A pharaonis phoborhodopsin mutant with the same retinal binding site residues as in bacteriorhodopsin

pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis. ppR has a blue-shifted absorption maximum (500 nm) relative those of other archaeal rhodopsins such as the proton-pump bacteriorhodopsin (BR; 570 nm). Among the 25 amino acids that are within 5 A of the retinal chromophore, 10 are different in BR and ppR, and they are presumed to be crucial in determining the color of their chromophores. However, the spectral red shift in a multiple mutant of ppR, in which the retinal binding site was made similar to that of BR (BR/ppR), was smaller than 40% (lambda(max) = 524 nm) than expected. In the paper presented here, we report on low-temperature Fourier transform infrared (FTIR) spectroscopy of BR/ppR, and compare the infrared spectral changes before and after photoisomerization with those for ppR and BR. The C[bond]C stretch and hydrogen out-of-plane (HOOP) vibrations of BR/ppR were similar to those of BR, suggesting that the surrounding protein moiety of BR/ppR becomes like BR. However, BR/ppR exhibited a unique IR band regarding the hydrogen bond of the protonated Schiff base. It has been known that ppR has a stronger hydrogen bond for the Schiff base than BR as judged from the frequency difference between their C[double bond]NH and C[double bond]ND stretches. We now find that replacement of the 10 amino acids of BR with ppR (BR/ppR) does not weaken the hydrogen bond of the Schiff base. Rather, the hydrogen bond in BR/ppR is stronger than that in the native ppR. We conclude that the principal factor of the smaller than expected opsin shift in BR/ppR is the strong association of the Schiff base with the surrounding counterion complex.     

Hydrogen-bonding interaction of the protonated schiff base with halides in a chloride-pumping bacteriorhodopsin mutant

Bacteriorhodopsin (BR) and halorhodopsin (HR) are light-driven proton and chloride ion pumps, respectively, in Halobacterium salinarum. The amino acid identity of these proteins is about 25%, suggesting that each has been optimized for their own functions during evolution. However, it is known that the BR mutants, D85T and D85S, can pump chloride ions. This fact implies that the Schiff base region is important in determining ionic selectivity. The X-ray crystallographic structure of D85S(Br(-)) showed the presence of a bromide ion in the Schiff base region (Facciotti, M. T., Cheung, V. S., Nguyen, D., Rouhani, S., and Glaeser, R. M. (2003) Biophys. J. 85, 451-458). In this article, we report on the study of hydrogen bonds of the Schiff base and water molecules in D85S in the absence and presence of various halides, assigning their N-D and O-D stretching vibrations in D(2)O, respectively, in low-temperature Fourier-transform infrared (FTIR) spectroscopy. We found that the hydrogen bond of the Schiff base in D85S(Cl(-)) is much stronger than that in HR, being as strong as that in wild-type BR. Similar halide dependence in D85S and in solution implies that the Schiff base forms a direct hydrogen bond with a halide, consistent with the X-ray structure. Photoisomerization causes a weakened hydrogen bond of the Schiff base, and halide dependence on the stretching frequency is lost. These spectral features are similar to those in the photocycle of proton-pumping BR, though the weakened hydrogen bond is more significant for BR. However, the spectral features of water bands in D85S are closer to chloride-pumping HR because O-D stretching vibrations of water are observed only at >2500 cm(-)(1). Unlike in BR, we did not observe strongly hydrogen-bonded water molecules for halide-pumping D85S mutants. This observation agrees with our recent hypothesis that strongly hydrogen-bonded water molecules are required for the proton-pumping activity of archaeal rhodopsins. Hydrogen-bonding conditions in the Schiff base region of D85S are discussed on the basis of the spectral comparison with those of wild-type BR and HR.     

