Resonance Raman studies of the HOOP modes in octopus bathorhodopsin with deuterium-labeled retinal chromophores

Resonance Raman spectra of the hydrogen out-of-plane (HOOP) vibrational modes in the retinal chromophore of octopus bathorhodopsin with deuterium label(s) along the polyene chain have been obtained. In clear contrast with bovine bathorhodopsin's HOOP modes, there are only two major HOOP bands at 887 and 940 cm-1 for octopus bathorhodopsin. On the basis of their isotopic shifts upon deuterium labeling, we have assigned the band at 887 cm-1 to C10H and C14H HOOP modes, and the band at 940 cm-1 to C11H = C12H Au-like HOOP mode. Except for a 26 cm-1 downward shift, the C11H = C12H Au-like wag appears to be little disturbed in octopus bathorhodopsin from the chromophore in solution since its changes upon deuterium labeling are close to those found in solution model-compound studies. We found also that the C10H and C14H HOOP wags are also similar to those in the model-compound studies. However, we have found that the interaction between the C7H and C8H HOOP internal coordinates of the chromophore in octopus bathorhodopsin is different from that of the chromophore in solution. The intensity of the C11H = C12H and the other HOOP modes suggests that the chromophore of octopus bathorhodopsin is somewhat torsionally distorted from a planar trans geometry. Importantly, a twist about C11 = C12 double bond is inferred. Such a twist breaks the local symmetry, resulting in the observation of the normally Raman-forbidden C11H = C12H Au-like HOOP mode. The twisted nature of the chromophore, semiquantitatively discussed here, likely affects the lambda max of the chromophore and its enthalpy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resonance Raman analysis of the mechanism of energy storage and chromophore distortion in the primary visual photoproduct

The vibrational structure of the chromophore in the primary photoproduct of vision, bathorhodopsin, is examined to determine the cause of the anomalously decoupled and intense C(11)=C(12) hydrogen-out-of-plane (HOOP) wagging modes and their relation to energy storage in the primary photoproduct. Low-temperature (77 K) resonance Raman spectra of Glu181 and Ser186 mutants of bovine rhodopsin reveal only mild mutagenic perturbations of the photoproduct spectrum suggesting that dipolar, electrostatic, or steric interactions with these residues do not cause the HOOP mode frequencies and intensities. Density functional theory calculations are performed to investigate the effect of geometric distortion on the HOOP coupling. The decoupled HOOP modes can be simulated by imposing approximately 40 degrees twists in the same direction about the C(11)=C(12) and C(12)-C(13) bonds. Sequence comparison and examination of the binding site suggests that these distortions are caused by three constraints consisting of an electrostatic anchor between the protonated Schiff base and the Glu113 counterion, as well as steric interactions of the 9- and 13-methyl groups with surrounding residues. This distortion stores light energy that is used to drive the subsequent protein conformational changes that activate rhodopsin.     

Assignment of fingerprint vibrations in the resonance Raman spectra of rhodopsin, isorhodopsin, and bathorhodopsin: implications for chromophore structure and environment

13C- and 2H-labeled retinal derivatives have been used to assign normal modes in the 1100-1300-cm-1 fingerprint region of the resonance Raman spectra of rhodopsin, isorhodopsin, and bathorhodopsin. On the basis of the 13C shifts, C8-C9 stretching character is assigned at 1217 cm-1 in rhodopsin, at 1206 cm-1 in isorhodopsin, and at 1214 cm-1 in bathorhodopsin. C10-C11 stretching character is localized at 1098 cm-1 in rhodopsin, at 1154 cm-1 in isorhodopsin, and at 1166 cm-1 in bathorhodopsin. C14-C15 stretching character is found at 1190 cm-1 in rhodopsin, at 1206 cm-1 in isorhodopsin, and at 1210 cm-1 in bathorhodopsin. C12-C13 stretching character is much more delocalized, but the characteristic coupling with the C14H rock allows us to assign the "C12-C13 stretch" at approximately 1240 cm-1 in rhodopsin, isorhodopsin, and bathorhodopsin. The insensitivity of the C14-C15 stretching mode to N-deuteriation in all three pigments demonstrates that each contains a trans (anti) protonated Schiff base bond. The relatively high frequency of the C10-C11 mode of bathorhodopsin demonstrates that bathorhodopsin is s-trans about the C10-C11 single bond. This provides strong evidence against the model of bathorhodopsin proposed by Liu and Asato [Liu, R., & Asato, A. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 259], which suggests a C10-C11 s-cis structure. Comparison of the fingerprint modes of rhodopsin (1098, 1190, 1217, and 1239 cm-1) with those of the 11-cis-retinal protonated Schiff base in methanol (1093, 1190, 1217, and 1237 cm-1) shows that the frequencies of the C-C stretching modes are largely unperturbed by protein binding. In particular, the invariance of the C14-C15 stretching mode at 1190 cm-1 does not support the presence of a negative protein charge near C13 in rhodopsin. In contrast, the frequencies of the C8-C9 and C14-C15 stretches of bathorhodopsin and the C10-C11 and C14-C15 stretches of isorhodopsin are significantly altered by protein binding. The implications of these observations for the mechanism of wavelength regulation in visual pigments and energy storage in bathorhodopsin are discussed.     

