Crystal Structure of the 11-cis Isomer of Pharaonis Halorhodopsin

Article pubs.acs.org/biochemistry

Crystal Structure of the 11-cis Isomer of Pharaonis Halorhodopsin: Structural Constraints on Interconversions among Different Isomeric States Siu Kit Chan,† Haruki Kawaguchi,† Hiroki Kubo,† Midori Murakami,† Kunio Ihara,‡ Kosuke Maki,† and Tsutomu Kouyama*,†,§ †

Department of Physics, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan Center for Gene Research, Nagoya University, Nagoya 464-8602, Japan § RIKEN Harima Branch, 1-1-1, Kouto, Sayo, Hyogo, Japan ‡

ABSTRACT: Like other microbial rhodopsins, the light driven chloride pump halorhodopsin from Natronomonas pharaonis (pHR) contains a mixture of all-trans/ 15-anti and 13-cis/15-syn isomers in the dark adapted state. A recent crystallographic study of the reaction states of pHR has shown that reaction states with 13-cis/15-syn retinal occur in the anion pumping cycle that is initiated by excitation of the all-trans isomer. In this study, we investigated interconversions among different isomeric states of pHR in the absence of chloride ions. The illumination of chloride free pHR with red light caused a large blue shift in the absorption maximum of the retinal visible band. During this “red adaptation”, the content of the 11-cis isomer increased significantly, while the molar ratio of the 13-cis isomer to the all-trans isomer remained unchanged. The results suggest that the thermally activated interconversion between the 13-cis and the all-trans isomers is very rapid. Diffraction data from red adapted crystals showed that accommodation of the retinal chromophore with the 11-cis/15-syn configuration was achieved without a large change in the retinal binding pocket. The measurement of absorption kinetics under illumination showed that the 11-cis isomer, with a λmax at 565 nm, was generated upon excitation of a red-shifted species (λmax = 625 nm) that was present as a minor component in the dark adapted state. It is possible that this red-shifted species mimics an O-like reaction state with 13-cis/15-syn retinal, which was hypothesized to occur at a late stage of the anion pumping cycle.

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Halorhodopsin from Natronomonas pharaonis (pHR), which functions as a light driven chloride ion pump, also contains a mixture of all-trans and 13-cis isomers in the resting state.11,12 However, the so-called light/dark adaptation is not detected in pHR; i.e., the molar ratio of the all-trans to 13-cis isomer is scarcely affected by illumination.13 This phenomenon has previously been explained by assuming that the 13-cis isomer of pHR is photochemically silent, and the anion pumping cycle is initiated only when the all-trans isomer is excited. On the basis of this assumption, Váró et al. analyzed flash-induced absorption kinetics of pHR and proposed the following reaction scheme: pHR(hν)→ K ↔ L ↔ N ↔ O ↔ pHR′ → pHR.14,15 Key reactions included in this scheme are (1) the photoisomerization of the all-trans isomer to the 13-cis/15-anti configuration initiates a photocycle; (2) re-isomerization of retinal to all-trans/15-anti configuration occurs in the N-to-O transition, during which the chloride release into the cytoplasmic medium takes place; (3) the chloride uptake from the extracellular side takes place in the O-to-pHR′ transition.

he major chromophore in animal rhodopsin is 11-cis retinal, whose photoisomerization into the all-trans configuration initiates a series of protein conformational changes leading to formation of an active state.1,2 By contrast, microbial rhodopsins isolated from many species contain alltrans and/or 13-cis retinal in the resting state.3−6 In the case of the light driven proton pump bacteriorhodopsin (bR), the dark adapted state contains a mixture of all-trans and 13-cis/15-syn isomers and, owing to a photoreaction of the 13-cis isomer into the trans isomer, the content of the all-trans isomer increases significantly under illumination.7,8 This light initiated interconversion, which is called the light adaptation, is physiologically meaningful because the photoisomerization of the alltrans retinal into the 13-cis/15-anti configuration initiates the proton pumping cycle, during which one proton is actively translocated across the cell membrane.3 In some microbial rhodopsins (e.g., Anabaena sensory rhodopsin), on the other hand, light initiated interconversions between 13-cis and alltrans isomers occur frequently in both directions, and, as a consequence, the photostationary equilibrium between these isomers is dependent on the wavelength of illumination.5,9 This photochromic property has been reported to be relevant for color sensitive gene expression.10 © 2016 American Chemical Society

Received: March 28, 2016 Revised: June 24, 2016 Published: June 28, 2016 4092

DOI: 10.1021/acs.biochem.6b00277 Biochemistry 2016, 55, 4092−4104

Article

Biochemistry

pHR.18 It seems possible that O2 corresponds to O shown in Figure 1, but the question as to how O1 is related to the reaction states N′ and O′ has remained unanswered. It is noteworthy that the reaction scheme shown in Figure 1 is not radically different from the previously reported reaction scheme of the HR homologue (sHR) from Halobactrium salinarum.19−21 According to the reaction scheme reported by Ames et al.,21 a red-shifted intermediate with 13-cis retinal (sHR640) occurs at a late stage of the anion pumping cycle in sHR. It seems possible that the hypothetical O′ state of pHR corresponds to sHR640. Elucidation of the structural difference between O and O′ is necessary in order to prove or disprove this hypothesis. The reaction states with no chloride ion inside the protein (N′, O′, and O) would be expected to be stabilized at low chloride concentrations.22 With the aim of clarifying the spectroscopic properties of these reaction states, we investigated light induced absorption changes in a chloride free suspension of pHR rich claret membrane. We unexpectedly observed that the 11-cis isomer accumulated significantly under red illumination. Diffraction data from partially red adapted crystals of pHR showed that the formation of the 11-cis isomer was accompanied by no significant structural change in the retinal binding pocket. Interestingly, the molar ratio of the 13cis isomer to the all-trans isomer remained unchanged during the red adaptation. This result implies that the thermally activated interconversion between 13-cis and all-trans isomers is very rapid. The absorption kinetics data suggested that the 11cis isomer was generated upon excitation of a red-shifted species with an absorption maximum at 625 nm, which was present as a minor component in the dark adapted state. On the basis of these observations, we discuss how this red-shifted species is related to the hypothetical O′ state.

Although it was hypothesized in this reaction scheme that the photoconversion between all-trans and 13-cis isomers did not occur, this basic hypothesis was recently challenged by a crystallographic study on reaction intermediates of bromide bound pHR.16 In particular, an N-like state (N′) with the 13cis/15-syn retinal configuration is shown to occur after the N state with the 13-cis/15-anti retinal configuration. As observed for the N state, the protein conformation of the N′ state is such that the cytoplasmic interhelical space is open. However, unlike the N state which contains a halide ion in the cytoplasmic vicinity of the Schiff base, the N′ state contains no halide ion within the protein. That is, the translocated halide ion is released into the cytoplasmic medium when the Schiff base linkage isomerizes resulting in the Schiff base NH bond being directed toward the extracellular side. On the other hand, the O state was shown to resemble the anion depleted blue form, in which the cytoplasmic interhelical space is closed and the alltrans retinal is present as the major isomer. This implies that the double isomerization around the C13C14 double bond and the Schiff base linkage takes place during the transition from N′ to O. If this isomerization is slower than the closing of the cytoplasmic interhelical space, an O-like species (the O′ state) with the 13-cis/15-syn retinal configuration would be expected to occur after the N′ state. This hypothetical O′ state is expected to decay into O at low chloride concentrations. It is possible that at high chloride concentrations, the chloride uptake from the extracellular medium becomes faster than the retinal re-isomerization and, hence, a large fraction of O′ decays into a chloride bound 13-cis isomer (pHR′). A branching reaction (at or after the L2 state) in the anion pumping cycle of pHR was previously pointed out by Hackmann et al.17 When the reaction scheme shown in Figure 1 is constructed on the

