FTIR study of the retinal Schiff base and internal water molecules of proteorhodopsin

Proteorhodopsin (PR), an archaeal-type rhodopsin found in marine bacteria, is a light-driven proton pump similar to bacteriorhodopsin (BR). It is known that Asp97, a counterion of the protonated Schiff base, possesses a higher pKa ( approximately 7) compared to that of homologous Asp85 in BR (<3). This suggests that PR has a hydrogen-bonding network different from that of BR. We previously reported that a strongly hydrogen-bonded water molecule is observed only in the alkaline form of PR, where Asp97 is deprotonated (Furutani, Y., Ikeda, D., Shibata, M., and Kandori, H. (2006) Chem. Phys. 324, 705-708). This is probably correlated with the pH-dependent proton pumping activity of PR. In this work, we studied the water-containing hydrogen-bonding network in the Schiff base region of PR by means of Fourier-transform infrared (FTIR) spectroscopy at 77 K. [zeta-15N]Lys-labeling and 18O water were used for assigning the Schiff base N-D and water O-D stretching vibrations in D2O, respectively. The frequency upshift of the N-D stretch in the primary K intermediate is much smaller for PR than for BR, indicating that the Schiff base forms a hydrogen bond after retinal photoisomerization. We then measured FTIR spectra of the mutants of Asp97 (D97N and D97E) and Asp227 (D227N and D227E) to identify the amino acid interacting with the Schiff base in the K state. The PRK minus PR spectra of D97N and D97E were similar to those of the acidic and alkaline forms, respectively, of the wild type implying that the structural changes upon retinal photoisomerization are not influenced by the mutation at Asp97. In contrast, clear spectral differences were observed in D227N and D227E, including vibrational bands of the Schiff base and water molecules. It is concluded that Asp227 plays a crucial role during the photoisomerization process, though Asp97 acts as the primary counterion in the unphotolyzed state of PR.     

Characterisation of Schiff base and chromophore in green proteorhodopsin by solid-state NMR

The proteorhodopsin family consists of hundreds of homologous retinal containing membrane proteins found in bacteria in the photic zone of the oceans. They are colour tuned to their environment and act as light-driven proton pumps with a potential energetic and regulatory function. Precise structural details are still unknown. Here, the green proteorhodopsin variant has been selected for a chemical shift analysis of retinal and Schiff base by solid-state NMR. Our data show that the chromophore exists in mainly all-trans configuration in the proteorhodopsin ground state. The optical absorption maximum together with retinal and Schiff base chemical shifts indicate a strong interaction network between chromophore and opsin. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Mark Lorch and Andreas C. Woerner contributed equally to this work.     

Changing the location of the Schiff base counterion in rhodopsin.

Rhodopsin and all of the vertebrate visual pigments have a carboxylic acid residue, Glu113, in the third transmembrane segment that serves as a counterion to the protonated Schiff base nitrogen of the chromophore. We show here that the counterion in bovine rhodopsin can be moved from position 113 to 117 without significantly changing the wild-type spectral properties of the protein. A series of double mutants were constructed where the Glu113 counterion was changed to Gln and an Asp residue was substituted for amino acid residues from position 111 to 121 in the third transmembrane segment of the protein. Only at position 117 can an Asp fully substitute for the counterion at position 113. The double mutant E113Q,-A117D has an absorption maximum at 493 nm which is independent of pH in the range 5.6-8.4 and independent of the presence of external chloride anions. An Asp at no other position tested in the third transmembrane segment can fully substitute for the Glu counterion at position 113. Partial substitution is observed for an Asp at position 120. Residues 113, 117, and 120 are expected to lie along the same face of an alpha-helix. These results suggest that the Schiff base nitrogen in rhodopsin is located between residues 113 and 117 but there is enough flexibility in the protein to allow partial interaction with an Asp at position 120. Position 117 is the same location of the counterion in the related biogenic amine receptors.     

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.     

