Resonance Raman study of intermediates of the halorhodopsin photocycle

The resonance Raman (RR) study of the retinal protein halorhodopsin (HR₅₇₈) was extended to two of its photoproducts: HR and HR^L₄₁₀ RR spectra of both species were recorded in H₂O and D₂O and compared with the RR spectra of the intermediates L₅₅₀ and M₄₁₂ from the bacteriorhodopsin photocycle. HR₅₂₀ was found to be a protonated Schiff base in the 13-cis configuration and HR^L₄₁₀ a deprotonated Schiff base in the 13-cis configuration.     

细菌视紫红质的质子传输机理

细菌视紫红质（bR）是嗜盐菌紫膜中的唯一蛋白质成分, 具有质子泵、电荷分离和光致变色功能. bR分子中的发色团视黄醛通过质子化席夫碱以共价键与Lys216相连. bR分子受可见光照射后, 视黄醛发生从全-反到13-顺式构型的异构化, 导致席夫碱的去质子化,继之以可极化基团位置的改变. 力场的变化引起包括蛋白质三级结构在内的诸多变化, 这些变化促进并保证了质子从细胞质侧向细胞外侧的定向传输.     

Effect of iodination on the pK of Schiff base deprotonation and M412 production in purple membrane

Previously, kinetic resonance Raman measurements as a function of pH have been used to demonstrate that, microseconds after light absorption, the pK of Schiff base deprotonation during the bacteriorhodopsin photocycle is 10.2 ± 0.3, whereas before the light event, the pK is > 12 (2). In this investigation, we have iodinated purple membrane suspensions and have found that the pK of Schiff base deprotonation in the photocycle has been lowered to between 7 and 8 for iodinated bacteriorhodopsin. These results, together with our previous data on the pK of Schiff base deprotonation, suggest that the amino acid tyrosine could be a critical component in the deprotonation mechanism.     

Structure of the retinal chromophore in sensory rhodopsin I from resonance Raman spectroscopy

Sensory rhodopsin I (SR-I) is a retinal-containing pigment which functions as a phototaxis receptor in Halobacterium halobium. We have obtained resonance Raman vibrational spectra of the native membrane-bound form of SR587 and used these data to determine the structure of its retinal prosthetic group. The similar frequencies and intensities of the skeletal fingerprint modes in SR587, bacteriorhodopsin (BR568), and halorhodopsin (HR578) as well as the position of the dideuterio rocking mode when SR-I is regenerated with 12,14-D2 retinal (915 cm-1) demonstrate that the retinal chromophore has an all-trans configuration. The shift of the C = N stretching mode from 1628 cm-1 in H2O to 1620 cm-1 in D2O demonstrates that the chromophore in SR587 is bound to the protein by a protonated Schiff base linkage. The small shift of the 1195 cm-1 C14-C15 stretching mode in D2O establishes that the protonated Schiff base bond has an anti configuration. The low value of the Schiff base stretching frequency together with its small 8 cm-1 shift in D2O indicates that the Schiff base proton is weakly hydrogen bonded to its protein counterion. This suggests that the red shift in the absorption maximum of SR-I (587 nm) compared with HR (578 nm) and BR (568 nm) is due to a reduction of the electrostatic interaction between the protonated Schiff base group and its protein counterion.     

Resonance enhanced Raman spectrum of all-trans anhydrovitamin A.

The Raman spectrum of all-trans anhydrovitamin A in hexane at 77 degrees K is presented. The similarity of the Raman spectra of anhydrovitamin A and the protonated Schiff base of retinal is striking. The implications of this for visual pigment studies and bacteriorhodopsin are discussed. Tentative assignments of geometry for four cis-trans isomers of anhydrovitamin A are made on the basis of the observed room-temperature absorption spectra.     

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

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

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

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

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

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

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

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

Time-resolved ultraviolet resonance Raman studies of protein structure: application to bacteriorhodopsin.

Time-resolved ultraviolet resonance Raman spectra of bacteriorhodopsin are used to study protein structural changes on the nanosecond and millisecond time scales. Excitation at 240 nm is used to selectively enhance vibrational scattering from tyrosine so that changes in its hydrogen bonding and protonation state can be examined. Both nanosecond and millisecond UV Raman difference spectra indicate that none of the tyrosine residues change ionization state during the BR----K and BR----M transitions. However, intensity changes are observed at 1172 and 1615 cm-1 in the BR----M UV Raman difference spectra. The 1615-cm-1 feature shifts down 25 cm-1 in tyrosine-d4-labeled BR, consistent with its assignment as a tyrosine vibration. The intensity changes in the BR----M UV Raman difference spectra most likely reflect an increase in resonance enhancement that occurs when one or more tyrosine residues interact more strongly with a hydrogen-bond acceptor in M412. The frequency of the v7a feature (1172 cm-1) in the BR----M UV Raman difference spectra supports this interpretation. The proximity of Tyr-185 and Asp-212 in the retinal binding pocket suggests that deprotonation of the Schiff base in M412 causes Tyr-185 to stabilize ionized Asp-212 by forming a stronger hydrogen bond.     

