pH Dependence of Anabaena Sensory Rhodopsin: Retinal Isomer

Jul 8, 2014 - Microbial rhodopsins are photoactive proteins, and their binding site can accommodate either all-trans or 13-cis retinal chromophore. Th...
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pH Dependence of Anabaena Sensory Rhodopsin: Retinal Isomer Composition, Rate of Dark Adaptation, and Photochemistry Rinat Rozin,† Amir Wand,‡ Kwang-Hwan Jung,§ Sanford Ruhman,*,‡ and Mordechai Sheves*,† †

Department of Organic Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel Institute of Chemistry, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem 91904, Israel § Department of Life Science and Institute of Biological Interfaces, Sogang University, Shinsu-Dong 1, Mapo-Gu, Seoul 121-742, South Korea ‡

S Supporting Information *

ABSTRACT: Microbial rhodopsins are photoactive proteins, and their binding site can accommodate either all-trans or 13-cis retinal chromophore. The pH dependence of isomeric composition, dark-adaptation rate, and primary events of Anabaena sensory rhodopsin (ASR), a microbial rhodopsin discovered a decade ago, are presented. The main findings are: (a) Two pKa values of 6.5 and 4.0 assigned to two different protein residues are observed using spectroscopic titration experiments for both ground-state retinal isomers: all-trans, 15-anti (AT) and 13-cis, 15-syn (13C). The protonation states of these protein residues affect the absorption spectrum of the pigment and most probably the isomerization process of the retinal chromophore. An additional pKa value of 8.5 is observed only for 13C-ASR. (b) The isomeric composition of ASR is determined over a wide pH range and found to be almost pH-independent in the dark (>96% AT isomer) but highly pH-dependent in the light-adapted form. (c) The kinetics of dark adaptation is recorded over a wide pH range, showing that the thermal isomerization from 13C to AT retinal occurs much faster at high pH rather than under acidic conditions. (d) Primary photochemical events of ASR at pH 5 are recorded using VIS hyperspectral pump−probe spectroscopy with 585 nm cutoff filter for 30 s. HPLC Analysis. HPLC analysis was performed as previously described,31 with a few modifications. Retinal extraction was carried out under red light at room temperature. The samples were analyzed on a Purospher STAR Si-5 μm (LichroCART 250-4, Merck) analytical column on a Waters 1525 HPLC equipped with a Waters 2487 Dual λ absorbance detector. The solvent was composed of 12% (v/v) ethyl acetate and 0.12% (v/v) ethanol in hexane, and the flow rate was 1.0 mL/min. Extraction of retinal oxime from the sample was carried out with hexane after adding hydroxylamine (at least 1000-fold molar excess with respect to the pigment) and denaturation with ethanol. This procedure was carried out in red light, either directly (for DA samples) or 0.5 min after light adaptation (for LA samples). The molar compositions of the retinal isomers were calculated from integration over the peak areas in the HPLC patterns. Retinal oxime isomers exist in 15-syn and 15anti forms; therefore, there are two isomers for all-trans (with retention times of 6.1 and 12.8 min) and two for 13-cis (6.9 and 8.0 min). The sum of integrated areas of both 15-syn and 15-anti was used for the isomeric ratio calculations. The detection was carried out by measuring the absorbance at 360 nm. Assignment of the peaks was performed by comparing them with HPLC pattern of retinal oximes extracted from BR in dark- and light- adapted states by the same method. UV−vis Absorption Spectroscopy. Steady-state absorption measurements were carried out with an Agilent 4583 diode-array spectrophotometer (Agilent Technologies, Palo Alto, CA) equipped with an Agilent 89090A thermostated cuvette holder.

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EXPERIMENTAL SECTION Sample Preparation. ASR samples were prepared as previously described.1,30 A truncated form of ASR (amino acids 8996

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Figure 2. pH-induced absorption changes of DA (left panels) and LA (right panels) samples of ASR in 300 mM NaCl in the pH range of 5.1−8.2. (a) Absorption spectra in the pH range of 5.1 (λmax = 553 nm) to 8.2 (λmax = 548 nm) for DA-ASR. The inset presents an expanded view around the absorption peak. Difference absorption spectra with extrema at 583 and 507 nm. All spectra were subtracted from the spectrum that was taken at pH 8.2. (c) Normalized titration curve at 583 nm, where the solid line represents the best fit with pKa 6.5 ± 0.1 (average with standard deviation of at least three experiments). (d) Absorption spectra in the pH range of 5.4 (λmax = 544 nm) to 7.6 (λmax = 539 nm) for LA-ASR. The inset presents an expanded view around the absorption peak. (e) Difference absorption spectra with extrema at 578 and 484 nm. All spectra were subtracted from the spectrum that was measured at pH 7.6. (f) Normalized titration curve at 578 nm, where the solid line represents the best fit with pKa 6.4 ± 0.2 (average with standard deviation of three experiments). The arrows in panels a, b, d, and e represent the direction of the titration from high to low pH.

