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.     

Deprotonation of the Schiff base of bacteriorhodopsin is obligate in light-induced proton pumping

Bacteriorhodopsin (bR) in purple membranes was permethylated with formaldehyde and pyridine-borane with the incorporation of approximately 12 methyl groups. This new pigment, PMbR, absorbed light in the dark-adapted state with a lambda max at 558 nm, virtually the same as that of bR. Light adaptation of PMbR produced a lambda max of 564 nm with a slightly elevated epsilon. Similar changes occurred with bR. When incorporated into asolectin vesicles, PMbR was able to pump protons in the light with an efficiency similar to that of bR itself. Bleaching of PMbR exposed its active site lysine residue, which was monomethylated to form active site methylated bR (AMbR) after regeneration with all-trans-retinal. This blue pigment, which is a cyanopsin rather than a rhodopsin, showed an extraordinary red shift, absorbing light with a lambda max of 620 nm in the dark-adapted state. Light adaptation of AMbR resulted in a spectral shift to 616 nm with a decrease in epsilon. This change was completely reversible in the dark. This shift was interpreted to mean that an L-like intermediate was accumulating, as would be expected if deprotonation of the protonated Schiff base could not occur to produce the M intermediate. Furthermore, when incorporated into asolectin vesicles, AMbR proved incapable of pumping protons in the light. It was concluded from these experiments that deprotonation of the Schiff base of bR is obligate for light-induced proton pumping.     

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.     

Connectivity of the retinal Schiff base to Asp85 and Asp96 during the bacteriorhodopsin photocycle: the local-access model.

In the recently proposed local-access model for proton transfers in the bacteriorhodopsin transport cycle (Brown et al. 1998. Biochemistry. 37:3982-3993), connection between the retinal Schiff base and Asp85 (in the extracellular direction) and Asp96 (in the cytoplasmic direction)is maintained as long as the retinal is in its photoisomerized state. The directionality of the proton translocation is determined by influences in the protein that make Asp85 a proton acceptor and, subsequently, Asp96 a proton donor. The idea of concurrent local access of the Schiff base in the two directions is now put to a test in the photocycle of the D115N/D96N mutant. The kinetics had suggested that there is a single sequence of intermediates, L<-->M1<-->M2<-->N, and the M2-->M1 reaction depends on whether a proton is released to the extracellular surface. This is now confirmed. We find that at pH 5, where proton release does not occur, but not at higher pH, the photostationary state created by illumination with yellow light contains not only the M1 and M2 states, but also the L and the N intermediates. Because the L and M1 states decay rapidly, they can be present only if they are in equilibrium with later intermediates of the photocycle. Perturbation of this mixture with a blue flash caused depletion of the M intermediate, followed by its partial recovery at the expense of the L state. The change in the amplitude of the C=O stretch band at 1759 cm-1 demonstrated protonation of Asp85 in this process. Thus, during the reequilibration the Schiff base lost its proton to Asp85. Because the N state, also present in the mixture, arises by protonation of the Schiff base from the cytoplasmic surface, these results fulfill the expectation that under the conditions tested the extracellular access of the Schiff base would not be lost at the time when there is access in the cytoplasmic direction. Instead, the connectivity of the Schiff base flickers rapidly (with the time constant of the M1<-->M2 equilibration) between the two directions during the entire L-to-N segment of the photocycle.     

The pK of Schiff base deprotonation in bacteriorhodopsin.

Kinetic resonance Raman spectroscopy as a function of pH has been utilized to determine the pK of Schiff base deprotonation during the bacteriorhodopsin photochemical cycle. It is shown that the pK of Schiff base deprotonation is between 9.9 and 10.3, microseconds after light absorption and is >12 before photon initiation of photochemical cycling associated with proton pumping.     

Protein changes associated with reprotonation of the Schiff base in the photocycle of Asp96-->Asn bacteriorhodopsin. The MN intermediate with unprotonated Schiff base but N-like protein structure.

The difference Fourier transform infrared spectrum for the N intermediate in the photoreaction of the light-adapted form of bacteriorhodopsin can be recorded at pH 10 at 274 K (Pfefferlé, J.-M., Maeda, A., Sasaki, J., and Yoshizawa, T. (1991) Biochemistry 30, 6548-6556). Under these conditions, Asp96-->Asn bacteriorhodopsin gives a photoproduct which shows changes in protein structure similar to those observed in N of wild-type bacteriorhodopsin. However, decreased intensity of the chromophore bands and the single absorbance maximum at about 400 nm indicate that the Schiff base is unprotonated, as in the M intermediate. This photoproduct was named MN. At pH 7, where the supply of proton is not as restricted as at pH 10, Asp96-->Asn bacteriorhodopsin yields N with a protonated Schiff base. The Asn96 residue, which cannot deprotonate as Asp96 in wild-type bacteriorhodopsin, is perturbed upon formation of both MN at pH 10 and N at pH 7. We suggest that the reprotonation of the Schiff base is preceded by a large change in the protein structure including perturbation of the residue at position 96.     

