Implications for the Light-Driven Chloride Ion Transport Mechanism of

Article pubs.acs.org/JPCB

Implications for the Light-Driven Chloride Ion Transport Mechanism of Nonlabens marinus Rhodopsin 3 by Its Photochemical Characteristics Takashi Tsukamoto,*,† Susumu Yoshizawa,‡ Takashi Kikukawa,§,∥ Makoto Demura,§,∥ and Yuki Sudo*,† †

Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, 700-8530 Okayama, Japan Atmosphere and Ocean Research Institute, The University of Tokyo, Chiba 277-8564, Japan § Faculty of Advanced Life Science and ∥Global Station for Soft Matter, Global Institution for Collaborative Research and Education, Hokkaido University, Sapporo 060-0810, Japan ‡

S Supporting Information *

ABSTRACT: Several new retinal-based photoreceptor proteins that act as light-driven electrogenic halide ion pumps have recently been discovered. Some of them, called “NTQ” rhodopsins, contain a conserved Asn−Thr−Gln motif in the third or C-helix. In this study, we investigated the photochemical characteristics of an NTQ rhodopsin, Nonlabens marinus rhodopsin 3 (NM-R3), which was discovered in the N. marinus S1-08T strain, using static and time-resolved spectroscopic techniques. We demonstrate that NM-R3 binds a Cl− in the vicinity of the retinal chromophore accompanied by a spectral blueshift from 568 nm in the absence of Cl− to 534 nm in the presence of Cl−. From the Cl− concentration dependence, we estimated the affinity (dissociation constant, Kd) for Cl− in the original state as 24 mM, which is ca. 10 times weaker than that of archaeal halorhodopsins but ca. 3 times stronger than that of a marine bacterial Cl− pumping rhodopsin (C1R). NM-R3 showed no dark−light adaptation of the retinal chromophore and predominantly possessed an all-trans-retinal, which is responsible for the light-driven Cl− pump function. Flash-photolysis experiments suggest that NM-R3 passes through five or six photochemically distinct intermediates (K, L(N), O1, O2, and NM-R3′). From these results, we assume that the Cl− is released and taken up during the L(N)−O1 transition from a transiently formed cytoplasmic (CP) binding site and the O2−NMR3′ or the NM-R3′−original NM-R3 transitions from the extracellular (EC) side, respectively. We propose a mechanism for the Cl− transport by NM-R3 based on our results and its recently reported crystal structure.

■

INTRODUCTION Almost all living organisms on earth use light as an information or an energy source. Rhodopsins are one type of photoreceptor protein that mediates such biological functions.1 Rhodopsins are widespread across the varied environments on earth and are found in all three domains of life, that is, archaea, bacteria, and eukaryotes.2 According to their conserved amino acids, rhodopsins are classified into two types: type-2 animal rhodopsins, which representatively act as visual photoreceptors in the eyes, and type-1 microbial rhodopsins, which act as ion transporters, including pumps and channels, and light sensors in microorganisms.1 Microbial rhodopsins were originally found in halophilic archaea in the 1970s and 1980s. Bacteriorhodopsin (BR) was the first rhodopsin discovered in a highly halophilic archaeon Halobacterium salinarum (formerly halobium) in 1971, and it functions as an outward electrogenic light-driven proton (H+) pump across the membrane.3 In 1977, the second microbial © 2017 American Chemical Society

rhodopsin, named halorhodopsin (HR), was discovered in the same archaeon and it functions as an inward chloride ion (Cl−) pump.4,5 Since then, another HR from the highly haloalkaliphilic archaeon Natronomonas pharaonis (formerly Natronobacterium), named NpHR, has been discovered6−8 and these two archaeal HRs have been investigated thoroughly. Within the three decades since the discovery of BR and HR, various other microbial rhodopsins, especially those derived from bacteria, have been discovered.2,9 Most of them are outward light-driven H+ pumps such as BR. In addition, inward light-driven Cl− pumps, such as HR, and outward light-driven sodium ion (Na+) pumps have also been discovered from marine bacteria (Figure 1A). Received: November 4, 2016 Revised: February 14, 2017 Published: February 14, 2017 2027

DOI: 10.1021/acs.jpcb.6b11101 J. Phys. Chem. B 2017, 121, 2027−2038

Article

The Journal of Physical Chemistry B

In 2014, a HR-like Cl− pumping rhodopsin was reported in a marine flavobacterium Nonlabens marinus S1-08T and was named Nonlabens marinus rhodopsin 3 (NM-R3) (Figure 1A).10 In addition, the rhodopsin genes for a H+ pump (NM-R1) and a Na+ pump (NM-R2) were also found in the same bacterial strain. The Cl− pump NM-R3 has a unique amino acid sequence in the third or C-helix near the retinal protonated Schiff base called the “NTQ-motif” (Asn98, Thr102, and Gln109), which corresponds to Asp85, Thr89, and Asp96 in BR, respectively (Figure 1B). On the other hand, in the case of HR, those residues are substituted by Thr, Ser, and Ala, which are completely different from the residues in NM-R3 with regard to the size of their side chain, charge, and hydrophilicity (Figure 1A,B). In addition, two Glu residues (Glu194 and Glu204) that compose the protonreleasing group in BR are replaced by Phe213 and Arg223 in NM-R3, respectively, indicating that these residues play different functional roles in the H+ pump BR and Cl− pump NM-R3. Moreover, Thr218 of NpHR, which is a hydrophilic residue located in the hydrophobic CP half channel and plays an important role in Cl− transport,11−15 is replaced with a hydrophobic amino acid Met197 in NM-R3, indicating that the Cl− transport mechanism of NM-R3 is different from that of NpHR. In 2016, Hosaka and co-workers determined the X-ray crystal structure of NM-R3 at 1.58 Å resolution.16 They suggested the roles of the Cl−-releasing and Cl−-uptake regions in the CP and EC sides, respectively, at the amino acid level. In the same year, Kim and co-workers also determined the crystal structure of NM-R3 at 1.56 Å resolution and they precisely discussed the Cl − -pumping mechanism based on their structure.17 However, those structures are from the original state and therefore the photoreaction dynamics directly connected to the Cl− pump function are still unknown. In this study, we performed static and time-resolved spectroscopic measurements to investigate the photochemical properties and photoreaction kinetics of NM-R3. Moreover, we compared them to those of the archaeal halide ion pump NpHR and the bacterial halide ion pump Fluvimarina pelagi rhodopsin (FR) from the marine alphaproteobacterium F. pelagi (Figure 1A,B).18 Combining our results with the crystal structure information of NM-R3, we propose a mechanism for its lightdriven electrogenic Cl− pumping function.

Figure 1. Evolutionary and sequential characteristics of NM-R3. (A) Phylogenetic tree of known microbial rhodopsins. The names of each clade are described: marine bacterial Cl− pumping rhodopsins (ClRs), marine bacterial Na+ pumping rhodopsins (NaRs), bacterial H+ pumping proteorhodpsins (PR), archaeal Cl− pumping HRs, archaeal H+ pumping BR, and archaeal sensory rhodopsins (SR). The tree was generated by the tree mode in ClustalW. All amino acid sequences of microbial rhodopsins used were obtained from the NCBI database. The parentheses denote the motif sequences in the third or C-helix. (B) Highlighted conserved amino acids of BR, PR, NpHR, Mastigocladopsins repens HR (MrHR), FR, and Krokinobacter eikastus rhodopsin 2 (KR2).