Titration Experiments. All titrations were conducted in red-lighting conditions using NaOH or HCl to achieve the desired pH. To minimize the process of light adaptation of DAASR samples during the measurements due to the spectrophotometer irradiation, we used a filter that reduces the light intensity and the spectrophotometer ultraviolet lamp was turned off. The pKa values were calculated using the following relation, derived from the Henderson−Hasselbach equation ΔA =

of DA-ASR samples during the measurements due to the spectrophotometer irradiation, we used a filter that reduces the light intensity, and the spectrophotometer ultraviolet lamp was turned off. Pump−Probe Experiments. The pump−probe experimental setup has been described in detail elsewhere.10 A homemade multipass amplified Ti:Sapph pumping a TOPAS optical parametric amplifier produced pump pulses of ∼30 fs duration and centered at ∼560 nm, and multichannel probing was conducted with a broadband supercontinuum in a double spectrometer system. Signal amplitudes were demonstrated to be linear in pump intensity up to two times that used in experiments. Dispersion of pump pulses was compensated for in a slightly misaligned zero-dispersion grating pulse shaper. The obtained spectra were time-corrected for group dispersion of the probe continuum based on Kerr scans in water. Roomtemperature samples with a nominal OD of ∼0.4 were syringe pumped through a ∼0.3 mm path length cell, equipped with 0.2 mm fused silica windows. Sample integrity was determined spectrophotometrically before and after each day’s runs. DAASR samples were kept in the dark for 48 h (room temperature) and protected from photoconversion during experimental runs by eliminating photochemically effective ambient light. LA-ASR was prepared by irradiation with an

ΔA max [1 + 10n(pKa − pH)]

where ΔA and ΔAmax are the absorbance difference and the maximum absorbance difference between two different states: protonated and deprotonated, respectively; n is the number of protons participating in the transition; pKa is the midpoint of the observed transition; and pH is the measured pH.32,33 For pKa determination, at least three independent measurements were averaged. Kinetics of Dark Adaptation. Kinetic measurements of dark-adaptation were conducted at a specific wavelength for each pH value (specified in the Results section). The samples were illuminated at 25 °C using a cutoff filter of λ>585 nm for 30 s; then, the absorption spectrum of ASR was measured in the dark over time. To minimize the process of light-adaptation 8997

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Figure 3. pH-induced absorption changes of DA (left panels) and LA (right panels) samples of ASR in 300 mM NaCl in the pH range of 2.9 to 4.7. (a) Absorption spectra in the pH range of 3.1 (λmax = 550 nm) to 4.7 (λmax = 553 nm). The inset presents an expanded view around the absorption peak. (b) Difference absorption spectra with extrema at 576 and 489 nm. All spectra were subtracted from the spectrum that was taken at pH 4.7. (c) Normalized titration curve at 576 nm, where the solid line represents the best fit with pKa 4.0 ± 0.1 (average with standard deviation of at least three experiments). (d) Absorption spectra in the pH range of 2.9 (λmax = 539 nm) to 4.6 (λmax = 543 nm). The inset presents an expanded view around the absorption peak. (e) Difference absorption spectra with extrema at 577 and 494 nm. All spectra were subtracted from the spectrum that was measured at pH 4.6. (f) Normalized titration curve at 577 nm, where the solid line represents the best fit with pKa 3.6 ± 0.1 (average with standard deviation of three experiments). The arrows in panels a, b, d, and e represent the direction of the titration from high to low pH.

interference-filtered halogen lamp at 600 ± 35 nm (CORION P70−600-F) for ∼30 min prior to ultrafast measurements.

been previously reported.29,30 The difference absorption spectra for both DA-ASR and LA-ASR, presented in panels b and e, are similar and depict the concomitant growth of a positive lobe and a negative lobe on the red wing and blue wing of the absorption spectra, respectively. The two include isosbestic points that are located at 550 nm for DA-ASR and 525 nm for LA-ASR. From the difference absorption spectra curves, titration curves are obtained. (See panels c and f in Figure 2.) These titration curves have pKa values of 6.5 ± 0.1 and 6.4 ± 0.2 for DA-ASR and LA-ASR, respectively. We note that in the pH range of ∼8−10 (not shown), no significant change in absorption was observed. The second titration process was observed at the pH range 4.7 to 2.9 and was detected in both DA and LA samples (Figure 3). Even though the observed change in the absorption maximum between the acidic and the basic forms is only 3 to 4 nm (Figure 3a,d), it was clearly pH-dependent. The extracted pKa values are 4.0 ± 0.1 and 3.6 ± 0.1 for DA-ASR and LAASR, respectively (Figure 3c,f). In this pH range, the difference spectra of DA and LA samples were very similar including an isosbestic point at 537 nm (Figure 3b,e). The similar observations for DA and LA samples in both pH ranges can be rationalized as resulting from similar effects of specific protein residues protonation state on both AT and 13C retinal isomers. This, however, should be handled with caution,

