materials, Nagoya Institute of Technology, Nagoya 466-8555, Japan PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-

Faculty of Pharmaceutical Sciences, Toho University, Funabashi 274-8510, Japan Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka 560-0043,

ACS Paragon Plus Environment

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§

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Okayama 700-8530, Japan

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Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University,

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AUTHOR INFORMATION

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Corresponding Author

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*

[email protected]

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ORCID

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Yuki Sudo: 0000-0001-8155-9356

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AUTHOR CONTRIBUTIONS

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┴

These authors contributed equally to this work.

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ABSTRACT. Rhodopsin is widely distributed in organisms as a membrane-embedded

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photoreceptor protein, consisting of the apoprotein opsin and vitamin-A aldehyde retinal, A1-

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retinal and A2-retinal being the natural chromophores. Modifications of opsin (e.g., by

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mutations) have provided insights into the molecular mechanism of the light-induced functions

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of rhodopsins as well as providing tools in chemical biology to control cellular activity by light.

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Instead of the apoprotein opsin, in this study, we focused on the retinal chromophore and

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synthesized three vinylene derivatives of A2-retinal. One of them, C(14)-vinylene A2-retinal


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(14V-A2), was successfully incorporated into the opsin of a light-driven proton pump

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archaerhodopsin-3 (AR3). Electrophysiological experiments revealed that the opsin of AR3

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(archaeopsin3, AO3) with 14V-A2 functions as a light-gated proton channel. The engineered

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proton channel showed the characteristic photochemical properties, which are significantly

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different from AR3. Thus, we successfully produced a proton channel by replacing the

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chromophore of AR3.

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TOC GRAPHICS

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KEYWORDS Photoactive protein • Ion transport • Rhodopsin.


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Rhodopsin, a seven-transmembrane photoreceptor protein, is widely distributed in all domains of

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life, including archaea, bacteria and eukaryotes, indicating its biological significance for

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organisms.1, 2 Rhodopsin consists of an apoprotein opsin and vitamin-A aldehyde retinal, with

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A1-retinal and A2-retinal being the natural chromophores (Figure 1A).3 Those retinals bind

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covalently to a conserved Lys residue on the seventh (or G) helix of opsin via a protonated

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retinal Schiff base (PRSB) linkage, where the positive charge is stabilized by a negatively

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charged calboxylate called the counterion (Figure 1B).4 Light absorption by rhodopsins triggers

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the isomerization of the retinal chromophore within several hundred femtoseconds and the stored

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energy in the excited state induces a stepwise photoreaction with structural changes of the opsin,

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that lead to a variety of photobiological functions including photo-energy conversion and photo-

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signal transduction.

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utilized as tools for optogenetics, a method to control cellular activity by light in vivo.5 On the

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basis of that background, to understand and utilize rhodopsins, researchers are extensively trying

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to modify the protein moiety (i.e., insertion, deletion and/or replacement) and both the

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production of color variants and functional conversions have been achieved by strategic

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mutations.6-9 Alternatively, in this study, we focused on the “chromophore” to modify the

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functional and photochemical properties of rhodopsins.

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In addition to their biological significance, rhodopsins have been widely

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Three derivatives of A2-retinal, C(6)-Vinylene A2-retinal (6V-A2), C(10)-Vinylene A2-

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retinal (10V-A2) and C(14)-Vinylene A2-retinal (14V-A2), each of which possess a long π-

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conjugation system on the polyene chain (Figure 1A), were newly synthesized by an organic

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chemistry method with overall yields of 12, 4 and 9%, respectively (Supporting Figures S1-S4).

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Due to the extension of the π-conjugation system, the absorption maxima of these derivatives

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(420 nm for 6V-A2, 418 nm for 10V-A2 and 421 nm for 14V-A2) were largely shifted to a


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longer wavelength than that of A2-retinal (376 nm). The spectral red-shifts are explained by a

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reduction of the energy gap between the electronic ground- and excited- states of the retinal

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chromophore.10 The opsin of Middle rhodopsin (Middle opsin, MO) was firstly employed as a

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protein template because MO characteristically accepts a variety of retinal isomers, 9-cis, 11-cis,

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13-cis and all-trans, which suggests that it has a large cavity around the chromophore.11,

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According to standard methods and procedures, MO was expressed in Escherichia coli cells with

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natural retinals (A1 or A2) and the synthesized A2-retinal derivatives (6V-, 10V- or 14V-A2). E.

