Properties of Asp212----Asn bacteriorhodopsin suggest that Asp212 and Asp85 both participate in a counterion and proton acceptor complex near the Schiff base.

The gene coding for bacteriorhodopsin was modified in vitro to replace Asp212 with asparagine and expressed in Halobacterium halobium. X-ray diffraction measurements showed that the major lattice dimension of purple membrane containing the mutated bacteriorhodopsin was the same as wild type. At pH greater than 7, the Asp212----Asn chromophore was blue (absorption maximum at 585 nm) and exhibited a photocycle containing only the intermediates K and L, i.e. a reaction sequence very similar to that of wild-type bacteriorhodopsin at pH less than 3 and the blue form of the Asp85----Glu protein at pH less than 9. Since in the latter cases these effects are attributed to protonation of residue 85, it now appears that removal of the carboxylate of Asp212 has similar consequences as removing the carboxylate of Asp85. However, an important difference is that only Asp85 affects the pKa of the Schiff base. At pH less than 7, the Asp212----Asn protein was purple (absorption maximum at 569 nm) but photoexcitation produced only 15% of the normal amount of M and the transport activity was partial. The reactions of the blue and purple forms after photoexcitation are both quantitatively accounted for by a proposed scheme, K in equilibrium with L1 in equilibrium with L2----BR, but with the addition of an L1 in equilibrium with M reaction with unfavorable pKa for Schiff base deprotonation in the purple form. The latter hinders the transient accumulation of M, and the consequent branching at L1 allows only partial proton transport activity. The results are consistent with the existence of a complex counterion for the Schiff base proposed earlier (De Groot, H. J. M., Harbison, G. S., Herzfeld, J., and Griffin, R. G. (1989) Biochemistry 28, 3346-3353) and suggest that Asp85, Asp212, and at least one other protonable residue participate in it.     

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.     

The retinylidene Schiff base counterion in bacteriorhodopsin

Previous studies of bacteriorhodopsin have indicated interactions between Asp-85, Asp-212, Arg-82, and the retinylidene Schiff base. The counterion environment of the Schiff base has now been further investigated by using single and double mutants of the above amino acids. Chromophore regeneration from bacterioopsin proceeds to a normal extent in the presence of a single aspartate or glutamate residue at position 85 or 212, whereas replacement of both charged amino acids in the mutant Asp-85----Asn/Asp-212----Asn abolishes the binding of retinal. This indicates that a carboxylate group at either residue 85 or 212 is required as counterion for formation and for stabilization of the protonated Schiff base. Measurements of the pKa of the Schiff base reveal reductions of greater than 3.5 units for neutral single mutants of Asp-85 but only decreases of less than 1.2 units for corresponding substitutions of Asp-212, relative to the wild type. Substitutions of Asp-85 show large red shifts in the absorption spectrum that are partially reversible upon addition of anions, whereas mutants of Asp-212 display minor red shifts or blue shifts. We conclude, therefore, that Asp-85 is the retinylidene Schiff base counterion in wild-type bacteriorhodopsin. In the mutant Asp-85----Asn/Asp-212----Asn formation of a protonated Schiff base chromophore is restored in the presence of salts. The spectral properties of the double mutant are similar to those of the acid-purple form of bacteriorhodopsin. Upon addition of salts the folded structure of wild-type and mutant proteins can be stabilized at low pH in lipid/detergent micelles. The data indicate that exogenous anions serve as surrogate counterions to the protonated Schiff base, when the intrinsic counterions have been neutralized by mutation or by protonation.     

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.     

FTIR spectroscopy of the M photointermediate in pharaonis rhoborhodopsin

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

Effects of Asp-96----Asn, Asp-85----Asn, and Arg-82----Gln single-site substitutions on the photocycle of bacteriorhodopsin