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RESULTS Effects of pH on the Absorption Spectrum of ASR Protein. The results of broad range pH titrations on samples of ASR are presented in Figures 2 (for pH range of 5−8) and 3 (for pH range of 2−5) for both DA (left panels) and LA (right panels) samples. These depict the ground-state absorption spectra (upper panels, a and d), the difference absorption spectra (middle panels, b and e), and the respective spectroscopic titration curves (lower panels, c and f). Because the isomeric compositions of the DA and LA samples are different (the DA sample is almost pure AT-ASR and the LA sample contains significant contribution of the 13C-ASR, as is further discussed) and the absorption maximum of ASR is known to be light-dependent and shifts from λmax = 548 nm in the dark to λmax = 542 nm following pigment irradiation (pH 7, 300 mM NaCl), we have carried out broad range pH titrations both in DA and LA ASR samples. Figure 2 presents our findings for pH ∼5−8. Results shown in panels a and d demonstrate a small red shift (of ∼5 nm across the entire pH range) of the absorption maximum of ASR following pH decrease. Similar small effects on the absorption maximum of ASR following pH increase from 4 to 11 have 8998

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that were used as well as an exemplary chromatogram at pH 5 are reported in the SI. For DA-ASR, the Figure presents the fraction of the AT isomer in the samples versus the pH. It is evident that the retinal isomers ratio is almost unaffected and consists of more than 96% AT over a wide pH range. Only at high pH (>9) the isomer ratio is affected, with the AT contribution decreasing to ∼85% at pH 11. This is not the case for LA-ASR, whose isomeric composition is dramatically changed by pH alterations. As can be seen in Figure 4b, the 13C fraction in the ASR samples varies from ∼30% to 92% over the entire studied pH range. The bell-shaped curve, with the highest 13C portion detected around pH 5 (more than 90%), indicates that at least two different processes affect the isomeric composition, one of which is dominant at pH < 5 and the other at pH > 5. This observation is in line with our spectroscopic titration results shown above, pointing at two residues with pKa values of ∼3.6 and ∼6.4. Therefore, it seems that the protonation states of these two residues probably have an effect not only on the absorption spectrum of the retinal chromophore (as detected above), but also on its isomeric composition. Furthermore, the curve that is observed at the pH range 5−9.5 in Figure 4b is very wide and thus might be associated with a change in the protonation states of more than one protein residue. The first process is expected to lie in the pH range of ∼5−7 and the second in ∼7−9.5. While the former can be correlated with the observable changes in absorption associated with the pKa value of 6.4, the pH range presented in the latter shows no analogous changes in the results presented so far. As will be shown below, we propose an additional titration curve with a pKa of ∼8.5 for the 13C isomer only, which lies exactly in this range. To conclude, we have revealed that the ratio between AT retinal and 13C retinal is strongly affected by the pH in LAASR and much less affected in DA samples. We note that the isomeric compositions were not affected by the illumination time or by the NaCl concentration in both LA and DA states. Absorption Spectra of 13C-ASR and AT-ASR at Different pH Values. The results presented hitherto demonstrate that both the isomeric composition and the ground-state absorption spectrum of ASR are pH-dependent. Because the former may affect the absorption of LA samples, it is useful to extract the absorption spectra of pure AT and 13C isomers. This can be done using the isomeric composition achieved from the HPLC analysis (Figure 4) and the measured spectra of the LA-ASR and DA-ASR at a variety of pH values, under the simple assumption of independent contributions from both isomers to the measured spectra. Figure 5 displays the extracted AT-ASR and 13C-ASR absorption spectra in the pH range of 3.6 to 9.6 (based on the results previously presented). Because of the dominant fraction (>96%) of the AT isomer in the DA sample, there is a high resemblance between the extracted spectra of AT-ASR (Figure 5a) and those of DA-ASR (Figures 2a and 3a). However, the calculated spectrum of 13C-ASR differs from that of LA-ASR (Figures 5a, 2d, and 3d) by at least two noticeable traits. First, there is a blue shift of 10 nm in the calculated spectrum of 13CASR between pH 5 and 7.6, whereas a shift of only 5 nm was revealed for LA-ASR. Second, remarkable changes are observed in the absorption maxima and extinction coefficients in the spectrum of 13C-ASR between pH 7.0 and 9.6 (Figure 5a), whereas no such changes were observed in the LA-ASR. The second behavior of 13C-ASR at high pH values is totally unexpected and cannot be explained easily. Figure 5b displays

