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.     

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.     

FTIR spectroscopy of the O photointermediate in pharaonis phoborhodopsin

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor protein for negative phototaxis in Natronobacterium pharaonis. During the photocycle of ppR, the retinal chromophore is thermally isomerized from the 13-cis to all-trans form. We employed FTIR spectroscopy of ppR at 260 K and pH 5 to reveal that this isomerization occurs upon formation of the O intermediate (ppR(O)) by using ppR samples reconstituted with 12,14-D(2)-labeled retinal. In ppR(O), C=O stretching vibrations of protonated carboxylates newly appear at 1757 (+)/1722 (-) cm(-1) in H(2)O and at 1747 (+)/1718 (-) cm(-1) in D(2)O in addition to the 1765 (+) cm(-1) band of Asp75. Amide I vibrations are basically similar between ppR(M) and ppR(O), whereas unique bands of ppR(O) are also observed such as the negative 1656 cm(-1) band in D(2)O and intense bands at 1686 (-)/1674 (+) cm(-1). In addition, O-D stretching vibrations of water molecules in the entire mid-infrared region are assigned for ppR(M) and ppR(O), the latter being unique for ppR, since it can be detected at low temperature (260 K). The ppR(M) minus ppR difference spectra lack the lowest frequency water band (2215 cm(-1)) observed in the ppR(K) minus ppR spectra, which is probably associated with water that interacts with the negative charges in the Schiff base region. It is likely that the proton transfer from the Schiff base to Asp75 in ppR(M) can be explained by a hydration switch of a water from Asp75 to Asp201, as was proposed for the light-driven proton-pump bacteriorhodopsin (hydration switch model) [Tanimoto, T., Furutani, Y., and Kandori, H. (2003) Biochemistry 42, 2300-2306]. In the transition from ppR(M) to ppR(O), a hydrogen-bonding alteration takes place for another water molecule that forms a strong hydrogen bond.     

Internal water molecules of pharaonis phoborhodopsin studied by low-temperature infrared spectroscopy.

In the Schiff base region of bacteriorhodopsin (BR), a light-driven proton-pump protein, three internal water molecules are involved in a pentagonal cluster structure. These water molecules constitute a hydrogen-bonding network consisting of two positively charged groups, the Schiff base and Arg82, and two negatively charged groups, Asp85 and Asp212. Previous infrared spectroscopy of BR revealed stretching vibrations of such water molecules under strong hydrogen-bonding conditions using spectral differences in D2O and D2(18O) [Kandori and Shichida (2000) J. Am. Chem. Soc. 122, 11745-11746]. The present study extends the infrared analysis to another archaeal rhodopsin, pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin-II, psR-II), involved in the negative phototaxis of Natronobacterium pharaonis. Despite functional differences between ppR and BR, similar spectral features of water bands were observed before and after photoisomerization of the retinal chromophore at 77 K. This implies that the structure and the structural changes of internal water molecules are similar between ppR and BR. Higher stretching frequencies of the bridged water in ppR suggest that the water-containing pentagonal cluster structure is considerably distorted in ppR. These observations are consistent with the crystallographic structures of ppR and BR. The water structure and structural changes upon photoisomerization of ppR are discussed here on the basis of their infrared spectra.     

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.     

Structural changes in the O-decay accelerated mutants of pharaonis phoborhodopsin

pharaonis phoborhodopsin ( ppR, also called pharaonis sensory rhodopsin II, psRII) is a receptor for negative phototaxis in Natronomonas pharaonis. The X-ray crystallographic structure of ppR is very similar to those of the ion-pumping rhodopsins, bacteriorhodopsin (BR) and halorhodopsin (hR). However, the decay processes of the photocycle intermediates such as M and O are much slower than those of BR and hR, which is advantageous for the sensor function of ppR. Iwamoto et al. previously found that, in a quadruple mutant (P182S/P183E/V194T/T204C; denoted as SETC) of ppR, the decay of the O intermediate was accelerated by approximately 100 times ( t 1/2 approximately 6.6 ms vs 690 ms for the wild type of ppR), being almost equal to that of BR (Iwamoto, M., et al. (2005) Biophys. J. 88, 1215-1223). The mutated residues are located on the extracellular surface (Pro182, Pro183, and Val194) and near the Schiff base (Thr204). The present Fourier-transform infrared (FTIR) spectroscopy of SETC revealed that protein structural changes in the K and M states were similar to those of the wild type. In contrast, the ppR O minus ppR infrared difference spectra of SETC are clearly different from those of the wild type in amide-I (1680-1640 cm (-1)) and S-H stretching (2580-2520 cm (-1)) vibrations. The 1673 (+) and 1656 (-) cm (-1) bands newly appear for SETC in the frequency region typical for the amide-I vibration of the alpha II- and alpha I-helices, respectively. The intensities of the 1673 (+) cm (-1) band of various mutants were well correlated with their O-decay half-times. Since the alpha II-helix possesses a considerably distorted structure, the result implies that distortion of the helix is required for fast O-decay. In addition, the characteristic changes in the S-H stretching vibration of Cys204 were different between SETC and T204C, suggesting that structural change near the Schiff base was induced by mutations of the extracellular surface. We conclude that the lifetime of the O intermediate in ppR is regulated by the distorted alpha-helix and strengthened hydrogen bond of Cys204.     