Bacteriorhodopsin (BR) with the single-site substitutions Arg-82----Gln (R82Q), Asp-85----Asn (D85N), and Asp-96----Asn (D96N) is studied with time-resolved absorption spectroscopy in the time regime from nanoseconds to seconds. Time-resolved spectra are analyzed globally by using multiexponential fitting of the data at multiple wavelengths and times. The photocycle kinetics for BR purified from each mutant are determined for micellar solutions in two detergents, nonyl glucoside and CHAPSO, and are compared to results from studies on delipidated BR (d-BR) in the same detergents. D85N has a red-shifted ground-state absorption spectrum, and the formation of an M intermediate is not observed. R82Q undergoes a pH-dependent transition between a purple and a blue form with different pKa values in the two detergents. The blue form has a photocycle resembling that for D85N, while the purple form of R82Q forms an M intermediate that decays more rapidly than in d-BR. The purple form of R82Q does not light-adapt to the same extent as d-BR, and the spectral changes in the photocycle suggest that the light-adapted purple form of R82Q contains all-trans- and 13-cis-retinal in approximately equal proportions. These results are consistent with the suggestions of others for the roles of Arg-82 and Asp-85 in the photocycle of BR, but results for D96N suggest a more complex role for Asp-96 than previously suggested. In nonyl glucoside, the apparent decay of the M-intermediate is slower in D96N than in d-BR, and the M decay shows biphasic kinetics. However, the role of Asp-96 is not limited to the later steps of the photocycle. In D96N, the decay of the KL intermediate is accelerated, and the rise of the M intermediate has an additional slow phase not observed in the kinetics of d-BR. The results suggest that Asp-96 may play a role in regulating the structure of BR and how it changes during the photocycle.     

Beta-amyloid 25 to 35 is intercalated in anionic and zwitterionic lipid membranes to different extents

D96N bacteriorhodopsin has two photointermediates with the deprotonated Schiff base: the M and MN intermediates. We measure the time-resolved x-ray diffraction of the D96N purple membrane after flash photoexcitation (pH 7.0, 25 degrees C). The data clearly show the M-MN transition during the D96N photocycle. Low-resolution projection maps of these states show that the F helix of the MN intermediate shifts from its original position and this shift is much larger than that of the M intermediate. This indicates that the F helix moves in the M-MN transition of the D96N bacteriorhodopsin photocycle. Moreover, the existence of the MN intermediate in the D96N photocycle under neutral pH indicates that the MN intermediate is not peculiar to the alkaline condition. It is notable that the structural transition of M-MN is independent of the protonation state of the Schiff base. Therefore, the F helix movement precedes reprotonation of the Schiff base in the bacteriorhodopsin photocycle. Our previous study showed that the M-MN transition is hydration-dependent and that the MN intermediate is more hydrated than the M intermediate. Considering this together with the present results, we conclude that the movement of the F helix causes hydration of the cytoplasmic side, which promotes the reprotonation of the Schiff base.     

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.     

Control of the pump cycle in bacteriorhodopsin: mechanisms elucidated by solid-state NMR of the D85N mutant.

By varying the pH, the D85N mutant of bacteriorhodopsin provides models for several photocycle intermediates of the wild-type protein in which D85 is protonated. At pH 10.8, NMR spectra of [zeta-(15)N]lys-, [12-(13)C]retinal-, and [14,15-(13)C]retinal-labeled D85N samples indicate a deprotonated, 13-cis,15-anti chromophore. On the other hand, at neutral pH, the NMR spectra of D85N show a mixture of protonated Schiff base species similar to that seen in the wild-type protein at low pH, and more complex than the two-state mixture of 13-cis,15-syn, and all-trans isomers found in the dark-adapted wild-type protein. These results lead to several conclusions. First, the reversible titration of order in the D85N chromophore indicates that electrostatic interactions have a major influence on events in the active site. More specifically, whereas a straight chromophore is preferred when the Schiff base and residue 85 are oppositely charged, a bent chromophore is found when both the Schiff base and residue 85 are electrically neutral, even in the dark. Thus a "bent" binding pocket is formed without photoisomerization of the chromophore. On the other hand, when photoisomerization from the straight all-trans,15-anti configuration to the bent 13-cis,15-anti does occur, reciprocal thermodynamic linkage dictates that neutralization of the SB and D85 (by proton transfer from the former to the latter) will result. Second, the similarity between the chromophore chemical shifts in D85N at alkaline pH and those found previously in the M(n) intermediate of the wild-type protein indicate that the latter has a thoroughly relaxed chromophore like the subsequent N intermediate. By comparison, indications of L-like distortion are found for the chromophore of the M(o) state. Thus, chromophore strain is released in the M(o)-->M(n) transition, probably coincident with, and perhaps instrumental to, the change in the connectivity of the Schiff base from the extracellular side of the membrane to the cytoplasmic side. Because the nitrogen chemical shifts of the Schiff base indicate interaction with a hydrogen-bond donor in both M states, it is possible that a water molecule travels with the Schiff base as it switches connectivity. If so, the protein is acting as an inward-driven hydroxyl pump (analogous to halorhodopsin) rather than an outward-driven proton pump. Third, the presence of a significant C [double bond] N syn component in D85N at neutral pH suggests that rapid deprotonation of D85 is necessary at the end of the wild-type photocycle to avoid the generation of nonfunctional C [double bond] N syn species.     