Structural changes in bacteriorhodopsin following retinal photoisomerization from the 13-cis form

Bacteriorhodopsin (BR), a light-driven proton pump in Halobacterium salinarum, accommodates two resting forms of the retinylidene chromophore, the all-trans form (AT-BR) and the 13-cis,15-syn form (13C-BR). Both isomers are present in thermal equilibrium in the dark, but only the all-trans form has proton-pump activity. In this study, we applied low-temperature Fourier-transform infrared (FTIR) spectroscopy to 13C-BR at 77 K and compared the local structure around the chromophore before and after photoisomerization with that in AT-BR. Strong hydrogen-out-of-plane (HOOP) vibrations were observed at 964 and 958 cm(-)(1) for the K state of 13C-BR (13C-BR(K)) versus a vibration at 957 cm(-)(1) for the K state of AT-BR (AT-BR(K)). In AT-BR(K), but not in 13C-BR(K), the HOOP modes exhibit isotope shifts upon deuteration of the retinylidene at C15 and at the Schiff base nitrogen. Whereas the HOOP modes of AT-BR(K) were significantly affected by the mutation of Thr89, this was not the case for the HOOP modes of 13C-BR(K). These observations imply that, while the chromophore distortion is localized near the Schiff base in AT-BR(K), it is located elsewhere in 13C-BR(K). By use of [zeta-(15)N]lysine-labeled BR, we identified the N-D stretching vibrations of the 13C-BR Schiff base (in D(2)O) at 2173 and 2056 cm(-)(1), close in frequency to those of AT-BR. These frequencies indicate strong hydrogen bonding of the Schiff base in 13C-BR, presumably with a water molecule as in AT-BR. In contrast, the N-D stretching vibration appears at 2332 and 2276 cm(-)(1) in 13C-BR(K) versus values of 2495 and 2468 cm(-)(1) for AT-BR(K), suggesting that the rupture of the Schiff base hydrogen bond that occurs in AT-BR(K) does not occur in 13C-BR(K). Rotational motion of the Schiff base upon retinal isomerization is probably smaller in magnitude for 13C-BR than for AT-BR. These differences in the primary step are possibly related to the absence of light-driven proton pumping by 13C-BR.     

Hydration switch model for the proton transfer in the Schiff base region of bacteriorhodopsin

In a light-driven proton-pump protein, bacteriorhodopsin (BR), protonated Schiff base of the retinal chromophore and Asp85 form ion-pair state, which is stabilized by a bridged water molecule. After light absorption, all-trans to 13-cis photoisomerization takes place, followed by the primary proton transfer from the Schiff base to Asp85 that triggers sequential proton transfer reactions for the pump. Fourier transform infrared (FTIR) spectroscopy first observed O-H stretching vibrations of water during the photocycle of BR, and accurate spectral acquisition has extended the water stretching frequencies into the entire stretching frequency region in D(2)O. This enabled to capture the water molecules hydrating with negative charges, and we have identified the water O-D stretch at 2171 cm(-1) as the bridged water interacting with Asp85. We found that retinal isomerization weakens the hydrogen bond in the K intermediate, but not in the later intermediates such as L, M, and N. On the basis of the observation particularly on the M intermediate, we proposed a model for the mechanism of proton transfer from the Schiff base to Asp85. In the "hydration switch model", hydration of a water molecule is switched in the M intermediate from Asp85 to Asp212. This will have raised the pK(a) of the proton acceptor, and the proton transfer is from the Schiff base to Asp85.     

Water-mediated hydrogen-bonded network on the cytoplasmic side of the Schiff base of the L photointermediate of bacteriorhodopsin

The L intermediate in the proton-motive photocycle of bacteriorhodopsin is the starting state for the first proton transfer, from the Schiff base to Asp85, in the formation of the M intermediate. Previous FTIR studies of L have identified unique vibration bands caused by the perturbation of several polar amino acid side chains and several internal water molecules located on the cytoplasmic side of the retinylidene chromophore. In the present FTIR study we describe spectral features of the L intermediate in D(2)O in the frequency region which includes the N-D stretching vibrations of the backbone amides. We show that a broad band in the 2220-2080 cm(-1) region appears in L. By use of appropriate (15)N labeling and mutants, the lower frequency side of this band in L is assigned to the amides of Lys216 and Gly220. These amides are coupled to each other, and interact with Thr46 and Val49 in helix B and Asp96 in helix C via weakly H-bonding water molecules that exhibit O-D stretching vibrations at 2621 and 2605 cm(-1). These water molecules are part of a hydrogen-bonded network characteristic of L which includes other water molecules located closer to the chromophore that exhibit an O-D stretching vibration at 2589 cm(-1). This structure, extending from the Schiff base to the internal proton donor Asp96, stabilizes L and affects the L-to-M transition.     