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 spectroscopy of the M photointermediate in pharaonis rhoborhodopsin

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis. During the photocycle of ppR, the Schiff base of the retinal chromophore is deprotonated upon formation of the M intermediate (ppR(M)). The present FTIR spectroscopy of ppR(M) revealed that the Schiff base proton is transferred to Asp-75, which corresponds to Asp-85 in a light-driven proton-pump bacteriorhodopsin (BR). In addition, the C==O stretching vibrations of Asn-105 were assigned for ppR and ppR(M). The common hydrogen-bonding alterations in Asn-105 of ppR and Asp-115 of BR were found in the process from photoisomerization (K intermediate) to the primary proton transfer (M intermediate). These results implicate similar protein structural changes between ppR and BR. However, BR(M) decays to BR(N) accompanying a proton transfer from Asp-96 to the Schiff base and largely changed protein structure. In the D96N mutant protein of BR that lacks a proton donor to the Schiff base, the N-like protein structure was observed with the deprotonated Schiff base (called M(N)) at alkaline pH. In ppR, such an N-like (M(N)-like) structure was not observed at alkaline pH, suggesting that the protein structure of the M state activates its transducer protein.     

Proton transfer reactions in the F86D and F86E mutants of pharaonis phoborhodopsin (sensory rhodopsin II)

pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII), a negative phototaxis receptor of Natronobacterium pharaonis, can use light to pump a proton in the absence of its transducer protein. However, the pump activity is much lower than that of the light-driven proton-pump bacteriorhodopsin (BR). ppR's pump activity is known to be increased in a mutant protein, in which Phe86 is replaced with Asp (F86D). Phe86 is the amino acid residue corresponding to Asp96 in BR, and we expect that Asp86 plays an important role in the proton transfer at the highly hydrophobic cytoplasmic domain of the F86D mutant ppR. In this article, we studied protein structural changes and proton transfer reactions during the photocycles of the F86D and F86E mutants in ppR by means of Fourier transform infrared (FTIR) spectroscopy and photoelectrochemical measurements using a tin oxide (SnO2) electrode. FTIR spectra of the unphotolyzed state and the K and M intermediates are very similar among F86D, F86E, and the wild type. Asp86 or Glu86 is protonated in F86D or F86E, respectively, and the pK(a) > 9. During the photocycle, the pK(a) is lowered and deprotonation of Asp86 or Glu86 is observed. Detection of both deprotonation of Asp86 or Glu86 and concomitant reprotonation of the 13-cis chromophore implies the presence of a proton channel between position 86 and the Schiff base. However, the photoelectrochemical measurements revealed proton release presumably from Asp86 or Glu86 to the cytoplasmic aqueous phase in the M state. This indicates that the ppR mutants do not have the BR-like mechanism that conducts a proton uniquely from Asp86 or Glu86 (Asp96 in BR) to the Schiff base, which is possible in BR by stepwise protein structural changes at the cytoplasmic side. In ppR, there is a single open structure at the cytoplasmic side (the M-like structure), which is shown by the lack of the N-like protein structure even in F86D and F86E at alkaline pH. Therefore, it is likely that a proton can be conducted in either direction, the Schiff base or the bulk, in the open M-like structure of F86D and F86E.     

FTIR spectroscopy of the complex between pharaonis phoborhodopsin and its transducer protein

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psRII) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis. ppR activates the cognate transducer protein, pHtrII, upon absorption of light. ppR and pHtrII form a tight 2:2 complex in the unphotolyzed state, and the interaction is somehow altered during the photocycle of ppR. In this paper, we studied the influence of pHtrII on the structural changes occurring upon retinal photoisomerization in ppR by means of low-temperature FTIR spectroscopy. We trapped the K intermediate at 77 K and compared the ppR(K) minus ppR spectra in the absence and presence of pHtrII. There are no differences in the X-D stretching vibrations (2700-1900 cm(-1)) caused by presence of pHtrII. This result indicates that the hydrogen-bonding network in the Schiff base region is not altered by interaction with pHtrII, which is consistent with the same absorption spectrum of ppR with or without pHtrII. In contrast, the ppR(K) minus ppR infrared difference spectra are clearly influenced by the presence of pHtrII in amide-I (1680-1640 cm(-1)) and amide-A (3350-3250 cm(-1)) vibrations. The identical spectra for the complex of the unlabeled ppR and (13)C- or (15)N-labeled pHtrII indicate that the observed structural changes for the peptide backbone originate from ppR only and are altered by retinal photoisomerization. The changes do not come from pHtrII, implying that the light signal is not transmitted to pHtrII in ppR(K). In addition, we observed D(2)O-insensitive bands at 3479 (-)/3369 (+) cm(-1) only in the presence of pHtrII, which presumably originate from an X-H stretch of an amino acid side chain inside the protein.     