Orientation of the protonated retinal Schiff base group in bacteriorhodopsin from absorption linear dichroism.

Linear dichroism experiments are performed on light-adapted bacteriorhodopsin (BR568) films containing native retinal (A1) and its 3,4-dehydroretinal (A2) analogue to measure the angle between the chromophore transition dipole moment and the membrane normal. QCFF/pi calculations show that the angle between the transition moment and the long axis of the polyene is changed by 3.4 degrees when the C3-C4 bond is unsaturated. The difference vector between the two transition moments points in the same direction as the Schiff base (N----H) bond for the all-trans BR568 chromophore. Because the plane of the chromophore is perpendicular to the membrane plane, a comparison of the transition moment orientations in the A1- and A2-pigments enables us to determine the orientation of the N----H bond with respect to the absolute chromophore (N----C5 vector) orientation. The angles of the transition moments are 70.3 degrees +/- 0.4 degrees and 67.8 degrees +/- 0.4 degrees for the A1- and A2-pigments, respectively. The fact that the change in the transition moment angle (2.5 degrees) is close to the predicted 3.4 degrees supports the idea that the chromophore plane is nearly perpendicular to the membrane plane. The decreased transition moment angle in the A2-analogue requires that the N----H bond and the N----C5 vector point toward the same membrane surface. Available results indicate that the N----C5 vector points toward the exterior in BR568. With this assignment, we conclude that the N----H bond points toward the exterior surface and its most likely counterion Asp-212. This information makes possible the construction of a computer graphics model for the active site in BR568.     

A defective proton pump, point-mutated bacteriorhodopsin Asp96----Asn is fully reactivated by azide.

Addition of azide fully restored the proton pump activity of defective bacteriorhodopsin (BR) mutant protein Asp96----Asn. The decay time of M of BR Asp96----Asn, the longest living intermediate, was decreased from 500 ms at pH 7.0 to approximately 1 ms under conditions of saturating azide concentrations. This decay was faster than the decay of M in the wild-type, where no such azide effect was detectable. Stationary photocurrents, measured with purple membranes immobilized and oriented in a polyacrylamide gel, increased upon addition of azide up to the level of the wild-type. Different small anions of weak acids restored the pump activity with decreasing affinity in the order: cyanate greater than azide greater than nitrite greater than formiate greater than acetate. The activation energy of the M decay in the mutant was higher in the presence (48 kJ/mol) than in the absence (27 kJ/mol) of 100 mM azide even though the absolute rate was dramatically increased by azide. This effect of azide is due to the substitution of a carboxamido group for a carboxylic group at position 96 which removes the internal proton donor and causes an increase in the entropy change of activation for proton transfer which is reversed by azide.     

Thermodynamics of proton transfer in carboxylic acid-retinal Schiff base hydrogen bonds with large proton polarizability

During the photocycle of bacteriorhodopsin (BR) the chromophore, a retinal Schiff base, is deprotonated. Simultaneously an asp residue is protonated. These results suggest that this deprotonation occurs via a Schiff base - asp hydrogen bond. Therefore, we studied carboxylic acid - retinal Schiff base model systems in CCl4 using IR spectroscopy. The IR spectra show that double minimum proton potentials are present in the OH ... N in equilibrium with O- ... HN+ H-bonds formed and that the proton can easily be shifted in these bonds by local electrical fields. The thermodynamic data of H-bond formation and proton transfer within these H-bonds are determined. On the basis of these data a hypothesis is developed with regard to the molecular mechanism of the deprotonation of the Schiff base of BR.     

Titration of aspartate-85 in bacteriorhodopsin: what it says about chromophore isomerization and proton release.