Structural changes of water in the Schiff base region of bacteriorhodopsin: proposal of a hydration switch model

In a light-driven proton-pump protein, bacteriorhodopsin (BR), three water molecules participate in a pentagonal cluster that stabilizes an electric quadrupole buried inside the protein. In low-temperature Fourier transform infrared (FTIR) K minus BR spectra, the frequencies of water bands suggest extremely strong hydrogen bonding conditions in BR. The three observed water O-D stretches, at 2323, 2292, and 2171 cm(-1), are probably associated with water that interacts with the negative charges in the Schiff base region. Retinal isomerization weakens these hydrogen bonds in the K intermediate, but not in the later intermediates such as L, M, and N. In these states, spectral changes of water bands appeared only in the >2500 cm(-1) region, which correspond to weak hydrogen bonds. This observation suggests that after the K state the water molecules in the Schiff base region find a hydrogen bonding acceptor. We propose here a model for the mechanism of proton transfer from the Schiff base to Asp85. In the "hydration switch model", hydration of a water molecule is switched in the M intermediate from Asp85 to Asp212. This will have increased the pK(a) of the proton acceptor, and the proton transfer is from the Schiff base to Asp85. The present results also suggest that the deprotonated Asp96 in the N intermediate is stabilized in a manner different from that of Asp85 in BR.     

Relocation of water molecules between the Schiff base and the Thr46-Asp96 region during light-driven unidirectional proton transport by bacteriorhodopsin: an FTIR study of the N intermediate

A key event in light-driven proton pumping by bacteriorhodopsin is the formation of the L intermediate, whose transition to M is accompanied by the first proton transfer step, from the Schiff base to Asp85 on the extracellular side. Subsequent reprotonation of the Schiff base from the other side of the membrane to form the N intermediate is crucial for unidirectional proton transport. Previous FTIR studies have suggested that the intense water O-D stretching vibration bands which appear in L at 2589, 2605, and 2621 cm(-)(1) are due to a cluster of polarized water molecules connecting the Schiff base to the Thr46-Asp96 region closer to the cytoplasmic surface. In the present study the difference spectrum was obtained of the N intermediate with its photoproduct N', formed after irradiating N at 80 K. The water O-D stretching vibrations of N appear as a broad feature in a similar frequency region with a similar intensity to those of L. This feature is also affected by T46V like in L. However, the intensities of these water vibrations of N nearly returned to the initial unphotolyzed state upon formation of N', unlike those of L which are preserved in L'. An exception was V49A, which preserved the intense water vibrations of N in N'. The results suggest that both L and N have a water cluster extending from the Schiff base to Thr46. The surrounding protein moiety stabilizes the water cluster in L, but in N it is stabilized mostly by interaction with the Schiff base.     

Chromophore-protein-water interactions in the L intermediate of bacteriorhodopsin: FTIR study of the photoreaction of L at 80 K.

Using FTIR spectroscopy, perturbations of several residues and internal water molecules have been detected when light transforms all-trans bacteriorhodopsin (BR) to its L intermediate having a 13-cis chromophore. Illumination of L at 80 K results in an intermediate L' absorbing around 550 nm. L' thermally converts to the original BR only at >130 K. In this study, we used the light-induced transformation of L to L' at 80 K to identify some amino acid residues and water molecules that closely interact with the chromophore and distinguish them from those residues not affected by the photoreaction. The L minus L' FTIR difference spectrum shows that the chromophore in L' is in the all-trans configuration. The perturbed states of Asp96 and Val49 and of the environment along the aliphatic part of the retinal and Lys216 seen in L are not affected by the L --> L' photoreaction. On the other hand, the environments of the Schiff base of the chromophore, of Asp115, and of water molecules close to Asp85 returned in L' to their state in which they originally had existed in BR. The water molecules that are affected by the mutations of Thr46 and Asp96 also change to a different state in the L --> L' transition, as indicated by transformation of a water O-H vibrational band at 3497 cm-1 in L into an intense peak at 3549 cm-1 in L'. Notably, this change of water bands in the L --> L' transition at 80 K is entirely different from the changes observed in the BR --> K photoreaction at the same temperature, which does not show such intense bands. These results suggest that these water molecules move closer to the Schiff base as a hydrogen bonding cluster in L and L', presumably to stabilize its protonated state during the BR to L transition. They may contribute to the structural constraints that prevent L from returning to the initial BR upon illumination at 80 K.     