■

EXPERIMENTAL METHODS Protein Expression and Purification. Escherichia coli BL21(DE3) cells were used for the functional expression of NM-R3 as a recombinant protein. The pET21a_NM-R3 plasmid vector10 was transformed into those cells, which were grown at 37 °C in 1 L 2 × YT medium supplemented with 50 μg/mL ampicillin (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Protein expression was induced at the optical density at 660 nm of ca. 1.6 by supplementation with 10 μM all-trans-retinal (Sigma-Aldrich) and 1 mM isopropyl β-D(-)-thiogalactopyranoside (IPTG, Wako Pure Chemical Industries, Ltd.) for 3−4 h at 37 °C. The harvested cells were resuspended in 50 mM Tris− HCl buffer (pH 8.0) containing 1 M NaCl (Wako Pure Chemical Industries, Ltd.) and were then disrupted by sonication (TOMY Seiko Co., Ltd., Tokyo, Japan). The membrane fraction was collected by ultracentrifugation (Hitachi Koki, P50A2 rotor, 4 °C, 40 000 rpm, 60 min) and then homogenized in the same buffer. The detergent n-dodecyl-β-D-maltoside (DDM; Dojindo Laboratories, Kumamoto, Japan) was added to the suspension at a final concentration of 1.5% (w/v) to solubilize the membranes containing NM-R3. After another ultracentrifugation, the

Regardless of the functional diversity in the substrate ions transported and the direction of transport, microbial rhodopsins are composed of seven transmembrane α-helices and a chromophore all-trans-retinal that binds to a conserved lysine residue in the seventh or G-helix via a protonated Schiff base linkage.1 Upon photoillumination, microbial rhodopsins commonly undergo a linear cyclic photoreaction called the photocycle, where the chromophore retinal absorbs light and is then isomerized from the all-trans to the 13-cis form, which converts the rhodopsin to the photoexcited state. The photoexcited molecules are then thermally relaxed to the original state as they pass through various photochemical intermediates, named, K, L, M, N, and O. During the relaxation process, the 13cis-retinal also returns to the original all-trans form. Especially in the ion pumping rhodopsins, the transported substrate ions are sequentially released and taken up during the photocycle and thus they transport one ion during each photoreaction cycle. 2028


Article


source and in front of the photomultiplier. For all samples, the time-dependent absorption changes were measured from 400 to 750 nm, with a 10 nm interval. The data before the laser pulse were adopted as a baseline. At each wavelength, the results of 30 flashes were averaged to improve the signal-to-noise ratio. No further data processing was applied. The temperature was kept at 20 °C using a thermostat. We analyzed the data using a sequential model based on previous reports21,22 as follows

supernatant was passed through a HisTrap FF prepacked column (GE Healthcare) to adsorb the C-terminal His6-tagged NM-R3 for purification. The protein was washed and eluted with 50 mM Tris−HCl buffers (pH 8.0) containing 1 M NaCl, 0.1% (w/v) DDM, and 70 mM or 1 M imidazole, respectively. For the following measurements, the buffer was sufficiently exchanged by centrifugation (AmiconUltra centrifuge unit, 30 000 molecular weight cutoff, Merck Millipore) and a PD-10 column (GE Healthcare) into 10 mM 3-(N-morpholino)-propanesulfonic acid (MOPS) buffer (pH 7.0, Dojindo Laboratories) containing 0.1% (w/v) DDM and NaCl, NaBr, NaI, and NaNO3 at the desired concentrations. To maintain the ionic strength at 4 M, Na2SO4, which is not a substrate ion for NM-R3,10 was added to the samples at the desired concentrations. The optical density of the DDM-solubilized NM-R3 was adjusted to ca. 0.5 at its absorption maximum. Lipid-reconstituted NM-R3 was prepared to compare the photoreaction with the DDM-solubilized NM-R3. For the reconstitution, phosphatidylcholine from egg yolk (EggPC) was purchased from Avanti Polar Lipids, Inc. The purified, DDM-solubilized NM-R3 in 10 mM MOPS (pH 7.0) buffer containing 100 mM NaCl and 0.05% DDM was mixed with EggPC dissolved in the same buffer at a molar ratio of 1:50, which is often used to reconstitute microbial rhodopsins.19 DDM was removed by dialysis for about 2 weeks using a 50 kDa molecular weight cutoff dialysis tubing (Thermo-Fischer Scientific, Co. Ltd.). The buffer without DDM (10 mM MOPS (pH 7.0), 50 mM NaCl) was exchanged once or twice a day, and the dialysis was carried out with stirring at 4 °C in the dark. The EggPCreconstituted NM-R3 was collected from the dialysis tubing and was then washed more than five times by centrifugation at 4 °C using 10 mM MOPS (pH 7.0) buffer containing 1 M NaCl. Finally, the sample was resuspended in the same buffer. The optical density of the reconstituted NM-R3 was adjusted to ca. 0.5 at its absorption maximum. UV−Vis Spectroscopy. All UV−Vis spectra were recorded at 25 °C using a UV-1800 spectrophotometer (Shimadzu Corp., Kyoto, Japan). For the anion titration experiment, the anioninduced absorbance changes (ΔA) of NM-R3 were plotted against the logarithm of the anion concentration and were analyzed by fitting the data to the Hill equation as follows

P0 → P1 → P2 → ... → Pi → P0

where P0 represents the unphotolyzed original pigment and Pi represents the ith kinetically defined state. In this model, the Pi states are allowed to contain a few physically defined photointermediates such as K−O with different absorption maxima when the quasi-equilibrium state exists between them. Briefly, the data obtained at all wavelengths were simultaneously fitted to this model using a multiexponential function. The appropriate number of exponents was determined as 5 in this study from the reductions in the standard deviation of the residuals. Using the fitting results, the time constants τi and the absorption differences (Δεi) between Pi and the original state P0 were determined. Independently, to determine the absolute spectra of each Pi state, the pure retinal spectrum was extracted by spectral decomposition as described previously21 and was used as the spectrum of P0. Finally, the absolute spectra of Pi states were obtained by adding the spectrum of P0 to the absorption differences, Δεi. Retinal Composition Analysis. Retinal isomer compositions were analyzed by normal-phase high-performance liquid chromatography (HPLC), as previously reported.23 An LC20AT HPLC system (Shimadzu Corp.) equipped with a YMCPack SIL HPLC column (YMC Co., Ltd., Kyoto, Japan) was used. The column was pre-equilibrated with a developing solvent (88% (v/v) n-hexane, 12% (v/v) ethyl acetate, and 0.12% (v/v) ethanol). Retinal was extracted as retinal oxime by adding 1 M hydroxylamine (pH 7.0, final concentration of 50 mM) and methanol (final concentration of 30% (v/v)). For measurements under dark conditions, the sample was kept in the dark for more than 1 week. For measurements under light conditions, the sample was irradiated with 540 nm light for 5 min before the retinal extraction. The retinal oxime extracted from the purple membrane (PM) containing BR was used for reference. The eluting retinal oxime was monitored by its absorption at 360 nm. The flow rate was set at 1 mL/min. All measurements were carried out at room temperature ca. 25 °C.

ΔA = ΔA max ·[Anion]n /(Kd n + [Anion]n )

where ΔAmax, [Anion], Kd, and n represent the maximum absorbance change, the concentration of anions, the dissociation constant for anions, and the Hill coefficient, respectively. Flash-Photolysis Measurements. Time-dependent absorption changes of NM-R3 were measured by flash-photolysis using a homemade computer-controlled apparatus equipped with an Nd:YAG laser (5 mJ/pulse, 532 nm, 7 ns) as an actinic light source as described previously.20 A 150 W Xe lamp (C4251, Hamamatsu Photonics, Hamamatsu, Japan) was used as a monitoring light source, the beam of which entered perpendicularly to that of the actinic laser flash. An R2949 photomultiplier (Hamamatsu Photonics) was used to detect the monitoring light passing through the sample. The output of the photomultiplier was amplified by a homemade I−V converter with an offset voltage and a response time of 0.5 μs. Because of the response time, a large scattering artifact from the laser flash appeared before 10 μs. Therefore, plots were started from 10 μs. To select the monitoring wavelength and exclude the light scattering caused by the actinic laser flash from the sample, two monochromators were placed behind the monitoring light