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coli cells expressing MO with the three synthesized derivatives showed an orange-red color

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(Supporting Figure S5A), indicating the successful incorporation of those derivatives into MO.

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During the purification, the color of MO with 6V-A2 or 10V-A2 changed to yellow due to

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denaturation, while MO with 14V-A2 was successfully purified with a visible color in the

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detergent n-dodecyl β-D-maltoside (DDM) and its absorption maximum appeared at 504 nm,

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comparable to that of MO with A2-retinal (508 nm). Those results suggest the breakage of the π-

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conjugation system on 14V-A2 in MO. The biological function of MR is still unclear, so we

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moved on to a second rhodopsin template, archaerhodopsin-3 (AR3).

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AR3 works as a light-driven outward proton pump, which is one of the most typical

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biological functions of rhodopsins, and is applicable for optogenetics as a neural silencer.13 Cells

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expressing archaeopsin-3 (AO3) with natural retinals (A1 or A2) or the three retinal derivatives

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(6V-, 10V- or 14V-A2) showed a slight orange color for AO3 with 6V- or 10V-A2 and a purple

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color for AO3 with 14V-A2 (Figure 1C), suggesting their successful expression in the cell

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membrane. As reported,8, 14 light-induced decreases in pH were observed for cells expressing

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AO3 with A1-retinal or A2-retinal, and treatment with the protonophore carbonyl cyanide 3-

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chlorophenylhydrazone (CCCP) strongly impaired those pH changes (Figure 2A), indicating


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their light-driven “outward” proton transport activities. On the other hand, for AO3 with 14V-

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A2, light-induced increases in pH and their inhibition by CCCP were observed, indicating a

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light-induced “inward” proton movement. Increases in pH were also observed for AO3 with 6V-

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A2 or 10V-A2 (Figure 2A). These results suggest that the inward proton movement of AO3 with

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6V-, 10V- or 14V- A2 retinal derivatives is caused by addition of the vinylene (-C=C-C-) group

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to any position on the polyene chain of the retinal. As occurred with MO, the color of AO3 with

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6V-A2 or 10V-A2 rapidly changed to yellow during purification, while the purple color of AO3

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with 14V-A2 was maintained for several days. Therefore, AO3 with 14V-A2 was used for

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further investigations. As shown in Figure 2B, the absorption maxima of purified AO3 with A1-

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retinal (i.e., wild-type AR3), A2-retinal or 14V-A2 were located at 556 nm, 584 nm and 543 nm,

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respectively. The spectral red-shift of A2-retinal (+28 nm) compared with A1-retinal was

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explained by the extended π-conjugation system on the polyene chain. On the other hand, a large

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spectral blue-shift was observed for 14V-A2 (-41 nm) compared with A2-retinal. Judging from

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the color of the cells (as shown in Figure 1C), a spectral blue-shift was also likely to be observed

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for AO3 with 6V-A2 or 10V-A2. These results suggest that addition of the vinylene group

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commonly induces breakage of the π-conjugation system on the polyene chain with different

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magnitudes.

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The dose-response relationship between light intensities and initial slope amplitudes of

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the light-induced pH changes showed a linear regression at a low light intensity below 6

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mW/cm2 (Supporting Figure S6). Therefore, we employed a light intensity of 6 mW/cm2 and

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obtained action spectra. The absorption spectra of AO3 with A1-retinal, A2-retinal or 14V-A2

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matched well with their light-induced proton transport activities (Figure 2C), indicating that light

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absorption of AO3 with 14V-A2 leads to its inward proton transportation. To confirm whether


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the proton movement is by active transport (pump) or by passive transport (channel), we

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performed electrophysiological experiments (Figures 2D-F). For illumination, we used a xenon

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light source through a 580 nm band-pass filter. Figure 2D shows a whole cell patch-clamp

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recording of murine ND7/23 cells expressing AO3 with A1-retinal (i.e., wild-type AR3, left) or

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AO3 with 14V-A2 (right) at different extracellular pHs (pHo = 7.2 and 4.5) and +20 mV of

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holding potential. Positive currents upon illumination were observed for AO3 with A1-retinal

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both at pHo 7.2 and 4.5, whereas positive and negative currents were observed for AO3 with