because the absorption of the LA samples consists of contributions from different mixtures of AT and 13C isomer. Additional transition of the absorption maximum occurs at high pH and is associated with deprotonation of the protonated Schiff base (PSB) moiety (data not shown). The pKa value of this transition is very high (above 12.5) in DA-ASR, resembling that of other MRPs.34,35 Retinal Isomer Composition of ASR. Inconclusive reports concerning the exact isomeric composition of the retinal chromophore in ASR have led us to revisit this issue by analyzing its pH dependence. For determination of the isomer ratio, the retinal molecule was extracted from the protein and analyzed by HPLC. To minimize the thermal isomerization process of the retinal isomers after extraction, they were converted to their oxime forms prior to the extraction.36,37 Consequently, each retinal isomer produces two stereoisomers, syn and anti oximes, which are considered later as originating from the same retinal isomer. (See typical HPLC curve in the SI.) LA samples were prepared by illumination with a cold light source, using a 585 nm cutoff filter, and DA samples were incubated in the dark for at least 24 h or 1 week for samples below pH 5 due to their very slow rate of dark-adaptation. (See later.) Summarized HPLC results are presented in Figure 4 for DA-ASR and LA-ASR. Tables containing the exact isomeric compositions found at all pH values and the appropriate buffers

Figure 4. pH dependence of isomeric composition in DA-ASR and LA-ASR. (a) AT isomer fraction in the DA-ASR sample. (b) 13C isomer fraction in the LA-ASR sample. Estimated errors in the DA and LA samples are ±1 and ±2%, respectively. 8999

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Figure 5. Extracted spectra of pure AT-ASR and 13C-ASR. (a) Ground-state spectra obtained for AT-ASR and 13C-ASR from the experimental spectra of DA-ASR and LA-ASR by proper weighting of the isomeric composition obtained by HPLC analysis for each pH value. The extraction was achieved by solving a system of two linear equations in two variables: SLA = aC + bT and SDA = aC + bT, where SLA and SDA are the measured spectra of LA-ASR and DA-ASR for a specific pH value, the variables C and T are the spectra of pure 13C-ASR and AT-ASR, respectively, and the coefficients a and b refer to the fractions of the 13C isomer and the AT isomer in the LA and DA samples according to the HPLC results. Isomeric composition of the DA-ASR was taken as constant for the chosen pH range because of almost unchangeable isomeric composition, with average of 97.1% of AT isomer. The inset presents an expanded view around the absorption peak for the pure spectra of AT-ASR. The scheme in the bottom emphasizes that there is a blue shift in the spectra as the pH increases. (b) Difference absorption spectra of 13C-ASR in the pH range of 7.0 to 9.6 with a minimum at 560 nm. All spectra were subtracted from that of pH 7.0. (c) Normalized titration curve at 560 nm (extracted from panel b), where the solid line represents the best fit with pKa 8.5.

isomer should in theory allow the extraction of its own titration curves and pKa values. However, it is hampered by S/N limitations due to the subtraction procedure and to the anyhow minute changes between the absorption curves. In the pH range relevant to these points (roughly 3 < pH < 7), the 13C isomer content in the mixture is ≥70%. Moreover, the absolute values of the difference spectra of LA-ASR are comparable to those of DA-ASR, rendering their attribution to the relatively low AT contents less probable. Therefore, we assign these two pKa values in the LA-ASR samples to the 13C isomer but note that their exact numeric values might be slightly shifted due to contributions from the AT isomer existing in the sample. pH Dependence of the Rate of Dark Adaptation in ASR. The pH dependence of the rate constant of dark adaptation, that is, the thermal isomerization of the chromophore from 13C to AT, is presented in Figure 6. It shows that (a) the dark-adaptation proceeds as a first-order process in all pH experiments, in accordance with previous

the difference spectra that were calculated from the absorption spectra of 13C-ASR in Figure 5a at the pH range of 7.0 to 9.6. It demonstrates the existence of a single and continuous trend, that is, domination of one process. A characteristic titration curve with a pKa value of 8.5 (Figure 5c) is extracted from Figure 5b and can be related to this transition. Interestingly, such a behavior in this pH range is observed only for the 13C isomer absorption and not for AT; that is, the protonation state of this protein residue affects only the absorption of the 13C isomer. An important issue that deserves further consideration is associated with the two pKa values observed experimentally for LA-ASR (around pH 6.4 and 3.6). Similar pKa values are observed for DA-ASR and can be assigned with certainty to the AT isomer, which uniquely dominates (>96%) in the isomeric composition throughout this pH range. The situation is more subtle for the LA sample, which contains varying ratios of AT/ 13C isomers. The extraction of the spectra of the pure 13C 9000

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Figure 6. Thermal isomerization of ASR in the dark-adaptation process. (a) Schematic presentation of the dark adaptation process in ASR. In the dark, 13C-ASR isomerizes to AT-ASR. The process is pH-dependent: Decrease in pH leads to an increase in the half-lifetime. (b) Kinetics of dark adaptation at pH 5, 7.5, and 8.6 normalized to the DA-LA ASR difference spectra at a specific wavelength for each pH experiment (566, 561, and 561 nm, respectively). The three representative pH values display first-order kinetics and pH dependence of the reaction half-life. (c) pH dependence of the rate constant of dark adaptation, kda, in ASR in 300 mM NaCl at 25 °C.