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MATERIALS AND METHODS Preparation and Crystallization of pHR. The claret membrane of the Natonomonas pharaonis strain KM-1 was isolated as previously described.23 For crystallization and spectroscopic investigation, the claret membrane was partially delipidated with 0.5 % Tween 20 in 0.1 M NaCl (pH 7). pHR was crystallized in space group C2 as described previously.24 Crystals of pHR grown into a size of ∼0.5 × 0.2 × 0.1 mm3 were used for X-ray crystallographic analysis. For the structural investigation of the dark adapted form of the chloride free pHR,25 a C2 crystal of pHR was first soaked in an alkaline postcrystallization solution consisting of 3.0 M (NH4)2SO4, 0.1 M glycine (pH 9) and 30% trehalose for half a day, and, after soaking in a solution with a similar composition but at pH 7, the crystal was flash cooled with liquid propane at its melting temperature (Tmelt = 86 K). For structural investigation of a partially red adapted state of the chloride free pHR, a C2 crystal in a postcrystallization solution at pH 7 was illuminated with orange light (>580 nm; ∼20 mW/cm2) for 5 min at 393 K. After incubation for ∼1 min in dim light, the illuminated crystal was flash cooled. Measurement of Absorption Spectra and Kinetics. Illumination induced absorption changes in the claret membrane were measured with a homemade cross-illumination spectrophotometer, in which actinic light and measuring light were alternately passed through the sample chamber at a frequency of 160 Hz.26 Emission from a 150-W xenon lamp was reflected by a dichroic mirror, passed through an interference filter with a bandwidth of 10 nm or a short wavelength-cut

Figure 1. A reaction scheme for the anion pumping cycle of pHR. The retinal-Lys-256 chain takes on the 13-cis/15-anti configuration in the early stages (K, L, and N) of the anion pumping cycle, whereas it takes on the 13-cis/15-syn configuration in N′, O′, and pHR′ and the 13trans/15-anti configuration in pHR and O. The cytoplasmic interhelical space is open in N and N′, whereas it is closed in the other states. The open blue and pink arrows indicate ion movements occurring on the extracellular side and the cytoplasmic side of the protein, respectively.

basis of their observation, it is supposed that the L2 state defined in their spectroscopic study corresponds to the N state defined in the crystallographic analysis, and that at high halide ion concentrations the decay rate of O′ (and N′) is much higher than that of N (or L2). Meanwhile, Chizhov et al. reported an experimental evidence for the occurrence of two substates of O (O1 and O2) in the anion pumping cycle of 4093


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Biochemistry Table 1. Data Collection and Final Refinement Statistics crystal id soaking solution pHa buffer (0.1 M) illuminationb Data collection resolution (Å) space group unit cell a (Å) unit cell b (Å) unit cell c (Å) unit cell β (deg) data completion (%) no. unique reflections multiplicity Rsymc (%) I/σ Refinement resolution limit (Å) protein residues no. lipid + detergent number of water Rcrystd (%) Rfree (%) B factor (Å2) protein lipid/detergent water

547a

824a

7 citrate yes

7 citrate no

45.1−1.7 (1.79−1.7) C2 154.32 97.93 101.53 128.67 96.8 (82.3) 125077 (15482) 3.3 (2.4) 5.5 (33.8) 12.6 (2.8)

50.8−1.8 (1.9−1.8) C2 154.48 97.8 101.63 128.82 91.3 (60.9) 99234 (9621) 3.5 (2.9) 6.5 (40.5) 12.7 (2.7)

15.0−1.7 259 × 3 × 2 12 + 4 236 + 208 20.3 21.7

15.0−1.8 259 × 3 10 + 4 227 21.9 23.6

19.5 72.6 25.7

21.7 76.9 29.6

a The C2 crystal grown in the presence of chloride was first soaked in a halide-free alkaline solution and then in the final soaking solution containing 3 M (NH4)2SO4, and 0.1 M Na-citrate at pH 7 and 30% trehalose. bThe C2 crystal was flash-cooled after 5 min illumination with red light (>580 nm; 10 mW/cm2). cRsym = ΣhklΣi |Ii − | /ΣhklΣi Ii, where Ii is the intensity of an individual reflection and is the mean intensity obtained from multiple observations of symmetry related reflections. dRcryst = Σhkl (|Fobs| − |Fcalc|)/Σhkl |Fobs| (5% randomly omitted reflections were used for Rfree).

filter, and directed to the sample chamber from the side opposite the measuring light. The time averaged intensity of monochromatic actinic light at the front of the sample was 0.22−0.35 mW/cm2. [The effective intensity of actinic light at the sample position could become 10 times higher when the power supply to the optical chopper system was turned off.] The measuring beam with a bandwidth of 2 nm was provided by a Shimadzu spectrophotometer (UV3500); its time averaged intensity at the sample position was less than 50 nW/cm2. The beam diameter of actinic light was expanded to 20 mm at the sample position, so that the whole area of a sample solution (5 mm in width and 8 mm in height) was uniformly illuminated. The measuring light was masked with a slit (3 mm in width and 5 mm in height) attached to a front face of the sample cuvette. High Performance Liquid Chromatography (HPLC). The isomeric composition of retinal was analyzed using HPLC following the procedure reported by Scherrer et al.27 Briefly, 50 μL of pHR rich claret membrane was mixed with 0.5 μL of 50% hydroxylamine and 125 μL of ethanol. The mixture solution was incubated at room temperature for 1 min, and then 125 μL of hexane was added to the mixture solution. The resulting emulsion was centrifuged at 15 000 rpm for 1 min, and the hexane phase (upper layer) was transferred to another container. The HPLC analysis was carried out using a silica column (6 × 150 mm; YMC-SL12S03-1506WT) with benzene containing 1.0% (v/v) diethyl ether and 0.2% (v/v) 2-propanol. The flow rate was set at 1.5 mL/min, and the monitoring wavelength was 360 nm. The assignment of each peak was

performed by comparing it with the HPLC patterns of retinal extracts from dark and light adapted bacteriorhodopsin. The retinal isomeric composition was calculated using the reported absorption coefficients of all-trans, 13-cis, 11-cis, and 9-cis retinal oximes.28 Data Collection and Scaling. X-ray diffraction measurements were performed at the beamline SPring8-BL38B1, where a frozen crystal kept at 100 K was exposed to a monochromatic X-ray beam at a wavelength of 1.0 Å with an X-ray flux rate of 2 × 1012 photons/mm2/s. Diffraction data were collected using an oscillation range of 1° and an X-ray flux of 4 × 1012 photons/mm2 per image. To collect a full diffraction data set, a single crystal was exposed to an X-ray flux of 7 × 1014 photons/ mm2. Indexing and integration of diffraction spots were carried out with iMosf lm 7.1.29 The scaling of data was carried out using SCALA in the CCP4 program suite.30 Crystal parameters and data collection statistics are summarized in Table 1. In this study, the size of the prepared crystal was large enough to allow for the collection of a full diffraction data set using a very low X-ray dose (0.03−0.1 MGy).31 It should be noted that a larger X-ray dose (0.2 MGy) caused a detectable alteration in the configuration of the retinal chromophore in the 11-cis isomer contained in this crystal (data not shown). Structural Refinement. A model of a chloride free blue form of pHR was built using the previously reported model of the chloride ion bound purple from of pHR (pdb id: 3A7K) as an initial search model. Structural refinement was completed using CNS-1.232 and XtalView-4.0.33 For the structural 4094