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.     

Photochemical reaction cycle and proton transfers in Neurospora rhodopsin.

It was recently found that NOP-1, a membrane protein of Neurospora crassa, shows homology to haloarchaeal rhodopsins and binds retinal after heterologous expression in Pichia pastoris. We report on spectroscopic properties of the Neurospora rhodopsin (NR). The photocycle was studied with flash photolysis and time-resolved Fourier-transform infrared spectroscopy in the pH range 5-8. Proton release and uptake during the photocycle were monitored with the pH-sensitive dye, pyranine. Kinetic and spectral analysis revealed six distinct states in the NR photocycle, and we describe their spectral properties and pH-dependent kinetics in the visible and infrared ranges. The phenotypes of the mutant NR proteins, D131E and E142Q, in which the homologues of the key carboxylic acids of the light-driven proton pump bacteriorhodopsin, Asp-85 and Asp-96, were replaced, show that Glu-142 is not involved in reprotonation of the Schiff base but Asp-131 may be. This implies that, if the NR photocycle is associated with proton transport, it has a low efficiency, similar to that of haloarchaeal sensory rhodopsin II. Fourier-transform Raman spectroscopy revealed unexpected differences between NR and bacteriorhodopsin in the configuration of the retinal chromophore, which may contribute to the less effective reprotonation switch of NR.     

Inversion of proton translocation in bacteriorhodopsin mutants D85N, D85T, and D85,96N.

Proton translocation activity of bacteriorhodopsin mutants lacking the proton acceptor Asp-85 was investigated using the black lipid membrane technique. Mutants D85N, D85T, and D85,96N were constructed and homologously expressed in Halobacterium salinarium to yield a membrane fraction with a buoyant density of 1.18 g/cm3, i.e., identical to that of wild-type purple membrane. In all mutants, the absorbance maximum was red-shifted between 27 and 49 nm compared with wild type, and the pKa values of the respective Schiff bases were reduced to between 8.3 and 8.9 compared with the value of > 13 in wild type. Therefore, a mixture of chromophores absorbing at 410 nm (deprotonated form) and around 600 nm (protonated form) exists at physiological pH. In continuous blue light, the deprotonated form generates stationary photocurrents. The currents are enhanced by a factor of up to 50 upon addition of azide in D85N and D85,96N mutants, whereas D85T shows no azide effect. The direction of these currents is the same as in wild type in yellow light. Yellow light alone is not sufficient to generate stationary currents in the mutants, but increasing yellow light intensity in the presence of blue light leads to an inversion of the current. Because all currents are carried by protons, this two-photon process demonstrates an inverted proton translocation by BR mutants.     

A bacteriorhodopsin analog reconstituted with a nonisomerizable 13-trans retinal derivative displays light insensitivity.

With the aim of preparing a light-insensitive bacteriorhodopsin-like pigment, bacterio-opsin expressed in Escherichia coli was treated in phospholipid-detergent micelles with the retinal analog II, in which the C13-C14 trans-double bond cannot isomerize due to inclusion in a cyclopentene ring. The formation of a complex with a fine structure (lambda max, 439 nm) was first observed. This partially converted over a period of 12 days to a bacteriorhodopsin-like chromophore (ebR-II) with lambda max, 555 nm. An identical behavior has been observed previously upon reconstitution of bleached purple membrane with the analog II. Purification by gel filtration gave pure ebR-II with lambda max, 558 nm, similar to that of light-adapted bacterio-opsin reconstituted with all-trans retinal (ebR-I). Spectrophotometric titration of ebR-II as a function of pH showed that the purple to blue transition of bacteriorhodopsin at acidic pH was altered, and the apparent pKa of Schiff base deprotonation at alkaline pH was lowered by 2.4 units, relative to that of ebR-I. ebR-II showed no light-dark adaptation, no proton pumping, and no intermediates characteristic of the bacteriorhodopsin photocycle. In addition, the rates of reaction with hydroxylamine in the dark and in the light were similar. These results show, as expected, that isomerization of the C13-C14 double bond is required for bacteriorhodopsin function and that prevention of this isomerization confers light insensitivity.     