Correlation of the O-intermediate rate with the pKa of Asp-75 in the dark, the counterion of the Schiff base of Pharaonis phoborhodopsin (sensory rhodopsin II)

Pharaonis phoborhodopsin (ppR), also called pharaonis sensory rhodopsin II, NpSRII, is a photoreceptor of negative phototaxis in Natronomonas (Natronobacterium) pharaonis. The photocycle rate of ppR is slow compared to that of bacteriorhodopsin, despite the similarity in their x-ray structures. The decreased rate of the photocycle of ppR is a result of the longer lifetime of later photo-intermediates such as M- (ppR(M)) and O-intermediates (ppR(O)). In this study, mutants were prepared in which mutated residues were located on the extracellular surface (P182, P183, and V194) and near the Schiff base (T204) including single, triple (P182S/P183E/V194T), and quadruple mutants. The decay of ppR(O) of the triple mutant was accelerated approximately 20-times from 690 ms for the wild-type to 36 ms. Additional mutation resulting in a triple mutant at the 204th position such as T204C or T204S further decreased the decay half-time to 6.6 or 8 ms, almost equal to that of bacteriorhodopsin. The decay half-times of the ppR(O) of mutants (11 species) and those of the wild-type were well-correlated with the pK(a) value of Asp-75 in the dark for the respective mutants as spectroscopically estimated, although there are some exceptions. The implications of these observations are discussed in detail.     

Regulation of phototransduction in short-wavelength cone visual pigments via the retinylidene Schiff base counterion.

Short-wavelength visual pigments (SWS1) have lambda(max) values that range from the ultraviolet to the blue. Like all visual pigments, this class has an 11-cis-retinal chromophore attached through a Schiff base linkage to a lysine residue of opsin apoprotein. We have characterized a series of site-specific mutants at a conserved acidic residue in transmembrane helix 3 in the Xenopus short-wavelength sensitive cone opsin (VCOP, lambda(max) approximately 427 nm). We report the identification of D108 as the counterion to the protonated retinylidene Schiff base. This residue regulates the pK(a) of the Schiff base and, neutralizing this charge, converts the violet sensitive pigment into one that absorbs maximally in the ultraviolet region. Changes to this position cause the pigment to exhibit two chromophore absorbance bands, a major band with a lambda(max) of approximately 352-372 nm and a minor, broad shoulder centered around 480 nm. The behavior of these two absorbance bands suggests that these represent unprotonated and protonated Schiff base forms of the pigment. The D108A mutant does not activate bovine rod transducin in the dark but has a significantly prolonged lifetime of the active MetaII state. The data suggest that in short-wavelength sensitive cone visual pigments, the counterion is necessary for the characteristic rapid production and decay of the active MetaII state.     

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

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

Formation of a long-lived photoproduct with a deprotonated Schiff base in proteorhodopsin, and its enhancement by mutation of Asp227

Proteorhodopsin, a retinal protein of marine proteobacteria similar to bacteriorhodopsin of the archaea, is a light-driven proton pump. Absorption of a light quantum initiates a reaction cycle (turnover time of ca. 50 ms), which includes photoisomerization of the retinal from the all-trans to the 13-cis form and transient deprotonation of the retinal Schiff base, followed by recovery of the initial state. We report here that in addition to this fast cyclic conversion, illumination at high pH results in accumulation of a long-lived photoproduct absorbing at 362 nm. This photoconversion is much more efficient in the D227N mutant in which the anionic Asp227, which together with Asp97 constitutes the Schiff base counterion, is replaced with a neutral residue. Upon illumination at pH 8.5, most of the D227N pigment is converted to the 362 nm species, with a quantum efficiency of ca. 0.2. The pK(a) for this transition in the wild type is 9.6, but decreased to 7.5 after mutation of Asp227. The short wavelength of the absorption maximum of the photoproduct indicates that it has a deprotonated Schiff base. In the dark, this photoproduct is converted back to the initial pigment with a time constant of 30 min (in D227N, at pH 8.5), but it can be reconverted more rapidly by illumination with near-UV light. Experiments with "locked" retinal analogues which selectively exclude rotation around either the C9=C10, C11=C12, or C13=C14 bond show that formation of the 362 nm species involves isomerization around the C13=C14 bond. In agreement with this, retinal extraction indicates that the 362 nm photoproduct is 13-cis whereas the initial state is predominantly all-trans. A rapid shift of the pH from 8.5 to 4 greatly accelerates thermal reconversion of the 362 nm species to the initial pigment, suggesting that its recovery involving the thermal isomerization of the chromophore is controlled by ionizable residues, primarily the Schiff base and Asp97. The transformation to the long-lived 362 nm photoproduct is apparently a side reaction of the photocycle, a response to high pH, caused by alteration of the normal reprotonation and reisomerization pathway of the Schiff base.     