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Biochemistry investigation of the accumulation of the 11-cis isomer under red light illumination of a chloride free pHR, the diffraction data set from a crystal that was illuminated with red light (Fred) was compared with that from an unilluminated crystal (Fdark), and the differences between the electron density maps of the two crystals (|Fred| − |Fdark| map) were evaluated using the phase derived from the structural model of the dark adapted state of chloride free pHR. The structural refinement of the 11-cis isomer contained in the red adapted crystal was performed based on the approximation that each subunit in the trimeric assembly of pHR (i.e., in the asymmetric unit) contains two conformers (the 11-cis isomer and the all-trans isomer). When the occupancy of the 11-cis isomer in the ith subunit is αi, the diffraction amplitude Fred is given as follows: |Fred| = Σi {αi·|F i_11‑cis| + (1-αi)·|Fi_trans|}, where Fi_11‑cis and Fi_trans are the structure factors of the 11-cis isomer and the all-trans isomer, respectively, in the ith subunit. The structure of the all-trans isomer was assumed to be identical to that observed in the unilluminated crystal, and the structure of the 11-cis isomer and the occupancies αi were refined as described previously.16 The crystallographic R value decreased to 0.203 when the optimal occupancies of the 11-cis isomer in the three subunits (0.35, 0.38, and 0.37 for subunits A, B and C, respectively) were used (Table 1).

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RESULTS Formation of the 11-cis and 9-cis Isomers in Chloride Free Claret Membrane. When the absorption spectrum of pHR rich claret membrane in a chloride free solution at pH 7 was recorded after illumination with green light at 570 nm, the visible absorption band of retinal was recognized as a shoulder at 600 nm (the blue line in Figure 2a). This absorption spectrum was nearly identical to that observed for the dark adapted chloride free claret membrane. This observation is consistent with the result of HPLC analysis showing that the isomeric composition of retinal in the “green adapted” claret membrane was nearly identical to that observed in the dark adapted claret membrane, which contained a mixture of 89% all-trans and 11% 13-cis retinal (Table 2). On the other hand, when “green adapted” or dark adapted claret membrane was exposed to red light, a large blue shift of the retinal visible band was observed (the red line in Figure 2a). The magnitude of the spectral shift was dependent on the wavelength of actinic light. The largest blue shift was observed when the claret membrane was exposed to far red light at 698 nm (the red line in Figure 2b). When this “red adapted” claret membrane was exposed to green light, the resulting absorption spectrum was similar to that observed for dark adapted claret membrane. At first sight, the observed photochromic property of pHR appeared to be explainable by light initiated interconversion between the chloride free blue form, with an absorption maximum at 600 nm, and a blue-shifted species, with an absorption maximum at or below 565 nm. However, closer inspection of the light induced absorption changes suggested that two distinct blueshifted species were produced under red illumination. First, the profile of the light induced spectral change varied with the cumulative exposure time to red light (Figure 2c). Second, the absorption kinetics recorded under red illumination were described with two time constants (Figure 2d). Indeed, HPLC analysis of retinal extracted from the illuminated claret membrane showed that the 11-cis isomer was predominantly generated at the initial phase of red illumination, whereas the 9-

Figure 2. Light induced absorption changes in chloride-free pHR. (a) The blue line represents the spectrum recorded after a membrane suspension in 5 mM Na citrate at pH 7 was exposed to green light ((λact = 570 nm, 0.3 mW/cm2) for 10 min, whereas the red line represents the spectrum recorded after the same membrane suspension was illuminated with red light ((λact = 698 nm; 0.3 mW/ cm2) for 10 h. The dotted line represents the absorption spectrum of the dark adapted chloride free claret membrane at pH 11.54, where the chloride free blue form was converted to the alkaline yellow form by deprotonation of the retinal Schiff base. The spectra are drawn after subtracting the contribution of light scattering, which was assumed to be proportional to λ−2.4. (b) The spectral profile of the retinal visible band in the red or green adapted membrane was made clearer by subtracting the vibronic bands of bacterioruberin at 541 nm, 505 and 475 nm from the spectra shown in panel a. This subtraction was achieved by taking into account the pH dependence of the bacterioruberin spectrum; i.e., the absorption spectrum of the anion depleted yellow form was modified (0.5 nm red shift and amplitude adjustment) before its usage for the subtraction. (c) Difference spectra, relative to the absorption spectrum of the green adapted membrane, were recorded after the membrane was exposed to red light for different periods. The blue line was recorded after a 3 min illumination with red light (λact = 698 nm; 3 mW/cm2), whereas the green line was recorded after a 100 min illumination with actinic light at λact = 698 nm (3 mW/cm2). The dotted lines represent the calculated difference absorption spectra between the 11-cis isomer and a mixture of all-trans and 13-cis isomers (the red dotted line) and between a mixture of the 11-cis and 9-cis isomers in red adapted membrane and a mixture of alltrans and 13-cis isomers in green adapted membrane (the purple dotted line). (d) The absorption change at λ = 625 nm was monitored under red illumination (λact > 670 nm; 3 mW/cm2) or under green illumination (λact = 570 nm; 0.34 mW/cm2). Before this recording, the membrane suspension was green adapted for 20 min. The absorption kinetics observed during the red and green adaptation were fitted by two exponential components (smooth red line).

cis isomer accumulated more slowly under red illumination (Figure 3b). 4095


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Biochemistry Table 2. Isomer Composition of Retinal Extracted from Dark-Adapted and Illuminated pHR under Various Conditionsa actinic light wavelength (nm) darkadapted 490 (±5) 590 (±5) 680 (±5)

intensity (mW/cm2)

2.4 2.8 3.2

retinal isomer

exposure time (min)

20 20 20

alltrans (%)

13-cis (%)

11-cis (%)

9-cis (%)

86.6

11.5

1.0

0.8

85.4 85.2 48.5

10.7 10.6 5.7

2.1 3.5 40.9

1.8 0.7 5.0

a

A suspension of Tween-treated claret membrane in 10 mM NaCitrate at pH 7 was dark adapted or illuminated with green or red light for various periods and was then subjected to retinal extraction and HPLC analysis. The retinal isomeric composition was calculated using the following absorption coefficients of retinal isomers:26 i.e., 51 600 and 54 600 cm−1 M−1 for anti and syn all-trans retinal oxime, respectively, 28 600 and 35 000 cm−1 M−1 for anti and syn 11-cis retinal oxime, respectively, and 47 900 cm−1 M−1 for other isomers.