The proton release group of bacteriorhodopsin controls the rate of the final step of its photocycle at low pH

The factors determining the pH dependence of the formation and decay of the O photointermediate of the bacteriorhodopsin (bR) photocycle were investigated in the wild-type (WT) pigment and in the mutants of Glu-194 and Glu-204, key residues of the proton release group (PRG) in bR. We have found that in the WT the rate constant of O --> bR transition decreases 30-fold upon decreasing the pH from 6 to 3 with a pKa of about 4.3. D2O slows the rise and decay of the O intermediate in the WT at pH 3.5 by a factor of 5.5. We suggest that the rate of the O --> bR transition (which reflects the rate of deprotonation of the primary proton acceptor Asp-85) at low pH is controlled by the deprotonation of the PRG. To test this hypothesis, we studied the E194D mutant. We show that the pKa of the PRG in the ground state of the E194D mutant, when Asp-85 is protonated, is increased by 1.2 pK units compared to that of the WT. We found a similar increase in the pKa of the rate constant of the O --> bR transition in E194D. This provides further evidence that the rate of the O --> bR transition is controlled by the PRG. In a further test, the E194Q mutation, which disables the PRG and slows proton release, almost completely eliminates the pH dependence of O decay at pHs below 6. A second phenomenon we investigated was that in the WT at neutral and alkaline pH the fraction of the O intermediate decreases with pKa 7.5. A similar pH dependence is observed in the mutants in which the PRG is disabled, E194Q and E204Q, suggesting that the decrease in the fraction of the O intermediate with pKa ca. 7.5 is not controlled by the PRG. We propose that the group with pKa 7.5 is Asp-96. The slowing of the reprotonation of Asp-96 at high pH is the cause of the decrease in the rate of the N --> O transition, leading to the decrease in the fraction of O.     

Vibrational spectroscopy of bacteriorhodopsin mutants. Evidence that Thr-46 and Thr-89 form part of a transient network of hydrogen bonds.

The role of Thr-46 and Thr-89 in the bacteriorhodopsin photocycle has been investigated by Fourier transform infrared difference spectroscopy and time-resolved visible absorption spectroscopy of site-directed mutants. Substitutions of Thr-46 and Thr-89 reveal alterations in the chromophore and protein structure during the photocycle, relative to wild-type bacteriorhodopsin. The mutants T89D and to a lesser extent T89A display red shifts in the visible lambda max of the light-adapted states compared with wild type. During the photocycle, T89A exhibits an increased decay rate of the K intermediate, while a K intermediate is not detected in the photocycle of T89D at room temperature. In the carboxyl stretch region of the Fourier transform infrared difference spectra of T89D, a new band appears as early as K formation which is attributed to the deprotonation of Asp-89. Along with this band, an intensity increase occurs in the band assigned to the protonation of Asp-212. In the mutant T46V, a perturbation in the environment of Asp-96 is detected in the L and M intermediates which corresponds to a drop in its pK alpha. These data indicate that Thr-89 is located close to the chromophore, exerts steric constraints on it during all-trans to 13-cis isomerization, and is likely to participate in a hydrogen-bonding network that extends to Asp-212. In addition, a transient interaction between Thr-46 and Asp-96 occurs early in the photocycle. In order to explain these results, a previously proposed model of proton transport is extended to include the existence of a transient network of hydrogen-bonded residues. This model can account for the protonation changes of key amino acid residues during the photocycle of bacteriorhodopsin.     

Fourier transform infrared study of the N intermediate of bacteriorhodopsin

Visible absorption spectroscopic experiments show that the N intermediate is the main photoproduct of a highly hydrated film of the light-adapted bacteriorhodopsin (70% water by weight) at pH 10 and 274 K. The difference Fourier transform infrared spectrum between the N intermediate and unphotolyzed light-adapted bacteriorhodopsin was recorded under these conditions. A small amount of the M intermediate present did not affect this spectrum significantly. The difference spectrum exhibited a positive band at 1755 cm-1 (probably due to Asp-85) and a negative band at 1742 cm-1 (due to Asp-96), neither of which was observed for the M intermediate. The spectrum of the N intermediate at pH 7 was nearly identical with that at pH 10. Spectra at pH 10 also were measured with isotope-substituted samples. A vibrational band at 1692 cm-1 due to the peptide bond disappeared, and a band at 1558 cm-1 emerged upon formation of the N intermediate. The spectrum also displayed bands containing the N-H and C15-H in-plane bending vibrational modes at 1394 and 1303 cm-1. These frequencies are similar to those of the L intermediate while the intensities of these bands are larger than those in the L intermediate, suggesting that the Schiff bases of both the L and N intermediates have a strong hydrogen-bonding interaction with the protein and that the C12-H to C15-H region of the chromophore is less twisted in the N intermediate than in the L intermediate.     