Water structural changes in the L and M photocycle intermediates of bacteriorhodopsin as revealed by time-resolved step-scan Fourier transform infrared (FTIR) spectroscopy

In previous Fourier transform infrared (FTIR) studies of the photocycle intermediates of bacteriorhodopsin at cryogenic temperatures, water molecules were observed in the L intermediate, in the region surrounded by protein residues between the Schiff base and Asp96. In the M intermediate, the water molecules had moved away toward the Phe219-Thr46 region. To evaluate the relevance of this scheme at room temperature, time-resolved FTIR difference spectra of bacteriorhodopsin, including the water O-H stretching vibration frequency regions, were recorded in the micro- and millisecond time ranges. Vibrational changes of weakly hydrogen-bonded water molecules were observed in L, M, and N. In each of these intermediates, the depletion of a water O-H stretching vibration at 3645 cm-1, originating from the initial unphotolyzed bacteriorhodopsin, was observed as a trough in the difference spectrum. This vibration is due to the dangling O-H group of a water molecule, which interacts with Asp85, and its absence in each of these intermediates indicates that there is perturbation of this O-H group. The formation of M is accompanied by the appearance of water O-H stretching vibrations at 3670 and 3657 cm-1, the latter of which persists to N. The 3670 cm-1 band of M is due to water molecules present in the region surrounded by Thr46, Asp96, and Phe219. The formation of L at 298 K is accompanied by the perturbations of Asp96 and the Schiff base, although in different ways from what is observed at 170 K. Changes in a broad water vibrational feature, centered around 3610 cm-1, are kinetically correlated with the L-M transition. These results imply that, even at room temperature, water molecules interact with Asp96 and the Schiff base in L, although with a less rigid structure than at cryogenic temperatures.     

Structural changes of Salinibacter sensory rhodopsin I upon formation of the K and M photointermediates

Sensory rhodopsin I (SRI) is one of the most interesting photosensory receptors in nature because of its ability to mediate opposite signals depending on light color by photochromic one-photon and two-photon reactions. Recently, we characterized SRI from eubacterium Salinibacter ruber (SrSRI). This protein allows more detailed information about the structure and structural changes of SRI during its action to be obtained. In this paper, Fourier transform infrared (FTIR) spectroscopy is applied to SrSRI, and the spectral changes upon formation of the K and M intermediates are compared with those of other archaeal rhodopsins, SRI from Halobacterium salinarum (HsSRI), sensory rhodopsin II (SRII), bacteriorhodopsin (BR), and halorhodopsin (HR). Spectral comparison of the hydrogen out-of-plane (HOOP) vibrations of the retinal chromophore in the K intermediates shows that extended choromophore distortion takes place in SrSRI and HsSRI, as well as in SRII, whereas the distortion is localized in the Schiff base region in BR and HR. It appears that sensor and pump functions are distinguishable from the spectral feature of HOOP modes. The HOOP band at 864 cm(-1) in SRII, important for negative phototaxis, is absent in SrSRI, suggesting differences in signal transfer mechanism between SRI and SRII. The strongly hydrogen-bound water molecule, important for proton pumps, is observed at 2172 cm(-1) in SrSRI, as well as in BR and SRII. The formation of the M intermediate accompanies the appearance of peaks at 1753 (+) and 1743 (-) cm(-1), which can be interpreted as the protonation signal of the counterion (Asp72) and the proton release signal from an unidentified carboxylic acid, respectively. The structure and structural changes of SrSRI are discussed on the basis of the present infrared spectral comparisons with other rhodopsins.     