Dark Adaptation of Chloride Free pHR. Figure 4 shows the absorption changes observed when a red adapted claret membrane was incubated at room temperature (∼24 °C) in the dark. The absorption kinetics associated with the dark adaption were described with two time constants (12 and 250 h) (Figure 4). HPLC analysis of extracted retinal showed that the fast phase of the dark adaptation was attributable to the interconversion from the 11-cis isomer to the 13-cis and/or all-trans isomer, whereas the slow phase represented the interconversion from the 9-cis isomer into the other isomers (Figure 3c). It is noteworthy that the molar ratio of the 13-cis isomer to the all-trans isomer scarcely changed during red adaptation, in spite of a large decrease in their summed content (Figure 3b). This result can be explained by supposing that the thermally activated interconversion between the 13-cis isomer and the alltrans isomer takes place very rapidly. That is, the rate of this thermal reaction seems to be so high (>1 s−1) that the light initiated interconversion between the all-trans and 13-cis isomers is undetectable at room temperature. At 0 °C, illumination of the chloride free claret membrane with green light (λact = 560 nm; 0.35 mW/cm2) resulted in the accumulation of a red-shifted state, which disappeared with a time constant of ∼10 s in the dark. This light induced absorption change was very small (ΔA630/A600 ≈ 0.01), making it difficult to identify the origin of this spectral shift. At this stage, we cannot exclude the possibility that, at this low temperature, the thermal equilibrium between the all-trans isomer and the 13-cis isomer was perturbed by the light initiated interconversion between them. Absorption Kinetics during Red Adaptation. To clarify whether the 11-cis isomer was generated upon excitation of the all-trans isomer or the 13-cis isomer, we investigated the time courses of absorption changes induced by actinic light at various wavelengths. Figure 5a shows typical examples of illumination induced absorption changes monitored at 625 nm, each of which was recorded after green adaptation of the claret membrane. These absorption changes are all well described with two exponential components, though their amplitudes and rate constants are significantly dependent on the wavelength of actinic light. In Figure 5c, the two rate constants (kf and ks) divided by the quantum flux of actinic light, Iact, are plotted

Figure 3. The isomeric composition of retinal in chloride-free claret membrane under various illumination conditions. The HPLC analysis was performed for retinal oximes extracted from the following samples: (1) Green adapted claret membrane; i.e., after a 20 min illumination with green light (λact = 480 ± 5 nm; 3 mW/cm2). (2) Red adapted claret membranes; i.e., after green adapted membrane was illuminated with red light (λact = 680 ± 5 nm, 3 mW/cm2) for 20 min. Ts, Ta, 13s, 13a, 11s, 11a, 9s and 9a stand for all-trans/15-syn-retinal oxime, all-trans/15-anti-retinal oxime, 13-cis/15-syn-retinal oxime, 13cis/15-anti-retinal oxime, 11-cis/15-syn-retinal oxime, 11-cis/15-antiretinal oxime, 9-cis/15-syn-retinal oxime, and 9-cis/15-anti-retinal oxime, respectively. (b) The contents of all-trans, 13-cis, 11-cis, and 9-cis isomers in the claret membrane that was kept in the dark or exposed to red light (λ = 698 nm, 3 mW/cm2) for various periods. The dashed curves represent the theoretical curves that were evaluated using the three-state kinetic model shown in Figure 6b. (c) The contents of all-trans, 13-cis, 11-cis, and 9-cis isomers in the claret membrane that was kept at ∼20 °C in the dark for various periods after a 20 min illumination with red light (λ = 688 nm, 3 mW/cm2) . The dashed curves represent the theoretical curves that were evaluated when 11-cis and 9-cis isomers were assumed to decay with time constants of 12.5 and 120 h, respectively.

against the wavelength of actinic light (the open circles and triangles). [Here, Iact is the light intensity averaged over the optical path of the sample cuvette.] In Figure 5d, the amplitudes (ΔAf and ΔAs) of the two exponential components 4096


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Biochemistry

Figure 4. Dark adaptation of chloride-free pHR. (a) Absorption changes associated with dark adaptation of pHR at pH 7. After a 10 h illumination with far red light (λact = 680 ± 5 nm, 0.29 mW/cm2), chloride free claret membrane was incubated at room temperature in the dark for various periods. The difference spectra shown were obtained by subtracting the absorption spectrum of green adapted claret membrane from the absorption spectra recorded after various periods of dark adaptation. (b) The time course of absorption change at 625 nm, relative to the absorbance measured for green adapted claret membrane, observed during dark adaptation. The two traces were recorded when chloride free claret membrane was kept at 24 °C in the dark after a 10 h illumination with red light at λact = 698 nm (open squares) or after a 20 min illumination with red light at λact > 670 nm (3 mW/cm2) (open circles). The absorption kinetics during dark adaption were described with two exponential components (τ1 ≈ 12.8 h; τ2 ≈ 250 h) (dashed lines). (c) Light induced absorption change (i.e., accumulation of a red-shifted species) observed at 0 °C. The absorption spectrum recorded in green light (λ = 560 nm, 0.35 mW/cm2) was subtracted by the spectrum that was recorded just after the green light was turned off.

Figure 5. Light initiated interconversion among different isomers under various illumination conditions. (a) Light induced absorption changes at 625 nm in a chloride free suspension of the claret membrane. Each trace was recorded when green adapted claret membrane at pH 7 was illuminated with red light at λact = 640, 660, 680, or 698 nm. Each experimental curve (solid lined) was fitted with two exponentials (dashed lines). (b) Absorption changes observed when partially red adapted claret membrane was illuminated at λact = 490 or 590 nm. (c) The circles and triangles represent the rate constants of the fast and the slow components of light induced absorption change at 625 nm divided by the effective photon flux density, Iact(λact), of the actinic light at λact. Here, Iact(λact) is given by I0(λact) × (1−10−A(λact))/2.3A(λact), where I0(λact) is the photon flux density at the front of the sample cuvette and A(λact) is the absorbance of the green adapted sample at λact. The open symbols represent the experimental values that were deduced from analyses of absorption kinetics associated with red adaptation, as shown in panel a. On the other hand, the closed symbols represent the rate constants that were deduced from analyses of absorption kinetics associated with green adaptation, i.e., absorption kinetics observed when partially red adapted membrane was illuminated with actinic light at λact ≤ 630 nm. The simulated rate constants (the dashed lines) are evaluated from the four microscopic rate constants (k12, k21, k23, and k32) appearing in the three-state kinetics model. Each of these microscopic rate constant (kij) is given by the product of the quantum efficiency, Φij, of the photoconversion of species i to j and the absorption coefficient of species i at λact. (d) The circles and triangles represent the amplitudes of the fast and the slow components of the light induced absorption change at 625 nm. Their sums are represented by the squares. The simulated rate constants (the dashed lines) were evaluated from the four microscopic rate constants (k12, k21, k23, and k32) outlined in panel c.

and their sum (ΔAf + ΔAs) are plotted. We observed that both ΔAf and ΔAs became very small at λact < 630 nm. This implies that the 11-cis and 9-cis isomers accumulated significantly only when the claret membrane was illuminated at the red edge of the visible absorption band. This observation cannot be explained by a simple reaction scheme where the 11-cis and 9-cis isomers are generated upon excitation of the major isomer (i.e., the all-trans isomer) in the dark adapted state, with a λmax at ∼600 nm. Instead, we suggest that the 11-cis isomer is generated upon excitation of a redshifted species (λmax > 620 nm) that is present as a minor species in the dark adapted chloride free claret membrane. A possible candidate for this red-shifted species is the 13-cis isomer with a distorted polyene chain. Hereafter the red-shifted species is referred to as the “13-cis” isomer, which may mimic the hypothetical reaction intermediate O′. Absorption Kinetics during Green Adaptation. In order to attain the spectral information about the “13-cis” 4097