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.     

Formation of the M(N) (M(open)) intermediate in the wild-type bacteriorhodopsin photocycle is accompanied by an absorption spectrum shift to shorter wavelength, like that in the mutant D96N bacteriorhodopsin photocycle

Maximum of the M intermediate difference spectrum in the wild-type Halobacterium salinarium purple membrane is localized at 405-406 nm under conditions favoring accumulation of the M(N) intermediate (6 M guanidine chloride, pH 9.6), whereas immediately after laser flash the maximum is localized at 412 nm. The maximum is also localized at 412 nm 0.1 msec after the flash in the absence of guanidine chloride at pH 11.3. Within several milliseconds the maximum is shifted to short-wavelength region by 5-6 nm. This shift is similar to that in the D96N mutant which accompanies the M(N) (M(open)) intermediate formation. The main two differences are: 1) the rate of the shift is slower in the wild-type bacteriorhodopsin, and is similar to the rate of the M to N intermediate transition (t1/2 approximately 2 msec); 2) the shift in the wild-type bacteriorhodopsin is observed at alkaline pH values which are higher than pK of the Schiff base (approximately 10.8 at 1 M NaCl) in the N intermediate with the deprotonated Asp-96. Thus, the M(N) (M(open)) intermediate with open water-permeable inward proton channel is observed only at high pH, when the Schiff base and Asp-96 are deprotonated. The data confirmed our earlier conclusion that the M intermediate observed at lower pH has the closed inward proton channel.     

The titrations of Asp-85 and of the cation binding residues in bacteriorhodopsin are not coupled

An outstanding problem relating to the structure and function of bacteriorhodopsin (bR), which is the only protein in the purple membrane of the photosynthetic microorganism Halobacterium salinarium, is the relation between the titration of Asp-85 and the binding/unbinding of metal cations. An extensively accepted working hypothesis has been that the two titrations are coupled, namely, protonation of Asp-85 (located in the vicinity of the retinal chromophore) and cation unbinding occur concurrently. We have carried out a series of experiments in which the purple blue equilibrium and the binding of Mn2+ ions (monitored by electron spin resonance) were followed as a function of pH for several (1-4) R = [Mn2+]/[bR] molar ratios. Data were obtained for native bR, bR mutants, artificial bR and chemically modified bR. We find that in the native pigment the two titrations are separated by more than a pKa unit [delta pKa = pKa(P/B)-pKa(Mn2+) = (4.2-2.8) = 1.4]. In the non-native systems, delta pKa values as high as 5 units, as well as negative delta pKas, are observed. We conclude that the pH titration of cation binding residues in bR is not directly related to the titration of Asp-85. This conclusion is relevant to the nature of the high affinity cation sites in bR and to their role in the photosynthetic function of the pigment.     

Chromophore structure in bacteriorhodopsin's N intermediate: implications for the proton-pumping mechanism

By elevating the pH to 9.5 in 3 M KCl, the concentration of the N intermediate in the bacteriorhodopsin photocycle has been enhanced, and time-resolved resonance Raman spectra of this intermediate have been obtained. Kinetic Raman measurements show that N appears with a half-time of 4 +/- 2 ms, which agrees satisfactorily with our measured decay time of the M412 intermediate (2 +/- 1 ms). This argues that M412 decays directly to N in the light-adapted photocycle. The configuration of the chromophore about the C13 = C14 bond was examined by regenerating the protein with [12,14-2H]retinal. The coupled C12-2H + C14-2H rock at 946 cm-1 demonstrates that the chromophore in N is 13-cis. The shift of the 1642-cm-1 Schiff base stretching mode to 1618 cm-1 in D2O indicates that the Schiff base linkage to the protein is protonated. The insensitivity of the 1168-cm-1 C14-C15 stretching mode to N-deuteriation establishes a C = N anti (trans) Schiff base configuration. The high frequency of the C14-C15 stretching mode as well as the frequency of the 966-cm-1 C14-2H-C15-2H rocking mode shows that the chromophore is 14-s-trans. Thus, N contains a 13-cis, 14-s-trans, 15-anti protonated retinal Schiff base.(ABSTRACT TRUNCATED AT 250 WORDS)