Effects of anion binding on the deprotonation reactions of halorhodopsin

The retinal Schiff base of halorhodopsin deprotonates with a pKa of 7.4 in 0.5 M Na2SO4 in the dark. In the presence of various anions, such as chloride or nitrate, etc., the pKa is raised by up to 1.5 units. Analysis of the dependency of the pKa on anion concentration favors the model in which the anions do not bind to the positively charged Schiff base nitrogen, but to a site near it, and exert their effect on the pKa by direct (perhaps electrostatic) interaction. Adding nitrate, or one of several other anions, causes also a small blueshift in the visible absorption band of the chromophore. These effects on the pKa and the absorption band define an anion binding site in halorhodopsin, termed Site I. Chloride and bromide apparently bind in addition to another site, which is associated with a small red-shift of the absorption band and changes in the photocycle. This other anion binding site is termed Site II. Illumination of halorhodopsin samples results in the deprotonation of the Schiff base with a much lowered pKa, but at very low rates probably determined by the generation of a deprotonating photointermediate. Binding of Site I anions increases the pKa of deprotonation in the light also. The similarity of the responses of the apparent pKa in the dark and in the light to anion concentration suggests that anion binding to Site I influences deprotonation of the Schiff base similarly in the photointermediate and in the parent halorhodopsin molecule.     

The pKa of the protonated Schiff bases of gecko cone and octopus visual pigments.

A visual pigment is composed of retinal bound to its apoprotein by a protonated Schiff base linkage. Light isomerizes the chromophore and eventually causes the deprotonation of this Schiff base linkage at the meta II stage of the bleaching cycle. The meta II intermediate of the visual pigment is the active form of the pigment that binds to and activates the G protein transducin, starting the visual cascade. The deprotonation of the Schiff base is mandatory for the formation of meta II intermediate. We studied the proton binding affinity, pKa, of the Schiff base of both octopus rhodopsin and the gecko cone pigment P521 by spectral titration. Several fluorinated retinal analogs have strong electron withdrawing character around the Schiff base region and lower the Schiff base pKa in model compounds. We regenerated octopus and gecko visual pigments with these fluorinated and other retinal analogs. Experiments on these artificial pigments showed that the spectral changes seen upon raising the pH indeed reflected the pKa of the Schiff base and not the denaturation of the pigment or the deprotonation of some other group in the pigment. The Schiff base pKa is 10.4 for octopus rhodopsin and 9.9 for the gecko cone pigment. We also showed that although the removal of Cl- ions causes considerable blue-shift in the gecko cone pigment P521, it affects the Schiff base pKa very little, indicating that the lambda max of visual pigment and its Schiff base pKa are not tightly coupled.     

Chloride binding regulates the Schiff base pK in gecko P521 cone-type visual pigment

The binding of chloride is known to shift the absorption spectrum of most long-wavelength-absorbing cone-type visual pigments roughly 30 nm to the red. We determined that the chloride binding constant for this color shift in the gecko P521 visual pigment is 0.4 mM at pH 6.0. We found an additional effect of chloride on the P521 pigment: the apparent pKa of the Schiff base in P521 is greatly increased as the chloride concentration is increased. The apparent Schiff base pKa shifts from 8.4 for the chloride-free form to >10.4 for the chloride-bound form. We show that this shift is due to chloride binding to the pigment, not to the screening of the membrane surface charges by chloride ions. We also found that at high pH, the absorption maximum of the chloride-free pigment shifts from 495 to 475 nm. We suggest that the chloride-dependent shift of the apparent Schiff base pKa is due to the deprotonation of a residue in the chloride binding site with a pKa of ca. 8.5, roughly that of the Schiff base in the absence of chloride. The deprotonation of this site results in the formation of the 475 nm pigment and a 100-fold decrease in the pigment's ability to bind chloride. Increasing the concentration of chloride results in the stabilization of the protonated state of this residue in the chloride binding site and thus increased chloride binding with an accompanying increase in the Schiff base pK.     

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.     

Environmental effects on the protonation states of active site residues in bacteriorhodopsin.

Finite difference solutions of the Poisson-Boltzmann equation are used to calculate the pKa values of the functionally important ionizable groups in bacteriorhodopsin. There are strong charge-charge interactions between the residues in the binding site leading to the possibility of complex titration behavior. Structured water molecules, if they exist in the binding site, can have significant effects on the calculated pKa by strongly stabilizing ionized species. The ionization states of the Schiff base and Asp-85 are found to be strongly coupled. Small environmental changes, which might occur as a consequence of trans-cis isomerization, are capable of causing large shifts in the relative pKa values of these two groups. This provides an explanation for the protonation of Asp-85 and the deprotonation of the Schiff base in the M state of bacteriorhodopsin. The different behavior of Asp-85 and Asp-212 is discussed in this regard.     