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14V-A2 at pHo 7.2 and 4.5, respectively. Similar experiments were then performed under various

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membrane holding potentials ranging from -100 to 60 mV to obtain the current-voltage

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relationship (I-V plot) of the photocurrent (Figures 2E and F). Positive currents were observed

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for AO3 with A1-retinal at all holding potentials (Figure 2E), representing the active proton

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“pump” activity from the inside to the outside of the cell. In contrast, the direction of the currents

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for AO3 with 14V-A2 was influenced by the membrane potential and a reversal of the potential

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appeared around zero at pH0 of 7.2, where the proton gradient (delta pH) between the inside and

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the outside of the cell is expected to be around zero (Figure 2F), suggesting a passive proton

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“channel” activity. From these results, we concluded that AO3 with 14V-A2 works as a light-

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gated proton “channel”. Thus, the addition of a vinylene group into the retinal chromophore

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converts a proton pump into a proton channel. A transient outward current and a rapid current

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reduction concomitant with the start and stop of illumination were observed in the current traces

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of cells expressing AO3 with 14V-A2 (Figure 2D), which suggests that AO3 with 14V-A2 also

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works as a leaky outward proton pump similar to natural light-gated proton channels.15, 16

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To characterize the photochemical properties of AO3 with 14V-A2, we performed

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further spectroscopic analysis (Figure 3). Firstly, we carried out pH titration experiments to


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estimate the pKa of the counterion (Asp95) of the PRSB (Figure 1B). Upon acidification, the

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absorption maxima were shifted from 556 nm to 573 nm for AO3 with A1-retinal (i.e., wild-type

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AR3), from 584 nm to 600 nm for AO3 with A2 and from 543 nm to 553 nm for AO3 with 14V-

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A2 (Figures 3A-C). These large spectral red-shifts were explained by protonation of the

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counterion, which drops the energy gap between the electronic ground state and the excited

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state.10 In the difference spectra, the maximal absorption changes were located at 618 nm for

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AO3 with A1-retinal, at 668 nm for AO3 with A2-retinal and at 641 nm for AO3 with 14V-A2.

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The absorption changes were then plotted against various pH values and the data were fitted by

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the Henderson-Hasselbalch equation with a single pKa value of 3.10 ± 0.13 for AO3 with A1-

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retinal, 3.05 ± 0.03 for AO3 with A2-retinal and 3.39 ± 0.08 for AO3 with 14V-A2 (Figure 3D).

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The slight upshift of the pKa in AO3 with 14V-A2 suggests that the environment of the PRSB of

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AO3 with 14V-A2 is slightly different from those of AR3 with A1-retinal or A2-retinal. We then

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performed resonance Raman spectroscopy to investigate the chromophore structure (Figure 3E).

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As seen, hydrogen-out-of-plane (HOOP) vibrations appearing at 950-1000 cm-1 were similar

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between the three retinals (A1-, A2- and 14V-A2). For C-C stretching vibrations appearing at

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around 1200 cm-1, the vibrational frequencies for AO3 with 14V-A2 were down-shifted from

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those for AO3 with A-2-retinal (1201 cm-1 1177 cm-1, 1180 cm-1 1159 cm-1 and 1170 cm-1

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1149 cm-1) (Figure 3E). It has been reported that torsions around single bonds of the retinal

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chromophore could be seen as down-shifts of vibrational frequencies in the finger print region,17

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suggesting the torsion of the conjugated polyene chain around a single bond(s) in the binding

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pocket of AO3 with 14V-A2 which is expected to give rise to a blue shift of the absorption

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maximum. For C=C stretching vibrations appearing at 1500-1550 cm-1, when C(3) and C(4) are

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dehydrogenated (i.e., AO3 with A2 or 14V-A2), a splitting in this mode was observed with the


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new peaks lower than the band around 1530 cm-1 in trans-retinal. This phenomenon has already

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been reported both for bacteriorhodopsin and for bovine rhodopsin and has been interpreted that

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it results from a carbon-carbon hydrogen bond contribution involving the hydrogen at C(9) or

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C(13) and the nearest double bond.18 This contribution makes it possible to alter the two lowest

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energy C=C stretching modes. These bands have been reported to be generally deuteration

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insensitive.18 Of note, the amplitudes of the deuteration shift of the C=N stretch band of the