Structural changes due to the deprotonation of the proton release group in the M-photointermediate of bacteriorhodopsin as revealed by time-resolved FTIR spectroscopy

One of the steps in the proton pumping cycle of bacteriorhodopsin (BR) is the release of a proton from the proton-release group (PRG) on the extracellular side of the Schiff base. This proton release takes place shortly after deprotonation of the Schiff base (L-to-M transition) and results in an increase in the pKa of Asp85, which is a crucial mechanistic step for one-way proton transfer for the entire photocycle. Deprotonation of the PRG can also be brought about without photoactivation, by raising the pH of the enzyme (pKa of PRG; approximately 9). Thus, comparison of the FTIR difference spectrum for formation of the M intermediate (M minus initial unphotolyzed BR state) at pH 7 to the corresponding spectrum generated at pH 10 may reveal structural changes specifically associated with deprotonation of the PRG. Vibrational bands of BR that change upon M formation are distributed across a broad region between 2120 and 1685 cm(-1). This broad band is made up of two parts. The band above 1780 cm(-1), which is insensitive to C15-deuteration of the retinal, may be due to a proton delocalized in the PRG. The band between 1725 and 1685 cm(-1), on the lower frequency side of the broad band, is sensitive to C15-deuteration. This band may arise from transition dipole coupling of the vibrations of backbone carbonyl groups in helix G with the side chain of Tyr57 and with the C15H of the Schiff base. In M, these broad bands are abolished, and the 3657 cm(-1) band, which is due to the disruption of the hydrogen bonding of a water molecule, probably with Arg82, appears. Loss of the interaction of the backbone carbonyl groups in helix G with Tyr57 and the Schiff base, and separation of Tyr57 from Arg82, may be causes of these spectral changes, leading to the stabilization of the protonated Asp85 in M.     

Strongly hydrogen-bonded water molecules in the Schiff base region of rhodopsins.

In many rhodopsins, a positively charged retinal chromophore is stabilized by a negatively charged carboxylate, and the presence of bound water molecules has been found in the Schiff base region by X-ray crystallography of various rhodopsins. Low-temperature Fourier-transform infrared (FTIR) spectroscopy can directly monitor hydrogen-bonding alterations of internal water molecules of rhodopsins. In particular, we found that a bridged water molecule between the Schiff base and Asp 85 in bacteriorhodopsin (BR), a light-driven proton-pump protein, forms an extremely strong hydrogen bond. It is likely that a hydration switch of the water from Asp 85 to Asp 212 plays an important role in the proton transfer in the Schiff base region of BR. Comprehensive studies of archaeal and visual rhodopsins have revealed that strongly hydrogen-bonded water molecules are only found in the proteins exhibiting proton-pump activities. Strongly hydrogen-bonded water molecules and its transient weakening may be essential for the proton-pump function of rhodopsins.     

FTIR studies of internal water molecules in the Schiff base region of bacteriorhodopsin

In a light-driven proton pump protein, bacteriorhodopsin (BR), three water molecules participate in a pentagonal cluster that stabilizes an electric quadrupole buried inside the protein. Previously, low-temperature Fourier-transform infrared (FTIR) difference spectra between BR and the K photointermediate in D(2)O revealed six O-D stretches of water in BR at 2690, 2636, 2599, 2323, 2292, and 2171 cm(-)(1), while five water bands were observed at 2684, 2675, 2662, 2359, and 2265 cm(-)(1) for the K intermediate. The frequencies are widely distributed over the possible range of stretching vibrations of water, and water molecules at <2400 cm(-)(1) were suggested to hydrate negative charges because of their extremely strong hydrogen bonds. In this paper, we aimed to reveal the origin of these water bands in the K minus BR spectra by use of various mutant proteins. The water bands were not affected by the mutations at the cytoplasmic side, such as T46V, D96N, and D115N, implying that the water molecules in the cytoplasmic domain do not change their hydrogen bonds in the BR to K transition. In contrast, significant modifications of the water bands were observed for the mutations in the Schiff base region and at the extracellular side, such as R82Q, D85N, T89A, Y185F, D212N, R82Q/D212N, and E204Q. From these results, we concluded that the six O-D stretches of BR originate from three water molecules, water401, -402, and -406, involved in the pentagonal cluster. Two stretching modes of each water molecule are highly separate (300-470 cm(-)(1) for O-D stretches and 500-770 cm(-)(1) for O-H stretches), which is consistent with the previous QM/MM calculation. The small amplitudes of vibrational coupling are presumably due to strong association of the waters to negative charges of Asp85 and Asp212. Among various mutant proteins, only D85N and D212N lack strongly hydrogen-bonded water molecules (<2400 cm(-)(1)) and proton pumpimg activity. We thus infer that the presence of a strong hydrogen bond of water is a prerequisite for proton pumping in BR. Internal water molecules in such a specific environment are discussed in terms of functional importance for rhodopsins.     