■

RESULTS Spectral Change upon Cl− Binding. First of all, we prepared and purified recombinant NM-R3 in the presence of the detergent DDM. Using the DDM-solubilized NM-R3, we measured the UV−Vis absorption spectrum of NM-R3 in the absence and the presence of 1 M Cl− as shown in Figure 2. The ionic strength was kept at 4 M by the addition of Na2SO4 at appropriate concentrations because the Cl− binding reaches a plateau at 4 M in many HRs and SO42− is not a transport substrate ion for NM-R3.10 As shown in Figure 2A, NM-R3 exhibited a maximum absorption wavelength (hereafter λmax) at 568 nm in the absence of Cl− and at 534 nm in the presence of 1 M Cl−. This result indicates that NM-R3 binds Cl− with a spectral blueshift as does NpHR24 and FR.18 Using the Cl−-induced spectral change, we measured the UV− Vis spectrum of NM-R3 at various Cl− concentrations and then estimated the Cl− binding affinity (practically the dissociation 2029


Article


Figure 2C shows the difference absorption spectra calculated from the UV-Vis spectra shown in Figure 2B. The difference spectra show an isosbestic point at around 550 nm, indicating that NM-R3 directly changes from the Cl−-free to the Cl−-bound form without any intermediate state. The difference absorbance at 592 nm was plotted against the NaCl concentration as shown in Figure 2D and was analyzed by the Hill equation as described in the Experimental Methods section. As a result, the estimated Kd value was 24 mM, indicating that the Cl− binding affinity of NM-R3 is ca. 10 times weaker than that of NpHR (ca. 2 mM)25 but ca. 3 times stronger than that of FR (84 mM).18 Other than Cl−, we estimated the Kd values for Br−, I−, and NO3− in the original state of NM-R3, which are all of the transport substrate ions reported previously,10 as shown in Figure 2D and summarized in Table 1. The estimated Kd values were 10 mM Table 1. Summary of Absorption Properties of NM-R3 in the Presence of Transport Substrate Ions anions −

Cl Br− I− NO3−

Kd (mM)

λmaxa (nm)

Δλmaxb (nm)

Δν (cm−1)

24 10 2.5 17

534 536 542 535

34 32 26 33

1121 1051 845 1086

The listed λmax values were determined at the following anion concentrations: Cl−, 4 M; Br−, 3 M; I−, 300 mM; NO3−, 3 M. The λmax shifts were almost saturated at these anion concentrations. bΔλmax values were the λmax shifts from the anion-free state (568 nm). a

for Br−, 2.5 mM for I−, and 17 mM for NO3−, the order of which almost follows the Hofmeister series of monoatomic anions.26 The Hill coefficient for NO3− was 0.91, indicating almost no cooperative effect upon binding NO3−. On the other hand, the Hill coefficients for Cl−, Br−, and I− ranged from 0.70 to 0.78, indicating a negative cooperativity upon binding these anions. Retinal Isomer Composition under Dark and Light Conditions. Figure 3A shows HPLC chromatograms of the extracted retinal oxime from BR and NM-R3 under dark and light conditions. BR was used as a reference because the observed peaks in BR were already assigned. As shown in Figure 3A, under both dark and light conditions, NM-R3 predominantly possesses all-trans-retinal (denoted as Ts and Ta) as a chromophore. Unlike that in BR, no dark−light adaptive change in the retinal isomer composition was observed. From the chromatograms, the peak areas and molar ratios of the all-trans and 13-cis (denoted as 13s and 13a) retinal isomers were calculated and are summarized in Figure 3B. In summary, NM-R3 predominantly has all-transretinal as a chromophore, which is responsible for the lightdriven Cl− pump function, at 90.3 and 91.6% under dark and light conditions, respectively. Photocycle at 1 M Cl− Concentration. The photocycle kinetics of NM-R3 was investigated using the flash-photolysis technique in the presence of 1 M NaCl and 0.1% DDM. To maintain the ionic strength at 4 M, 1 M Na2SO4 was added to the solution. Figure 4A shows the light minus dark difference absorption spectra in three time regions. To avoid the large scattering artifact from the laser flash, plots were started from 10 μs. The times 0.61 and 4.3 ms were chosen because the accumulation of the characteristic photointermediates reached a maximum. As shown in the upper panel of Figure 4A (time region from 10 μs to 0.61 ms), the absorption band of the original state (λmax at ca. 530 nm) disappeared upon flash excitation and then decreased with a slight blueshift of the absorption. This

Figure 2. Static UV−Vis absorption measurements of NM-R3. The purified NM-R3 was suspended in 10 mM MOPS (pH 7.0) containing 0.1% DDM and certain concentrations of NaCl and Na2SO4. The ionic strength was kept at 4 M. (A) Absorption spectra in the absence (gray line) and presence of 4 M NaCl (black line). (B) Cl−-dependent spectral shifts from 0 to 4 M NaCl. The spectra of 0 and 4 M NaCl are illustrated by black lines. The arrow indicates the direction of the shift. (C) Cl−dependent difference absorption spectra calculated from the spectra in (B). The spectrum of 0 M NaCl is drawn as a baseline. Arrows indicate the increase and decrease in absorbance. (D) Hill plot of the difference absorbance at selected wavelengths (594 nm for Cl−, 595 nm for Br−, 598 nm for I−, and 594 nm for NO3−) against the logarithm of anion concentrations for Cl− (black circles), Br− (open squares), I− (open circles), and NO3− (open triangles). Fitting curves are illustrated by broken lines. Only the curve for Cl− is shown in black. The calculated Kd values are shown in parentheses. Following are the fitting parameters used: ΔAmax,Cl− = 1.02, nCl− = 0.78; ΔAmax,Br− = 1.00, nBr− = 0.78; ΔAmax,I− = 1.04, nI− = 0.70; ΔAmax,NO3− = 1.00, nNO3− = 0.91.

constant, Kd) in the original state. Figure 2B shows the absorption spectrum of NM-R3 at various Cl− concentrations. The spectrum was gradually blue-shifted from a λmax at 568 nm in the absence of Cl− to a λmax at 534 nm in the presence of 4 M Cl−, indicating that the spectral changes reached a plateau at 4 M Cl−. 2030


Article


Figure 3. Retinal isomer composition analysis by HPLC under dark and light conditions. (A) HPLC chromatograms of BR in the native PM and NM-R3 in DDM solution under dark and light conditions. Four peaks were observed and are assigned as Ts (all-trans, 15-syn), 13s (13-cis, 15syn), 13a (13-cis, 15-anti), and Ta (all-trans, 15-anti). (B) Bar graph representations of the retinal isomer compositions calculated from the peak areas of the chromatograms. The averages and standard deviations were calculated from three independent experiments. The calculated values were all-trans 90.3 ± 0.3% and 13-cis 9.7 ± 0.3% in the dark, and all-trans 91.6 ± 0.3%, 13-cis 8.0 ± 0.2%, and a small amount of 11-cis contamination 0.4 ± 0.2% in the light.

spectral change may indicate the decay of a photointermediate with a shorter absorption band (λmax at ca. 520 nm) compared to that of the original state. On the other hand, a photointermediate with a longer λmax at ca. 580 nm appeared soon after the flash excitation. After that, another photointermediate with a longer λmax at ca. 620 nm and a wide spectral bandwidth appeared, and then an increase in this band was observed. Judging from the time region and the locations of the absorption bands, the photointermediates with the 580, 520, and 620 nm bands were tentatively assigned as K-, L- or N-like, and O-like intermediates (abbreviated as K, L(N), and O hereafter), respectively. In the case of the archaeal NpHR, it is known that the L and N intermediates show similar absorption maxima that are blueshifted to that of the original state.20,21 The upper panel in the Supporting Figure shows the double difference absorption spectra, which were redrawn with the spectrum at 10 μs after flash excitation as a new baseline. Those spectra show that the photointermediate with a shorter λmax (L(N)) was converted to that with a longer λmax (O) with an isosbestic point on the baseline at around 550 nm. In the time region from 0.61 to 4.3 ms (middle panel in Figure 4A), a decrease in the 620 nm band of O and a concomitant increase in the 520 nm band were observed. The 620 nm band decreased with a blueshift, indicating the presence of another photointermediate with a λmax at ca. 600 nm. Therefore, judging from the time region and the locations of those absorption bands, we assumed that NM-R3 has two red-shifted O-like photointermediates, with λmax at around 620 and 600 nm, which are named O1 and O2, respectively. Consequently, the increase in the 520 nm band may be caused by the spectral blueshift from O1 to O2 but not the recovery of the original state with the 530 nm band. The double difference absorption spectra in this time region (middle panel in Supporting Figure), in which the spectrum at 0.61 ms is redrawn as a new baseline, show that O1 is converted with an isosbestic point on the baseline together with an apparent increase in the 520 nm band because O1 and O2