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retinal appearing around 1640 cm-1 was 11 cm-1 for AO3 with 14V-A2, which was significantly

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smaller than those for AO3 with A1-retinal and AO3 with A2-retinal (18 cm-1) (Figure 3E). It is

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known that a smaller deuteration shift of the C=N stretching frequency means a weaker hydrogen

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bond formation between the PRSB (Lys226) and its counterion (Asp95).19 Thus, these

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observations indicate that the hydrogen-bond strength of the Schiff base in AO3 with 14V-A2 is

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weaker than those of AO3 with A1-retinal or A2-retinal. From these results, we concluded that

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the structure around the Schiff base in AO3 with 14V-A2 is different from those with A1-retinal

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or with A2-retinal. Because the Schiff base region is altered upon illumination with

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isomerization of the retinal and plays an important role in the biological function of rhodopsins,

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the structural difference would be related to the functional conversion from the proton pump into

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the proton channel.

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Finally, to investigate the photoreaction kinetics, we carried out flash-photolysis

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experiments from millisecond to second time frames (Figure 4). The apparatus and procedures

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were essentially the same as previously reported.20 Upon illumination with light above 520 nm,

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the three variants commonly showed both a depression of the initial state absorption (540 – 590

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nm) and the formation of two photointermediates at shorter (410 – 430 nm) and longer (640 –

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670 nm) wavelengths (Figure 4A). Judging from the time region and the location of the


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absorption maxima, we tentatively assigned them as M and O intermediates, respectively. These

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results indicate that elementary reactions are common among the three derivatives of retinal. The

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M intermediate was formed with the decrease in the initial state absorption soon after the flash

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excitation and then decayed with formation of the O intermediate. The O intermediate then

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decayed with the recovery of the initial state and the cyclic reaction was completed (Figures 4B-

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C). To analyze the reaction kinetics more precisely, the data were fitted with the exponential

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decay function with a sum of two exponential terms, τ1 for the M intermediate and τ2 for the O

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intermediate (Figure 4B). From that analysis, we found that the photoreaction kinetics of AO3

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with A1-retinal or A2-retinal were similar (τ1 = 1.9 ms, τ2 = 119 ms for AO3 with A1-retinal,

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and τ1 = 1.9 ms, τ2 = 68 ms for AO3 with the A2-retinal) (Figures 4B-C). In contrast, the

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photoreaction kinetics of AO3 with 14V-A2 was significantly different from those, especially

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regarding the slow decay of the O intermediate (τ2 = 800 ms) (Figures 4B-C). As shown in

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Figure 2D, the recovery of the photocurrent for AO3 with 14V-A2 is slower than that of AO3

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with A1-retinal. The time constant of the recovery of the photocurrent for AO3 with 14V-A2 was

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estimated to be approx. 400 – 2200 ms, which is consistent with the decay of the O intermediate

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(800 ms). From these results, we hypothesized that the long-lived O intermediate corresponds to

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a proton conducting state for the proton channel function. Another explanation is that the

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rhodopsin undergoes two brunched routes, i.e. one with a fast H+ pumping and another with a

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slow H+ channel which is not visible in the spectroscopic measurement. Such phenomenon was

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already observed in the functional conversion of CsR (an algal proton pumping rhodopsin) where

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passive H+ transfer has much slower photocycle than that of the active proton transfer.21

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Thus, we demonstrated several characteristic features for AO3 with 14V-A2 as follows; (i)

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short π-conjugation system on the polyene chain, (ii) characteristic structure around the Schiff


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base region, and (iii) slow recovery of the photocurrent after turning off the light and a long-

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lived intermediate during the photoreaction. We previously converted AR3 into a light-gated

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proton channel by three mutations around the chromophore (M128A, G132V and A225T).8 In

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that study, a slow O-decay was observed with alteration of the hydrogen bond between the PRSB

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and its counterion.8 Thus, the determinant between the proton pump and the proton channel

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would be localized around the Schiff base region. We also demonstrated here that, in addition to

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the protein moiety, the chromophore moiety can be a target to produce a synthetic photoreceptor

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protein. In order to use our approach (i.e., addition of the synthetic chromophore) for optogenetic

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purposes, the synthetic chromophore needs to be injected into the organism. Therefore our

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approach can be directly applied to organisms without retinal synthetic pathway such as

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Caenorhabditis elegans and some of the bacteria, while the opsin should be protected from

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naturally occurring retinal for the other organisms. However, as we demonstrated here, when the

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opsin is overexpressed in the mammalian (murine ND7/23) cells, it successfully incorporates the

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synthetic retinal chromophore instead of naturally occurring retinal (Figure 2D). Thus our

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approach would be an alternative choice for optogenetics.