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Biochemistry isomer, we first attempted to determine the exact spectral properties of the 11-cis and 9-cis isomers. For this purpose, we analyzed the action spectra associated with the backward photoconversion from the 11-cis and 9-cis isomers to the “13cis” and/or all-trans isomers. In particular, chloride free claret membrane was preilluminated with monochromatic red light at 680 nm (2 mW/cm2) for >10 min, and the absorption changes induced by monochromatic yellow or green light (at a wavelength between 630 and 490 nm) were subsequently measured. Typical examples of the absorption kinetics monitored at 625 nm under yellow or green illumination are shown in Figure 5b. These absorption changes were all well described with two exponentials. In Figure 5c, the two rate constants (kf and ks) divided by the quantum flux of actinic light (Iact) are plotted against the wavelength of actinic light (the closed circles and triangles). Since the highest values of kf/Iact and ks/Iact were observed at λact ≈ 560 nm, both the 11-cis and 9-cis isomers were roughly estimated to exhibit absorption maxima at around 560 nm. This estimated wavelength of maximal absorption is close to that of the absorption spectrum recorded after far red illumination (the red line in Figure 2b), suggesting that far red illumination is effective at converting a large fraction of the protein into the 11-cis and 9-cis isomers. Although the data set shown in Figure 5 contains information regarding the spectral properties of the “13-cis” isomer, its absorption spectrum cannot be unambiguously determined unless we place some constraints on our consideration about interconversions among the four isomers contained in the red adapted claret membrane. Fortunately, we can make use of the structural data showing that the structure of the protein moiety in the 11-cis isomer is basically identical to that in the all-trans isomer (see Figure 9). On the basis of this structural information, we consider a reaction scheme as shown in Figure 6b, where it is assumed that double isomerization around two adjacent double bonds in the retinal polyene chain (i.e., “bicycle pedal” isomerization of retinal34) is allowed in the retinal binding pocket with a low flexibility; that is, the photoconversions between the “13-cis” isomer and the 11-cis isomer and between the 11-cis isomer and the 9-cis isomer take place frequently, whereas other conceivable interconversions are very rare. It should be mentioned that the absorption kinetics data could be explained by another reaction scheme as shown in Figure 6c. In this case, however, the molar ratio of the 9-cis to the 11-cis isomer in the red adapted claret membrane was predicted to be much higher than the experimental value determined by HPLC analysis. This does not necessarily mean that the direct interconversion between the “13-cis” isomer and the 9-cis isomer is completely inhibited. Instead, we argue that the 11-cis isomer is predominantly generated upon excitation of a red-shifted species that exists as a minor component in the dark adapted chloride free pHR. Three-State Kinetics Model. When the thermally activated conversion between the all-trans isomer and the “13-cis” isomer is extremely rapid, the reaction scheme shown in Figure 6b becomes analogous to a three-state kinetic model that has been utilized to analyze a protein folding/unfolding process in which the conversion from the native state (N) to the denatured state (D), and the reverse reaction, occur via one intermediate (I): i.e., N ↔ I ↔ D.35 When this three-state kinetic model is adopted, the illumination induced absorption

Figure 6. Interconversions among different isomers of pHR. (a) Schematic drawing of retinal in the all-trans/15-anti, 13-cis/15-syn, 11cis/15-syn, and 9-cis/15-syn configurations. (b) Possible interconversions among the four isomers coexisting in red adapted claret membrane (i.e., all-trans, “13-cis”, 11-cis, and 9-cis isomers). Here, a single interconversion step is assumed to take place in such a manner that two adjacent double bonds in the polyene chain isomerize simultaneously. The red arrows indicate the light initiated interconversions, the quantum efficiencies of which are deduced by analysis of the absorption kinetics data shown in Figure 5. The blue arrows represent thermally activated interconversions, the rate constants of which are deduced by analysis of the experimental data shown in Figure 2 and 5. (c) A different three-state kinetic model in which a single interconversion step is assumed to take place between the 13-cis isomer and the 9-cis isomer.

change observed at the exposure time t, ΔA(t), is given by two exponential components: ΔA(t ) = ΔA f (1 − exp(−k f t )) + ΔA s(1 − exp(−kst )) (1)

The rate constants of the fast and slow components, kf and ks, are given by the equation: k f,s = [k12 + k 21 + k 23 + k 32 ± {(k12 + k 21 + k 23 + k 32)2 − 4(k12k 23 + k12k 32 + k 21k 32)}0.5]/2

(2)

In the present case, the microscopic rate constants, k12, k21, k23, and k32, are dependent on the wavelength of actinic light (λact); that is, the transition rate from species i to j is given by kij(λactinic) = Φij × fi × εi(λact) × I(λact)

(3)

where species 1, 2, and 3 correspond to the “13-cis” isomer, 11cis isomer, and 9-cis isomer, respectively; Φij is the quantum efficiency of the transition of species i to j, εi(λact) is the absorption coefficient of species i at the wavelength of actinic light, and I(λact) is the quantum flux of the actinic light; f i=2 and f i=3 are equal to 1, whereas f i=1 is equal to the fraction (∼0.1) of the “13-cis” isomer in the dark adapted state. On the other hand, the amplitudes, ΔAf and ΔAs, are given as functions of 4098


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Biochemistry the wavelength of monitoring light (λ) and the wavelength of actinic light (λact):

Hence, the spectral data shown in Figure 2 can be used to determine the absorption spectra of the 11-cis and 9-cis isomers if their content in red adapted claret membrane is well estimated. To estimate the content of these isomers, however, we need to determine the absorption spectrum of the “13-cis” isomer, on which the microscopic rate constant k12 is dependent (eq 3). The Spectroscopic Properties of the “13-cis” Isomer. The absorption spectrum of the retinal chromophore in a bacterioruberin free sample of pHR has been reported to be well fitted by a skew Gaussian function.18 For the purpose of obtaining the spectral information for the “13-cis” isomer, the spectral data shown in Figure 2, and the kinetic data shown in Figure 5 were simultaneously analyzed on the assumption that the absorption spectra of the “13-cis” isomer and the other isomers are described with skew Gaussian functions. The most likely values for the bandwidth (Δν), the skewness (ρ) and the wavelength of absorption maximum (λmax) of each skew Gaussian function were evaluated when these parameters and the other variables in eq 3 were iteratively adjusted so as to fit all the spectral data shown in Figures 2 and 5. [The λmax for the 11-cis isomer can be estimated by analyzing the wavelength dependence of the fast rate constant of the green adaptation; i.e., kf ≈ k21 in the wavelength region where the microscopic rate constant k21 is much higher than the other microscopic rate constants. Similarly, the λmax for the 9-cis isomer can be estimated by analyzing the wavelength dependence of the slow rate constant; i.e, ks ≈ k32 in the wavelength region where k32 is much higher than k23 but much lower than k21.] The additional assumption used in this analysis is that the absorption spectra of the all-trans and the “13-cis” isomers are described by skew Gaussian functions with the same bandwidth (Δν = 3500 cm−1) and the same skewness (ρ = 1.55). In Figure 7b, the most likely absorption spectrum of the “13-cis” isomer is compared with those of the all-trans, 11-cis, and 9-cis isomers. It is worthwhile noting that the absorption spectrum of the “13cis” isomer is largely red shifted (λmax ≈ 625 nm) as compared with that of the all-trans isomer (λmax ≈ 595 nm). In Figure 2c, the difference spectra ΔA(t; λ) observed at different exposure times (at t ≪ kf and at t ≫ kf) (the solid line) were compared with those calculated using the most likely absorption spectra of the four isomers. In Figure 5c, the rate constants kf and ks that were calculated using the optimal values of the microscopic rate constants (the dashed lines) were compared with the experimental data (the circles and triangles). In Figure 5d, the calculated values for ΔAf (λact), ΔAs(λact) and their sum (the dashed lines) were compared with the experimental data (the open symbols). In Figure 7c, the calculated time course of the absorption change at 625 nm induced by illumination at λact = 680 nm (the dashed red line) was compared with the experimental curve (the solid blue line). All of the experimental data were well reproduced by the calculated curves. The quantum efficiencies of the photoconversions among the different isomers were calculated using the approximation that the photon cross section for absorption at λmax was similar (2Å2) for all the isomers. When the content of the “13-cis” isomer in the dark adapted or green adapted claret membrane was 10% (as suggested by the result of the HPLC analysis), the quantum efficiency of the photoconversion from the “13-cis” to the 11-cis isomer was estimated to be 0.04 (Figure 6b). This value is much lower than the quantum efficiency (∼0.15) of the backward photoconversion of the 11-cis isomer. The quantum