Effects of various anions on the Raman spectrum of halorhodopsin.

Resonance Raman experiments were conducted to probe and understand the effect of various anions on halorhodopsin. These included monoatomic anions Cl- and Br-, which bind to the so-called halorhodopsin binding sites I and II, and polyatomic anions NO3- and ClO4-, which bind to site I only. The two types of ions clearly show different effects on the vibrational spectrum of the chromophore. The differences are not localized to the Schiff base region of the molecule, but extend to the chromophore structure-sensitive fingerprint region as well. We find that the protonated Schiff base frequency is at 1,633 cm-1 for Cl- and Br- ions, as reported previously for Cl-. However, we find that two Schiff base frequencies characterize halorhodopsin upon binding of the polyatomic anions. One frequency lies at the same location as that found for the monoatomic anions and the other is at 1,645 cm-1. Halorhodopsin with bound NO3- and ClO4- thus may consist of two heterogeneous structures in equilibrium. This heterogeneity does not seem to correlate with a retinal isomeric heterogeneity, which we can also demonstrate in these samples. The results suggest that anions binding to site I do not bind to the Schiff base directly, but can influence chromophore and/or protein conformational states.     

Anion binding to the Schiff base of the bacteriorhodopsin mutants Asp-85----Asn/Asp-212----Asn and Arg-82----Gln/Asp-85----Asn/Asp-212----Asn.

Studies of bacteriorhodopsin have indicated that the charge environment of the protonated Schiff base consists of residues Asp-85, Asp-212, and Arg-82. As shown recently (Marti, T., R?sselet, S. J., Otto, H., Heyn, M. P., and Khorana, H. G. (1991) J. Biol. Chem. 266, 18674-18683), in the double mutant Asp-85----Asn/Asp-212----Asn chromophore formation is restored in the presence of salts, suggesting that exogenous anions function as counterions to the protonated Schiff base. To investigate the role of Arg-82 and of the Schiff base in anion binding, we have prepared the triple mutant Arg-82----Gln/Asp-85----Asn/Asp-212----Asn and compared its properties with those of the Asp-85----Asn/Asp-212----Asn double mutant. Regeneration of the chromophore with absorption maximum near 560 nm occurs in the triple mutant in the presence of millimolar salt, whereas in the double mutant molar salt concentrations are required. Spectrometric titrations reveal that the pKa of Schiff base deprotonation is markedly reduced from 11.3 for the wild type to 4.9 for the triple mutant in 1 mM NaCl and to 5.5 for the double mutant in 10 mM NaCl. In both mutants, increasing the chloride concentration promotes protonation of the chromophore and results in a continuous rise of the Schiff base pKa, yielding a value of 8.4 and 7.6, respectively, in 4 M NaCl. The absorption maximum of the two mutants shows a progressive red shift, as the ionic radius of the halide increases in the sequence fluoride, chloride, bromide, and iodide. An identical spectral correlation in the presence of halides is observed for the acid-purple form of bacteriorhodopsin. We conclude, therefore, that upon neutralization of the two counterions Asp-85 and Asp-212 by mutation or by protonation at low pH, exogenous anions substitute as counterions by directly binding to the protonated Schiff base. This interaction may provide the basis for the proposed anion translocation by the acid-purple form of bacteriorhodopsin as well as by the related halorhodopsin.     

Thermodynamics and energy coupling in the bacteriorhodopsin photocycle

Time-resolved absorption changes of photoexcited bacteriorhodopsin were measured with a gated multichannel analyzer between 100 ns and 100 ms at six temperatures between 5 and 30 degrees C. The energetics of the chromophore reaction cycle were analyzed on the basis of a model containing a single cycle and reversible reactions. The calculated thermodynamic parameters provide insights to general principles of the active transport. They indicate that in this light-driven proton pump the free energy is retained after absorption of the photon as the enthalpy of the pKa shift in the chromophore which allows deprotonation of the Schiff base. Part of the excess free energy is dissipated at the "switch" step where the reaction and transport cycles are coupled, and the rest at the chromophore recovery step. All other reactions take place near equilibrium. The "switch" step is the M1----M2 transition in the reaction cycle [Váró, G., & Lanyi, J. K. (1991) Biochemistry (preceeding paper in this issue)]. It provides for return of the chromophore pKa to its initial value so the Schiff base will become a proton acceptor, for reordering access of the Schiff base from one side of the membrane to the other, and for unidirectionality of the proton transfer. Conformational energy of the protein, acquired during the "switch" step, drives the completion of the photocycle.     