Figure 4. Photoreaction kinetics of NM-R3 at a 1 M NaCl concentration. (A) Light minus dark difference spectra in the presence of 0.1% DDM in three time domains. The spectra of the first and the last domains are shown as black lines. Arrows indicate the direction of the spectral changes. (B) Absorption spectra of the kinetically defined P1 (red), P2 (orange), P3 (green), P4 (blue), and P5 (purple) states in the presence of 0.1% DDM. P0 is shown as a gray broken line and is the pure absorption spectrum of NM-R3 obtained from the skewed Gaussian fitting of the absorption spectrum at 1 M NaCl. (C) (Upper panel) Time evolution of the flash-induced absorption changes of the DDMsolubilized NM-R3 in the presence of 1 M NaCl. (Lower panel) Time evolution of the flash-induced absorption changes of the EggPCreconstituted NM-R3 in the presence of 1 M NaCl. The selected wavelengths are at 500 (blue), 540 (green), 600 (orange), and 650 nm (red), which monitor L(N), the original, O2, and O1 states, respectively. Fitting curves are shown as black lines.

exhibit close λmax values at around 620 and 600 nm and a wide spectral bandwidth (at least 100 nm). 2031


Article

The Journal of Physical Chemistry B In the time region from 4.3 to 388 ms (lower panel in Figure 4A), the decrease in the 600 nm band of O2 and a concomitant increase in the 520 nm band were observed. In this case, the increase in the 520 nm band is attributable to the recovery of the original state with a λmax at ca. 530 nm because of the spectral overlap with the O2 with the wider spectral bandwidth. Therefore, the O2 declines completely and returns to the original pigment within several hundreds of milliseconds, which is also supported by the double difference absorption spectra in this time region, where the spectrum at 4.3 ms is redrawn as a new baseline (Supporting Figure). Next, we performed global fitting analysis, as described in the Experimental Methods section. We found that the experimental results were sufficient to be simulated by the sum of five exponential terms, indicating the existence of five photochemically defined states (designated as P1−P5) during the photocycle of NM-R3. The time constants, τ1−τ5, of P1−P5 were determined as 82.7 μs, 354 μs, 4.69 ms, 8.88 ms, and 29.5 ms, respectively. According to the sequential model, we calculated the absorption spectra of the P1−P5 states. In this model, the photocycle is represented by some kinetically defined states (Pi). Pi is estimated to decay by first-order kinetics and is allowed to contain a few physically defined photointermediates, such as K− O. The calculated absorption spectra of the P1−P5 states of NMR3 in the presence of 1 M NaCl are shown in Figure 4B. P0 with a λmax at 530 nm represents the original, pure retinal spectrum of NM-R3 under the same condition. The P1 state showed a broad absorption spectrum from 400 to 700 nm and seems not to follow a Gaussian or skewed Gaussian distribution, indicating the existence of several photointermediates. Thus, it is assumed that the P1 state contains intermediates with a longer λmax estimated to be around 580 nm and a shorter one at 520 nm. Judging from the time region, the locations of absorption bands, and the NaCl dependency of the P1 spectra as described below, we tentatively assigned the former intermediate as K (580 nm) and the latter one as L(N) (520 nm). Further studies monitoring the timedependent structural dynamics of the retinal configuration and the protein moiety will help clarify this. The spectrum of the P2 state, which is converted from P1 with a time constant of 82.7 μs, mainly contains L(N) and small fractions of K. The absorption spectra dramatically changed in the P3 state, which was converted from P2 with a time constant of 354 μs. The main absorption appeared at 610 nm, which we assigned as O1 showing a wide spectral bandwidth. In the P4 state, which is converted from P3 with a time constant of 4.69 ms, another O-like intermediate named O2 was seen as the main component. O2 is slightly blueshifted from O1. These two O-like intermediates are clearly different from each other, suggesting that they play different roles in the Cl− pump function of NM-R3. The P4 state is converted to P5 with a time constant of 8.88 ms. The spectrum of P5 was almost the same as that of the original state NM-R3 and thus we named P5 as NM-R3′, analogous to the NpHR′20,21 and FR′18 intermediates in NpHR and FR, respectively. Finally, the P5 state is converted to the original state with a time constant of 29.5 ms, which is the rate-limiting step of the photoreaction and thus the photocycle is finished. In summary, the photocycle scheme in the presence of 1 M NaCl is described in Scheme 1. The upper panel of Figure 4C shows the flash-induced, timedependent absorption changes of NM-R3 in the presence of 1 M NaCl and 0.1% DDM. The selected wavelengths are 500, 540, 600, and 650 nm, which correspond to the absorption wavelengths of L(N), the original state, O2, and O1, respectively. Because of the spectral overlap and the wide spectral bandwidth,

Scheme 1. Photocycle Model of NM-R3 in the Presence of 1 M NaCl at pH 7.0 and 20 °C

as shown in Figure 4B, every selected wavelength except for 650 nm contains a large amount of contamination, which is not negligible. In the early stage of the photoreaction, the L(N) (500 nm) decay with the time constant of 354 μs seems to match the rise of O1. On the other hand, in the late stage, the decays of O1 and O2 seem to be biphasic. As described in the following section, the late stage of the photoreaction of NM-R3 is largely affected by the NaCl concentration (see Figure 5). The lower panel of Figure 4C shows the flash-induced, time-dependent absorption changes of NM-R3 reconstituted into EggPC membranes. Compared to that in the DDM-solubilized NM-R3 (the upper panel of Figure 4C), there seems to be a slight change in the timedependent absorption changes of the original (540 nm) and L(N) (500 nm) states. Although further analysis may be needed to reveal the reason for these changes, we conclude that the basic components in the photocycles of the DDM-solubilized and the membrane-reconstituted NM-R3 are not remarkably altered under our experimental conditions. For further spectroscopic experiments, the DDM-solubilized NM-R3 was used in this study. Photocycle at Various Cl− Concentrations. To elucidate which intermediate is involved in the uptake and release of Cl− by NM-R3, we performed flash-photolysis measurements at various Cl− concentrations. Figure 5A shows the flash-induced timedependent absorption changes (left panels) and the calculated absorption spectra of the P1−P5 states (right panels) at 400 mM (upper panels) and 4 M (lower panels). The corresponding figures for the NaCl concentration at 1 M are shown in panels B and C (upper) in Figure 4. As shown in Figure 2D, almost 100% of NM-R3 is in the Cl−-bound form at above 400 mM NaCl concentration. Therefore, we observed and analyzed the physiological photoreactions of the Cl−-bound NM-R3 under these conditions. The selected wavelengths were the same as described in Figure 4C. As can be seen, the duration of the photocycle was shortened (left panels) and characteristic absorption decrease in P4 and shift in P5 were observed (right panels) at a higher NaCl concentration (see details described below). Figure 5B shows the relationships between the photocycle time constants (τ1−τ5) and NaCl concentrations. The global fitting analysis was successfully performed by the same exponential function with the sum of five exponents as described above. From Figure 5B, the photoreaction of NM-R3 in the early stage, which corresponds to the P1−P2 and P2−P3 transitions, with time constants of τ1 and τ2, respectively, was slightly delayed at higher NaCl concentrations. However, the time constants τ4 and τ5, which correspond to the P4−P5 and P5− P0 transitions, respectively, were more sensitively affected by the NaCl concentration and thus the photoreaction in the later stage was accelerated at a higher NaCl concentration. These results suggest that the release and uptake of Cl− occur in the early and in the later stages of the photocycle, respectively. Only τ3 was almost independent from NaCl concentration, indicating that the P3−P4 transition may be the state without release or uptake of Cl− to or from the bulk space. Figure 6A shows the NaCl concentration dependence of the absorption spectra in the five kinetically defined states, P1−P5. The dashed and gray lines correspond to the original P0 state of 2032