Titration of Asp-85, the proton acceptor and part of the counterion in bacteriorhodopsin, over a wide pH range (2-11) leads us to the following conclusions: 1) Asp-85 has a complex titration curve with two values of pKa; in addition to a main transition with pKa = 2.6 it shows a second inflection point at high pH (pKa = 9.7 in 150-mM KCl). This complex titration behavior of Asp-85 is explained by interaction of Asp-85 with an ionizable residue X'. As follows from the fit of the titration curve of Asp-85, deprotonation of X' increases the proton affinity of Asp-85 by shifting its pKa from 2.6 to 7.5. Conversely, protonation of Asp-85 decreases the pKa of X' by 4.9 units, from 9.7 to 4.8. The interaction between Asp-85 and X' has important implications for the mechanism of proton transfer. In the photocycle after the formation of M intermediate (and protonation of Asp-85) the group X' should release a proton. This deprotonated state of X' would stabilize the protonated state of Asp-85.2) Thermal isomerization of the chromophore (dark adaptation) occurs on transient protonation of Asp-85 and formation of the blue membrane. The latter conclusion is based on the observation that the rate constant of dark adaptation is directly proportional to the fraction of blue membrane (in which Asp-85 is protonated) between pH 2 and 11. The rate constant of isomerization is at least 10(4) times faster in the blue membrane than in the purple membrane. The protonated state of Asp-85 probably is important for the catalysis not only of all-trans <=> 13-cis thermal isomerization during dark adaptation but also of the reisomerization of the chromophore from 13-cis to all-trans configuration during N-->O-->bR transition in the photocycle. This would explain why Asp-85 stays protonated in the N and O intermediates.     

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.     

Proton transfer along the external proton channel of bacteriorhodopsin

The process of proton transfer along a proton channel is considered using bacteriorhodopsin as a model system, for which a large body of experimental data is available. The possible amino acid composition of the external proton half-channel of bacteriorhodopsin and the stepwise scheme of proton transfer consistent with experimental data are proposed. The rate of proton transfer between fixed centers is assessed for certain regions of this channel for which spectroscopic data are available.     

Proton transfer via the external proton channel of bacteriorhodopsin

The process of proton transfer across the membrane via the external proton channel in bacteriorhodopsin is considered. A possible amino acid composition of the channel is suggested and the step-by-step mechanism of proton transfer is proposed which agrees with the experimental data. The rate of proton transfer between fixed centers at several chains of the channel was estimated for which the spectroscopic data are available.     

Evidence for a bound water molecule next to the retinal Schiff base in bacteriorhodopsin and rhodopsin: a resonance Raman study of the Schiff base hydrogen/deuterium exchange.

The retinal chromophores of both rhodopsin and bacteriorhodopsin are bound to their apoproteins via a protonated Schiff base. We have employed continuous-flow resonance Raman experiments on both pigments to determine that the exchange of a deuteron on the Schiff base with a proton is very fast, with half-times of 6.9 +/- 0.9 and 1.3 +/- 0.3 ms for rhodopsin and bacteriorhodopsin, respectively. When these results are analyzed using standard hydrogen-deuteron exchange mechanisms, i.e., acid-, base-, or water-catalyzed schemes, it is found that none of these can explain the experimental results. Because the exchange rates are found to be independent of pH, the deuterium-hydrogen exchange can not be hydroxyl (or acid-)-catalyzed. Moreover, the deuterium-hydrogen exchange of the retinal Schiff base cannot be catalyzed by water acting as a base because in that case the estimated exchange rate is predicted to be orders of magnitude slower than that observed. The relatively slow calculated exchange rates are essentially due to the high pKa values of the Schiff base in both rhodopsin (pKa > 17) and bacteriorhodopsin (pKa approximately 13.5). We have also measured the deuterium-hydrogen exchange of a protonated Schiff base model compound in aqueous solution. Its exchange characteristics, in contrast to the Schiff bases of the pigments, is pH-dependent and consistent with the standard base-catalyzed schemes. Remarkably, the water-catalyzed exchange, which has a half-time of 16 +/- 2 ms and which dominates at pH 3.0 and below, is slower than the exchange rate of the Schiff base in rhodopsin and bacteriorhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural and energetic determinants of primary proton transfer in bacteriorhodopsin.

In the light-driven bacteriorhodopsin proton pump, the first proton transfer step is from the retinal Schiff base to a nearby carboxylate group. The mechanism of this transfer step is highly controversial, in particular whether a direct proton jump is allowed. Here, we review the structural and energetic determinants of the direct proton transfer path computed by using a combined quantum mechanical/molecular mechanical approach. Both protein flexibility and electrostatic interactions play an important role in shaping the proton transfer energy profile. Detailed analysis of the energetics of putative transitions in the first half of the photocycle focuses on two elements that determine the likelihood that a given configuration of the active site is populated during the proton-pumping cycle. First, the rate-limiting barrier for proton transfer must be consistent with the kinetics of the photocycle. Second, the active-site configuration must be compatible with a productive overall pumping cycle.     

