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.     

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 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.     

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 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.     

Crystallographic structures of the M and N intermediates of bacteriorhodopsin: assembly of a hydrogen-bonded chain of water molecules between Asp-96 and the retinal Schiff base

An M intermediate of wild-type bacteriorhodopsin and an N intermediate of the V49A mutant were accumulated in photostationary states at pH 5.6 and 295 K, and their crystal structures determined to 1.52A and 1.62A resolution, respectively. They appear to be M(1) and N' in the sequence, M(1)<-->M(2)<-->M'(2)<-->N<-->N'-->O-->BR, where M(1), M(2), and M'(2) contain an unprotonated retinal Schiff base before and after a reorientation switch and after proton release to the extracellular surface, while N and N' contain a reprotonated Schiff base, before and after reprotonation of Asp96 from the cytoplasmic surface. In M(1), we detect a cluster of three hydrogen-bonded water molecules at Asp96, not present in the BR state. In M(2), whose structure we reported earlier, one of these water molecules intercalates between Asp96 and Thr46. In N', the cluster is transformed into a single-file hydrogen-bonded chain of four water molecules that connects Asp96 to the Schiff base. We find a network of three water molecules near residue 219 in the crystal structure of the non-illuminated F219L mutant, where the residue replacement creates a cavity. This suggests that the hydration of the cytoplasmic region we observe in N' might have occurred spontaneously, beginning at an existing water molecule as nucleus, in the cavities from residue rearrangements in the photocycle.     

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.     

Coupling photoisomerization of retinal to directional transport in bacteriorhodopsin

In order to understand how isomerization of the retinal drives unidirectional transmembrane ion transport in bacteriorhodopsin, we determined the atomic structures of the BR state and M photointermediate of the E204Q mutant, to 1.7 and 1.8 A resolution, respectively. Comparison of this M, in which proton release to the extracellular surface is blocked, with the previously determined M in the D96N mutant indicates that the changes in the extracellular region are initiated by changes in the electrostatic interactions of the retinal Schiff base with Asp85 and Asp212, but those on the cytoplasmic side originate from steric conflict of the 13-methyl retinal group with Trp182 and distortion of the pi-bulge of helix G. The structural changes suggest that protonation of Asp85 initiates a cascade of atomic displacements in the extracellular region that cause release of a proton to the surface. The progressive relaxation of the strained 13-cis retinal chain with deprotonated Schiff base, in turn, initiates atomic displacements in the cytoplasmic region that cause the intercalation of a hydrogen-bonded water molecule between Thr46 and Asp96. This accounts for the lowering of the pK(a) of Asp96, which then reprotonates the Schiff base via a newly formed chain of water molecules that is extending toward the Schiff base.     

Crystal structure of the L intermediate of bacteriorhodopsin: evidence for vertical translocation of a water molecule during the proton pumping cycle

For structural investigation of the L intermediate of bacteriorhodopsin, a 3D crystal belonging to the space group P622 was illuminated with green light at 160 K and subsequently with red light at 100 K. This yielded a approximately 1:4 mixture of the L intermediate and the ground-state. Diffraction data from such crystals were collected using a low flux of X-rays ( approximately 2 x 10(15) photons/mm2 per crystal), and their merged data were compared with those from unphotolyzed crystals. These structural data, together with our previous data, indicate that the retinal chromophore, which is largely twisted in the K-intermediate, takes a more planar 13-cis, 15-anti configuration in the L intermediate. This configurational change, which is accompanied by re-orientation of the Schiff base N-H bond towards the intracellular side, is coupled with a large rotation of the side-chain of an amino acid residue (Leu93) making contact with the C13 methyl group of retinal. Following these motions, a water molecule, at first hydrogen-bonded to the Schiff base and Asp85, is dragged to a space that is originally occupied by Leu93. Diffraction data from a crystal containing the M intermediate showed that this water molecule moves further towards the intracellular side in the L-to-M transition. It is very likely that detachment of this water molecule from the protonated Schiff base causes a significant decrease in the pKa of the Schiff base, thereby facilitating the proton transfer to Asp85. On the basis of these observations, we argue that the vertical movement of a water molecule in the K-to-L transition is a key event determining the directionality of proton translocation in the protein.     