Article


Figure 5. Photoreaction kinetics of NM-R3 at various NaCl concentrations. (A) (Left panels) Time evolution of flash-induced absorption changes in the presence of 400 mM and 4 M NaCl. The selected wavelengths are at 500 (blue), 540 (green), 600 (orange), and 650 nm (red), which monitor L(N), the original, O2, and O1 states, respectively. Fitting curves are shown as black lines. (Right panels) Calculated absorption spectra of the kinetically defined P1 (red), P2 (orange), P3 (green), P4 (blue), and P5 (purple) states in the presence of 400 mM and 4 M NaCl. P0 is shown as a gray broken line and is the pure absorption spectrum of NM-R3 obtained from the skewed Gaussian fitting of the absorption spectrum at each NaCl concentration. (B) NaCl concentration dependence of time constants τ1 (open circles), τ2 (open squares), τ3 (open triangles), τ4 (black circles) and τ5 (black squares) obtained from the global fitting analysis. The broken lines show the linear correlations of Cl−-dependent changes in the τ constants.

Figure 6. NaCl concentration dependency of P1−P5 states of NM-R3. (A) Absorption spectra of P1−P5 states at various NaCl concentrations. The spectrum of P0 in the presence of 1 M NaCl is shown as a gray broken line. The spectra at 400 mM and 4 M NaCl concentrations are shown as black lines. Arrows indicate the directions of the spectral changes. Difference spectra of P4 and P5 states are shown in each inset. The spectra at 400 mM NaCl were redrawn as a baseline. (B) Hill plot of the difference absorbance at 590 nm against the logarithm of NaCl concentrations for P4 (open circles) and P5 (closed circles) states. Fitting curves for P4 and P5 states are illustrated by broken and solid lines, respectively. The calculated Kd values are shown in parentheses. Following are the fitting parameters used: ΔAmax,P4 = 1.21, nP4 = 1.10; ΔAmax,P5 = 1.02, nP5 = 1.63.

NM-R3 in the presence of 1 M NaCl. All absorption spectra in each state are normalized as that the absorption at λmax of 530 nm in the P0 state equals 1. The P1 state shows a broad absorption spectrum at lower NaCl concentrations, which then becomes narrow at higher NaCl concentrations, suggesting that the spectrum in the P1 state may be composed of at least two photointermediates, whose λmax values were estimated to be 580 and 520 nm by deconvolution of the spectra. The P2 state also contains intermediates with λmax values of 580 and 520 nm. The population of the former

intermediate was decreased and the latter one was increased in the P2 state compared to that of the P1 state. Therefore, we assigned these intermediates as K (580 nm) and L(N) (520 nm), respectively, by taking into account the time region and the 2033


Article


absence of Cl−. The λmax values of Cl−-bound and Cl−-free NMR3 were determined as 534 and 568 nm, respectively (see Figure 2A). The energy of this shift was calculated as 1121 cm−1 (see Table 1), which is larger than that of NpHR (578 and 600 nm, 634 cm−1)12 and FR (518 and 542 nm, 855 cm−1).18 The crystal structure of NM-R3 reveals that the initial Cl− binding site is in the vicinity of the protonated Schiff base (Figure 7).16,17 The Cl−-dependent absorption shift described above is coupled with Cl− binding to the initial binding site. The bound Cl− is surrounded by Arg95, Asn98, Trp99, Thr102, Asp231, the protonated Schiff base (Lys235), and two or three water molecules and forms hydrogen bonds with Asn98, Thr102, the protonated Schiff base, and the nearest water molecule. In NpHR, the bound Cl− forms an electric quadrupolar complex together with the protonated Schiff base, Arg123, and Asp252, and is also stabilized by three water molecules.27 However, in the structure of NM-R3, such a quadrupolar complex is missing. In addition, compared to that in the initial Cl− binding site of NpHR, the number of OH-group bearing residues (Ser and Thr) in NM-R3 is less, as seen in FR.18 These structural differences in the initial binding site between NpHR and NM-R3 may affect the Cl− binding affinity (the dissociation constant, Kd). In fact, we determined the Kd of NM-R3 as ca. 24 mM (see also Figure 2 and Table 1), which is ca. 10 times larger than that of NpHR (ca. 2 mM) but ca. 3 times smaller than that of FR (ca. 84 mM). We suppose that marine bacterial Cl− pumping rhodopsins (ClRs), such as NM-R3 and FR, bind Cl− with a weaker affinity than do archaeal HRs. However, from the biological and environmental point of view, the Kd of 24 mM for NM-R3 is sufficient under physiological conditions of the marine environment (Cl− concentration is ca. 600 mM). In addition to the Cl− binding site around the Schiff base, Kim and co-workers proposed in their crystal structure that NM-R3 has a secondary Cl− binding site in the CP surface composed of Ala44, Pro45, and Lys46 (Figure 7).17 Our Cl− titration experiments using the UV−Vis absorption spectra did not show any additional change even at 4 M Cl− concentration (Figure 2B). On the other hand, a negative cooperativity effect upon Cl− binding was observed as well as with Br− and I− binding (Figure 2D). It is speculated that the Cl− binding to the initial binding site near the protonated Schiff base inhibits the additional Cl− binding to the secondary binding site near the CP surface. Interestingly, the negative cooperativity was not observed for the NO3− binding to NM-R3 (Hill coefficient is 0.91, Figure 2D). Such a tendency for the anion binding may have originated from the structural differences between the anions (monoatomic or polyatomic) and may imply anion specificity for the secondary binding site. The significance of the negative cooperativity of NM-R3 upon anion binding may be important to understand the molecular mechanisms in detail and thus requires further study. As the authors mentioned, we suppose that Cl− binding to the secondary binding site may not necessarily be required for the Cl− pump activity; however, this site may be involved in a potential Cl− transport pathway. The binding affinities of anions other than Cl− were also examined (see Figure 2D) and are summarized in Table 1. The Kd values were determined as 24 mM for Cl−, 17 mM for NO3−, 10 mM for Br−, and 2.5 mM for I−, and that order almost follows the Hofmeister series of monovalent anions (Cl− > Br− > NO3− > I−), which represents their order of hydrophobicity and their tendency to stabilize structured low-density water.26 Such a tendency also indicates that NM-R3 can bind NO3− and I− more strongly than Cl− but cannot transport them as strongly as Cl−