Figure 7. Transient absorption differences for DA-ASR (left) and LA-ASR (right) at pH 5. The data are presented as color maps depicting the pump induced difference of optical density (ΔOD, see color-bar inset), plotted against probe wavelength (horizontal axis) and pump−probe time delays (vertical axis). The delays are shown on a split time scale with an expanded scale up to 2 ps and a logarithmic scale for longer times.

findings at neutral pH9 (see panel b of the Figure for a few representative kinetic curves) and (b) the dark-adaptation rate constant, kda, is significantly affected by the pH value and changes by ca. five orders of magnitude between pH 4 and 12 (Figure 6c). Figure 6c summarizes the results of kda versus pH on a logarithmic scale. (A detailed table with t1/2, kda, and the respective buffers that were used can be found in the SI.) The curve can be roughly divided into three different regions: almost linear increase between pH 4 and 5.5, a plateau between pH 5.5 and 7.5, and linear increase again above pH 7.5. Rate

constants for pH below 4.0 are not shown due to the difficulty to carry out precise measurements for such a long-lived process. Significant differences of a few orders of magnitude were observed for the time constants of dark adaptation in acidic and basic pH: from ∼56 h at pH 4 to 2.1 h at pH 6 and to 7 s at pH 10. Our titration curves show that at least three ionizable groups of the protein have an effect on the chromophore: a residue with pKa of 6.5 (possibly Asp217) and two yet unidentified residues with pKa values of 4.0 and 8.5. These groups, whose three pKa values lie exactly within the three 9001

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segments of the graph previously described, probably affect the thermal isomerization process in the ASR protein as well. Similar pH rate dependence was already observed in BR. However, the effect on the rate constant was inversed, namely, high kda for acidic pH and low kda for basic pH.38,39 Femtosecond Pump−Probe Experiments. Figure 7 presents time-corrected transient absorption spectra obtained from DA-ASR and LA-ASR samples at pH 5 as color-coded contour maps, representing the absorption differences as a function of wavelength and time: ΔOD(λ,t). The first 2 ps of delay are expanded to clarify the rapid spectral changes taking place during this period, and later delay times are presented on a logarithmic time scale. As at neutral pH,10 the gross appearance of transient spectral features is similar in both samples and reminiscent of data recorded for other MRPs, exhibiting a rapid buildup of excitedstate absorption and emission to the blue and to the red of the ∼550 nm ground-state bleach, respectively. Aside from some early spectral shifting, these features decay gradually within a few picoseconds with multiexponential internal conversion kinetics, leaving behind the weak signature of the red-shifted K photoproduct around 600 nm. Despite the similarities in both panels, careful analysis shows some shortening of decay times in LA-ASR, a trend that is in accordance with the studies of ASR and BR at neutral pH.10,25 As in the previous study, the pure 13C-ASR transient spectra can be isolated from the LA-ASR scans, which represent contributions from mixtures of AT-ASR/13C-ASR by properly weighted subtractions. To that end, the HPLC-extracted isomeric compositions were used along with the relative extinction coefficients of both isomers. The latter is required even though the acidic sample at pH 5 is highly enriched in 13C- isomer (∼90%) due to its much smaller extinction coefficient at the pump wavelength employed. To characterize the pH dependence of the initial dynamics of ASR, we present in Figure 8 a series of single-wavelength kinetics (vertical cuts in Figure 7 above) at both pH values: 5 (solid lines) and 7.5 (dashed lines, based on ref 10). The kinetic traces for DA-ASR (blue lines), which is almost pure AT-ASR at both pH values, are practically identical, and no noticeable differences exist in the initial photochemistry of ATASR in the pH range of 5−7.5. In this range, significant kinetic variations were observed by Tahara et al. for later stages in the photocycle.29,40 One difference between the two pH values is the “K” difference spectrum, which has a shallower negative lobe at the acidic pH, as depicted in Figure 9. This is likely the result of differences in the absorption spectra of both the ground state (mapped above) and K photoproduct of AT-ASR. The green curves in Figure 8 represent the pure 13C-ASR cuts, which were extracted as previously described. They show that in contrast with the behavior of the 13C-ASR in neutral suspensions, at the higher acidity most of the IC takes place nearly on the same time scales as the AT-ASR, differing only by a shallow initial rapid phase of decay. Accordingly, the strong kinetic differences observed for the two “half cycles” of photoswitching in ASR have been nearly washed out by increasing the proton concentration 100-fold.

Figure 8. A series of single-wavelength kinetics (vertical cuts in the contour map in Figure 7) at representative probing wavelengths of the transient difference spectra of DA-ASR (blue), LA-ASR (red), and 13C-ASR (green) shown for pH 5 (solid lines) and 7.5 (dashed lines, taken from ref 10). The time axis is shown on a split scale with an expanded scale up to 2 ps and a logarithmic scale for longer times.