Suppression of the back proton-transfer from Asp85 to the retinal Schiff base in bacteriorhodopsin: a theoretical analysis of structural elements

The transfer of a proton from the retinal Schiff base to the nearby Asp85 protein group is an essential step in the directional proton-pumping by bacteriorhodopsin. To avoid the wasteful back reprotonation of the Schiff base from Asp85, the protein must ensure that, following Schiff base deprotonation, the energy barrier for back proton-transfer from Asp85 to the Schiff base is larger than that for proton-transfer from the Schiff base to Asp85. Here, three structural elements that may contribute to suppressing the back proton-transfer from Asp85 to the Schiff base are investigated: (i) retinal twisting; (ii) hydrogen-bonding distances in the active site; and (iii) the number and location of internal water molecules. The impact of the pattern of bond twisting on the retinal deprotonation energy is dissected by performing an extensive set of quantum-mechanical calculations. Structural rearrangements in the active site, such as changes of the Thr89:Asp85 distance and relocation of water molecules hydrogen-bonding to the Asp85 acceptor group, may participate in the mechanism which ensures that following the transfer of the Schiff base proton to Asp85 the protein proceeds with the subsequent photocycle steps, and not with back proton transfer from Asp85 to the Schiff base.     

Atomic resolution structures of bacteriorhodopsin photocycle intermediates: the role of discrete water molecules in the function of this light-driven ion pump

High-resolution X-ray crystallographic studies of bacteriorhodopsin have tremendously advanced our understanding of this light-driven ion pump during the last 2 years, and emphasized the crucial role of discrete internal water molecules in the pump cycle. In the extracellular region an extensive three-dimensional hydrogen-bonded network of protein residues and seven water molecules leads from the buried retinal Schiff base via water 402 and the initial proton acceptor Asp85 to the membrane surface. Near Lys216 where the retinal binds, transmembrane helix G contains a pi-bulge that causes a non-proline kink. The bulge is stabilized by hydrogen bonding of the main chain carbonyl groups of Ala215 and Lys216 with two buried water molecules located in the otherwise very hydrophobic region between the Schiff base and the proton donor Asp96 in the cytoplasmic region. The M intermediate trapped in the D96N mutant corresponds to a late M state in the transport cycle, after protonation of Asp85 and release of a proton to the extracellular membrane surface, but before reprotonation of the deprotonated retinal Schiff base. The M intermediate from the E204Q mutant corresponds to an earlier M, as in this mutant the Schiff base deprotonates without proton release. The structures of these two M states reveal progressive displacements of the retinal, main chain and side chains induced by photoisomerization of the retinal to 13-cis,15-anti, and an extensive rearrangement of the three-dimensional network of hydrogen-bonded residues and bound water that accounts for the changed pK(a)s of the Schiff base, Asp85, the proton release group and Asp96. The structure for the M state from E204Q suggests, moreover, that relaxation of the steric conflicts of the distorted 13-cis,15-anti retinal plays a critical role in the reprotonation of the Schiff base by Asp96. Two additional waters now connect Asp96 to the carbonyl of residue 216, in what appears to be the beginning of a hydrogen-bonded chain that would later extend to the retinal Schiff base. Based on the ground state and M intermediate structures, models of the molecular events in the early part of the photocycle are presented, including a novel model which proposes that bacteriorhodopsin pumps hydroxide (OH(-)) ions from the extracellular to the cytoplasmic side.     