Energetics of primary processes in visula escitation: photocalorimetry of rhodopsin in rod outer segment membranes.

A sensitive technique for the direct calorimetric determination of the energetics of photochemical reactions under low levels of illumination, and its application to the study of primary processes in visula excitation, are described. Enthlpies are reported for various steps in the bleaching of rhodopsin in intact rod outer segment membranes, together with the heats of appropriate model reactions. Protonation changes are also determined calorimetrically by use of buffers with differing heats of proton ionization. Bleaching of rhodopsin is accompanied by significant uptake of heat energy, vastly in excess of the energy required for simple isomerization of the retinal chromophore. Metarhodopsin I formation involves the uptake of about 17 kcal/mol and no net change in proton ionization of the system. Formation of metarhodopsin II requires an additional energy of about 10 kcal/mol and involves the uptake on one hydrogen ion from solution. The energetics of the overall photolysis reaction, rhodopsin leads to opsin + all-trans-retinal, are pH dependent and involve the exposure of an additional titrating group on opsin. This group has a heat of proton ionization of about 12 kcal/mal, characteristic of a primary amine, but a pKa in the region of neutrality. We suggest that this group is the Schiff base lysine of the chromophore binding site of rhodopsin which becomes exposed on photolysis. The low pKa for this active lysine would result in a more stable retinal-opsin linkage, and might be induced by a nearby positively charged group on the protein (either arginine or a second lysine residue). This leads to a model involving intramolecular protonation of the Schiff base nitrogen in the retinal-opsin linkage of rhodopsin, which is consistent with the thermodynamic and spectroscopic properties of the system. We further propose that the metarhodopsin I leads to metarhodopsin II step in the bleaching sequence involves reversible hydrolysis of the Schiff base linkage in the chromophore binding site, and that subsequent steps are the result of migration of the chromophore from this site.     

Induced chirality of the light-harvesting carotenoid salinixanthin and its interaction with the retinal of xanthorhodopsin

In xanthorhodopsin, a retinal protein-carotenoid complex of Salinibacter ruber, the carotenoid salinixanthin functions as a light-harvesting antenna in supplying additional excitation energy for retinal isomerization and proton transport. Another retinal protein, archaerhodopsin, has been shown to contain a carotenoid, bacterioruberin, but without an antenna function. We report here that the binding site confers a chiral geometry on salinixanthin in xanthorhodopsin and confirm that the same is true for bacterioruberin in archaerhodopsin. Cell membranes containing these rhodopsins exhibit CD spectra with sharp positive bands in the visible region where the carotenoids absorb, and in the case of xanthorhodopsin a negative band at 536 nm, as well as bands in the UV region. The carotenoid in ethanol has very weak optical activity in the visible region of the spectrum. Denaturation of the opsin upon deprotonation of the Schiff base at pH 12.5 eliminates the induced CD bands in both proteins. In one of these proteins, but not in the other, the carotenoid binding site depends entirely on the retinal. Hydrolysis of the retinal Schiff base of xanthorhodopsin with hydroxylamine eliminates the induced CD bands of salinixanthin. In contrast, hydrolysis of the Schiff base in archaerhodopsin does not abolish the CD bands of bacterioruberin. Thus, consistent with its antenna function, the carotenoid binding site interacts closely with the retinal only in xanthorhodopsin, and this interaction is the major source of the CD bands. In this protein, protonation of the counterion with a decrease in pH from 8 to 5 causes significant changes in the CD spectrum. The observed spectral features suggest that binding of salinixanthin in xanthorhodopsin involves the cyclohexenone ring of the carotenoid and its conformational heterogeneity is restricted.     