Figure 9. “K” photoproduct difference absorption spectrum for DAASR in pH 5 (solid line) and pH 7.5 (dashed line), obtained by averaging the transient spectra for probe delays of 50−100 ps in both cases.

similarly by the protonation states of the relevant protein residues. Despite the relatively small changes in the absorption spectra upon pH alterations, two transitions with pKa values of 6.5 (6.4) and 4.0 (3.6) have been detected in DA (and LA) samples. Several protein residues in the cytoplasmic side of the protein play a pivotal role in the biological activity of ASR. Nonetheless, the pKa values of these residues are still not unequivocally

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DISCUSSION Complex pH Dependence of ASR Protein. Our broadrange titrations show that the change of pH leads to similar shifts in the absorption spectra of both LA and DA samples (Figures 2 and 3), suggesting that both forms are affected 9002

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determined. It is plausible that the first transition previously described with a pKa value of 6.5 is correlated with the protonation/deprotonation process of the Asp217 residue. FTIR studies of ASR proposed that the pKa values of Asp217 and of Glu36 are between 6 and 7.5,40,41 Our results indicate that indeed a protein residue, possibly Asp217, experiences a protonation/deprotonation process in this pH range. The retinal PSB and Asp217 residues are connected by a hydrogenbonding network,5 and as was previously shown, deprotonation of the PSB moiety leads to the protonation of Asp217 during the photocycle.40 Therefore, it is plausible that although Asp217 and the PSB are separated by ∼15 Å,5 protonation of Asp217 perturbs the hydrogen-bonding network in the retinal PSB vicinity and affects its absorption (Figure 2).42 The nature of the second transition with a pKa value of 4.0 is unclear. In contrast with other MRPs, the Asp75 counterion is not protonated during the photocycle in ASR, an observation to which a few tentative rationalizations were proposed.40,43−47 In the case of BR, Asp85, which serves as a major component of retinal PSB counterion, has a pKa value of 2.6.48,49 In the photoreceptors like SRII (Sensory Rhodopsin II) and ppR (Pharaonis phoborhodopsin), the pKa values of the counterions were determined to be 3.0 (Asp73)50 and 3.5 (Asp75),51 respectively. Therefore, it is plausible that the second observed shift in the absorption spectrum in ASR is associated with the change in the protonation state of the Asp75 residue, which changes the electrostatic interactions in the retinal binding site. However, the observed change in absorption is smaller than expected from an adjacent residue (only 3 to 4 nm). Furthermore, recently reported QM/MM and electrostatic calculations have proposed that the pKa of Asp75 in ASR is significantly lower than the pKa of Asp85 in BR.52 Other calculations showed that mutated far side chains in ASR affect the absorption maxima of the chromophore22 and thus may have an effect also on the pKa value of the counterion. Therefore, the observed transition in this pH range can represent a change in protonation states of other residues, like the more distant Asp198 or His8 (both of which share possible hydrogen-bonding network with the PSB). A third transition with a pKa of 8.5 was detected only after extracting the absorption of pure 13C-ASR (Figure 5). We propose that this process is not evident in the LA-ASR experiments probably due to coexistence of two competing processes in this pH range − increase in the pH leads to: (1) Deprotonation of a protein residue that blue-shifts the absorption spectrum of the 13C retinal chromophore. (2) Increase in the AT retinal fraction in the LA-ASR form, as was indicated from the HPLC analysis. Because λmax,AT‑ASR > λmax,13C‑ASR, the deprotonation process leads to a red-shifting of the absorption maximum. Consequently, when the pH is raised from 5 to 7.6 and from 7.6 to 9.6, the absorption spectrum of the LA protein is only slightly changed. Moreover, the results indicate that the AT retinal absorption (the main component in the DA samples) is not affected by pH in the pH range of ∼7.5−9.5, so that titration of the residue with pKa of 8.5 probably has no effect on the AT absorption. A recent study that dealt with pH dependence of intermediates in the ASR protein proposed the existence of a proton acceptor with a pKa between 7 and 9.29 Why such a transition affects only the 13CASR isomer remains, however, unclear. To conclude, while the first transition can be associated with Asp217, further mutational studies are required to support this and shed light on the nature and source of the second and third transitions.