FTIR analysis of the SII540 intermediate of sensory rhodopsin II: Asp73 is the Schiff base proton acceptor

Sensory rhodopsin II (SRII), a repellent phototaxis receptor found in Halobacterium salinarum, has several homologous residues which have been found to be important for the proper functioning of bacteriorhodopsin (BR), a light-driven proton pump. These include Asp73, which in the case of bacteriorhodopsin (Asp85) functions as the Schiff base counterion and proton acceptor. We analyzed the photocycles of both wild-type SRII and the mutant D73E, both reconstituted in Halobacterium salinarum lipids, using FTIR difference spectroscopy under conditions that favor accumulation of the O-like, photocycle intermediate, SII540. At both room temperature and -20 degrees C, the difference spectrum of SRII is similar to the BR-->O640 difference spectrum of BR, especially in the configurationally sensitive retinal fingerprint region. This indicates that SII540 has an all-trans chromophore similar to the O640 intermediate in BR. A positive band at 1761 cm-1 downshifts 40 cm-1 in the mutant D73E, confirming that Asp73 undergoes a protonation reaction and functions in analogy to Asp85 in BR as a Schiff base proton acceptor. Several other bands in the C=O stretching regions are identified which reflect protonation or hydrogen bonding changes of additional Asp and/or Glu residues. Intense bands in the amide I region indicate that a protein conformational change occurs in the late SRII photocycle which may be similar to the conformational changes that occur in the late BR photocycle. However, unlike BR, this conformational change does not reverse during formation of the O-like intermediate, and the peptide groups giving rise to these bands are partially accessible for hydrogen/deuterium exchange. Implications of these findings for the mechanism of SRII signal transduction are discussed.     

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.     

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.     

Halide binding by the D212N mutant of Bacteriorhodopsin affects hydrogen bonding of water in the active site

Bacteriorhodopsin (BR), a membrane protein found in Halobacterium salinarum, functions as a light-driven proton pump. The Schiff base region has a quadrupolar structure with positive charges located at the protonated Schiff base and Arg82, and the counterbalancing negative charges located at Asp85 and Asp212. The quadropole inside the protein is stabilized by three water molecules, forming a roughly planar pentagonal cluster composed of these waters and two oxygens of Asp85 and Asp212 (one from each carboxylate side chain). It is known that BR lacks proton-pumping activity if Asp85 or Asp212 is neutralized by mutation, but binding of Cl- has different functional effects in mutants at these positions. Binding of Cl- to D85T converts into a chloride ion pump (Sasaki, J., Brown, L. S., Chon, Y.-S., Kandori, H., Maeda, A., Needleman, R., and Lanyi, J. K. (1995) Science 269, 73-75). On the other hand, photovoltage measurements suggested that binding of Cl- to D212N restores the proton-pumping activity at low pH (Moltke, S., Krebs, M. P., Mollaaghababa, R., Khorana, H. G., and Heyn, M. P. (1995) Biophys. J. 69, 2074-2083). In this paper, we studied halide-bound D212N mutant BR in detail. Light-induced pH changes in a suspension of proteoliposomes containing D212N(Cl-) at pH 5 clearly showed that Cl- restores the proton-pumping activity. Spectral blue-shift induced by halide binding to D212N indicates that halides affect the counterion of the protonated Schiff base, whereas much smaller halide dependence of the lambdamax than in D85T suggests that the binding site is distant from the chromophore. In fact, the K minus BR difference Fourier-transform infrared (FTIR) spectra of D212N at 77 K exhibit little halide dependence for vibrational bands of retinal and protein. The only halide-dependent bands were the C=N stretch of Arg82 and some water O-D stretches, suggesting that these groups constitute a halide-binding pocket. A strongly hydrogen-bonded water molecule is observed for halide-bound D212N, but not for halide-free D212N, which is consistent with our hypothesis that such a water molecule is a prerequisite for proton-pumping activity of rhodopsins. We concluded that halide binding near Arg82 in D212N restores the water-containing hydrogen-bonding network in the Schiff base region. In particular, the ion pair formed by the Schiff base and Asp85 through a strongly hydrogen-bonded water is essential for the proton-pumping activity of this mutant and may be controlled by the halide binding to the distant site.     