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ACKNOWLEDGMENT

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This work was financially supported by JSPS KAKENHI Grant Numbers JP15K18519 to TT

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and JP15H04363, JP15H00878, JP25104005 and JP17H05726 to YS. This research was partially

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supported by CREST-JST (16815580) and AMED (17933570) to YS, and by PRESTO-JST

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(JPMJPR1688)

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ms.com/home.html) for the English language review.

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ASSOCIATED CONTENT

to

SPT.

We

also

thank

“DASS

Manuscript”

(http://www.dass-


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Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at DOI:

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xxxxxx.

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Experimental details of chemical synthesis, protein preparation and spectroscopic

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characterization (PDF).

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NOTES

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The authors declare no competing financial interests.

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REFERENCES

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long-wavelength sensitivity of optogenetic microbial rhodopsins by 3,4-dehydroretinal.

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Biochemistry 2012, 51, 4499-4506.


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(15) Bamann, C., Kirsch, T., Nagel, G., and Bamberg, E., Spectral characteristics of the

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photocycle of channelrhodopsin-2 and its implication for channel function. J. Mol. Biol. 2008,

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375, 686-694.

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(16) Feldbauer, K., Zimmermann, D., Pintschovius, V., Spitz, J., Bamann, C., and Bamberg, E.,

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Channelrhodopsin-2 is a leaky proton pump. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 12317-

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12322.

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(17) Palings, I., Pardoen, J. A., van den Berg, E., Winkel, C., Lugtenburg, J., and Mathies, R. A.,

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Assignment of fingerprint vibrations in the resonance Raman spectra of rhodopsin,

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isorhodopsin, and bathorhodopsin: implications for chromophore structure and environment.

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Biochemistry 1987, 26, 2544-2556.

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(18) Cookingham, R. E., Lewis, A., and Lemley, A. T., A vibrational analysis of rhodopsin and

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bacteriorhodopsin chromophore analogues: resonance Raman and infrared spectroscopy of

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chemically modified retinals and Schiff bases. Biochemistry 1978, 17, 4699-4711.

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(19) Baasov, T., Friedman, N., and Sheves, M., Factors affecting the C = N stretching in

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protonated retinal Schiff base: a model study for bacteriorhodopsin and visual pigments.

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Biochemistry 1987, 26, 3210-3217.

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(20) Kanehara, K., Yoshizawa, S., Tsukamoto, T., and Sudo, Y., A phylogenetically distinctive

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and extremely heat stable light-driven proton pump from the eubacterium Rubrobacter

291

xylanophilus DSM 9941T. Sci. Rep. 2017, 7, 44427.

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(21) Vogt, A., Guo, Y., Tsunoda, S. P., Kateriya, S., Elstner, M., and Hegemann, P., Conversion of a light-driven proton pump into a light-gated ion channel. Sci. Rep. 2015, 5, 16450.


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Page 15 of 21 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


294 295

Figure 1. Expression of AO3 with vinylene derivatives. (A) Chemical structures of natural

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retinals (A1- and A2-) and three newly synthesized vinylene derivatives (6V-A2, 10V-A2 and

297

14V-A2). Absorption maxima (λmax) of A1-retinal, A2-retinal and the three synthesized

298

derivatives in EtOH solution are shown in parentheses. (B) Schematic of an outward proton

299

pump. All-trans A1-retinal binds to the cognate apoprotein opsin via the protonated Schiff base

300

linkage with the conserved lysine residue. EC and CP indicate extracellular side and cytoplasmic

301

side, respectively. (C) Visible color of E. coli BL21(DE3) cells expressing AO3 with natural or

302

synthesized derivatives of retinal.


