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.     

Aspartic acid 85 in bacteriorhodopsin functions both as proton acceptor and negative counterion to the Schiff base.

In bacteriorhodopsin Asp85 has been proposed to function both as a negative counterion to the Schiff base and as proton acceptor in the early stages of the photocycle. To test this proposal further, we have replaced Asp85 by His. The rationale for this replacement is that although His can function as a proton acceptor, it cannot provide a negative charge at residue 85 to serve as a counterion to the protonated Schiff base. We show here that the absorption spectrum of the D85H mutant is highly sensitive to the pH of the external medium. From spectroscopic titrations, we have determined the apparent pK for deprotonation of the Schiff base to be 8.8 +/- 0.1 and the apparent pK for protonation of the His85 side chain to be approximately 3.5. Between pH 3.5 and 8.8, where the Schiff base is protonated, and the His side chain is deprotonated, the D85H mutant is completely inactive in proton transport. Time-resolved studies show that there is no detectable formation of an M-like intermediate in the photocycle of the D85H mutant. These experiments show that the presence of a neutral proton-accepting moiety at residue 85 is not sufficient for carrying out light-driven proton transport. The requirements at residue 85 are therefore for a group that serves both as a negatively charged counterion and as a proton acceptor.     

Structural changes of water molecules during the photoactivation processes in bovine rhodopsin

Internal water molecules of rhodopsins play an important role in stabilizing the crucial ion pair comprised by the protonated retinal Schiff base and its counterion. Previous low-temperature FTIR spectroscopy of archaeal rhodopsins observed water O-D stretching vibrations at 2400-2100 cm(-1) in D(2)O, corresponding to strong hydrogen bonds. Since a water molecule bridges the protonated Schiff base and an aspartate in archaeal rhodopsins, the observed water molecules presumably hydrate the negative charges in the Schiff base region. In contrast, the FTIR spectroscopy data of bovine rhodopsin presented here revealed that there are no spectral changes of water molecules under strongly hydrogen-bonding conditions (in the range <2400 cm(-1) for O-D stretch) during the photoactivation processes. The only observed water bands were located in the >2500 cm(-1) region that corresponds to weak hydrogen bonding. These results imply that the ion pair state in vertebrate visual rhodopsins is stabilized in a manner different from that in archaeal rhodopsins. In addition, the internal water molecules that hydrate the negative charges do not play important role in the photoactivation processes of rhodopsin that involve proton transfer from the Schiff base to Glu113 upon formation of Meta II. Structural changes of the H-D exchangeable peptide amide of a beta-sheet are observed upon formation of metarhodopsin II, suggesting that motion of a beta-sheet is coupled to the proton transfer reaction from the Schiff base to its counterion.     

Bacteriorhodopsin: a high-resolution structural view of vectorial proton transport

Recent 3-D structures of several intermediates in the photocycle of bacteriorhodopsin (bR) provide a detailed structural picture of this molecular proton pump in action. In this review, we describe the sequence of conformational changes of bR following the photoisomerization of its all-trans retinal chromophore, which is covalently bound via a protonated Schiff base to Lys216 in helix G, to a 13-cis configuration. The initial changes are localized near the protein's active site and a key water molecule is disordered. This water molecule serves as a keystone for the ground state of bR since, within the framework of the complex counter ion, it is important both for stabilizing the structure of the extracellular half of the protein, and for maintaining the high pK(a) of the Schiff base (the primary proton donor) and the low pK(a) of Asp85 (the primary proton acceptor). Subsequent structural rearrangements propagate out from the active site towards the extracellular half of the protein, with a local flex of helix C exaggerating an early movement of Asp85 towards the Schiff base, thereby facilitating proton transfer between these two groups. Other coupled rearrangements indicate the mechanism of proton release to the extracellular medium. On the cytoplasmic half of the protein, a local unwinding of helix G near the backbone of Lys216 provides sites for water molecules to order and define a pathway for the reprotonation of the Schiff base from Asp96 later in the photocycle. A steric clash of the photoisomerized retinal with Trp182 in helix F drives an outward tilt of the cytoplasmic half of this helix, opening the proton transport channel and enabling a proton to be taken up from the cytoplasm. Although bR is the first integral membrane protein to have its catalytic mechanism structurally characterized in detail, several key results were anticipated in advance of the structural model and the general framework for vectorial proton transport has, by and large, been preserved.     

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.     