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.     

FTIR spectroscopy of the K photointermediate of Neurospora rhodopsin: structural changes of the retinal, protein, and water molecules after photoisomerization

Neurospora rhodopsin (NR, also known as NOP-1) is the first rhodopsin of the haloarchaeal type found in eucaryotes. NR demonstrates a very high degree of conservation of the amino acids that constitute the proton-conducting pathway in bacteriorhodopsin (BR), a light-driven proton pump of archaea. Nevertheless, NR does not appear to pump protons, suggesting the absence of the reprotonation switch that is necessary for the active transport. The photocycle of NR is much slower than that of BR, similar to the case of pharaonis phoborhodopsin (ppR), an archaeal photosensory protein. The functional and photochemical differences between NR and BR should be explained in the structural context. In this paper, we studied the structural changes of NR following retinal photoisomerization by means of low-temperature Fourier transform infrared (FTIR) spectroscopy and compared the obtained spectra with those for BR. For the spectroscopic analysis, we established the light-adaptation procedure for NR reconstituted into 1,2-dimyristoyl-sn-glycero- 3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phosphate (DMPC/DMPA) liposomes, which takes approximately 2 orders of magnitudes longer than in BR. The structure of the retinal chromophore and the hydrogen-bonding strength of the Schiff base in NR are similar to those in BR. Unique spectral features are observed for the S-H stretching vibrations of cysteine and amide-I vibrations for NR before and after retinal isomerization. In NR, there are no spectral changes assignable to the amide bands of alpha helices. The most prominent difference between NR and BR was seen for the water O-D stretching vibrations (measured in D(2)O). Unlike for haloarchaeal rhodopsins such as BR and ppR, no O-D stretches of water under strong hydrogen-bonded conditions (<2400 cm(-1)) were observed in the NR(K) minus NR difference spectra. This suggests a unique hydrogen-bonded network of the Schiff base region, which may be responsible for the lack of the reprotonation switch in NR.     

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.     

Interaction of Asn105 with the retinal chromophore during photoisomerization of pharaonis phoborhodopsin

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 spectrum with a spectral shoulder, which is highly unique for the archaeal rhodopsin family. The primary reaction of ppR is a cis-trans photoisomerization of the retinal chromophore to form the K intermediate, like the well-studied proton pump bacteriorhodopsin (BR). Recent comparative FTIR spectroscopy of the K states in ppR and BR revealed that more extended structural changes take place in ppR than in BR with respect to chromophore distortion and protein structural changes [Kandori, H., Shimono, K., Sudo, Y., Iwamoto, M., Shichida, Y., and Kamo, N. (2001) Biochemistry 40, 9238-9246]. FTIR spectroscopy of the N105D mutant protein reported here assigns the vibrational bands at 1704 and 1700 cm(-1) as C=O stretches of Asn105 in ppR and ppR(K), respectively. A comparative investigation between ppR and BR further reveals that the structure at position 105 in ppR is similar to that of the corresponding position (Asp115) in BR; this observation is supported by the recent X-ray crystallographic structures of ppR [Luecke, H., Schobert, B., Lanyi, J. K., Spudich, E. N., and Spudich, J. L. (2001) Science 293, 1499-1503; Royant, A., Nollert, P., Edman, K., Neutze, R., Landau, E. M., Pebay-Peyroulla, E., and Navarro, J. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 10131-10136]. Nevertheless, structural changes upon photoisomerization at position 105 in ppR are greater than those at position 115 in BR. As a consequence of a unique chromophore-protein interaction in ppR, extended protein structural changes accompanying retinal photoisomerization occur, and these include Asn105 which is approximately 7 A from the retinal chromophore.     

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.     