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Page 16 of 21

303 304

Figure 2. Light-induced proton transport activity in AO3 with A1-retinal, A2-retinal, 6V-

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A2, 10V-A2 or 14V-A2. (A) Light-induced pH changes of E. coli cells expressing AO3 with

306

A1-retinal, A2-retinal, 6V-A2, 10V-A2 and 14V-A2. The cell suspensions were illuminated with

307

light (9-12 mW/cm2) for 3 min. The wavelengths of light were roughly chosen from the visible

308

color of the cells. The thick and thin gray lines indicate pH changes in the absence or presence of

309

CCCP (10 µM), respectively. (B) Absorption spectra of the purified proteins in a buffer

310

containing 50 mM Tris-HCl (pH 7.0), 1 M NaCl and 0.05 % DDM. (C) Action spectra of AO3

311

with A1-retinal, A2-retinal or 14V-A2 with offsets. The initial slope amplitudes of the light-

312

induced pH changes of E. coli cell suspensions are plotted against varying wavelengths of light

313

(6 mW/cm2). Data points represent the averages of 3 independent experiments; error bars

314

represent standard deviations (SD). Absorption spectra were superimposed into the figure (solid


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315

lines). (D) Whole cell patch-clamp recordings of the ion transport activities of AO3 with A1-

316

retinal (black, left panel) or 14V-A2 (blue, right) in ND7/23 cells. Representative current traces

317

of cells expressing AO3 with A1-retinal or 14V-A2 are shown. The holding potential was kept at

318

+20 mV. Upper and lower panels show traces at extracellular pHo of 7.2 and 4.5, respectively.

319

The 580 nm light was illuminated during the time indicated by the grey bars on the traces. (E, F)

320

Current-voltage relationship (I-V plot) of the photocurrent of AO3 with (E) A1-retinal (black) or

321

(F) 14V-A2 (blue) at pHo of 7.2 (filled circles) and 4.5 (open circles). For AO3 with A1-retinal

322

(panel E), the current amplitude was normalized to the condition of pHo 7.2 at 0 mV as 1.0. Error

323

bars represent SD (n = 4 for pHo 7.2 and 3 for pHo 4.5). For AO3 with 14V-A2 (panel F), the

324

current amplitude was normalized to the condition of pHo 7.2 at -80 mV as 1.0. Error bars

325

represent SD (n = 3 for pHo 7.2 and 5 for pHo 4.5).


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Page 18 of 21

326 327

Figure 3. Photochemical properties of AO3 with A1-retinal, A2-retinal or 14V-A2. (A)

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Absorption spectra of AO3 with A1-retinal ranging pH7 to pH2. (B) Absorption spectra of AO3

329

with A2-retinal ranging pH7 to pH2. (C) Absorption spectra of AO3 with 14V-A2 ranging pH7

330

to pH3. (D) Estimation of the pKa of the counterion Asp95 by pH-induced absorbance change of

331

AO3 with A1-retinal (black open circle), A2-retinal (red) or 14V-A2 (blue). Solid lines represent

332

fitting curves by the Henderson-Hasselbalch function with a single pKa. The experiments were

333

performed at room temperature (ca. 25˚C). (E) Resonance Raman spectra of AO3 with A1-

334

retinal (black line), A2-retinal (red) or 14V-A2 (blue). The solid and dotted lines indicate the


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335

spectra of retinals hydrated with H2O or D2O, respectively. Numbers marked by asterisks are

336

values for the D2O sample. Expanded views of the resonance Raman spectra of AO3 with A1-

337

retinal, A2-retinal and 14V-A2 were also represented as insets. The probe wavelength was 532

338

nm and the concentration of the sample was approximately 20 µM. The experiments were

339

performed at approximately 20˚C. The data for AO3 with A2-retinal or 14V-A2 were scaled by

340

2.2 and 1.3, respectively, for comparison.


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Page 20 of 21

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342 343

Figure 4. Photochemical kinetics of AO3 with A1-retinal, A2-retinal or 14V-A2. (A) Time-

344

resolved transient absorption spectra of AO3 with A1-retinal (upper panel), A2-retinal (middle

345

panel) or 14V-A2 (lower panel) against wavelength ranging from t = 0.80 msec to 8110 msec

346

time frame. (B) Time-resolved transient absorption changes of AO3 with A1-retinal (upper

347

panel), A2-retinal (middle panel) or 14V-A2 (lower panel) against time. The dotted grey lines

348

represent the fitting curves with the sum of two exponential terms. The temperature was kept at

349

15˚C. (C) A model for the photoreaction. Subscribed numbers indicate the absorption maxima of

350

each state.


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