ΔA f,s(λ ; λact) = Δε12(λ)[11C]f,s (λact) + Δε13(λ)[9C]f,s (λact)

(4)

where Δε12(λ) and Δε13(λ) are the difference absorption spectra associated with the transitions of the dark adapted state (a mixture of the all-trans and “13-cis” isomers) to the 11-cis isomer and the 9-cis isomer, respectively, and [11C]f,s and [9C] f,s are the magnitudes of the fast and slow components in the functions describing the illumination induced change in the content of the 11-cis and 9-cis isomer, respectivelyi.e., the content of the 11-cis and 9-cis isomers at the exposure time t is given by [9C](t ) = [9C]f exp( −k f t ) + [9C]s exp( −kst ) + [9C]∞ [11C](t ) = [11C]f exp( −k f t ) + [11C]s exp( −kst ) + [11C]∞

(5)

In general cases, [11C]f,s and [9C]f,s are dependent on the initial content of the 9-cis and 11-cis isomers as well as the microscopic rate constants kij(λact).36 [9C]f = ξf {ξs([11C]∞ − [11C]o ) + ([9C]∞ − [9C]o ) (ξs − 1)}/(ξs − ξf )

[9C]s = ξs{ξf ([11C]∞ − [11C]o ) + ([9C]∞ − [9C]o ) (ξf − 1)}/(ξf − ξs) [11C]f,s = 1 − (1 + 1/ξf,s)[9C]f,s

[9C]∞ = [9C]o − [9C]f − [9C]s = k12k 23/(k12k 23 + k12k 32 + k 21k 32)

[11C]∞ = [11C]o − [11C]f − [11C]s = k12k 32/(k12k 23 + k12k 32 + k 21k 32) ξf,s = (k f,s − k12 − k 21)/k 21

(6)

Here, [9C]o and [11C]o are the initial content of 9-cis and 11cis isomers, respectively. To analyze the dependences of ΔAf and ΔAs on the wavelength of actinic light in the red region (λactinic ≥ 630 nm) (the closed symbols in Figure 5c), we made use of the HPLC data showing that the content of the 11-cis and 9-cis isomers in green adapted claret membrane (i.e., [9C]o and [11C]o in eq 6) was very small. In the analysis of the spectral change induced by a short exposure to red light (the solid blue line in Figure 2c), we made use of the experimental data showing that kf is ∼10 times higher than ks (Figure 5c). In this case, ΔA(t; λ) at t ≪1/kf is proportional to Δε12(λ). On the other hand, the spectral change induced by a very long illumination (the green solid line in Figure 2c) is approximated by ΔA(t ≫ ks ; λ) ≈ Δε12(λ)([11C]f + [11C]s ) + Δε13(λ) ([9C]f + [9C]s )

(7) 4099


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efficiency (∼0.02) of the photoconversion from the 11-cis to the 9-cis isomer was also much smaller than that (∼0.05) of its backward photoconversion. The imbalances in the rate constants for the forward and backward interconversions explain why the contents of the 11-cis and 9-cis isomers in the green adapted membrane were very small. Structural Changes Induced by Red Illumination. Figure 8a shows the protein packing in the C2 crystal of

Figure 8. Illumination-induced structural change in chloride-free pHR. (a) The crystal packing in the C2 crystal of pHR is viewed along the b axis. The asymmetric unit contains three subunits (i.e., one trimer). (b) Difference electron density map between a partially red adapted crystal (|Fred|) and a dark adapted crystal (|Fdark|), contoured at + 3.6 σ (blue) and − 3.6 σ (red) and overlaid on the structural model of the all-trans isomer of chloride free pHR. For preparation of the partially red adapted crystal, the C2 crystal soaked in a chloride free postcrystallization solution was illuminated at room temperature with red light (>580 nm) for 5 min and flash cooled with liquid propane at 85 K. (c) |Fred| − |Fdark| map around the retinal chromophore in subunit C, contoured at 1.2 σ (brown) and −1.2 σ (green) and overlaid on the structural models of the 11-cis isomer (orange) and the all-trans isomer (cyan). (d) 2Fo − Fc map in the dark adapted crystal, contoured at 1.8 σ and overlaid on the model of the all-trans isomer in subunit C. (e) 2Fo − Fc map in a red adapted crystal, contoured at 1.2 σ and overlaid on the structural model of a mixture of the 11-cis isomer (gold) and the all-trans isomer (cyan) coexisting in subunit C. (f) 2Fo − Fc map evaluated using the quantity 3 × Fred − 2 × Fdark, contoured at 1.2 σ and overlaid on the model of the 11-cis isomer trapped in subunit C.

Figure 7. Absorption spectra of the all-trans, “13-cis”, 11-cis, and 9-cis isomers of pHR. (a) The dashed lines represent the most likely spectral profiles of the visible absorption band of retinal in the all-trans isomer (AT, the cyan line), the 13-cis isomer (the red line), the 11-cis isomer (the blue line) and the 9-cis isomer (the green line) coexisting in chloride-free claret membrane. For derivation of these spectra, the spectral profile of the retinal visible band in each isomer was approximated by a skew Gaussian function, and the parameters (i.e., the wavelength of the absorption maximum λmax, the bandwidth Δν, and the skewness ρ) included in this function were iteratively adjusted until the spectra shown in Figure 2 and the absorption kinetics data shown in Figure 5 were best fitted. The most likely λmax values are 595 nm, 625 nm, 565 and 560 nm for the all-trans, 13-cis, 11-cis and 9-cis isomers, respectively; the most likely ρ value is 1.55 for the all-trans and 13-cis isomers and 1.35 for the 11-cis and 9-cis isomers; the most likely Δν value is 2857 cm−1 for all the isomers. The relative amplitude of the absorption spectrum of the 13-cis isomer to that of the all-trans isomer was adjusted so as to explain the isomeric composition of retinal in green adapted membrane. The calculated absorption spectrum of the retinal visible band in a 9:1 mixture of the all-trans and the 13-cis isomer (the dotted red line) was compared with the observed spectrum shown in Figure 2b, which is redrawn by the solid line in this panel. (b) The most likely absorption spectra of the four isomers were used to simulate the contents of the 11-cis and 9-cis isomers that would be expected to exist in the claret membrane exposed to actinic light at λact for an infinite time period are plotted against λact. (c) The observed time course of absorption change under red illumination at λact = 680 nm (the solid blue line) was compared with the simulated curve (the dashed red line) that was evaluated using the microscopic rate constants (k12, k21, k23, and k32) that were used to fit the absorption kinetics data in Figure 5. (d) The simulated time course of the accumulation of the 11-cis and 9-cis isomers under red illumination at λact = 680 nm.