Resonance Raman analysis of the Pr and Pfr forms of phytochrome

Resonance Raman vibrational spectra of the Pr and Pfr forms of oat phytochrome have been obtained at room temperature. When Pr is converted to Pfr, new bands appear in the C = C and C = N stretching region at 1622, 1599, and 1552 cm-1, indicating that a major structural change of the chromophore has occurred. The Pr to Pfr conversion results in an 11 cm-1 lowering of the N-H rocking band from 1323 to 1312 cm-1. Normal mode calculations correlate this frequency drop with a Z----E isomerization about the C15 = C16 bond. A line at 803 cm-1 in Pr is replaced by an unusually intense mode at 814 cm-1 in Pfr. Calculations on model tetrapyrrole chromophores suggest that these low-wavenumber modes are hydrogen out-of-plane (HOOP) wagging vibrations of the bridging C15 methine hydrogen and that both the intensity and frequency of the C15 HOOP mode are sensitive to the geometry around the C14-C15 and C15 = C16 bonds. The large intensity of the 814-cm-1 mode in Pfr indicates that the chromophore is highly distorted from planarity around the C15 methine bridge. If the Pr----Pfr conversion does involve a C15 = C16 Z----E isomerization, then the intensity of the C15 HOOP mode in Pfr argues that the chromophore has an E,anti conformation. On the basis of a comparison with the vibrational calculations, the low frequency (803 cm-1) and the reduced intensity of the C15 HOOP mode in Pr suggest that the chromophore in Pr adopts the C15-Z,syn conformation.     

Chromophore structure in lumirhodopsin and metarhodopsin I by time-resolved resonance Raman microchip spectroscopy.

Time-resolved resonance Raman microchip flow experiments have been performed on the lumirhodopsin (Lumi) and metarhodopsin I (Meta I) photointermediates of rhodopsin at room temperature to elucidate the structure of the chromophore in each species as well as changes in protein-chromophore interactions. Transient Raman spectra of Lumi and Meta I with delay times of 16 micros and 1 ms, respectively, are obtained by using a microprobe system to focus displaced pump and probe laser beams in a microfabricated flow channel and to detect the scattering. The fingerprint modes of both species are very similar and characteristic of an all-trans chromophore. Lumi exhibits a relatively normal hydrogen-out-of-plane (HOOP) doublet at 951/959 cm(-1), while Meta I has a single HOOP band at 957 cm(-1). These results suggest that the transitions from bathorhodopsin to Lumi and Meta I involve a relaxation of the chromophore to a more planar all-trans conformation and the elimination of the structural perturbation that uncouples the 11H and 12H wags in bathorhodopsin. Surprisingly, the protonated Schiff base C=N stretching mode in Lumi (1638 cm(-1)) is unusually low compared to those in rhodopsin and bathorhodopsin, and the C=ND stretching mode shifts down by only 7 cm(-1) in D2O buffer. This indicates that the Schiff base hydrogen bonding is dramatically weakened in the bathorhodopsin to Lumi transition. However, the C=N stretching mode in Meta I is found at 1654 cm(-1) and exhibits a normal deuteration-induced downshift of 24 cm(-1), identical to that of the all-trans protonated Schiff base. The structural relaxation of the chromophore-protein complex in the bathorhodopsin to Lumi transition thus appears to drive the Schiff base group out of its hydrogen-bonded environment near Glu113, and the hydrogen bonding recovers to a normal solvated PSB value but presumably a different hydrogen bond acceptor with the formation of Meta I.     