pH-Dependent Isomeric Composition of ASR. The present study shows that while the DA-ASR consists of more than 95% protein with the AT retinal chromophore over a wide pH range (Figure 4a), the LA-ASR consists of varying compositions of 13C and AT isomers (Figure 4b). Consequently, the absorption spectrum of an illuminated ASR sample is affected by at least two factors: the pH-dependent retinal isomeric composition leading to different mixtures of AT/13C retinals and the protonation state of several amino acids in the vicinity of the chromophore. Our results show that following exposure to light in the pH range of 5 to 5.4, the protein is converted from almost 100% AT-ASR to more than 90% 13C-ASR. The bell-shaped curve (Figure 4b), indicates that protonation of the protein residues with pKa of 6.5 and 8.5 favors the 13C isomer, whereas protonation of the protein residue with a pKa of ca. 4.0 favors the AT isomer. Such a behavior is quite unusual for MRPs, most of which possess two configurations: photosensors that accommodate mainly AT retinal in both DA and LA forms (HsSRI, HsSRII, SrSRI, NpSRII)12−15 and light-driven ion pumps that accommodate AT and 13C retinal in the dark and usually increasing fraction of AT retinal after irradiation (BR, HwBR, PR, ChRII).16−19 The unique behavior of ASR raises questions regarding its functionality and the role of 13C isomer in the protein. A recent study has proposed that AT-ASR functions as a repressor of the chromatic adaptive gene, whereas the light activation of ASR leads to the expression of the pigment protein phycocyanin, which is involved in the chromatic adaptation of the cyanobacteria.2 According to our results, at pH ∼5.5, the LA form of Anabaena contains in its membrane ASR protein with high contents of the 13C-retinal isomer. We thus speculate that in acidic environment there is an increased expression of the phycocyanin, which in turn may lead to more efficient growth and development of the organism. Anabaena is a representative of cyanobacteria, ancient bacteria capable of performing oxygenic photosynthesis. Thus, the ability to evolve efficiently in low pH can possibly reflect an evolutionary residue of an organism that lived in acidic pH environment in the ancient word. In addition, one cannot exclude the possibility that incomplete photosynthesis cycles can cause by leakage an acidification of the cell.53 While it is clear that AT-ASR is the thermodynamically stable form of ASR, photoactivation of the protein leads to a photostationary equilibrium, which depends on the pH and consists of a mixture of AT-ASR and 13C-ASR. Both species are photointerconvertible and have their own photoreaction pathway.21 Kandori and coworkers have shown that the photoreaction of ASR is completely photochromic and the stable photoproduct of the AT-ASR is 100% 13C-ASR, and vice versa. (K and L intermediates of AT-ASR are completely converted into 13C-ASR, and the K intermediate formed from 13C-ASR is completely converted into AT-ASR.) We show here that the conversion between the two photoreactions is pH-dependent. The existence of a pH-dependent isomeric ratio implies that there is a “leak” between the two imaginary photocycles (imaginary because the photoreactions are not really cyclic21). Presumably, the environmental pH affects this leak through the protonation states of protein residues, which in turn affects the photostationary stage without changing the thermodynamic stability. We assume that protonation of two groups with pKa of 8.5 and 6.5 favors 13C form, while protonation of the group with the lower pKa favors AT. 9003

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Insights from Femtosecond Pump−Probe Experiments. The main findings of our ultrafast spectroscopic study are that (a) primary events of AT-ASR are almost pHindependent in the range 5 to 7.5 and are reminiscent of other MRPs such as BR, and that (b) the vast kinetic differences observed in neutral suspensions are nearly washed out in acidic pH, where the photochemistry of 13C-ASR exhibits only an initial shallow phase of IC that is faster than in AT ASR. Finding (a) is surprising, given that a protein residue with a pKa of 6.5 exists that directly affects absorptive properties of the retinal and that later stages of the ASR cycle were reported to be extremely affected by pH within this range.29 The fact that such a change in pH affects both the ground-state absorption and the later stages of the cycle but barely affects the photoinduced dynamics probably suggests that it does not significantly alter the excited-state properties, such as its topology. Interestingly, this pH range (5−7.5) lies in the plateau region of dark-adaptation rates (see Figure 6), suggesting that the protonation state of Asp75 might control here much of the photochemistry, akin to similar scenarios proposed for BR.56−58 Moreover, comparison of primary events at pH 5 and pH 7.5 does not suggest the existence of different ground states, nor multiple “K” photoproducts, as interpreted in the work by Tahara and coworkers, suggesting that further study is needed to clarify this point. In an attempt to rationalize the significantly reduced average rate of IC in 13C-ASR, we note that the unique transition that was found only for 13C-ASR that lies in pKa 8.5 is irrelevant for the studied pH range, in which only the transition at pH 6.5 should be relevant. Since this transition is common to both ATASR and 13C-ASR, it is unclear why it should alter primary events only in the latter. For BR, Song et al. have studied the photochemistry of wild-type and mutated species, concluding that replacement of charged residues affected the photoisomerization rates of 13C-BR and AT-BR differently, in their case affecting the latter more.56 Taking into account that LAASR and DA-ASR isomer compositions are roughly inverted with respect to BR, our current observation is in line with theirs. One option that will have to be studied in future studies is that the QY for photoisomerization of the 13C-ASR is also pH-dependent and is reflected in the amplitude of different pathways on the excited state. This brings into mind the case of rhodopsin versus iso-rhodopsin, which has recently been revisited by Ishida and coworkers59 and Cerullo and coworkers.60 They have shown that previous models for the differences of these two proteins were oversimplified and that complex excited-state dynamics (e.g., bifurcation into reactive and nonreactive channels), induced by steric interactions of the retinal, take place and affect the QYs and excited-state decays. Finally, understanding the mechanism underlying the effects of pH on the primary events of both isomers would be assisted greatly by an identification of the residues whose protonation states are switched by pH lowering in ASR. Current suggested mechanisms, such as alteration of the topology of the excitedstate potential surface near the conical intersection28 (e.g., existence of a barrier), or different geometries of the ground state retinal near the C11C12−C13C14−C15 area (twisting of single bonds), would have to be tested accordingly.