pHR that was soaked in a chloride free postcrystallization solution and then flash cooled in the dark. The asymmetric unit of this crystal contains three subunits with different environments. For the structural investigation of red illumination induced structural changes, the C2 crystal of chloride free pHR was illuminated with red light (>600 nm; ∼20 mW/cm2) for 5 min and then flash cooled to 85 K. Since the crystal used was optically thick (OD ≈ 10 at 600 nm), most proteins in the inner part of the crystal were excited at the red edge of the visible absorption band of retinal. A full data set of X-ray diffraction from this illuminated crystal (Fred) was collected 4100


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the 11-cis isomer (Figure 9). It is noteworthy that unlike the 11cis isomer, the all-trans isomer has no water molecule in the

using a low dose of X-rays (0.1 MGy) and compared with the diffraction data from an unilluminated crystal (Fdark). Figure 8b shows the |Fred| − |Fdark| difference map from the cytoplasmic side of pHR. A large difference in the profile of the difference density map among the three subunits contained in the asymmetric unit is evident. This result suggests that the formation efficiencies of the 11-cis and 9-cis isomers are influenced by the crystal lattice force. The structural difference observed in subunit B or C, which are confined to the retinal binding site, suggests that the 11-cis isomer was preferentially produced under red illumination (Figure 8c). On the other hand, the difference map in subunit A suggests that, in addition to the 11-cis isomer, another species with an alteration in the middle moiety of helix C was produced under red illumination. One possible explanation is that a non-negligible amount of the 9-cis isomer accumulated in subunit A. An alternative explanation is that the unilluminated crystal contained a small amount of an anion bound purple form (or an acidic purple form) of pHR. In the following analysis, we describe the structural models of the all-trans isomer and the 11-cis isomer that were constructed on the basis of the electron density map within subunit C. Structure of the 11-cis Isomer of pHR. Figure 8d,e shows the 2F0 − Fc maps within subunit C in the unilluminated and illuminated crystals, respectively. These maps were evaluated on the following approximations: (1) only the all-trans isomer was contained in the unilluminated crystal; (2) a mixture of the all-trans and the 11-cis isomers was contained in the illuminated crystal. These approximations were adopted because it was difficult to determine the structure of the “13-cis” isomer, which was expected to coexist as a minor component in the unilluminated crystal as well as in the illuminated crystals. This difficulty cannot be overcome as long as the thermal interconversion between the all-trans and the “13-cis” isomer takes place very rapidly. Meanwhile, a previous crystallographic study of the dark and light adapted states of bacteriorhodopsin demonstrated that the structural difference between the 13-cis isomer and the all-trans isomer is very small and confined to the region of the Schiff base linkage.37 Hence, it can be argued that the structural model of the all-trans isomer shown in Figure 8d actually represents a weighted average of the structures of the all-trans and the “13-cis” isomers. It should be emphasized that the coexistence of the “13-cis” isomer in the unilluminated or illuminated crystal would not impair our interpretation of the |F red| − |Fdark| difference map unless the molar ratio of the “13-cis” isomer to the all-trans isomer was greatly altered under the red illumination. From the analysis of the 2F0 − Fc map shown in Figure 8e, the occupancy of the 11-cis isomer in a red adapted crystal was estimated to be 32−38%. Figure 8f shows the 2F0 − Fc map that was evaluated using the quantity 3 × Fred − 2 × Fdark, in which the contribution of the all-trans isomer in subunit C to the diffraction amplitude Fred was nearly zero; i.e., 3 × Fred − 2 × Fdark ∼ F11‑cis, where F11‑cis is the diffraction amplitude that would be expected for a crystal containing only the 11-cis isomer. This map suggests that the 11-cis isomer has the following features; (1) the retinal-Lys256 chain takes on the 11cis/15-syn configuration; (2) the Schiff base is hydrogenbonded to a water molecule that is inserted into an open space created by a large conformational change of the side chain of Lys256; (3) the orientation of the indole ring of Trp127 is slightly altered; (4) the other residues in the retinal binding site scarcely move in the transformation from the all-trans isomer to

Figure 9. Structural comparison between the 11-cis isomer and the alltrans isomer of chloride-free pHR. The carbon atoms in the 11-cis isomer are drawn in gold or purple, whereas the carbon atoms and water molecules in the all-trans isomer are in cyan or light blue.

cytoplasmic vicinity of the Schiff base. This observation implies that the water distribution around the Schiff base is an important structural factor affecting the peak wavelength of the visible absorption band of the retinal chromophore.

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DISCUSSION Red Shifted Reaction States in the Anion Pumping Cycle of pHR. The present analysis of illumination induced absorption changes in chloride free pHR showed that, in addition to the all-trans isomer with a λmax at 595 nm, the “13cis” isomer, with a λmax at 625 nm, also made up a minor component of the dark adapted claret membrane. We also demonstrated that the thermally activated interconversion between the all-trans and “13-cis” isomers occurs very rapidly (10%) in pHR′ than in pHR. This spectral property of pHR′ can be explained by supposing that pHR′ is a chloride bound 13-cis isomer. Since the magnitude of the illumination induced absorption decrease at 587 nm increased with the increasing concentration of halide ion (Br− or Cl−), we suggest that at high halide ion concentrations, a large fraction of O′ decays into pHR′, which subsequently relaxes into pHR with a time constant of ∼50 ms at room temperature. At low halide ion concentrations, on the other hand, O′ is likely to decay into pHR via the O state. In the anion pumping cycle of the HR homologue (sHR) from H. salinarum, a red-shifted intermediate with the 13-cis retinal (sHR640) was considered to occur after sHR520.19−21 If the fundamental concept of the anion pumping mechanism is not different for sHR and pHR, it can be argued that the reaction intermediates sHR520, sHR640, and sHR565 for sHR correspond to N, O′, and O, respectively, for pHR. For a detailed comparison, it is necessary to take into account the following differences between sHR and pHR: (1) removal of a chloride ion from sHR is accompanied by a 10 nm blue shift, whereas removal of a chloride ion from pHR is accompanied by a 20 nm red shift;22,40 (2) the content of the 13-cis isomer in the dark adapted state is much higher in sHR than in pHR;41 (3) unlike pHR, sHR possesses a secondary chloride binding site in the chloride uptake pathway.42 Formation Mechanism of 11-cis and 9-cis Isomers of Microbial Rhodopsin. The present study showed that the

Figure 10. Illumination induced absorption changes in the presence of halide ions. (a) Absorption spectra of the claret membrane in 4 M NaBr at pH 7 recorded at 0 °C under illumination (the blue line) with orange light (λact > 570 nm; 3 mW/cm2) and after the illumination (the orange line) is drawn after subtraction of the contribution of light scattering. The visible absorption band of retinal in each spectrum is approximated by a skew Gaussian function (the dotted lines). (b) Difference spectrum between the two absorption spectra shown in panel a. (c) Difference absorption spectrum associated with the red adaptation of bromide bound pHR. This difference spectrum is derived by subtracting the spectrum of the dark adapted state of bromide bound pHR from the spectrum recorded after a 20 min illumination with orange light (the blue line in panel a).