A comparative study of the infrared difference spectra for octopus and bovine rhodopsins and their bathorhodopsin photointermediates

Fourier-transform infrared difference spectroscopy has been used to detect the vibrational modes in the chromophore and protein that change in position and intensity between octopus rhodopsin and its photoproducts formed at low temperature (85 K), bathorhodopsin and isorhodopsin. The infrared difference spectra between octopus rhodopsin and octopus bathorhodopsin, octopus bathorhodopsin and octopus isorhodopsin, and octopus isorhodopsin and octopus rhodopsin are compared to analogous difference spectra for the well-studied bovine pigments, in order to understand the similarities in pigment structure and photochemical processes between the vertebrate and invertebrate systems. The structure-sensitive fingerprint region of the infrared spectra for octopus bathorhodopsin shows strong similarities to spectra of both all-trans-retinal and bovine bathorhodopsin, thus confirming chemical extraction data that suggest that octopus bathorhodopsin contains an all-trans-retinal chromophore. In contrast, we find dramatic differences in the hydrogen out-of-plane modes of the two bathorhodopsins, and in the fingerprint lines of the rhodopsins and isorhodopsins for the two pigments. These observations suggest that while the primary effect of light in the octopus rhodopsin system, as in the bovine rhodopsin system, is 11-cis/11-trans isomerization, the protein-chromophore interactions for the two systems are quite different. Finally, striking similarities and differences in infrared lines attributable to changes in amino acid residues in the opsin are found between the two pigment systems. They suggest that no carboxylic acid or tyrosine residues are affected in the initial changes of light-energy transduction in octopus rhodopsin. Comparing the amino acid sequences for octopus and bovine pigments also allows us to suggest that the carboxylic acid residues altered in the bovine transitions are Glu-122 and/or Glu-134.     

Time-resolved resonance raman structural studies of the pB' intermediate in the photocycle of photoactive yellow protein

Time-resolved resonance Raman spectroscopy is used to obtain chromophore vibrational spectra of the pR, pB', and pB intermediates during the photocycle of photoactive yellow protein. In the pR spectrum, the C8-C9 stretching mode at 998 cm(-1) is approximately 60 cm(-1) lower than in the dark state, and the combination of C-O stretching and C7H=C8H bending at 1283 cm(-1) is insensitive to D2O substitution. These results indicate that pR has a deprotonated, cis chromophore structure and that the hydrogen bonding to the chromophore phenolate oxygen is preserved and strengthened in the early photoproduct. However, the intense C7H=C8H hydrogen out-of-plane (HOOP) mode at 979 cm(-1) suggests that the chromophore in pR is distorted at the vinyl and adjacent C8-C9 bonds. The formation of pB' involves chromophore protonation based on the protonation state marker at 1174 cm(-1) and on the sensitivity of the COH bending at 1148 cm(-1) as well as the combined C-OH stretching and C7H=C8H bending mode at 1252 cm(-1) to D2O substitution. The hydrogen out-of-plane Raman intensity at 985 cm(-1) significantly decreases in pB', suggesting that the pR-to-pB' transition is the stage where the stored photon energy is transferred from the distorted chromophore to the protein, producing a more relaxed pB' chromophore structure. The C=O stretching mode downshifts from 1660 to 1651 cm(-1) in the pB'-to-pB transition, indicating the reformation of a hydrogen bond to the carbonyl oxygen. Based on reported x-ray data, this suggests that the chromophore ring flips during the transition from pB' to pB. These results confirm the existence and importance of the pB' intermediate in photoactive yellow protein receptor activation.     