Trans-cis isomerization of retinal and a mechanism for ion translocation in halorhodopsin

The chromophore in halorhodopsin (HR) which acts as a light-driven chloride pump in halobacteria shares many properties with its counterpart in bacteriorhodopsin (BR): (i) a similar retinal protein interaction, (ii) trans to cis isomerization and (iii) similar intermediates of its photocycle. One major difference between the two chromoproteins is that the HR chromophore does not become deprotonated during its photocycle. A mechanism for the photocycle of HR is presented, which, in close analogy to an earlier proposed mechanism for BR, involves the sequence of all-trans 13-cis, 14s-cis 13-cis all-trans isomerizations of the chromophore, a Schiff base of retinal. In contrast to the situation in BR the 13-cis, 14s-cis13-cis isomerization is induced not by deprotonation of the retinal Schiff base chromophore but rather by the movement of an anion (Cl^-) towards the protonated nitrogen of the Schiff's base. The suggested mechanism involves the Schiff base directly in the chloride translocation in halorhodopsin.     

Vibrational analysis of the all-trans retinal protonated Schiff base.

We have obtained Raman spectra of a series of all-trans retinal protonated Schiff-base isotopic derivatives. 13C-substitutions were made at the 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 positions while deuteration was performed at position 15. Based on the isotopic shifts, the observed C--C stretching vibrations in the 1,100-1,400 cm-1 fingerprint region are assigned. Normal mode calculations using a modified Urey-Bradley force field have been refined to reproduce the observed frequencies and isotopic shifts. Comparison with fingerprint assignments of all-trans retinal and its unprotonated Schiff base shows that the major effect of Schiff-base formation is a shift of the C14--C15 stretch from 1,111 cm-1 in the aldehyde to approximately 1,163 cm-1 in the Shiff base. This shift is attributed to the increased C14--C15 bond order that results from the reduced electronegativity of the Schiff-base nitrogen compared with the aldehyde oxygen. Protonation of the Schiff base increases pi-electron delocalization, causing a 6 to 16 cm-1 frequency increase of the normal modes involving the C8--C9, C10--C11, C12--C13, and C14--C15 stretches. Comparison of the protonated Schiff base Raman spectrum with that of light-adapted bacteriorhodopsin (BR568) shows that incorporation of the all-trans protonated Schiff base into bacterio-opsin produces an additional approximately 10 cm-1 increase of each C--C stretching frequency as a result of protein-induced pi-electron delocalization. Importantly, the frequency ordering and spacing of the C--C stretches in BR568 is the same as that found in the protonated Schiff base.     

Determination of retinal chromophore structure in bacteriorhodopsin with resonance Raman spectroscopy

The analysis of the vibrational spectrum of the retinal chromophore in bacteriorhodopsin with isotopic derivatives provides a powerful "structural dictionary" for the translation of vibrational frequencies and intensities into structural information. Of importance for the proton-pumping mechanism is the unambiguous determination of the configuration about the C13=C14 and C=N bonds, and the protonation state of the Schiff base nitrogen. Vibrational studies have shown that in light-adapted BR568 the Schiff base nitrogen is protonated and both the C13=C14 and C=N bonds are in a trans geometry. The formation of K625 involves the photochemical isomerization about only the C13=C14 bond which displaces the Schiff base proton into a different protein environment. Subsequent Schiff base deprotonation produces the M412 intermediate. Thermal reisomerization of the C13=C14 bond and reprotonation of the Schiff base occur in the M412------O640 transition, resetting the proton-pumping mechanism. The vibrational spectra can also be used to examine the conformation about the C--C single bonds. The frequency of the C14--C15 stretching vibration in BR568, K625, L550 and O640 argues that the C14--C15 conformation in these intermediates is s-trans. Conformational distortions of the chromophore have been identified in K625 and O640 through the observation of intense hydrogen out-of-plane wagging vibrations in the Raman spectra (see Fig. 2). These two intermediates are the direct products of chromophore isomerization. Thus it appears that following isomerization in a tight protein binding pocket, the chromophore cannot easily relax to a planar geometry. The analogous observation of intense hydrogen out-of-plane modes in the primary photoproduct in vision (Eyring et al., 1982) suggests that this may be a general phenomenon in protein-bound isomerizations. Future resonance Raman studies should provide even more details on how bacterio-opsin and retinal act in concert to produce an efficient light-energy convertor. Important unresolved questions involve the mechanism by which the protein catalyzes deprotonation of the L550 intermediate and the mechanism of the thermal conversion of M412 back to BR568. Also, it has been shown that under conditions of high ionic strength and/or low light intensity two protons are pumped per photocycle (Kuschmitz & Hess, 1981). How might this be accomplished?(ABSTRACT TRUNCATED AT 400 WORDS)     

Time-resolved resonance Raman characterization of the bL550 intermediate and the two dark-adapted bRDA/560 forms of bacteriorhodopsin.