FTIR study of the L intermediate of Anabaena sensory rhodopsin: structural changes in the cytoplasmic region

Anabaena sensory rhodopsin (ASR) is an archaeal-type rhodopsin found in eubacteria. The gene encoding ASR forms a single operon with ASRT (ASR transducer) that is a 14 kDa soluble protein, suggesting that ASR functions as a photochromic sensor by activating the soluble transducer. One of the characteristics of ASR is that the formation of the M intermediate accompanies a proton transfer from the Schiff base to Asp217 in the cytoplasmic side [Shi, L., Yoon, S. R., Bezerra, A. G., Jr., Jung, K. H., and Brown, L. S. (2006) J. Mol. Biol. 358, 686-700], in remarkable contrast to other archaeal-type rhodopsins such as a light-driven proton-pump, bacteriorhodopsin (BR). In this study, we applied low-temperature Fourier transform infrared (FTIR) spectroscopy to the all- trans form of ASR at 170 K, and compared the structural changes in the L intermediate with those of BR. The ASR L minus ASR difference spectra were essentially similar to those for BR, suggesting common structures for the L state in ASR and BR. On the other hand, unique CO stretching bands of a protonated carboxylic acid were observed at 1722 (+) and 1703 (-) cm (-1) at pH 5 and 7, and assigned to Glu36 by use of mutants. Glu36 is located at the cytoplasmic side, and the distance from the Schiff base is about 20 A. This result shows the structural changes at the cytoplasmic surface in ASR L. pH-dependent frequency change was also observed for a water stretching vibration, suggesting that the water molecule is involved in a hydrogen-bonding network with Glu36 and Asp217. Unique hydrogen-bonding network in the cytoplasmic domain of ASR will be discussed.     

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.     

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.     

Structural changes in the L photointermediate of bacteriorhodopsin

The L to M reaction of the bacteriorhodopsin photocycle includes the crucial proton transfer from the retinal Schiff base to Asp85. In spite of the importance of the L state in deciding central issues of the transport mechanism in this pump, the serious disagreements among the three published crystallographic structures of L have remained unresolved. Here, we report on the X-ray diffraction structure of the L state, to 1.53-1.73 A resolutions, from replicate data sets collected from six independent crystals. Unlike earlier studies, the partial occupancy refinement uses diffraction intensities from the same crystals before and after the illumination to produce the trapped L state. The high reproducibility of inter-atomic distances, and bond angles and torsions of the retinal, lends credibility to the structural model. The photoisomerized 13-cis retinal in L is twisted at the C(13)=C(14) and C(15)=NZ double-bonds, and the Schiff base does not lose its connection to Wat402 and, therefore, to the proton acceptor Asp85. The protonation of Asp85 by the Schiff base in the L-->M reaction is likely to occur, therefore, via Wat402. It is evident from the structure of the L state that various conformational changes involving hydrogen-bonding residues and bound water molecules begin to propagate from the retinal to the protein at this stage already, and in both extracellular and cytoplasmic directions. Their rationales in the transport can be deduced from the way their amplitudes increase in the intermediates that follow L in the reaction cycle, and from the proton transfer reactions with which they are associated.     

Localization and orientation of functional water molecules in bacteriorhodopsin as revealed by polarized Fourier transform infrared spectroscopy.

Linear dichroic difference Fourier transform infrared spectra upon formation of the M photointermediate were recorded with oriented purple membranes. The purpose was to determine the angle of the directions of the dipole moments of 1) the water molecule whose O-H stretching vibration appears at 3643 cm-1 for the unphotolyzed state and 3671 cm-1 for the M intermediate, and 2) the C=O bond of protonated Asp85 in the M intermediate. The angle of 36 degrees we find for the C=O of the protonated Asp85 in the M intermediate is not markedly different from 26 degrees for unprotonated Asp85 in the model based on cryoelectron diffraction, indicating the absence of gross orientation changes in Asp85 upon its protonation. The O-H band at 3671 cm-1 of a water molecule in the M intermediate, although its position has not determined, is fixed almost parallel to the membrane plane. For the unphotolyzed state the angle of the water O-H to the membrane normal was determined to be 60 degrees. On the basis of these data and the structural model, we place the water molecule in the unphotolyzed state at a position where it forms hydrogen bonds with the Schiff base, Asp85, Asp212, and Trp86.