locations of the absorption bands. The P3 state showed broad and the most red-shifted absorption spectra and contains an intermediate with a λmax of 610 nm, O1. We suppose that O1 is a Cl−-free form because of the Cl− independence in the absorption of O1. Therefore, we assumed that the Cl− is released during the transition from L(N) to O1, which was also supported by the Cl− dependent delay of the time constant τ2 (Figure 5B). Another red-shifted intermediate with a λmax of 580 nm, O2, which was blue-shifted 30 nm from O1, was observed in the P4 state. From the Cl− independence of the time constant τ3 (Figure 5B), exchange of Cl− with the bulk space does not seem to occur during the O1 (P3)−O2 (P4) transition as described above. On the other hand, the spectral amplitudes but not the absorption maxima of O2 decrease with the Cl− concentration. The P5 spectra were also sensitively dependent on NaCl concentration. The spectra were blue-shifted with increase in the Cl − concentration, and at higher concentrations, the spectra were almost the same as the original state, which we assigned as NMR3′. Difference spectra of the P4 and P5 states (see each inset) showed a Cl− dependent increase at 500 nm and decrease at 590 nm with an isosbestic point at 520 nm. From the difference spectra, we plotted the difference absorbance at 590 nm against the NaCl concentration as shown in Figure 6B and analyzed using the Hill equation. As a result, the apparent Kd values were estimated as 1097 mM for the P4 state and 541 mM for the P5 state, respectively. Taking into account the Cl− concentration in the marine environment (ca. 600 mM), the former value seemed not to be significant in physiological conditions. Moreover, the Hill coefficients were estimated as 1.1 for the P4 state and 1.6 for the P5 state, respectively, indicating positive cooperativity for Cl− binding in the P5 state. Therefore, we speculated the possibilities that the O2 is mainly a Cl−-free form as well as the O1 and the Cl− uptake from the bulk space in the EC side occurs during the O2− NM-R3′ transition, which is also supported by the Cl−dependent acceleration of the time constant τ4 (Figure 5B). In addition, a Cl−-dependent acceleration of the time constant τ5 was observed (Figure 5B), which may indicate the possibility that the Cl− uptake occurs during the transition from NM-R3′ without Cl− to the original NM-R3. Finally, the photocycle is completed. In summary, the photocycle scheme including the Cl− uptake and release is described in Scheme 2. Scheme 2. Photocycle Model of NM-R3 Including Cl− Uptake and Release at pH 7.0 and 20 °Ca

a

The time constants in the presence of 1 M NaCl were used.

■

DISCUSSION In this study, we investigated the photochemical characteristics of NM-R3 using static and time-resolved spectroscopic techniques and characterized its light-driven electrogenic Cl− pumping mechanism. Figure 7 summarizes and illustrates the Cl− pumping photoreaction mechanism based on our results together with the recently revealed X-ray crystal structure of NM-R3.16,17 Cl− Binding in the Original State. We measured the UV− Vis absorption spectrum of NM-R3 in the presence and in the 2034


Article


Figure 7. Proposed models for the photoreaction and the mechanism of the inward Cl− pump. All close-up views are illustrated using one of the crystal structures of NM-R3 (PDB ID 5B2N).16 The crystallization condition of pH 8.0 is close to our experimental condition of pH 7.0. In the original state P0, NM-R3 binds Cl− in the vicinity of the protonated Schiff base as shown at the upper-right. The binding site is composed of Arg95, Asn98, Trp99, Thr102, Asp231, Lys235, and two water molecules (denoted as Wat.). During the transition from the original to the P1 states, the photoisomerization of the retinal chromophore and the formation of K occur as shown by the broken arrow. P1 and P2 contain K and L(N), which are in quasi-equilibrium, indicated by the double-headed arrow. The transiently formed CP binding site in L(N) and O1 and the Cl−-release during the L(N)−O1 transition are illustrated at the lower-right. Two hydrophilic residues (Ser54 and Gln109) and three water molecules are located in the hydrophobic CP half channel. The hydrophilic Thr218 in NpHR is replaced by the hydrophobic Met197 in NM-R3. Ala44, Pro45, and Lys46 are suggested to be involved in a potential Cl− transport pathway. The O1 in the P3 state and O2 in the P4 state are assigned as Cl−-free forms, respectively. In L(N)−O2, some structural changes are speculated, which play important roles in the vectorial transport of Cl−. NM-R3′ in P5 is a Cl−-bound form. From the Cl− dependency in the P5 state, a Kd of 541 mM was estimated, which is lower than the Cl− concentration in the marine environment (ca. 600 mM) and thus this indicates the functional significance of the Cl− uptake at this state. Cl− would be taken up from the hydrophilic EC region to the initial binding site near the protonated Schiff base. The EC region is composed of Lys2, Asn3, Asn92, Gln143, Glu146, Arg223, and four water molecules and is connected to the initial binding site by hydrogen bonds, as suggested by the crystal structure.16,17 Finally, the photocycle is completed. As a result, one Cl− is transported inwardly by each photocycle reaction.

Cl− Movement in Photointermediate States during the Cl -Pumping Photocycle. The photocycle of NM-R3 in the presence of Cl− was precisely analyzed on the basis of the results of flash-photolysis measurements (Figures 4−6). We checked the photocycle of both NM-R3 suspended in the DDM solution and NM-R3 reconstituted into the EggPC membrane fraction (Figure 4C) and concluded that the photocycle of the DDMsolubilized NM-R3 was not remarkably different from that of the EggPC-reconstituted one. Our flash-photolysis measurements and the following kinetic analysis suggested that the photocycle of NM-R3 is composed of five kinetically defined states (P1−P5), which contain physically defined photointermediates named K,

(see Figure S5 in ref 10). These larger anions may be difficult to move toward the protonated Schiff base region and to pass through the CP half channel during the photocycle. On the other hand, in the cases of NpHR and FR, the order of Kd of these anions were opposite or totally different, and were reported as NO3− (14.5 mM) > I− (4.1 mM) > Cl− (1.6 mM) > Br− (1.0 mM) for NpHR28 and NO3− (129 mM) > Cl− (84 mM) > Br− (81 mM) > I− (44 mM) for FR.18 These results suggest that the structure and interactions in the initial Cl− binding site are different among these three molecules. Further studies will be needed to elucidate the precise mechanism of anion binding.

−

2035


Article


Pro45, and Lys46 on the CP surface, which compose the secondary Cl−-binding site suggested by the crystal structure,17 may be involved in a potential Cl−-transport pathway. Therefore, the bound Cl− may be released by passing through these three residues during the L(N)−O1 transition. During the following O1 to O2 transition, a structural change may occur to avoid the back-flow of Cl− from the CP side and to accept the next uptake of Cl− from the EC bulk space, which may achieve the vectorial Cl− transport of NM-R3 from the EC to the CP side of biological membranes. The Cl− uptake will occur during the transition from O2 in P4 to NM-R3′ in P5 because the time constant τ4 is accelerated in a Cl− concentration-dependent manner (see Figure 5B). From the Cl− dependence of the P5 spectra (see Figure 6), we estimated the apparent Kd value as 541 mM, which is smaller than the Cl− concentration in the marine environment (ca. 600 mM). In addition, the Hill coefficient of 1.6 indicates the positive cooperativity upon Cl− uptake in the P5 state. Therefore, NMR3′ is attributable to a Cl−-bound form. In addition, NM-R3′ in the P5 state decays to the original state with the time constant τ5, which also depends on the Cl− concentration (see Figure 5B). Therefore, we speculated the possibility that the Cl− uptake occurs during the O2−NM-R3′ and NM-R3′−original NM-R3 transitions. The crystal structure shows the hydrophilic EC region is composed of Lys2, Asn3, Asn92, Gln143, Glu146, and Arg223, and four water molecules.17 The structure also implies that the EC region connects to the initial Cl− binding site near the protonated Schiff base through a hydrogen bond network.16,17 Therefore, upon Cl− uptake during the O2−NM-R3′ and NM-R3′-the original NM-R3 transitions, we speculated that Cl− in the EC bulk space enters through the hydrophilic EC region to the initial binding site. Note that, as shown in the crystal structure,16,17 one Cl− is located at the initial binding site near the protonated Schiff base in the original state. Further study is awaited to identify the role of these two transitions and the positive cooperativity for the Cl− uptake in NM-R3. In the case of FR, it has been reported that a transient Cl− binding site is formed in the EC side of the molecule.18 We speculate that NMR3 also has such a transient Cl− binding mechanism during the photocycle because of high sequential similarity with FR (ca. 74%), which may correspond to the decay of O2 as with FR.18 We also speculate that the Cl− bound to the transient EC binding site moves toward the initial binding site near the protonated Schiff base through the hydrophilic EC half channel in the late photocycle as suggested by the crystal structure.16,17 On the other hand, the formation of such a transient binding site is unclear in NpHR. Therefore, we estimate that the formation of the transient binding site upon Cl− uptake from the EC bulk space may be a common mechanism for marine bacterial Cl− pumps, including NM-R3 and FR.