Bacteriorhodopsin: a high-resolution structural view of vectorial proton transport

Recent 3-D structures of several intermediates in the photocycle of bacteriorhodopsin (bR) provide a detailed structural picture of this molecular proton pump in action. In this review, we describe the sequence of conformational changes of bR following the photoisomerization of its all-trans retinal chromophore, which is covalently bound via a protonated Schiff base to Lys216 in helix G, to a 13-cis configuration. The initial changes are localized near the protein's active site and a key water molecule is disordered. This water molecule serves as a keystone for the ground state of bR since, within the framework of the complex counter ion, it is important both for stabilizing the structure of the extracellular half of the protein, and for maintaining the high pK_a of the Schiff base (the primary proton donor) and the low pK_a of Asp85 (the primary proton acceptor). Subsequent structural rearrangements propagate out from the active site towards the extracellular half of the protein, with a local flex of helix C exaggerating an early movement of Asp85 towards the Schiff base, thereby facilitating proton transfer between these two groups. Other coupled rearrangements indicate the mechanism of proton release to the extracellular medium. On the cytoplasmic half of the protein, a local unwinding of helix G near the backbone of Lys216 provides sites for water molecules to order and define a pathway for the reprotonation of the Schiff base from Asp96 later in the photocycle. A steric clash of the photoisomerized retinal with Trp182 in helix F drives an outward tilt of the cytoplasmic half of this helix, opening the proton transport channel and enabling a proton to be taken up from the cytoplasm. Although bR is the first integral membrane protein to have its catalytic mechanism structurally characterized in detail, several key results were anticipated in advance of the structural model and the general framework for vectorial proton transport has, by and large, been preserved.     

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.     

Effect of introducing different carboxylate-containing side chains at position 85 on chromophore formation and proton transport in bacteriorhodopsin.

During the initial stages of the bacteriorhodopsin photocycle, a proton is transferred from the Schiff base to the deprotonated carboxylate of Asp85. Earlier studies have shown that replacement of Asp85 by Asn completely abolishes proton transport activity, whereas extension of the side chain by an additional carbon-carbon bond (Asp85-->Glu) results in a functional proton pump. Here we show that extension of the Asp85 side chain by two additional bond lengths also results in a functional proton pump as long as the terminal group is a carboxylate moiety. These side chains were created by modification of the cysteine residue in the Asp85-->Cys mutant with either iodoacetic acid or iodoacetamide. In vitro chromophore formation studies show that the rate of Schiff base protonation in mutants that contain a carboxylate at residue 85 is invariably faster than in mutants that contain neutral substitutions at this position. We conclude that in bacteriorhodopsin, there is considerable tolerance in the volume of the side chain that can be accommodated at position 85 and that the presence of a carboxylate at residue 85 is important both for proton pumping and for stabilizing the protonated Schiff base.     

Fourier transform infrared evidence for Schiff base alteration in the first step of the bacteriorhodopsin photocycle

The first step of the bacteriorhodopsin (bR) photocycle involves the formation of a red-shifted product, K. Fourier transform infrared difference spectra of the bR570 to K630 transition at 81 K has been measured for bR containing different isotopic substitutions at the retinal Schiff base. In the case of bacteriorhodopsin containing a deuterium substitution at the Schiff base nitrogen, carbon 15, or both, we find spectral changes in the 1600-1610- and 1570-1580-cm-1 region consistent with the hypothesis that the K630 C=N stretching mode of a protonated Schiff base is located near 1609 cm-1. A similar set of Schiff base deuterium substitutions for retinal containing a 13C at the carbon 10 position strongly supports this conclusion. This assignment of the K630 C=N stretching vibration provides evidence that the bR Schiff base proton undergoes a substantial environmental change most likely due to separation from a counterion. In addition, a correlation is found between the C=N stretching frequency and the maximum wavelength of visible absorption, suggesting that movement of a counterion relative to the Schiff base proton is the main source of absorption changes in the early stages of the photocycle. Such a movement is a key prediction of several models of proton transport and energy transduction. Evidence is also presented that one or more COOH groups are involved in the formation of the K intermediate.     

Effect of intermolecular orientation upon proton transfer within a polarizable medium.