The resonance Raman spectrum of the second intermediate in the bacteriorhodopsin cycle, bL550, is obtained by a simple flow technique. The Schiff base linkage in this intermediate appears to be protonated, contrary to previous suggestion. The fingerprint region of the spectrum of bL550 does not closely match those of any presently available model Schiff bases of retinal isomers, though some comparisons can be made. The resonance Raman spectrum of dark-adapted bacteriorhodopsin is obtained and decomposed by computer subtraction of the spectrum of bR570. The remaining spectrum does not match the spectra of any model compounds presently in the literature. The spectra of bL550 and dark-adapted bRDA/560 from purple membrane in H2O are compared to those in D2O. It is found that changes in the spectrum occur in the 1,600 - 1,650 cm-1 region as well as in the 800 - 1,000 cm-1 region, but apparently not in the fingerprint region (1,100 - 1,400 cm-1). The possibilities of conformational changes of the retinal chromophore in the light adaptation process as well as the photosynthetic cycle are discussed.     

The pK of Schiff base deprotonation in bacteriorhodopsin.

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

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.     

Evidence for a 13,14-Cis Cycle in Bacteriorhodopsin

We discuss to what extent the vibrational spectra of bacteriorhodopsin that have been observed and assigned by Smith et al. (1, 2) by means of resonance Raman and by Gerwert and Siebert (EMBO (Eur. Mol. Biol. Organ.) J. In press) by means of infrared absorption experiments are in agreement with a photo-cycle of bacteriorhodopsin that involves the sequence BR, IO(all-trans) → K(13,14-cis) → L(13,14-cis) → M(13-cis) → N(13-cis) → O(all-trans). Our discussion is based on a quantumchemical modified neglect of diatomic overlap [MNDO] calculation of the vibrational spectra of the relevant isomers of the protonated retinal Schiff base. In particular, we investigated in these calculations the effects of different charge environments on the frequencies of the relevant C-C single bond stretching vibrations of these isomers.     

Evidence for light-induced 13-cis, 14-s-cis isomerization in bacteriorhodopsin obtained by FTIR difference spectroscopy using isotopically labelled retinals

We have obtained by Fourier transformed infra-red (FTIR)-spectroscopy BR-K, BR-L and BR-M difference spectra of bacteriorhodopsin regenerated with isotopically labelled retinals. Thereby, we are able to assign reliably the C₁₄–C₁₅ and C=N stretching vibrations of the various intermediates. The lower C₁₄–C₁₅ stretching vibration frequency in L as compared with 13-cis protonated Schiff base model compounds indicates a 13-cis, 14-s-cis configuration of the retinal in this species. The unusually low C=N stretching vibration in K at 1615 cm⁻¹ indicates less stabilization of the positive charge at the Schiff base by the protein environment. Based on these results, a mechanism is suggested by which the stored light energy is transformed into proton transfers.     

Vibrational spectroscopy of bacteriorhodopsin mutants: I. Tyrosine-185 protonates and deprotonates during the photocycle

The techniques of FTIR difference spectroscopy and site-directed mutagenesis have been combined to investigate the role of individual tyrosine side chains in the proton-pumping mechanism of bacteriorhodopsin (bR). For each of the 11 possible bR mutants containing a single Tyr----Phe substitution, difference spectra have been obtained for the bR----K and bR----M photoreactions. Only the Tyr-185----Phe mutation results in the disappearance of a set of bands that were previously shown to be due to the protonation of a tyrosinate during the bR----K photoreaction [Rothschild et al.: Proceedings of the National Academy of Sciences of the United States of America 83:347, (1986]). The Tyr-185----Phe mutation also eliminates a set of bands in the bR----M difference spectrum associated with deprotonation of a Tyr; most of these bands (e.g., positive 1272-cm-1 peak) are completely unaffected by the other ten Tyr----Phe mutations. Thus, tyrosinate-185 gains a proton during the bR----K reaction and loses it again when M is formed. Our FTIR spectra also provide evidence that Tyr-185 interacts with the protonated Schiff base linkage of the retinal chromophore, since the negative C = NH+ stretch band shifts from 1640 cm-1 in the wild type to 1636 cm-1 in the Tyr-185----Phe mutant. A model that is consistent with these results is that Tyr-185 is normally ionized and serves as a counter-ion to the protonated Schiff base. The primary photoisomerization of the chromophore translocates the Schiff base away from Tyr-185, which raises the pKa of the latter group and results in its protonation.     

Proton translocation in hydrogen bonds with large proton polarizability formed between a Schiff base and phenols

OH…N ? O^?…H⁺N hydrogen bonds formed between N-all-transretinylidene butylamine (Schiff base) and phenols (1:1) are studied by IR spectroscopy. It is shown that both proton limiting structures of these hydrogen bonds have the same weight with Δ pK_a (50%) = (pK_a protonated Schiff base minus pK_a phenol) = 5.5. With the largely symmetrical systems, continua demonstrate that these hydrogen bonds show great proton polarizability. In the Schiff base + tyrosine system in a non-polar solvent the residence time of the proton at the tyrosine residue is much larger than that at the Schiff base. In CH₂CCl₂ these hydrogen bonds show, however, still proton polarizability, i.e., the position of the proton transfer equilibrium OH…N ? O^?…H⁺N is shifted to and fro as function of the nature of the environment of this hydrogen bond. Consequences regarding bacteriorhodopsin are discussed.