The influence of pH on antioxidant properties and the mechanism of antioxidant action of hydroxyflavones

The effect of the pH on antioxidant properties of a series of hydroxyflavones was investigated. The pKa of the individual hydroxyl moieties in the hydroxyflavones was compared to computer-calculated deprotonation energies. This resulted in a quantitative structure activity relationship (QSAR), which enables the estimation of pKa values of individual hydroxyl moieties, also in hydroxyflavones for which these pKa values are not available. Comparison of the pKa values to the pH-dependent antioxidant profiles, determined by the TEAC assay, reveals that for various hydroxyflavones the pH-dependent behavior is related to hydroxyl moiety deprotonation, resulting in an increase of the antioxidant potential upon formation of the deprotonated forms. Comparison of these experimental results to computer calculated O-H bond dissociation energies (BDE) and ionization potentials (IP) of the nondeprotonated and the deprotonated forms of the various hydroxyflavones indicates that especially the parameter reflecting the ease of electron donation, i.e., the IP, and not the BDE, is greatly influenced by the deprotonation. Based on these results it is concluded that upon deprotonation the TEAC value increases (radical scavenging capacity increases) because electron-, not H*-, donation becomes easier. Taking into account that the mechanism of radical scavenging antioxidant activity of the neutral form of the hydroxyflavones is generally considered to be hydrogen atom donation, this implies than not only the ease of radical scavenging, but also the mechanism of antioxidant action changes upon hydroxyflavone deprotonation.     

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.     

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.     

Crystal structure analysis of oxidized Pseudomonas aeruginosa azurin at pH 5.5 and pH 9.0. A pH-induced conformational transition involves a peptide bond flip

The X-ray crystal structure of recombinant wild-type azurin from Pseudomonas aeruginosa was determined by difference Fourier techniques using phases derived from the structure of the mutant His35Leu. Two data sets were collected from a single crystal of oxidized azurin soaked in mother liquor buffered at pH 5.5 and pH 9.0, respectively. Both data sets extend to 1.93 A resolution. The two pH forms were refined independently to crystallographic R-factors of 17.6% (pH 5.5) and 17.5% (pH 9.0). The conformational transition previously attributed to the protonation/deprotonation of residue His35 (pKa(red) = 7.3, pKa(ox) = 6.2), which lies in a crevice of the protein close to the copper binding site, involves a concomitant Pro36-Gly37 main-chain peptide bond flip. At the lower pH, the protonated imidazole N delta 1 of His35 forms a strong hydrogen bond with the carbonyl oxygen from Pro36, while at alkaline pH the deprotonated N delta 1 acts as an acceptor of a weak hydrogen bond from HN Gly37. The structure of the remainder of the azurin molecule, including the copper binding site, is not significantly affected by this transition.     

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.     

Protein structural change at the cytoplasmic surface as the cause of cooperativity in the bacteriorhodopsin photocycle.

The effects of excitation light intensity on the kinetics of the bacteriorhodopsin photocycle were investigated. The earlier reported intensity-dependent changes at 410 and 570 nm are explained by parallel increases in two of the rate constants, for proton transfers to D96 from the Schiff base and from the cytoplasmic surface, without changes in the others, as the photoexcited fraction is increased. Thus, it appears that the pKa of D96 is raised by a cooperative effect within the purple membrane. This interpretation of the wild-type kinetics was confirmed by results with several mutant proteins, where the rates are well separated in time and a model-dependent analysis is unnecessary. Based on earlier results that demonstrated a structural change of the protein after deprotonation of the Schiff base that increases the area of the cytoplasmic surface, and the effects of high hydrostatic pressure and lowered water activity on the photocycle steps in question, we suggest that the pKa of D96 is raised by a lateral pressure that develops when other bacteriorhodopsin molecules are photoexcited within the two-dimensional lattice of the purple membrane. Expulsion of no more than a few water molecules bound near D96 by this pressure would account for the calculated increase of 0.6 units in the pKa.     

Monitoring the conformational changes of photoactivated rhodopsin from microseconds to seconds by transient fluorescence spectroscopy

The transient changes of the tryptophan fluorescence of bovine rhodopsin in ROS membranes were followed in time from 1 micros to 10 s after flash excitation of the photoreceptor. Up to about 100 micros the fluorescence did not change, suggesting that the tryptophan lifetimes in rhodopsin and the M(I) intermediate are similar. The fluorescence then decreases on the millisecond time scale with kinetics that match the rise of the M(II) state as measured on the same sample by the transient absorption increase at 360 nm. Both the sign and kinetics of the fluorescence change strongly suggest that it is due to an increase in energy transfer to the retinylidene chromophore caused by the increased spectral overlap in M(II). Calculation of the Forster radius of each tryptophan from the high-resolution crystal structure suggests that W265 and W126 are already completely quenched in the dark, whereas W161, W175, and W35 are located at distances from the retinal chromophore that are comparable to their Forster radii. The fluorescence from these residues is thus sensitive to an increase in energy transfer in M(II). Similar results were obtained at other temperatures and with monomeric rhodopsin in dodecyl maltoside micelles. A large light-induced transient fluorescence increase was observed with ROS membranes that were selectively labeled with Alexa594 at cysteine 316 in helix 8. Using transient absorption spectroscopy the kinetics of this structural change at the cytoplasmic surface was compared to the formation of the signaling state M(II) (360 nm) and to the kinetics of proton uptake as measured with the pH indicator dye bromocresol purple (605 nm). The fluorescence kinetics lags behind the deprotonation of the Schiff base. The proton uptake is even further delayed. These observations show that in ROS membranes (at pH 6) the sequence of events is Schiff base deprotonation, structural change, and proton uptake. From the temperature dependence of the kinetics we conclude that the Schiff base deprotonation and the transient fluorescence have comparable activation energies, whereas that of proton uptake is much smaller.     