pH Dependence of Dark Adaptation Rate. The rate of thermal isomerization of ASR was measured over a wide pH range. The results show a change of more than four orders of magnitude in the rate of the dark adaptation process following pH alteration, demonstrating that isomerization of 13C retinal to AT retinal occurs faster at high pH. Therefore, protonation of a protein residue increases the barrier for thermal isomerization at low pH. It is conceivable that this protein residue is Asp75 (the counterion of the retinal PSB), as it is in keeping with previously recorded results of BR, in which thermal isomerization of the retinal chromophore was shown to be strongly affected by the protonation states of Asp 85.38,39,49 The pH dependence of the rate constant (kda) in ASR has an opposite trend than that measured for BR in which kda is high in acidic pH and lower in basic pH. However, dark adaptation in BR refers to isomerization of AT retinal to 13C retinal, whereas in ASR the process direction is inverted. The complex pH dependence of kda in BR was explained by the existence of two pKa values of the major counterion component, Asp85. These two pKa values are associated with the interaction of Asp85 with another ionizable residue, X′ (probably Glu204).38,49,54 When the residue X′ is protonated, the pKa of Asp85 is 2.6, but once X′ deprotonates, the pKa of Asp85 increases by several pH units. It was shown that the kda is proportional to the fraction of transient protonated Asp85 even at high pH.49 The curve shape of the dark adaptation rate of ASR (Figure 6C) resembles that of BR; therefore, analogous explanation can be proposed, for example, that Asp75 interacts with Asp217, which affects its pKa in a similar manner to that of Asp85 and X′ in BR, respectively. Accordingly, when Asp217 is protonated, the pKa of Asp75 is 4 or much lower, as was proposed in previous studies,52 and deprotonation of Asp75 increases the rate of thermal isomerization. Deprotonation of Asp217 (pKa of 6.5) may increase the pKa of Asp75 by several pH units (e.g., by alteration of the retinal pocket environment through the helices motion or the hydrogen bond network that includes the retinal moiety). Consequently, as the pH increases, the fraction of deprotonated Asp75 does not increase, which is manifested as a plateau in a log10kda−pH curve. As the pH further increases, the fraction of deprotonated Asp75 with a possible transient pKa value of 8.5 increases, thereby increasing the kda. The sharp increase in the rate, almost 10 times per pH unit, at both low and high pH, indicates that the rate constant is inversely proportional to the fraction of protonated residue, possibly Asp75; therefore, deprotonation of this residue catalyzes the dark adaptation process in ASR. Electrostatic interaction of the PSB and its counterion can affect the rate of the retinal double-bond thermal isomerization, as was demonstrated in solution55 and suggested for BR, in which protonation of Asp85 catalyzes the dark adaptation process. This protonation process increases the π-electron delocalization along the retinal polyene and decreases the barrier for thermal isomerization.38,55 However, additional factors can affect the barrier for thermal isomerization such as steric factors originating from retinal−protein interactions. These interactions can affect the retinal conformation or packing within its binding site and affect the barrier for thermal isomerization. Therefore, it is possible that protonation of Asp75 in ASR induces protein conformational changes that increase the barrier for retinal double-bond thermal isomerization.

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ASSOCIATED CONTENT

S Supporting Information *

Detailed information about the pH-dependent retinal isomer composition of DA-ASR and LA-ASR and the pH-dependent 9004

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dark-adaptation rates. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*S.R.: Tel: +972-2-6585326. Fax: +972-2-5618033. E-mail: [email protected]. *M.S.: Tel: +972-8-9344320. Fax: +972-8-9343026. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Vlad Brumfeld for helping with mathematical calculations. This work was supported by the Israel Science Foundation (ISF), which is administered by the Israel Academy of Sciences and Humanities, the US-Israel Binational Science Foundation (BSF), and the Kimmelman center for Biomolecular Structure and Assembly. A.W. is supported by the Adams Fellowship Program of the Israel Academy of Sciences and Humanities. M.S. holds the Katzir-Makineni chair in chemistry.

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ABBREVIATIONS ASR, anabaena sensory rhodopsin; AT, all-trans, 15-anti; 13C, 13-cis, 15-syn; MRP, microbial retinal protein; BR, bacteriorhodopsin; DA, dark-adapted; LA, light-adapted; PSB, protonated Schiff base; kda, dark-adaptation rate constant

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REFERENCES

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dx.doi.org/10.1021/jp504688y | J. Phys. Chem. B 2014, 118, 8995−9006