Conformational changes in the photocycle of Anabaena sensory rhodopsin: absence of the Schiff base counterion protonation signal

Anabaena sensory rhodopsin (ASR) is a novel microbial rhodopsin recently discovered in the freshwater cyanobacterium Anabaena sp. PCC7120. This protein most likely functions as a photosensory receptor as do the related haloarchaeal sensory rhodopsins. However, unlike the archaeal pigments, which are tightly bound to their cognate membrane-embedded transducers, ASR interacts with a soluble cytoplasmic protein analogous to transducers of animal vertebrate rhodopsins. In this study, infrared spectroscopy was used to examine the molecular mechanism of photoactivation in ASR. Light adaptation of the pigment leads to a phototransformation of an all-trans/15-anti to 13-cis/15-syn retinylidene-containing species very similar in chromophore structural changes to those caused by dark adaptation in bacteriorhodopsin. Following 532 nm laser-pulsed excitation, the protein exhibits predominantly an all-trans retinylidene photocycle containing a deprotonated Schiff base species similar to those of other microbial rhodopsins such as bacteriorhodopsin, sensory rhodopsin II, and Neurospora rhodopsin. However, no changes are observed in the Schiff base counterion Asp-75, which remains unprotonated throughout the photocycle. This result along with other evidence indicates that the Schiff base proton release mechanism differs significantly from that of other known microbial rhodopsins, possibly because of the absence of a second carboxylate group at the ASR photoactive site. Several conformational changes are detected during the ASR photocycle including in the transmembrane helices E and G as indicated by hydrogen-bonding alterations of their native cysteine residues. In addition, similarly to animal vertebrate rhodopsin, perturbations of the polar head groups of lipid molecules are detected.     

Aspartate-histidine interaction in the retinal schiff base counterion of the light-driven proton pump of Exiguobacterium sibiricum

One of the distinctive features of eubacterial retinal-based proton pumps, proteorhodopsins, xanthorhodopsin, and others, is hydrogen bonding of the key aspartate residue, the counterion to the retinal Schiff base, to a histidine. We describe properties of the recently found eubacterium proton pump from Exiguobacterium sibiricum (named ESR) expressed in Escherichia coli, especially features that depend on Asp-His interaction, the protonation state of the key aspartate, Asp85, and its ability to accept a proton from the Schiff base during the photocycle. Proton pumping by liposomes and E. coli cells containing ESR occurs in a broad pH range above pH 4.5. Large light-induced pH changes indicate that ESR is a potent proton pump. Replacement of His57 with methionine or asparagine strongly affects the pH-dependent properties of ESR. In the H57M mutant, a dramatic decrease in the quantum yield of chromophore fluorescence emission and a 45 nm blue shift of the absorption maximum with an increase in the pH from 5 to 8 indicate deprotonation of the counterion with a pK(a) of 6.3, which is also the pK(a) at which the M intermediate is observed in the photocycle of the protein solubilized in detergent [dodecyl maltoside (DDM)]. This is in contrast with the case for the wild-type protein, for which the same experiments show that the major fraction of Asp85 is deprotonated at pH >3 and that it protonates only at low pH, with a pK(a) of 2.3. The M intermediate in the wild-type photocycle accumulates only at high pH, with an apparent pK(a) of 9, via deprotonation of a residue interacting with Asp85, presumably His57. In liposomes reconstituted with ESR, the pK(a) values for M formation and spectral shifts are 2-3 pH units lower than in DDM. The distinctively different pH dependencies of the protonation of Asp85 and the accumulation of the M intermediate in the wild-type protein versus the H57M mutant indicate that there is strong Asp-His interaction, which substantially lowers the pK(a) of Asp85 by stabilizing its deprotonated state.     