Raman microscope studies on the primary photochemistry of vertebrate visual pigments with absorption maxima from 430 to 502 nm

Raman microscope vibrational spectra have been recorded from single photoreceptor cells frozen at 77 K. Spectra of photostationary steady-state mixtures of visual pigments and their primary photoproducts were obtained from toad red rods (lambda max 502 nm), angelfish rods (lambda max 500 nm), gecko blue rods (lambda max 467 nm), and bullfrog green rods (lambda max 430 nm). All four photoproducts have enhanced low-wavenumber Raman lines at approximately 850, 875, and 915 cm-1 and show the anomalous decoupling of the 11- and 12-hydrogen out-of-plane (HOOP) wagging vibrations, as is observed in the bovine primary photoproduct. The low-wavenumber lines are enhanced in the resonance Raman spectrum by conformational distortion, and the uncoupling of the 11- and 12-hydrogen wags is caused by additional protein perturbations. The similarity of the HOOP modes in all four photoproducts indicates that the protein perturbations that uncouple the 11- and 12-hydrogen wags and that enhance the HOOP modes are very similar. Thus, these perturbations of the photoproduct Raman spectrum cannot be caused by the same protein-chromophore interactions that are responsible for wavelength regulation in these pigments.     

Resonance Raman microprobe spectroscopy of rhodopsin mutants: effect of substitutions in the third transmembrane helix.

A microprobe system has been developed that can record Raman spectra from as little as 2 microL of solution containing only micrograms of biological pigments. The apparatus consists of a liquid nitrogen (l-N2)-cooled cold stage, an epi-illumination microscope, and a substractive-dispersion, double spectrograph coupled to a l-N2-cooled CCD detector. Experiments were performed on native bovine rhodopsin, rhodopsin expressed in COS cells, and four rhodopsin mutants: Glu134 replaced by Gln (E134Q), Glu122 replaced by Gln (E122Q), and Glu113 replaced by Gln (E113Q) or Ala (E113A). Resonance Raman spectra of photostationary steady-state mixtures of 11-cis-rhodopsin, 9-cis-isorhodopsin, and all-trans-bathorhodopsin at 77 K were recorded. The Raman spectra of E134Q and the wild-type are the same, indicating that Glu134 is not located near the chromophore. Substitution at Glu122 also does not affect the C = NH stretching vibration of the chromophore. The fingerprint and Schiff base regions of the Raman spectra of the 380-nm, pH 7 forms of E113Q and E113A are characteristic of unprotonated retinal Schiff bases. The C = NH modes of the approximately 500-nm, pH 5 forms of E113Q and E113A in H2O (D2O) are found at 1648 (1629) and 1645 (1630) cm-1, respectively. These frequencies indicate that the protonated Schiff base interacts more weakly with its protein counterion in the Glu113 mutants than it does in the native pigment. Furthermore, perturbations of the unique bathorhodopsin hydrogen out-of-plane (HOOP) vibrations in E113Q and E113A indicate that the strength of the protein perturbation near C12 is weakened compared to that in native bathorhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fourier-transform infrared difference spectroscopy of rhodopsin and its photoproducts at low temperature

Fourier-transform infrared difference spectroscopy has been used to detect the vibrational modes in the chromophore and protein that change in position or intensity between rhodopsin and the photoproducts formed at low temperature (70 K), bathorhodopsin and isorhodopsin. A method has been developed to obtain infrared difference spectra between rhodopsin and bathorhodopsin, bathorhodopsin and isorhodopsin, and rhodopsin and isorhodopsin. To aid in the identification of the vibrational modes, we performed experiments on deuterated and hydrated films of native rod outer segments and rod outer segments regenerated with either retinal containing 13C at carbon 15 or 15-deuterioretinal. Our infrared measurements provide independent verification of the resonance Raman result that the retinal in bathorhodopsin is distorted all-trans. The positions of the C = N stretch in the deuterated pigment and the deuterated pigments regenerated with 11-cis-15-deuterioretinal or 11-cis-retinal containing 13C at carbon 15 are indicative that the Schiff-base linkage is protonated in rhodopsin, bathorhodopsin, and isorhodopsin. Furthermore, the C = N stretching frequency occurs at the same position in all three species. The data indicate that the protonated Schiff base has a C = N trans conformation in all three species. Finally, we present evidence that, even in these early stages of the rhodopsin photosequence, changes are occurring in the opsin and perhaps the associated lipids.     