Vibrational modes of the protonated Schiff base in pharaonis phoborhodopsin

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis. Recent X-ray crystallographic structures showed that ppR and bacteriorhodopsin (BR), a light-driven proton pump, possess similar molecular environments of the retinal Schiff base. Nevertheless, absorption spectra are different by 70 nm between ppR and BR, suggesting the different chromophore-protein interactions involving the Schiff base region. In this article, we identify frequencies of the Schiff base vibrations in the ppR(K) minus ppR difference spectra by means of low-temperature FTIR spectroscopy of [zeta-(15)N]lysine-labeled ppR. The N-D stretch in D(2)O was found at 2140 and 2091 cm(-1) for ppR, which are shifted to a lower frequency by 32-33 cm(-1) compared to those for BR. This observation indicates the stronger hydrogen bond of the Schiff base in ppR than in BR. The N-D stretch of the Schiff base and O-D stretch of water molecules are located at the different frequencies in ppR, while they appear in the same frequency region in BR [Kandori, H., Belenky, M., and Herzfeld, J. (2002) Biochemistry 41, 6026-6031]. These differences could be correlated with the distorted pentagonal cluster structure in ppR. In contrast, the N-D stretch of ppR(K) was found at 2474 cm(-1), which is close in frequency to that of BR(K). The O-D stretch of Thr79 was also assigned at 2512 and 2474 cm(-1) for ppR and ppR(K), respectively. These frequencies are close to those of BR, suggesting the interaction of Thr79 and Asp75 in ppR is similar to that of Thr89 and Asp85 in BR.     

FTIR study of the retinal Schiff base and internal water molecules of proteorhodopsin

Proteorhodopsin (PR), an archaeal-type rhodopsin found in marine bacteria, is a light-driven proton pump similar to bacteriorhodopsin (BR). It is known that Asp97, a counterion of the protonated Schiff base, possesses a higher pKa ( approximately 7) compared to that of homologous Asp85 in BR (<3). This suggests that PR has a hydrogen-bonding network different from that of BR. We previously reported that a strongly hydrogen-bonded water molecule is observed only in the alkaline form of PR, where Asp97 is deprotonated (Furutani, Y., Ikeda, D., Shibata, M., and Kandori, H. (2006) Chem. Phys. 324, 705-708). This is probably correlated with the pH-dependent proton pumping activity of PR. In this work, we studied the water-containing hydrogen-bonding network in the Schiff base region of PR by means of Fourier-transform infrared (FTIR) spectroscopy at 77 K. [zeta-15N]Lys-labeling and 18O water were used for assigning the Schiff base N-D and water O-D stretching vibrations in D2O, respectively. The frequency upshift of the N-D stretch in the primary K intermediate is much smaller for PR than for BR, indicating that the Schiff base forms a hydrogen bond after retinal photoisomerization. We then measured FTIR spectra of the mutants of Asp97 (D97N and D97E) and Asp227 (D227N and D227E) to identify the amino acid interacting with the Schiff base in the K state. The PRK minus PR spectra of D97N and D97E were similar to those of the acidic and alkaline forms, respectively, of the wild type implying that the structural changes upon retinal photoisomerization are not influenced by the mutation at Asp97. In contrast, clear spectral differences were observed in D227N and D227E, including vibrational bands of the Schiff base and water molecules. It is concluded that Asp227 plays a crucial role during the photoisomerization process, though Asp97 acts as the primary counterion in the unphotolyzed state of PR.     