The protonation-deprotonation kinetics of the protonated Schiff base in bicelle bacteriorhodopsin crystals

In the recently published x-ray crystal structure of the "bicelle" bacteriorhodopsin (bbR) crystal, the protein has quite a different structure from the native and the in cubo bacteriorhodopsin (cbR) crystal. Instead of packing in parallel trimers as do the native membrane and the cbR crystals, in the bbR crystal the protein packs as antiparallel monomers. To date, no functional studies have been performed, to our knowledge, to investigate if the photocycle is observed in this novel protein packing structure. In this study, both Raman and time-resolved transient absorption spectroscopy are used to both confirm the presence of the photocycle and investigate the deprotonation-reprotonation kinetics of the Schiff base proton in the bbR crystal. The observed rates of deprotonation and reprotonation processes of its Schiff base have been compared to those observed for native bR under the same conditions. Unlike the previously observed similarity of the rates of these processes for cbR crystals and those for native bacteriorhodopsin (bR), in bbR crystals the rate of deprotonation has increased by 300%, and the rate of reprotonation has decreased by nearly 700%. These results are discussed in light of the changes observed when native bR is delipidated or monomerized by detergents. Both the change of the hydrophobicity of the environment around the protonated Schiff base and Asp85 and Asp96 (which could change the pKa values of proton donor-acceptor pairs) and the water structure in the bbR crystal are offered as possible explanations for the different observations.     

The site of attachment of retinal in bacteriorhodopsin. The epsilon-amino group in Lys-41 is not required for proton translocation

Chymotryptic fragments C-1 (amino acids 72-248) and C-2 (amino acids 1-71) of bacteriorhodopsin have been shown previously to reassociate so as to regenerate the native bacteriorhodopsin chromophore in lipid/detergent mixtures and to form functional proton-translocating vesicles. The fragment C-2 has now been selectively methylated with formaldehyde and sodium cyanoborohydride to give the epsilon-dimethylamino derivatives of Lys-30, 40, and 41 in 96-99% average yield. The methylated and unmethylated C-2 fragments were identical in their ability to reassociate with fragment C-1 and retinal to regenerate the bacteriorhodopsin chromophore and to form functional proton-translocating vesicles. In contrast, dimethylation of the lysine residues of the C-1 fragment gave a derivative which did not form an active complex with unmethylated C-2. We conclude that the epsilon-amino group in Lys-41 is not required for Schiff's base formation with retinal at any step in the light-driven proton-translocation cycle.     

Time-resolved resonance Raman characterization of the bO640 intermediate of bacteriorhodopsin. Reprotonation of the Schiff base.

The resonance Raman spectrum of photolyzed bacteriorhodopsin under conditions known to increase the concentration of the bO640 intermediate in both H2O and D2O is presented. By use of computer subtraction techniques and a knowledge of the Raman spectra of the unphotolyzed bacteriorhodopsin as well as the other intermediates in the cycle, a qualitative spectrum of bO640 is determined. The shift of a band at 1630 cm-1 in H2O to 1616 cm-1 in D2O suggests that the Schiff base of bO640 is protonated. Additional bands at 947, 965, and 992 cm-1 that appear only in D2O suspensions confirm that a proton is coupled to the retinal chromophore of bO640. The reprotonation of the Schiff base thus occurs during the bM412 to bO640 step. The fingerprint region, sensitive to the isomeric configuration of the retinal chromophore of bO640, is dissimilar to the fingerprint regions of published model compounds and other forms of bacteriorhodopsin.     

Titration of the bacteriorhodopsin Schiff base involves titration of an additional protein residue

The retinal protein protonated Schiff base linkage plays a key role in the function of bacteriorhodopsin (bR) as a light-driven proton pump. In the unphotolyzed pigment, the Schiff base (SB) is titrated with a pK(a) of approximately 13, but following light absorption, it experiences a decrease in the pK(a) and undergoes several alterations, including a deprotonation process. We have studied the SB titration using retinal analogues which have intrinsically lower pK(a)'s which allow for SB titrations over a much lower pH range. We found that above pH 9 the channel for the SB titration is perturbed, and the titration rate is considerably reduced. On the basis of studies with several mutants, it is suggested that the protonation state of residue Glu204 is responsible for the channel perturbation. We suggest that above pH 12 a channel for the SB titration is restored probably due to titration of an additional protein residue. The observations may imply that during the bR photocycle and M photointermediate formation the rate of Schiff base protonation from the bulk is decreased. This rate decrease may be due to the deprotonation process of the "proton-releasing complex" which includes Glu204. In contrast, during the lifetime of the O intermediate, the protonated SB is exposed to the bulk. Possible implications for the switch mechanism, and the directionality of the proton movement, are discussed.     

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

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