13C magic-angle spinning NMR studies of bathorhodopsin, the primary photoproduct of rhodopsin.

Magic-angle spinning NMR spectra have been obtained of the bathorhodopsin photointermediate trapped at low temperature (less than 130 K) by using isorhodopsin samples regenerated with retinal specifically 13C-labeled at positions 8, 10, 11, 12, 13, 14, and 15. Comparison of the chemical shifts of the bathorhodopsin resonances with those of an all-trans-retinal protonated Schiff base (PSB) chloride salt show the largest difference (6.2 ppm) at position 13 of the protein-bound retinal. Small differences in chemical shift between bathorhodopsin and the all-trans PSB model compound are also observed at positions 10, 11, and 12. The effects are almost equal in magnitude to those previously observed in rhodopsin and isorhodopsin. Consequently, the energy stored in the primary photoproduct bathorhodopsin does not give rise to any substantial change in the average electron density at the labeled positions. The data indicate that the electronic and structural properties of the protein environment are similar to those in rhodopsin and isorhodopsin. In particular, a previously proposed perturbation near position 13 of the retinal appears not to change its position significantly with respect to the chromophore upon isomerization. The data effectively exclude charge separation between the chromophore and a protein residue as the main mechanism for energy storage in the primary photoproduct and argue that the light energy is stored in the form of distortions of the bathorhodopsin chromophore.     

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)     

Effect of anion binding on iodopsin studied by low-temperature fourier transform infrared spectroscopy.

The effect of anion binding on iodopsin, the chicken red-sensitive cone visual pigment, was studied by measurements of the Fourier transform infrared spectra of chloride- and nitrate-bound forms of iodopsin at 77 K. In addition to the blue shift of the absorption maximum upon substituting nitrate for chloride, the C=C stretching vibrations of iodopsin and its photoproducts were upshifted 5-6 cm(-)(1). The C=NH and C=ND stretching vibrations were the same in wavenumber between the chloride- and nitrate-bound forms, indicating that the binding of either chloride or nitrate has no effect on the interaction between the protonated Schiff base and the counterion. The vibrational bands of iodopsin in the fingerprint and the hydrogen out-of-plane wagging regions were insensitive to anion substitution, suggesting that local chromophore interactions with the anions are not crucial for the absorption spectral shift. In contrast, bathoiodopsin in the chloride-bound form exhibited an intense C(14)H wagging mode, whose intensity was considerably weakened upon substitution of nitrate for chloride. These results suggest that binding of chloride changes the environment near the C(14) position of the chromophore, which could be one of the factors in the thermal reverse reaction of bathoiodopsin to iodopsin in the chloride-bound form.     

Orientation of the bacteriorhodopsin chromophore probed by polarized Fourier transform infrared difference spectroscopy

Polarized, low-temperature Fourier transform infrared (FTIR) difference spectroscopy has been used to investigate the structure of bacteriorhodopsin (bR) as it undergoes phototransitions from the light-adapted state, bR570, to the K630 and M412 intermediates. The orientations of specific retinal chromophore and protein groups relative to the membrane plane were calculated from the linear dichroism of the infrared bands, which correspond to the vibrational modes of those groups. The linear dichroism of the chromophore C=C and C-C stretching modes indicates that the long axis of the polyene chain is oriented at 20-25 degrees from the membrane plane at 250 K and that it orients more in-plane when the temperature is reduced to 81 K. The polyene plane is found to be approximately perpendicular to the membrane plane from the linear dichroism calculations of the HOOP (hydrogen out-of-plane) wags. The orientation of the transition dipole moments of chromophore vibrations in the K630 and M412 intermediates has been probed, and the dipole moment direction of the C=O bond of an aspartic acid that is protonated in the bR570----M412 transition has been measured.     