Light activation of the isomerization and deprotonation of the protonated Schiff base retinal

We perform an ab initio analysis of the photoisomerization of the protonated Schiff base of retinal (PSB-retinal) from 11-cis to 11-trans rotating the C10-C11=C12-C13 dihedral angle from 0° (cis) to -180° (trans). We find that the retinal molecule shows the lowest rotational barrier (0.22 eV) when its charge state is zero as compared to the barrier for the protonated molecule which is ∼0.89 eV. We conclude that rotation most likely takes place in the excited state of the deprotonated retinal. The addition of a proton creates a much larger barrier implying a switching behavior of retinal that might be useful for several applications in molecular electronics. All conformations of the retinal compound absorb in the green region with small shifts following the dihedral angle rotation; however, the Schiff base of retinal (SB-retinal) at trans-conformation absorbs in the violet region. The rotation of the dihedral angle around the C11=C12 π-bond affects the absorption energy of the retinal and the binding energy of the SB-retinal with the proton at the N-Schiff; the binding energy is slightly lower at the trans-SB-retinal than at other conformations of the retinal.     

Serine 85 in transmembrane helix 2 of short-wavelength visual pigments interacts with the retinylidene Schiff base counterion.

Short-wavelength cone visual pigments (SWS1) are responsible for detecting light from 350 to 430 nm. Models of this class of pigment suggest that TM2 has extensive contacts with the retinal binding pocket and stabilizes interhelical interactions. The role of TM2 in the structure-function of the Xenopus SWS1 (VCOP, lambda(max) = 427 nm) pigment was studied by replacement of the helix with that of bovine rhodopsin and also by mutagenesis of highly conserved residues. The TM2 chimera and G78D, F79L, M81E, P88T, V89S, and F90V mutants did not produce any significant spectral shift of the dark state or their primary photointermediate formed upon illumination at cryogenic temperatures. The mutant G77R (responsible for human tritanopia) was completely defective in folding, while C82A and F87T bound retinal at reduced levels. The position S85 was crucial for obtaining the appropriate spectroscopic properties of VCOP. S85A and S85T did not bind retinal. S85D bound retinal and had a wild-type dark state at room temperature and a red-shifted dark state at 45 K and formed an altered primary photointermediate. S85C absorbed maximally at 390 nm at neutral pH and at 365 nm at pH >7.5. The S85C dark state was red shifted by 20 nm at 45 K and formed an altered primary photointermediate. These data suggest that S85 is involved in a hydrogen bond with the protonated retinylidene Schiff base counterion in both the dark state and the primary photointermediate.     

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.     

Nuclear magnetic resonance study of the Schiff base in bacteriorhodopsin: counterion effects on the 15N shift anisotropy

High-resolution, solid-state 15N NMR has been used to study the chemical shift anisotropies of the Schiff bases in bacteriorhodopsin (bR) and in an extensive series of model compounds. Using slow-spinning techniques, we are able to obtain sufficient rotational sideband intensity to determine the full 15N chemical shift anisotropy for the Schiff base nitrogen in bR548 and bR568. Comparisons are made between all-trans-bR568 and N-all-trans-retinylidene butylimine salts with halide, phenolate, and carboxylate counterions. It is argued that for the model compounds the variation in 15N chemical shift reflects the variation in (hydrogen) bond strength with the various counterions. The results suggest that carboxylates and tyrosinates may form hydrogen bonds of comparable strength in a hydrophobic environment. Thus, the hydrogen bonding strength of a counterion depends on factors that are not completely reflected in the solution pKa of its conjugate acid. For the model compounds, the two most downfield principal values of the 15N chemical shift tensor, sigma 22 and sigma 33, vary dramatically with different counterions, whereas sigma 11 remains essentially unaffected. In addition, there exists a linear correlation between sigma 22 and sigma 33, which suggests that a single mechanism is responsible for the variation in chemical shifts present in all three classes of model compounds. The data for bR568 follow this trend, but the isotropic shift is 11 ppm further upfield than any of the model compounds. This extreme value suggests an unusually weak hydrogen bond in the protein.     