“13-cis” isomer, with a λmax at 625 nm, was first photoconverted into the 11-cis isomer, with a λmax at 565 nm, a fraction of which was then slowly photoconverted into the 9-cis isomer, with a λmax at 560 nm. This “red adaptation” is not a unique property of chloride free pHR. Maeda et al. previously reported that the 11-cis isomer, with a λmax at 560 nm, and the 9-cis isomer, with a λmax at 495 nm, were generated when the acidic blue form of bacteriorhodopsin was illuminated with far red light.43 Under their experimental condition, a much larger amount of the 9-cis isomer than the 11-cis isomer accumulated after a long illumination. This is explainable because the absorption maximum of the 9-cis isomer is largely blue-shifted so that the backward photoconversion of the 9-cis isomer to the 11-cis isomer is seldom induced by far red illumination. The accumulation of the 9-cis isomer, with a λmax at 478 nm, was also observed in sHR under red illumination.44 By contrast, the absorption maximum of the 9-cis isomer of pHR is only slightly blue-shifted as compared with that of the 11-cis isomer, so that the backward photoconversion of the 9-cis isomer into the 11cis isomer occurs frequently under red illumination. Interestingly, preferential accumulation of the 11-cis isomer under orange illumination was also reported for middle rhodopsin, a microbial rhodopsin from Haloquadratum walsby.45 4102


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(8) Hofrichter, J., Henry, E. R., and Lozier, H. (1989) Photocycles of bacteriorhodopsin in light- and dark-adapted purple membrane studied by time-resolved absorption spectroscopy. Biophys. J. 56, 693−706. (9) Kawanabe, A., Furutani, Y., Jung, K. H., and Kandori, H. (2007) Photochromism of Anabaena sensory rhodopsin. J. Am. Chem. Soc. 129, 8644−8649. (10) Irieda, H., Morita, T., Maki, K., Homma, M., Aiba, H., and Sudo, Y. (2012) Photo-induced regulation of the chromatic adaptive gene expression by Anabaena sensory rhodopsin. J. Biol. Chem. 287, 32485− 32493. (11) Bivin, D. B., and Stoeckenius, W. (1986) Photoactive retinal pigments in haloalkaliphilic bacteria. Microbiology 132, 2167−2177. (12) Gerscher, S., Mylrajan, M., Hildebrandt, P., Baron, M. H., Müller, R., and Engelhard, M. (1997) Chromophore-anion interactions in halorhodopsin from Natronobacterium pharaonis probed by time-resolved resonance Raman spectroscopy. Biochemistry 36, 11012−11020. (13) Zimányi, L., and Lanyi, J. K. (1997) Fourier transform Raman study of retinal isomeric composition and equilibration in halorhodopsin. J. Phys. Chem. B 101, 1930−1933. (14) Váró, G., Brown, L. S., Sasaki, J., Kandori, H., Maeda, A., Needleman, R., and Lanyi, J. K. (1995) Light-driven chloride ion transport by halorhodopsin from Natronobacterium pharaonis. I. The photochemical cycle. Biochemistry 34, 14490−14499. (15) Váró, G., Needleman, R., and Lanyi, J. K. (1995) Light-driven chloride ion transport by halorhodopsin from Natronobacterium pharaonis. 2. Chloride release and uptake, protein conformation change, and thermodynamics. Biochemistry 34, 14500−14507. (16) Kouyama, T., Kawaguchi, H., Nakanishi, T., Kubo, H., and Murakami, M. (2015) Crystal Structures of the L1, L2, N, and O States of pharaonis Halorhodopsin. Biophys. J. 108, 2680−2690. (17) Hackmann, C., Guijarro, J., Chizhov, I., Engelhard, M., Rödig, C., and Siebert, F. (2001) Static and time-resolved step-scan Fourier transform infrared investigations of the photoreaction of halorhodopsin from Natronobacterium pharaonis: consequences for models of the anion translocation mechanism. Biophys. J. 81, 394−406. (18) Chizhov, I., and Engelhard, M. (2001) Temperature and halide dependence of the photocycle of halorhodopsin from Natronobacterium pharaonis. Biophys. J. 81, 1600−1612. (19) Lanyi, J. K., and Vodyanoy, V. (1986) Flash spectroscopic studies of the kinetics of the halorhodopsin photocycle. Biochemistry 25, 1465−1470. (20) Tittor, J., Oesterhelt, D., Maurer, R., Desel, H., and Uhl, R. (1987) The photochemical cycle of halorhodopsin: absolute spectra of intermediates obtained by flash photolysis and fast difference spectra measurements. Biophys. J. 52, 999−1006. (21) Ames, J. B., Raap, J., Lugtenburg, J., and Mathies, R. A. (1992) Resonance Raman study of halorhodopsin photocycle kinetics, chromophore structure, and chloride-pumping mechanism. Biochemistry 31, 12546−12554. (22) Scharf, B., and Engelhard, M. (1994) Blue halorhodopsin from Natronobacterium pharaonis: wavelength regulation by anions. Biochemistry 33, 6387−6393. (23) Ihara, K., Narusawa, A., Maruyama, K., Takeguchi, M., and Kouyama, T. (2008) A halorhodopsin-overproducing mutant isolated from an extremely haloalkaliphilic archaeon Natronomonas pharaonis. FEBS Lett. 582, 2931−2936. (24) Kouyama, T., Kanada, S., Takeguchi, U., Narusawa, A., Murakami, M., and Ihara, K. (2010) Crystal structure of the lightdriven chloride pump halorhodopsin from Natronomonus pharaonic. J. Mol. Biol. 396, 564−579. (25) Kanada, S., Takeguchi, Y., Murakami, M., Ihara, K., and Kouyama, T. (2011) Crystal structures of an O-like blue form and an anion-free yellow form of pharaonis halorhodopsin. J. Mol. Biol. 413, 162−176. (26) Kouyama, T., Nasuda-Kouyama, A., Ikegami, A., Mathew, M. K., and Stoeckenius, W. (1988) Bacteriorhodopsin photoreaction:

It should be mentioned that the red adaptation of pHR (i.e., accumulation of a blue-shifted species) was also observed in the presence of halide ions (Figure 10c). At this stage, we cannot exclude the possibility that the 11-cis and/or 9-cis isomer is generated upon excitation of pHR′. Although the red adaptation is less significant in the presence of halide ions than in the absence of halide ions, we need to take into account the possible effects of the red adaptation on light induced absorption changes in halide bound pHR.

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ASSOCIATED CONTENT

Accession Codes

Crystallographic coordinates of the 11-cis and all-trans isomers of the anion depleted form of pHR are deposited in the Protein Data Bank with accession codes 5B0W and 5ETZ.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +81-52-789-5108. Fax: +81-52-789-2436. E-mail: [email protected]. Funding

This work was supported by a grant-in-aid from the Japanese Ministry of Education, Culture, Sports, Science and Technology to TK (16K07267). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the staff of BL38B1 of SPring-8 (Harima, Japan) for their technical assistance during data collection.

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ABBREVIATIONS pHR, pharaonis halorhodopsin; sHR, salinarum halorhodopsin; HPLC, high performance liquid chromatography

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REFERENCES

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Crystal Structure of the 11-cis Isomer of Pharaonis Halorhodopsin

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