Vibrational motions associated with primary processes in bacteriorhodopsin studied by coherent infrared emission spectroscopy

The primary energetic processes driving the functional proton pump of bacteriorhodopsin take place in the form of complex molecular dynamic events after excitation of the retinal chromophore into the Franck-Condon state. These early events include a strong electronic polarization, skeletal stretching, and all-trans-to-13-cis isomerization upon formation of the J intermediate. The effectiveness of the photoreaction is ensured by a conical intersection between the electronic excited and ground states, providing highly nonadiabatic coupling to nuclear motions. Here, we study real-time vibrational coherences associated with these motions by analyzing light-induced infrared emission from oriented purple membranes in the 750–1400 cm⁻¹ region. The experimental technique applied is based on second-order femtosecond difference frequency generation on macroscopically ordered samples that also yield information on phase and direction of the underlying motions. Concerted use of several analysis methods resulted in the isolation and characterization of seven different vibrational modes, assigned as C-C stretches, out-of-plane methyl rocks, and hydrogen out-of-plane wags, whereas no in-plane H rock was found. Based on their lifetimes and several other criteria, we deduce that the majority of the observed modes take place on the potential energy surface of the excited electronic state. In particular, the direction sensitivity provides experimental evidence for large intermediate distortions of the retinal plane during the excited-state isomerization process.     

Inherent chirality of the retinal chromophore in rhodopsin-A nonempirical theoretical analysis of chiroptical data

CASSCF and GAUSSIAN CIS calculations were performed on ground and excited states of different conformations of 11-cis-retinal protonated Schiff bases, the chromophore of rhodopsin, in order to study their chiroptical properties and attempt a correlation between absolute conformation and CD-spectral data. Geometries were taken from molecular models, from published rhodopsin models, and from retinal conformations obtained from molecular dynamics with geometry restraints. In all the cases studied we find that a positive sense of twist about the C12-C13 bond correlates with a calculated positive CD of the long wavelength absorption band; the twist of the C6-C7 bond modulates this primary contribution of the C12-C13 bond. The correlation of the beta-band with structural features is not straightforward. Calculations on bathorhodopsin lend support to the idea that this intermediate is a highly twisted all-trans-conformation.     

Resonance Raman spectroscopy of octopus rhodopsin and its photoproducts

We report here the resonance Raman spectra of octopus rhodopsin and its photoproducts, bathorhodopsin and acid metarhodopsin. These studies were undertaken in order to make comparisons with the well-studied bovine pigments, so as to understand the similarities and the differences in pigment structure and photochemical processes between vertebrates and invertebrates. The flow method was used to obtain the Raman spectrum of rhodopsin at 13 degrees C. The bathorhodopsin spectrum was obtained by computer subtraction of the spectra containing different photostationary mixtures of rhodopsin, isorhodopsin, hypsorhodopsin, and bathorhodopsin, obtained at 12 K using the pump-probe technique and from measurements at 80 K. Like their bovine counterparts, the Schiff base vibrational mode appears at approximately 1660 cm-1 in octopus rhodopsin and the photoproducts, bathorhodopsin and acid metarhodopsin, suggesting a protonated Schiff base linkage between the chromophore and the protein. Differences between the Raman spectra of octopus rhodopsin and bathorhodopsin indicate that the formation of bathorhodopsin is associated with chromophore isomerization. This inference is substantiated by the chromophore chemical extraction data which show that, like the bovine system, octopus rhodopsin is an 11-cis pigment, while the photoproducts contain an all-trans pigment, in agreement with previous work. The octopus rhodopsin and bathorhodopsin spectra show marked differences from their bovine counterparts in other respects, however. The differences are most dramatic in the structure-sensitive fingerprint and the HOOP regions. Thus, it appears that although the two species differ in the specific nature of the chromophore-protein interactions, the general process of visual transduction is the same.     

Probing the photoreaction mechanism of phytochrome through analysis of resonance Raman vibrational spectra of recombinant analogues