Estimated acid dissociation constants of the Schiff base, Asp-85, and Arg-82 during the bacteriorhodopsin photocycle.

The pK(a) values of D85 in the wild-type and R82Q, as well as R82A recombinant bacteriorhodopsins, and the Schiff base in the D85N, D85T, and D85N/R82Q proteins, have been determined by spectroscopic titrations in the dark. They are used to estimate the coulombic interaction energies and the pK(a) values of the Schiff base, D85, and R82 during proton transfer from the Schiff base to D85, and the subsequent proton release to the bulk in the initial part of the photocycle. The pK(a) of the Schiff base before photoexcitation is calculated to be in effect only 5.3-5.7 pH units higher than that of D85; overcoming this to allow proton transfer to D85 requires about two thirds of the estimated excess free energy retained after absorption of a photon. The proton release on the extracellular surface is from an unidentified residue whose pK(a) is lowered to about 6 after deprotonation of the Schiff base (Zimanyi, L., G. Varo, M. Chang, B. Ni, R. Needleman, and J.K. Lanyi, 1992. Biochemistry. 31:8535-8543). We calculate that the pK(a) of the R82 is 13.8 before photoexcitation, and it is lowered after proton exchange between the Schiff base and D85 only by 1.5-2.3 pH units. Therefore, coulombic interactions alone do not appear to change the pK(a) of R82 as much and D85 only by 1.5-2.3 pH units. Therefore, coulombic interactions alone do not appear to change the pK(a) of R82 as much as required if it were the proton release group.     

Solid state 15N NMR evidence for a complex Schiff base counterion in the visual G-protein-coupled receptor rhodopsin.

Using the baculovirus/Sf9 cell expression system, we have incorporated 99% 15N-enriched [alpha,epsilon-15N2]-L-lysine into the rod visual pigment rhodopsin. We have subsequently investigated the protonated Schiff base (pSB) linkage in the [alpha, epsilon-15N2]Lys-rhodopsin with cross-polarization magic angle spinning (CP/MAS) 15N NMR. The Schiff base (SB) 15N in [alpha, epsilon-15N2]Lys-rhodopsin resonates with an isotropic shift sigmaI of 155.9 ppm, relative to 5.6 M 15NH4Cl. This suggests that the SB in rhodopsin is protonated and stabilized by a complex counterion. The 15N shifts of retinal SBs correlate with the energy difference between the ground and excited states and the frequency of maximum visible absorbance, numax, associated with the pi-pi transition of the polyene chromophore. Experimental modeling of the relation between the numax and the size of the counterion with a set of pSBs provides strong evidence that the charged chromophore in rhodopsin is stabilized by a counterion with an estimated effective center-center distance (deff) between the counterion and the pSB of 0.43 +/- 0.01 nm. While selected prokaryotic proteins and complexes have been labeled before, this is the first time to our knowledge that a 15N-labeled eukaryotic membrane protein has been generated in sufficient amount for such NMR investigations.     

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.     