Effect of lipid-protein interaction on the color of bacteriorhodopsin

Detergent solubilization and subsequent delipidation of bacteriorhodopsin (bR) results in the formation of a new species absorbing maximally at 480 nm (bR480). Upon lowering the pH, its absorption shifts to 540 nm (bR540). The pK of this equilibrium is 2.6, with the higher pH favoring bR480 (Baribeau, J. and Boucher, F. (1987) Biochim. Biophysica Acta, 890, 275-278). Resonance Raman spectroscopy shows that bR480, like the native bR, contains a protonated Schiff base (PSB) linkage between the chromophore and the protein. However, the Schiff base vibrational frequency in bR480, and its shift upon deuteration, are quite different from these in the native bR, suggesting changes in the Schiff base environment upon delipidation. Infrared absorption and circular-dichroism (CD) spectral studies do not show any net change in the protein secondary structure upon formation of bR480. It is shown that deprotonation of the Schiff base is not the only mechanism of producing hypsochromic shift in the absorption maximum of bR-derived pigments, subtle changes in the protein tertiary structure, affecting the Schiff base environment of the chromophore, may play an equally significant role in the color regulation of bR-derived pigments.     

Role of the cytoplasmic domain in Anabaena sensory rhodopsin photocycling: vectoriality of Schiff base deprotonation

Light-induced electric signals in intact E. coli cells generated by heterologously expressed full-length and C-terminally truncated versions of Anabaena sensory rhodopsin (ASR) demonstrate that the charge movements within the membrane-embedded part of the molecule are stringently controlled by the cytoplasmic domain. In particular, truncation inverts the direction of proton movement during Schiff base deprotonation from outward to cytoplasmic. Truncation also alters faster charge movements that occur before Schiff base deprotonation. Asp(217) as previously shown by FTIR serves as a proton acceptor in the truncated ASR but not in the full-length version, and its mutation to Asn restores the natural outward direction of proton movement. Introduction of a potential negative charge (Ser(86) to Asp) on the cytoplasmic side favors a cytoplasmic direction of proton release from the Schiff base. In contrast, mutation of the counterion Asp(75) to Glu reverses the photocurrent to the outward direction in the truncated pigment, and in both truncated and full-length versions accelerates Schiff base deprotonation more than 10-fold. The communication between the cytoplasmic domain and the membrane-embedded photoactive site of ASR demonstrated here is likely to derive from the receptor's use of a cytoplasmic protein for signal transduction, as has been suggested previously from binding studies.     

Deprotonation of tyrosines in bacteriorhodopsin as studied by Fourier transform infrared spectroscopy with deuterium and nitrate labeling

Fourier transform infrared (FTIR) difference spectra are presented for bacteriorhodopsin (BR) at low temperature. Previous FTIR measurements have identified several tyrosine residues that change their absorption characteristics between light-adapted BR and dark-adapted BR, or between intermediates K and M [Dollinger, G., Eisenstein, L., Lin, S.-L., Nakanishi, K., Odashima, K., & Termini, J. (1986) Methods Enzymol. 127, 649-662]. These changes were explained by protonation/deprotonation of tyrosine moieties and perturbation of the protein environment surrounding tyrosines. A tyrosine deprotonation was observed to occur between intermediates K and M. The present studies confine the deprotonation to being between intermediates L and M and show that no tyrosines undergo changes between the K and the L states. Evidence is presented that none of the tyrosines undergoing changes at low temperature can be assigned to tyrosine-64. The environmental changes of these tyrosines are discussed in relation to the proton pumping mechanism. Their spatial relation to the chromophore is also discussed. At least two tyrosines are suggested to reside close to the retinal binding site. The reactive groups of the nitrated tyrosine-64 are speculated to be remote from the Schiff base and the active tyrosines but can possibly interact sterically with the ionone ring of the retinal.