Resonance Raman spectra of native and recombinant analogues of oat phytochrome have been obtained and analyzed in conjunction with normal mode calculations. On the basis of frequency shifts observed upon methine bridge deuteration and vinyl and C(15)-methine bridge saturation of the chromophore, intense Raman lines at 805 and 814 cm(-)(1) in P(r) and P(fr), respectively, are assigned as C(15)-hydrogen out-of-plane (HOOP) wags, lines at 665 cm(-)(1) in P(r) and at 672 and 654 cm(-)(1) in P(fr) are assigned as coupled C=C and C-C torsions and in-plane ring twisting modes, and modes at approximately 1300 cm(-)(1) in P(r) are coupled N-H and C-H rocking modes. The empirical assignments and normal mode calculations support proposals that the chromophore structures in P(r) and P(fr) are C(15)-Z,syn and C(15)-E,anti, respectively. The intensities of the C(15)-hydrogen out-of-plane, C=C and C-C torsional, and in-plane ring modes in both P(r) and P(fr) suggest that the initial photochemistry involves simultaneous bond rotations at the C(15)-methine bridge coupled to C(15)-H wagging and D-ring rotation. The strong nonbonded interactions of the C- and D-ring methyl groups in the C(15)-E,anti P(fr) chromophore structure indicated by the intense 814 cm(-1) C(15) HOOP mode suggest that the excited state of P(fr) and its photoproduct states are strongly coupled.     

Assignment of the hydrogen-out-of-plane and -in-plane vibrations of the retinal chromophore in the K intermediate of pharaonis phoborhodopsin

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor protein for negative phototaxis in Natronomonas pharaonis. Photoisomerization of the retinal chromophore from all-trans to 13-cis initiates conformational changes of the protein leading to activation of the cognate transducer protein (pHtrII). Elucidation of the initial photoreaction, formation of the K intermediate of ppR, is important for understanding the mechanism of storage of photon energy. We have reported the K minus ppR Fourier transform infrared (FTIR) spectra, including several vibrational bands of the retinal, the protein, and internal water molecules. It is interesting that more vibrational bands were observed in the hydrogen-out-of-plane (HOOP) region than for the light-driven proton pump, bacteriorhodopsin. This result implied that the steric constraints on the retinal chromophore in the binding pocket of ppR are distributed more widely upon formation of the initial intermediate. In this study, we assigned the HOOP and hydrogen-in-plane vibrations by means of low-temperature FTIR spectroscopy applied to ppR reconstituted with retinal deuterated at C7, C8, C10-C12, C14, and C15. As a result, the 966 (+)/971 (-) and 958 (+)/961 (-) cm(-1) bands were assigned to the C7=C8 and C11=C12 Au HOOP modes, respectively, suggesting that the structural changes spread to the middle part of the retinal. The positive bands at 1001, 994, 987, and 979 cm(-1) were assigned to the C15-HOOP vibrations of the K intermediate, whose frequencies are similar to those of the K(L) intermediate of bacteriorhodopsin trapped at 135 K. Another positive band at 864 cm(-1) was assigned to the C14-HOOP vibration. Relatively many positive bands of hydrogen-in-plane vibrations supported the wide distribution of structural changes of the retinal as well. These results imply that the light energy was stored mainly in the distortions around the Schiff base region while some part of the energy was transferred to the distal part of the retinal.     

Relative orientation between the beta-ionone ring and the polyene chain for the chromophore of rhodopsin in native membranes

Rotational resonance solid state nuclear magnetic resonance has been used to determine the relative orientation of the beta-ionone ring and the polyene chain of the chromophore 11-Z-retinylidene of rhodopsin in rod outer segment membranes from bovine retina. The bleached protein was regenerated with either 11-Z-[8,18-(13)C(2)]retinal or 11-Z-[8,16/17(13)C(2)]retinal, the latter having only one (13)C label at either of the chemically equivalent positions 16 and 17. Observation of (13)C selectively enriched in the ring methyl groups, C16/17, revealed alternative conformational states for the ring. Minor spectral components comprised around 26% of the chromophore. The major conformation (approximately 74%) has the chemical shift resolution required for measuring internuclear distances to (13)C in the retinal chain (C8) separately from each of these methyl groups. The resulting distance constraints, C8 to C16 and C17 (4.05 +/- 0.25 A) and from C8 to C18 (2.95 +/- 0.15 A), show that the major portion of retinylidene in rhodopsin has a twisted 6-s-cis conformation. The more precise distance measurement made here between C8 and C18 (2.95 A) predicts that the chain is twisted out-of-plane with respect to the ring by a modest amount (C5-C6-C7-C8 torsion angle = -28 +/- 7 degrees ).