Ultrafast excited state dynamics of the protonated Schiff base of all-trans retinal in solvents

We present a comparative study of the ultrafast photophysics of all-trans retinal in the protonated Schiff base form in solvents with different polarities and viscosities. Steady-state spectra of retinal in the protonated Schiff base form show large absorption-emission Stokes shifts (6500-8100 cm(-1)) for both polar and nonpolar solvents. Using a broadband fluorescence up-conversion experiment, the relaxation kinetics of fluorescence is investigated with 120 fs time resolution. The time-zero spectra already exhibit a Stokes-shift of approximately 6000 cm(-1), indicating depopulation of the Franck-Condon region in < or =100 fs. We attribute it to relaxation along skeletal stretching. A dramatic spectral narrowing is observed on a 150 fs timescale, which we assign to relaxation from the S(2) to the S(1) state. Along with the direct excitation of S(1), this relaxation populates different quasistationary states in S(1), as suggested from the existence of three distinct fluorescence decay times with different decay associated spectra. A 0.5-0.65 ps decay component is observed, which may reflect the direct repopulation of the ground state, in line with the small isomerization yield in solvents. Two longer decay components are observed and are attributed to torsional motion leading to photo-isomerization. The various decay channels show little or no dependence with respect to the viscosity or dielectric constant of the solvents. This suggests that in the protein, the bond selectivity of isomerization is mainly governed by steric effects.     

Role of the retinal hydrogen bond network in rhodopsin Schiff base stability and hydrolysis

Little is known about the molecular mechanism of Schiff base hydrolysis in rhodopsin. We report here our investigation into this process focusing on the role of amino acids involved in a hydrogen bond network around the retinal Schiff base. We find conservative mutations in this network (T94I, E113Q, S186A, E181Q, Y192F, and Y268F) increase the activation energy (E(a)) and abolish the concave Arrhenius plot normally seen for Schiff base hydrolysis in dark state rhodopsin. Interestingly, two mutants (T94I and E113Q) show dramatically faster rates of Schiff base hydrolysis in dark state rhodopsin, yet slower hydrolysis rates in the active MII form. We find deuterium affects the hydrolysis process in wild-type rhodopsin, exhibiting a specific isotope effect of approximately 2.5, and proton inventory studies indicate that multiple proton transfer events occur during the process of Schiff base hydrolysis for both dark state and MII forms. Taken together, our study demonstrates the importance of the retinal hydrogen bond network both in maintaining Schiff base integrity in dark state rhodopsin, as well as in catalyzing the hydrolysis and release of retinal from the MII form. Finally, we note that the dramatic alteration of Schiff base stability caused by mutation T94I may play a causative role in congenital night blindness as has been suggested by the Oprian and Garriga laboratories.     

Regulation of photoactivation in vertebrate short wavelength visual pigments: protonation of the retinylidene Schiff base and a counterion switch

Xenopus violet cone opsin (VCOP) and its counterion variant (VCOP-D108A) are expressed in mammalian COS1 cells and regenerated with 11-cis-retinal. The phototransduction process in VCOP-D108A is investigated via cryogenic electronic spectroscopy, homology modeling, molecular dynamics, and molecular orbital theory. The VCOP-D108A variant is a UV-like pigment that displays less efficient photoactivation than the mouse short wavelength sensitive visual pigment (MUV) and photobleaching properties that are significantly different. Theoretical calculations trace the difference to the protonation state of the nearby glutamic acid residue E176, which is the homology equivalent of E181 in rhodopsin. We find that E176 is negatively charged in MUV but neutral (protonated) in VCOP-D108A. In the dark state, VCOP-D108A has an unprotonated Schiff base (SB) chromophore (lambdamax = 357 nm). Photolysis of VCOP-D108A at 70 K generates a bathochromic photostationary state (lambdamax = 380 nm). We identify two lumi intermediates, wherein the transitions from batho to the lumi intermediates are temperature- and pH-dependent. The batho intermediate decays to a more red-shifted intermediate called lumi I. The SB becomes protonated during the lumi I to lumi II transition. Decay of lumi II forms meta I, followed by the formation of meta II. We conclude that even in the absence of a primary counterion in VCOP-D108A, the SB becomes protonated during the photoactivation cascade. We examine the relevance of this observation to the counterion switch mechanism of visual pigment activation.