L(N), O1, O2 and NM-R3′ (Figure 7). Here, we explain the photocycle of NM-R3 with respect to that of NpHR. It should be noted that these two molecules have been investigated using the same instrument, experimental conditions, and analysis procedures by our group.20,21,25 The start of the photocycle of NM-R3 is similar to other microbial rhodopsins. The Cl−-bound original NM-R3 that predominantly possesses over 90% all-trans-retinal (P0) receives visible light (λmax at 534 nm) which induces the photoisomerization of the chromophore retinal from the all-trans to the 13-cis configuration. We observed K (580 nm) after the photoisomerization with our laser flash-photolysis system. K (580 nm) and L(N) (520 nm) form both in the P1 and P2 states, which are in quasi-equilibrium. We assigned the photointermediate with a λmax at 520 nm as L(N) because L and N were not distinguishable in this study because their absorption bands were similar. According to studies of NpHR, in L (especially L1), Cl− is thought to still be located near the initial binding site in the EC side, whereas the structures of retinal and the protein moiety are changed.29−31 On the other hand, the accessibility change of the Cl− from the EC side in L1 to the CP side in L2 and N occurs with a conformational change of the protein moiety.29−31 It is difficult to identify the location of Cl− in the L(N) state of NM-R3 at present. Therefore, further study is needed to reveal the detailed Cl− translocation in the early photointermediates. In the later L(N), Cl− is transferred from the initial binding site to a transiently formed binding site in the hydrophobic CP half channel. In this channel, two hydrophilic amino acid residues, Ser54 (Thr71 in NpHR) and Gln109 (Ala137), and three water molecules are located. We speculated that these residues and water molecules play an important role in the transient Cl− binding, however Ala and Glu mutants of Gln109 (Q109A and Q109E) have been shown not to be responsible for the Cl− pump activity.16 Further, the role of Ser54 in NM-R3 for the transient Cl− binding is unclear. Note that in the case of NpHR, Thr218 is located in the CP half channel and is the most likely candidate for the transient Cl−-binding site in the channel.11−15 However, the hydrophilic Thr218 in NpHR is replaced by the hydrophobic Met197 in NM-R3. Therefore, the hydrophilicity and/or hydrophobicity in the transiently formed Cl−-binding site in the CP half channel in L(N) may be a key to understanding the detailed mechanism of Cl− transport in NM-R3 as well as in NpHR. We also speculated that during the quasi-equilibrium transition between K and L(N) a less restricted pathway may be formed, which connects the initial Cl− binding site in the EC side and the transient binding site in the CP side. As a result, the external Cl− concentration may affect the intramolecular movement of the bound Cl− and therefore the time constants τ1 and τ2 show a Cl− concentration dependence (Figure 5B). We found two red-shifted intermediates, O1 and O2, in the P3 and P4 states, respectively. It is noted that such multiple O intermediates are not seen in FR or in NpHR18,20,21 and therefore the O1 and O2 in NM-R3 are each unique and they play important roles in the Cl− pump function of NM-R3. We assigned O1 and O2 as the Cl−-free forms because no Cl−dependent absorption change of O1 and no Cl−-dependent acceleration or deceleration of the time constant τ3 for the P3 (O1)−P4 (O2) transition were observed (Figures 5B and 6). During the transition from L(N) to O1, we speculated that the Cl− is released from the transiently formed CP binding site to the CP bulk space, which is different from the fact that in FR the Cl−release occurs with the decay of O.18 As described above, Ala44,

■

CONCLUSIONS Our spectroscopic results combined with the recently revealed Xray crystal structure of NM-R3 suggest the mechanism of Cl− pumping by NM-R3, which has an NTQ-motif sequence different from that of conventional archaeal HRs with the TSA motif. The affinity for Cl− in the initial state is determined to be Kd = 24 mM, which is ca. 10 times weaker than that of NpHR but is ca. 3 times stronger than that of FR. NM-R3 also has a unique anion selectivity (the order of Kd is Cl− > NO3− > Br− > I−), the reason for which will be determined on further study. The functional characteristics and photochemistry of NM-R3 are revealed, especially in the two O-like red-shifted photo2036


Article


halobium Differing in Pigmentation. Biochem. Biophys. Res. Commun. 1977, 78, 237−243. (5) Matsuno-Yagi, A.; Mukohata, Y. ATP Synthesis Linked to LightDependent Proton Uptake in a Rad Mutant Strain of Halobacterium Lacking Bacteriorhodopsin. Arch. Biochem. Biophys. 1980, 199, 297− 303. (6) Bivin, D. B.; Stoeckenius, W. Photoactive Retinal Pigments in Haloalkaliphilic Bacteria. J. Gen. Microbiol. 1986, 132, 2167−2177. (7) Duschl, A.; Lanyi, J. K.; Zimányi, L. Properties and Photochemistry of a Halorhodopsin From the Haloalkalophile, Natronobacterium pharaonis. J. Biol. Chem. 1990, 265, 1261−1267. (8) Lanyi, J. K.; Duschl, A.; Hatfield, G. W.; May, K.; Oesterhelt, D. The Primary Structure of a Halorhodopsin From Natronobacterium pharaonis. Structural, Functional and Evolutionary Implications for Bacterial Rhodopsins and Halorhodopsins. J. Biol. Chem. 1990, 265, 1253−1260. (9) Brown, L. S. Eubacterial Rhodopsins - Unique Photosensors and Diverse Ion Pumps. Biochim. Biophys. Acta 2014, 1837, 553−561. (10) Yoshizawa, S.; Kumagai, Y.; Kim, H.; Ogura, Y.; Hayashi, T.; Iwasaki, W.; Delong, E. F.; Kogure, K. Functional Characterization of Flavobacteria Rhodopsins Reveals a Unique Class of Light-Driven Chloride Pump in Bacteria. Proc. Natl. Acad. Sci. U.S.A. 2014, 111, 6732−6737. (11) Rüdiger, M.; Oesterhelt, D. Specific Arginine and Threonine Residues Control Anion Binding and Transport in the Light-Driven Chloride Pump Halorhodopsin. EMBO J. 1997, 16, 3813−3821. (12) Sato, M.; Kubo, M.; Aizawa, T.; Kamo, N.; Kikukawa, T.; Nitta, K.; Demura, M. Role of Putative Anion-Binding Sites in Cytoplasmic and Extracellular Channels of Natronomonas pharaonis Halorhodopsin. Biochemistry 2005, 44, 4775−4784. (13) Kolbe, M.; Besir, H.; Essen, L. O.; Oesterhelt, D. Structure of the Light-Driven Chloride Pump Halorhodopsin at 1.8 Å Resolution. Science 2000, 288, 1390−1396. (14) Seki, A.; Miyauchi, S.; Hayashi, S.; Kikukawa, T.; Kubo, M.; Demura, M.; Ganapathy, V.; Kamo, N. Heterologous Expression of pharaonis Halorhodopsin in Xenopus laevis Oocytes and Electrophysiological Characterization of Its Light-Driven Cl− Pump Activity. Biophys. J. 2007, 92, 2559−2569. (15) Shibasaki, K.; Shigemura, H.; Kikukawa, T.; Kamiya, M.; Aizawa, T.; Kawano, K.; Kamo, N.; Demura, M. Role of Thr218 in the LightDriven Anion Pump Halorhodopsin From Natronomonas pharaonis. Biochemistry 2013, 52, 9257−9268. (16) Hosaka, T.; Yoshizawa, S.; Nakajima, Y.; Ohsawa, N.; Hato, M.; Delong, E. F.; Kogure, K.; Yokoyama, S.; Kimura-Someya, T.; Iwasaki, W.; et al. Structural Mechanism for Light-Driven Transport by a New Type of Chloride Ion Pump, Nonlabens marinus Rhodopsin-3. J. Biol. Chem. 2016, 291, 17488−17495. (17) Kim, K.; Kwon, S.-K.; Jun, S.-H.; Cha, J. S.; Kim, H.; Lee, W.; Kim, J. F.; Cho, H.-S. Crystal Structure and Functional Characterization of a Light-Driven Chloride Pump Having an NTQ Motif. Nat. Commun. 2016, 7, No. 12677. (18) Inoue, K.; Koua, F. H. M.; Kato, Y.; Abe-Yoshizumi, R.; Kandori, H. Spectroscopic Study of a Light-Driven Chloride Ion Pump From Marine Bacteria. J. Phys. Chem. B 2014, 118, 11190−11199. (19) Inoue, K.; Tsukamoto, T.; Shimono, K.; Suzuki, Y.; Miyauchi, S.; Hayashi, S.; Kandori, H.; Sudo, Y. Converting a Light-Driven Proton Pump Into a Light-Gated Proton Channel. J. Am. Chem. Soc. 2015, 137, 3291−3299. (20) Kikukawa, T.; Kusakabe, C.; Kokubo, A.; Tsukamoto, T.; Kamiya, M.; Aizawa, T.; Ihara, K.; Kamo, N.; Demura, M. Probing the Cl−Pumping Photocycle of pharaonis Halorhodopsin: Examinations with Bacterioruberin, an Intrinsic Dye, and Membrane Potential-Induced Modulation of the Photocycle. Biochim. Biophys. Acta 2015, 1847, 748− 758. (21) Hasegawa, C.; Kikukawa, T.; Miyauchi, S.; Seki, A.; Sudo, Y.; Kubo, M.; Demura, M.; Kamo, N. Interaction of the Halobacterial Transducer to a Halorhodopsin Mutant Engineered So as to Bind the Transducer: Cl− Circulation Within the Extracellular Channel. Photochem. Photobiol. 2007, 83, 293−302.