FTIR study of the photoisomerization processes in the 13-cis and all-trans forms of Anabaena sensory rhodopsin at 77 K

Archaeal-type rhodopsins can accommodate either all-trans- or 13-cis,15-syn-retinal in their chromophore binding site in the dark, but only the former isomer is functionally important. In contrast, Anabaena sensory rhodopsin (ASR), an archaeal-type rhodopsin found in eubacteria, exhibits a photochromic interconversion of both forms, suggesting that ASR functions as a photosensor which interacts with its 14 kDa soluble transducer differently in the all-trans and 13-cis,15-syn forms. In this study, we applied low-temperature Fourier transform infrared (FTIR) spectroscopy to the 13-cis,15-syn form of ASR (13C-ASR) at 77 K and compared the local structure around the chromophore and its structural changes upon retinal photoisomerization with those of the all-trans form (AT-ASR) [Furutani, Y., Kawanabe, A., Jung, K. H., and Kandori, H. (2005) Biochemistry 44, 12287-12296]. By use of [zeta-15N]lysine-labeled ASR, we identified the N-D stretching vibrations of the Schiff base (in D2O) at 2165 cm(-1) for 13C-ASR and at 2163 and 2125 cm(-1) for AT-ASR. The frequencies indicate strong hydrogen bonds of the Schiff base with a water molecule for both 13C-ASR and AT-ASR. In contrast, the N-D stretching vibration appears at 2351 cm(-1) and at 2483 cm(-1) for the K states of 13C-ASR (13C-ASR(K)) and AT-ASR (AT-ASR(K)), respectively, indicating that the Schiff base still forms a hydrogen bond in 13C-ASR(K). Rotational motion of the Schiff base upon retinal isomerization is probably smaller for 13C-ASR than for AT-ASR, the latter altering hydrogen bonding of the Schiff base similar to bacteriorhodopsin (BR), a light-driven proton pump. Appearance of several hydrogen-out-of-plane vibrations and amide I vibrations in 13C-ASR(K), but not in AT-ASR(K), suggests that structural changes are distributed widely along the polyene chain for 13C-ASR. On the other hand, retinal photoisomerization in AT-ASR breaks the hydrogen bond of the Schiff base, and localized structural changes in the Schiff base region are induced.     

Early photocycle structural changes in a bacteriorhodopsin mutant engineered to transmit photosensory signals

Bacteriorhodopsin (BR) and sensory rhodopsin II (SRII) function as a light-driven proton pump and a receptor for negative phototaxis in haloarchaeal membranes, respectively. SRII transmits light signals through changes in protein-protein interaction with its transducer HtrII. Recently, we converted BR by three mutations into a form capable of transmitting photosignals to HtrII to mediate phototaxis responses. The BR triple mutant (BR-T) provides an opportunity to identify structural changes necessary to activate HtrII by comparing light-induced infrared spectral changes of BR, BR-T, and SRII. The hydrogen out-of-plane (HOOP) vibrations of the BR-T were very similar to those of SRII, indicating that they are distributed more extensively along the retinal chromophore than in BR, as in SRII. On the other hand, the bands of the protein moiety in BR-T are similar to those of BR, indicating that they are not specific to photosensing. The alteration of the O-H stretching vibration of Thr-204 in SRII, which we had previously shown to be essential for signal relay to HtrII, occurs also in BR-T. In addition, 1670(+)/1664(-) cm(-1) bands attributable to a distorted alpha-helix were observed in BR-T in a HtrII-dependent manner, as is seen in SRII. Thus, we identified similarities and dissimilarities of BR-T to BR and SRII. The results suggest signaling function of the structural changes of the HOOP vibrations, the O-H stretching vibration of the Thr-215 residue, and a distorted alpha-helix for the signal generation. We also succeeded in measurements of L minus initial state spectra of BR-T, which are the first FTIR spectra of L intermediates among sensory rhodopsins.     

Hydrogen-bonding alterations of the protonated Schiff base and water molecule in the chloride pump of Natronobacterium pharaonis