Ab initio calculations are used to investigate the proton transfer process in bacteriorhodopsin. HN = CH2 serves as a small prototype of the Schiff base while HCOO- models its carboxylate-containing counterion and HO- the hydroxyl group of water of tyrosine, leading to the HCOO-..H+..NHCH2 and HO-..H+..NHCH2 complexes. In isolation, both complexes prefer a neutral pair configuration wherein the central proton is associated with the anion. However, the Schiff base may be protonated in the former complex, producing the HCOO-..+HNHCH2 ion pair, when there is a high degree of dielectric coupling with an external polarizable medium. Within a range of intermediate level coupling, the equilibrium position of the proton (on either the carboxylate or Schiff base) can be switched by suitable changes in the intermolecular angle. pK shift resulting from a 60 degrees reorientation are calculated to be some 5-12 pK U within the coupling range where proton transfers are possible. The energy barrier to proton transfer reinforces the ability of changes in angle and dielectric coupling to induce a proton transfer.     

FTIR studies of the photoactivation processes in squid retinochrome

Retinochrome is a photoisomerase of the invertebrate visual system, which converts all-trans-retinal to the 11-cis configuration and supplies it to visual rhodopsin. In this paper, we studied light-induced structural changes in squid retinochrome by means of low-temperature UV-visible and Fourier transform infrared (FTIR) spectroscopy. In PC liposomes, lumi-retinochrome was stable in the wide temperature range between 77 and 230 K. High thermal stability of the primary intermediate in retinochrome is in contrast to the case in rhodopsins. FTIR spectroscopy suggested that the chromophore of lumi-retinochrome is in a relaxed planar 11-cis form, being consistent with its high thermal stability. The chromophore binding pocket of retinochrome appears to accommodate both all-trans and 11-cis forms without a large distortion, and limited protein structural changes between all-trans and 11-cis chromophores may be suitable for the function of retinochrome as a photoisomerase. The analysis of N-D and O-D stretching vibrations in D(2)O revealed that the hydrogen bond of the Schiff base is weaker in retinochrome than in bovine rhodopsin and bacteriorhodopsin, while retinochrome has a water molecule under strongly hydrogen-bonded conditions (O-D stretch at 2334 cm(-)(1)). The hydrogen bond of the water is further strengthened in lumi-retinochrome. The formation of meta-retinochrome accompanies deprotonation of the Schiff base, together with the peptide backbone alterations of alpha-helices, and possible formation of beta-sheets. It was found that the Schiff base proton is not transferred to its counterion, Glu181, but directly released to the aqueous phase in PC liposomes (pH 7.5). This suggests that the Schiff base environment is exposed to solvent in meta-retinochrome, which may be advantageous for the hydrolysis reaction of the Schiff base in the transport of 11-cis-retinal to its shuttle protein.     

Proton transport by proteorhodopsin requires that the retinal Schiff base counterion Asp-97 be anionic

At pH >7, proteorhodopsin functions as an outward-directed proton pump in cell membranes, and Asp-97 and Glu-108, the homologues of the Asp-85 and Asp-96 in bacteriorhodopsin, are the proton acceptor and donor to the retinal Schiff base, respectively. It was reported, however [Friedrich, T. et al. (2002) J. Mol. Biol., 321, 821-838], that proteorhodopsin transports protons also at pH <7 where Asp-97 is protonated and in the direction reverse from that at higher pH. To explore the roles of Asp-97 and Glu-108 in the proposed pumping with variable vectoriality, we compared the photocycles of D97N and E108Q mutants, and the effects of azide on the photocycle of the E108Q mutant, at low and high pH. Unlike at high pH, at a pH low enough to protonate Asp-97 neither the mutations nor the effects of azide revealed evidence for the participation of the acidic residues in proton transfer, and as in the photocycle of the wild-type protein, no intermediate with unprotonated Schiff base accumulated. In view of these findings, and the doubts raised by absence of charge transfer after flash excitation at low pH, we revisited the question whether transport occurs at all under these conditions. In both oriented membrane fragments and liposomes reconstituted with proteorhodopsin, we found transport at high pH but not at low pH. Instead, proton transport activity followed the titration curve for Asp-97, with an apparent pK(a) of 7.1, and became zero at the pH where Asp-97 is fully protonated.     

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.     

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.     

Tunable laser resonance Raman spectroscopic investigations of the transduction process in vertebrate rod cells.