intermediates, O1 and O2, which are involved in the light-driven inward Cl− pumping. We assumed that NM-R3 releases the initially bound Cl− to the CP side upon the formation of O1 (decay of L(N)), which is similar to that for NpHR but different from that for FR, and then takes up the next Cl− from the EC bulk space upon the decay of O2 and NM-R3′. The structural changes between the O1 and O2 photointermediates are suggested to play important roles in the vectorial Cl− transport.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b11101. Light minus dark double difference spectra of NM-R3 at 1 M NaCl concentration (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (T.T.). *E-mail: [email protected] (Y.S.). ORCID

Takashi Tsukamoto: 0000-0002-6348-6664 Yuki Sudo: 0000-0001-8155-9356 Author Contributions

T.T., S.Y., and Y.S. designed the research. T.K. and M.D. set up the flash-photolysis apparatus. T.T. performed the experiments, collected and analyzed the data, and wrote the paper. All authors discussed the results of the paper. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This research was supported by grants from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (MEXT) to T.T. (JP15K18519), to S.Y. (JP15H02800) and to Y.S. (JP15H04363). This work was also supported by a Grant-inAid for Scientific Research on Innovative Areas (25104005) and CREST, JST to Y.S. The authors thank DASS Manuscript for English language editing.

■

ABBREVIATIONS NM-R3, Nonlabens marinus rhodopsin 3; NM-R1, N. marinus rhodopsin 1; NM-R2, N. marinus rhodopsin 2; HR, halorhodopsin; BR, bacteriorhodopsin; NpHR, Natronomonas pharaonis HR; FR, Fluvimarina pelagi rhodopsin; DDM, n-dodecyl-β-Dmaltoside; MOPS, 3-(N-morpholino)-propanesulfonic acid; EggPC, egg yolk phosphatidylcholine; CP, cytoplasmic; EC, extracellular; HPLC, high-performance liquid chromatography

■

REFERENCES

(1) Ernst, O. P.; Lodowski, D. T.; Elstner, M.; Hegemann, P.; Brown, L. S.; Kandori, H. Microbial and Animal Rhodopsins: Structures, Functions, and Molecular Mechanisms. Chem. Rev. 2014, 114, 126−163. (2) Inoue, K.; Tsukamoto, T.; Sudo, Y. Molecular and Evolutionary Aspects of Microbial Sensory Rhodopsins. Biochim. Biophys. Acta 2014, 1837, 562−577. (3) Oesterhelt, D.; Stoeckenius, W. Rhodopsin-Like Protein from the Purple Membrane of Halobacterium halobium. Nature (London), New Biol. 1971, 233, 149−152. (4) Matsuno-Yagi, A.; Mukohata, Y. Two Possible Roles of Bacteriorhodopsin; a Comparative Study of Strains of Halobacterium 2037


Article

The Journal of Physical Chemistry B (22) Chizhov, I.; Chernavskii, D. S.; Engelhard, M.; Mueller, K. H.; Zubov, B. V.; Hess, B. Spectrally Silent Transitions in the Bacteriorhodopsin Photocycle. Biophys. J. 1996, 71, 2329−2345. (23) Tsukamoto, T.; Inoue, K.; Kandori, H.; Sudo, Y. Thermal and Spectroscopic Characterization of a Proton Pumping Rhodopsin from an Extreme Thermophile. J. Biol. Chem. 2013, 288, 21581−21592. (24) Scharf, B.; Engelhard, M. Blue Halorhodopsin From Natronobacterium pharaonis: Wavelength Regulation by Anions. Biochemistry 1994, 33, 6387−6393. (25) Tsukamoto, T.; Sasaki, T.; Fujimoto, K. J.; Kikukawa, T.; Kamiya, M.; Aizawa, T.; Kawano, K.; Kamo, N.; Demura, M. Homotrimer Formation and Dissociation of pharaonis Halorhodopsin in Detergent System. Biophys. J. 2012, 102, 2906−2915. (26) Zhang, Y.; Cremer, P. S. Interactions Between Macromolecules and Ions: the Hofmeister Series. Curr. Opin. Chem. Biol. 2006, 10, 658− 663. (27) Kouyama, T.; Kanada, S.; Takeguchi, Y.; Narusawa, A.; Murakami, M.; Ihara, K. Crystal Structure of the Light-Driven Chloride Pump Halorhodopsin from Natronomonas pharaonis. J. Mol. Biol. 2010, 396, 564−579. (28) Hayashi, S.; Tamogami, J.; Kikukawa, T.; Okamoto, H.; Shimono, K.; Miyauchi, S.; Demura, M.; Nara, T.; Kamo, N. Thermodynamic Parameters of Anion Binding to Halorhodopsin from Natronomonas pharaonis by Isothermal Titration Calorimetry. Biophys. Chem. 2013, 172, 61−67. (29) Sato, M.; Kikukawa, T.; Araiso, T.; Okita, H.; Shimono, K.; Kamo, N.; Demura, M.; Nitta, K. Roles of Ser130 and Thr126 in Chloride Binding and Photocycle of pharaonis Halorhodopsin. J. Biochem. 2003, 134, 151−158. (30) Hackmann, C.; Guijarro, J.; Chizhov, I.; Engelhard, M.; Rödig, C.; Siebert, F. 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. 2001, 81, 394−406. (31) Kouyama, T.; Kawaguchi, H.; Nakanishi, T.; Kubo, H.; Murakami, M. Crystal Structures of the L1, L2, N, and O States of pharaonis Halorhodopsin. Biophys. J. 2015, 108, 2680−2690.

2038


Implications for the Light-Driven Chloride Ion Transport Mechanism of

Recommend Documents