Halorhodopsin is a light-driven chloride ion pump. Chloride ion is bound in the Schiff base region of the retinal chromophore, and unidirectional chloride transport is probably enforced by the specific hydrogen-bonding interaction with the protonated Schiff base and internal water molecules. In this article, we study hydrogen-bonding alterations of the Schiff base and water molecules in halorhodopsin of Natronobacterium pharaonis (pHR) by assigning their N-D and O-D stretching vibrations in D(2)O, respectively. Highly accurate low-temperature Fourier transform infrared spectroscopy revealed that hydrogen bonds of the Schiff base and water molecules are weak in the unphotolyzed state, whereas they are strengthened upon retinal photoisomerization. Halide dependence of the stretching vibrations enabled us to conclude that the Schiff base forms a direct hydrogen bond with Cl(-) only in the K intermediate. Hydrogen bond of the Schiff base is further strengthened in the L(1) intermediate, whereas the halide dependence revealed that the acceptor is not Cl(-), but presumably a water molecule. Thus, it is concluded that the hydrogen-bonding interaction between the Schiff base and Cl(-) is not a driving force of the motion of Cl(-). Rather, the removal of its hydrogen bonds with the Schiff base and water(s) makes the environment around Cl(-) less polar in the L(1) intermediate, which presumably drives the motion of Cl(-) from its binding site to the cytoplasmic domain.     

Structural changes of pharaonis phoborhodopsin upon photoisomerization of the retinal chromophore: infrared spectral comparison with bacteriorhodopsin

Archaeal rhodopsins possess a retinal molecule as their chromophores, and their light energy and light signal conversions are triggered by all-trans to 13-cis isomerization of the retinal chromophore. Relaxation through structural changes of the protein then leads to functional processes, proton pump in bacteriorhodopsin and transducer activation in sensory rhodopsins. In the present paper, low-temperature Fourier transform infrared spectroscopy is applied to phoborhodopsin from Natronobacterium pharaonis (ppR), a photoreceptor for the negative phototaxis of the bacteria, and infrared spectral changes before and after photoisomerization are compared with those of bacteriorhodopsin (BR) at 77 K. Spectral comparison of the C--C stretching vibrations of the retinal chromophore shows that chromophore conformation of the polyene chain is similar between ppR and BR. This fact implies that the unique chromophore-protein interaction in ppR, such as the blue-shifted absorption spectrum with vibrational fine structure, originates from both ends, the beta-ionone ring and the Schiff base regions. In fact, less planer ring structure and stronger hydrogen bond of the Schiff base were suggested for ppR. Similar frequency changes upon photoisomerization are observed for the C==N stretch of the retinal Schiff base and the stretch of the neighboring threonine side chain (Thr79 in ppR and Thr89 in BR), suggesting that photoisomerization in ppR is driven by the motion of the Schiff base like BR. Nevertheless, the structure of the K state after photoisomerization is different between ppR and BR. In BR, chromophore distortion is localized in the Schiff base region, as shown in its hydrogen out-of-plane vibrations. In contrast, more extended structural changes take place in ppR in view of chromophore distortion and protein structural changes. Such structure of the K intermediate of ppR is probably correlated with its high thermal stability. In fact, almost identical infrared spectra are obtained between 77 and 170 K in ppR. Unique chromophore-protein interaction and photoisomerization processes in ppR are discussed on the basis of the present infrared spectral comparison with BR.     

Vibrational frequency and dipolar orientation of the protonated Schiff base in bacteriorhodopsin before and after photoisomerization

Light-driven proton transport in bacteriorhodopsin (BR) is initiated by photoisomerization of the retinylidene chromophore, which perturbs the hydrogen bonding network in the Schiff base region of the active site. This study aimed to identify the frequency and dipolar orientation of the N-D stretching vibrations of the Schiff base before and after photoisomerization, by means of low-temperature polarized FTIR spectroscopy of [zeta-(15)N]lysine-labeled BR in D(2)O. (15)N-shifted modes were found at 2123 and 2173 cm(-1) for BR, and at 2468 and 2495 cm(-1) for the K intermediate. The corresponding N-H stretches are at approximately 2800 cm(-1) for BR and 3350-3310 cm(-1) for the K intermediate. The shift to a 350 cm(-1) higher frequency upon photoisomerization is consistent with loss of the hydrogen bond of the Schiff base. The N-D stretch frequencies of the Schiff base in BR and the K intermediate are close to the O-D stretch frequencies of strongly hydrogen bonded water and Thr89, respectively. The angles of the dipole moments of the N-D stretches to the membrane normal were determined to be 60-65 degrees for BR and approximately 90 degrees for the K intermediate. In the case of BR, the stretch orientation is expected to deviate from the N-D bond orientation due to vibrational mixing in the hydrogen bonding network. In contrast, the data for the K intermediate suggest that the N-D group is not hydrogen bonded and orients along the membrane.