Tunable laser resonance Raman spectroscopy has been applied to probe (in vivo) the role of rhodopsin in transducing light energy into the chemical necessary to generate a neural response. These in vivo experiments have suggested that the Schiff base linkage through which retinal is attached to opsin in rhodopsin is protonated. Furthermore, it appears that light eventually stimulates the deprotonation of the Schiff base linkage between the Meta I and Meta II steps in the intermediate sequence which is the result of light interacting with rhodopsin. Our data suggest that this deprotonation of the Schiff base occurs on the same time scale as overall proton release and uptake by the rhodopsin molecule. It is interesting to note that this series of protonations and deprotonations also occurs within the same time scale as the neural response generation in vertebrates and the generation of a proton gradient by bacteriorhodopsin, which is used by the bacterium, Halobacterium halobium, for ATP synthesis. If these data are analyzed within the context of the in vivo resonance Raman experiments (which seem to indicate that proton release is stimulated in the disc membrane during transduction) then there is a strong suggestion that the proton will assume an important role in any working hypothesis of visual transduction. In essence it appears that protons along with ATP and calcium ions must all be essential elements in the transduction process.     

Chloride ion binding to bacteriorhodopsin at low pH: an infrared spectroscopic study

Bacteriorhodopsin (bR) and halorhodopsin (hR) are light-induced ion pumps in the cell membrane of Halobacterium salinarium. Under normal conditions bR is an outward proton transporter, whereas hR is an inward Cl- transporter. There is strong evidence that at very low pH and in the presence of Cl-, bR transports Cl- ions into the cell, similarly to hR. The chloride pumping activity of bR is connected to the so-called acid purple state. To account for the observed effects in bR a tentative complex counterion was suggested for the protonated Schiff base of the retinal chromophore. It would consist of three charged residues: Asp-85, Asp-212, and Arg-82. This quadruplet (including the Schiff base) would also serve as a Cl- binding site at low pH. We used Fourier transform infrared difference spectroscopy to study the structural changes during the transitions between the normal, acid blue, and acid purple states. Asp-85 and Asp-212 were shown to participate in the transitions. During the normal-to-acid blue transition, Asp-85 protonates. When the pH is further lowered in the presence of Cl-, Cl- binds and Asp-212 also protonates. The binding of Cl- and the protonation of Asp-212 occur simultaneously, but take place only when Asp-85 is already protonated. It is suggested that HCl is taken up in undissociated form in exchange for a neutral water molecule.     

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.     

Understanding structure and function in the light-driven proton pump bacteriorhodopsin

The atomic structure of bacteriorhodopsin and the outlines of its proton transport mechanism are now available. Photoisomerization of the retinal in the chromophore creates a steric and electrostatic conflict at the retinal binding site. The free energy gain sets off a sequence of reactions in which directed proton transfers take place between the protonated retinal Schiff base, Asp-85, and Asp-96. These internal steps, and other proton transfers at and near the two aqueous interfaces, add up to the translocation of a proton from the cytoplasmic to the extracellular side of the membrane. Bound water plays a crucial role in proton conduction in both extracellular and cytoplasmic regions, but the means by which the protons move from site to site differ. Proton release to the extracellular surface is through interaction of a hydrogen-bonded chain of identified aspartic acid, arginine, water, and glutamic acid residues with Asp-85, while proton uptake from the cytoplasmic surface utilizes a single aspartic acid, Asp-96, whose protonation state appears to be regulated by the protein conformation dependent hydration of this region. The directionality of the translocation is ensured by the accessibility of the Schiff base to the extracellular and cytoplasmic directions after the retinal is photoisomerized, as well as the changing proton affinities of the acceptor Asp-85 and donor Asp-96.     

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.     

A large photolysis-induced pKa increase of the chromophore counterion in bacteriorhodopsin: implications for ion transport mechanisms of retinal proteins.

The proton-pumping mechanism of bacteriorhodopsin is dependent on a photolysis-induced transfer of a proton from the retinylidene Schiff base chromophore to the aspartate-85 counterion. Up until now, this transfer was ascribed to a > 7-unit decrease in the pKa of the protonated Schiff base caused by photoisomerization of the retinal. However, a comparably large increase in the pKa of the Asp-85 acceptor also plays a role, as we show here with infrared measurements. Furthermore, the shifted vibrational frequency of the Asp-85 COOH group indicates a transient drop in the effective dielectric constant around Asp-85 to approximately 2 in the M photointermediate. This dielectric decrease would cause a > 40 kJ-mol-1 increase in free energy of the anionic form of Asp-85, fully explaining the observed pK alpha increase. An analogous photolysis-induced destabilization of the Schiff base counterion could initiate anion transport in the related protein, halorhodopsin, in which aspartate-85 is replaced by Cl- and the Schiff base proton is consequently never transferred.