Structural changes in the Schiff base region of squid rhodopsin upon photoisomerization studied by low-temperature FTIR spectroscopy

Low-temperature Fourier transform infrared (FTIR) spectroscopy is used to study squid rhodopsin at 77 K in investigating structural changes in the Schiff base region upon photoisomerization. The analysis of O-D stretching vibrations in D(2)O revealed that there are more internal water molecules near the retinal chromophore in squid rhodopsin than in bovine rhodopsin. Among nine O-D stretching vibrations of water in squid rhodopsin, eight peaks are identical between rhodopsin and 9-cis-rhodopsin (Iso). On the other hand, the isomer-specific O-D stretch of water was observed for rhodopsin (2451 cm(-)(1)) and Iso (2382 cm(-)(1)). Low frequencies of these bands suggest that the water forms a strong hydrogen bond with a negatively charged counterion. In addition, it was suggested that the hydrogen bond of the Schiff base is weaker in squid rhodopsin than in bacteriorhodopsin and bovine rhodopsin, and squid rhodopsin possessed similar hydrogen bonding strength for the Schiff base among rhodopsin, Iso, and bathorhodopsin. Most vibrational bands in the X-D stretch region originate from water O-D or the Schiff base N-D stretches, suggesting that the hydrogen bonding network in the Schiff base region of squid rhodopsin is composed of only water molecules. On the basis of these results, we propose that squid rhodopsin possesses a "bridge" water between the Schiff base and its counterion as well as squid retinochrome [Furutani, Y., Terakita, A., Shichida, Y., and Kandori, H. (2005) Biochemistry 44, 7988-7997], which is absent in vertebrate rhodopsin [Furutani, Y., Shichida, Y., and Kandori, H. (2003) Biochemistry 42, 9619-9625].     

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.     

FTIR spectroscopy of the all-trans form of Anabaena sensory rhodopsin at 77 K: hydrogen bond of a water between the Schiff base and Asp75

Anabaena sensory rhodopsin (ASR) is an archaeal-type rhodopsin found in eubacteria, and is believed to function as a photosensor interacting with a 14 kDa soluble protein. Most of the residues in the retinal binding pocket are similar in ASR except proline 206, where the corresponding amino acid in other archaeal-type rhodopsins is highly conserved aspartate that constitutes the counterion complex of the positively charged protonated Schiff base. The recently determined X-ray crystallographic structure of ASR revealed a water molecule between the Schiff base and Asp75 [Vogeley, L., Sineshchekov, O. A., Trivedi, V. D., Sasaki, J., Spudich, J. L., and Luecke, H. (2004) Science 306, 1390-1393], as well as the case for bacteriorhodopsin (BR), a typical transport rhodopsin working as a proton pump. In this study, we applied low-temperature Fourier transform infrared (FTIR) spectroscopy to the all-trans form of ASR at 77 K, and compared the local structure around the chromophore and their structural changes upon retinal photoisomerization with those of BR. The K intermediate minus ASR difference spectra were essentially similar to those for BR, indicating that photoisomerization yields formation of the distorted 13-cis form. In contrast, little amide I bands were observed for ASR. The presence of the proline-specific vibrational bands suggests that peptide backbone alterations are limited to the Pro206 moiety in the K state of ASR. The N-D stretching of the Schiff base is presumably located at 2163 (-) and 2125 (-) cm(-)(1) in ASR, suggesting that the hydrogen bonding strength of the Schiff base in ASR is similar to that in BR. A remarkable difference between ASR and BR was revealed from water bands. Although ASR possesses a bridged water molecule like BR, the O-D stretching of water molecules was observed only in the >2500 cm(-)(1) region for ASR. We interpreted that the weak hydrogen bond of the bridged water between the Schiff base and Asp75 originates from their geometry. Since ASR does not pump protons, our result supports the working hypothesis that the existence of strongly hydrogen bonded water molecules is essential for proton pumping activity in archaeal rhodopsins.     

Proton release and uptake of pharaonis phoborhodopsin (sensory rhodopsin II) reconstituted into phospholipids

pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII) is a photo-receptor for negative phototaxis in Natronobacterium pharaonis. During the photoreaction cycle (photocycle), ppR exhibits intraprotein proton movements, resulting in proton pumping from the cytoplasmic to the extracellular side, although it is weak. In this study, light-induced proton uptake and release of ppR reconstituted with phospholipid were analyzed using a SnO(2) electrode. The reconstituted ppR exhibited properties in proton uptake and release that are different from those of dodecyl maltoside solubilized samples. It showed fast proton release before the decay of ppR(M) (M-photointermediate) followed by proton uptake, which was similar to that of bacteriorhodopsin (BR), a light-driven proton pump. Mutant analysis assigned Asp193 to one (major) of the members of the proton-releasing group (PRG). Fast proton release was observed only when the pH was approximately 5-8 in the presence of Cl(-). When Cl(-) was replaced with SO(4)(2-), the reconstituted ppR did not exhibit fast proton release at any pH, suggesting Cl(-) binding around PRG. PRG in BR consists of Glu204 (Asp193 in ppR) and Glu194 (Pro183 in ppR). Replacement of Pro183 by Glu/Asp, a negatively charged residue, led to Cl(-)-independent fast proton release. The transducer binding affected the properties of PRG in ppR in the ground state and in the ppR(M) state, suggesting that interaction with the transducer extends to the extracellular surface of ppR. Differences and similarities in the molecular mechanism of the proton movement between ppR and BR are discussed.     

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.