Raman microscope and quantum yield studies on the primary photochemistry of A2-visual pigments.

The 77-K resonance Raman vibrational spectrum of intact goldfish rod photoreceptors containing 3,4-dehydro (A2) retinal is dominated by scattering from the 9-cis component of the steady state at all excitation wavelengths. Intact goldfish photoreceptors were regenerated with an A1-retinal chromophore to determine whether this behavior is caused by the protein or the chromophore. The resulting Raman spectrum was typical of an A1-pigment exhibiting significant scattering from all three components of the steady state: rhodopsin, bathorhodopsin, and isorhodopsin. Furthermore, regeneration of bovine opsin with A2-retinal produces a characteristic "A2-Raman spectrum" that is dominated by scattering from the 9-cis pigment. We conclude that the differences between the Raman spectra of the A1-and A2-pigments are caused by some intrinsic difference in the photochemical properties of the retinal chromophores. To quantitate these observations, the 77-K adsorption spectra and the photochemical quantum yields (phi) of the native A2-goldfish and the regenerated A2-bovine pigments were measured. In the goldfish A2-pigment, the value of phi 4 (9-cis----trans) is 0.05; phi 3 (trans----9-cis) is 0.10; and phi 2 (trans----11-cis) is 0.35. By contrast, in the bovine A1-pigment, these quantum yields are 0.10, 0.053, and 0.50, respectively. The reduced value of phi 4 and the increased value of phi 3 in the goldfish pigment confirms that the 9-cis isomer is photochemically more stable in A2-pigments.

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.

Low-temperature solid-state 13C NMR studies of the retinal chromophore in rhodopsin.

Magic angle sample spinning (MASS) 13C NMR spectra have been obtained of bovine rhodopsin regenerated with retinal prosthetic groups isotopically enriched with 13C at C-5 and C-14. In order to observe the 13C retinal chromophore resonances, it was necessary to employ low temperatures (-15-----35 degrees C) to restrict rotational diffusion of the protein. The isotropic chemical shift and principal values of the chemical shift tensor of the 13C-5 label indicate that the retinal chromophore is in the twisted 6-s-cis conformation in rhodopsin, in contrast to the planar 6-s-trans conformation found in bacteriorhodopsin. The 13C-14 isotropic shift and shift tensor principal values show that the Schiff base C = N bond is anti. Furthermore, the 13C-14 chemical shift (121.2 ppm) is within the range of values (120-123 ppm) exhibited by protonated (C = N anti) Schiff base model compounds, indicating that the C = N linkage is protonated. Our results are discussed with regard to the mechanism of wavelength regulation in rhodopsin.

High-resolution solid-state 13C-NMR study of carbons C-5 and C-12 of the chromophore of bovine rhodopsin. Evidence for a 6-S-cis conformation with negative-charge perturbation near C-12.

Solid-state 13C magic-angle spinning NMR spectroscopy has been employed to study the conformation of the 11-cis-retinylidene Schiff base chromophore in bovine rhodopsin. Spectra were obtained from lyophilized samples of bovine rhodopsin selectively 13C-labeled at position C-5 or C-12 of the retinyl moiety, and reconstituted in the fully saturated branched-chain phospholipid diphytanoyl glycerophosphocholine. Comparison of the NMR parameters for carbon C-5 presented in this paper with those published for retinyl Schiff base model compounds and bacteriorhodopsin by Harbison and coworkers [Harbison et al. (1985) Biochemistry 24, 6955-6962], indicate that in bovine rhodopsin the C-6-C-7 single bond has the unperturbed cis conformation. This is in contrast to the 6-S-trans conformation found in bacteriorhodopsin. The NMR parameters for bovine [12-13C]rhodopsin present evidence for the presence of a negative charge interacting with the retinyl moiety near C-12, in agreement with the model for the opsin shift presented by Honig and Nakanishi and coworkers [Kakitani et al. (1985) Photochem. Photobiol. 41, 471-479].

Are C14-C15 single bond isomerizations of the retinal chromophore involved in the proton-pumping mechanism of bacteriorhodopsin?

Resonance Raman spectroscopy is used to examine the possibility that C14-C15 single bond isomerizations of the retinal prosthetic group are involved in the photochemical reactions of bacteriorhodopsin. Normal mode calculations show that the vibration that contains predominantly C14-C15 stretch character is approximately equal to 70 cm-1 lower in frequency in the 14-s-cis conformer than in the s-trans case. This geometric effect is insensitive to out-of-plane twists and should be observed in the sterically hindered 13-cis, 14-s-cis retinal protonated Schiff base, which has been proposed as the chromophore in the K and L intermediates of bacteriorhodopsin. Resonance Raman spectra were obtained of K625 by using the low temperature (77 K) spinning-cell technique. Isotopic substitutions with 13C and 2H show that significant C14-C15 stretch character is observed in normal modes at approximately equal to 1185-1195 cm-1. The relatively high frequency of the C14-C15 stretch argues that K625 contains a 13-cis, 14-s-trans chromophore. Similarly, isotopic derivatives show that L550 has a localized C14-C15 stretch at 1172 cm-1, consistent with a 14-s-trans chromophore. These results argue that the primary step in bacteriorhodopsin is a C13=C14 trans----cis photoisomerization that does not involve C14-C15 s-cis structures.

Solid-state 13C NMR detection of a perturbed 6-s-trans chromophore in bacteriorhodopsin.

Solid-state 13C magic angle sample spinning NMR spectroscopy has been used to study the ionone ring portion of the chromophore of bacteriorhodopsin. Spectra were obtained from fully hydrated samples regenerated with retinals 13C labeled at positions C-5, C-6, C-7, C-8, and C-18 and from lyophilized samples regenerated with retinals labeled at C-9 and C-13. C-15-labeled samples were studied in both lyophilized and hydrated forms. Three independent NMR parameters (the downfield element of the C-5 chemical shift tensor, the C-8 isotropic chemical shift, and the C-18 longitudinal relaxation time) indicate that the chromophore has a 6-s-trans conformation in the protein, in contrast to the 6-s-cis conformation that is energetically favored for retinoids in solution. We also observe an additional 27 ppm downfield shift in the middle element of the C-5 shift tensor, which provides support for the existence of a negatively charged protein residue near C-5. Evidence for a positive charge near C-7, possibly the counterion for the negative charge, is also discussed. On the basis of these results, we present a new model for the retinal binding site, which has important implications for the mechanism of the "opsin shift" observed in bacteriorhodopsin.

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.

Fourier transform infrared evidence for Schiff base alteration in the first step of the bacteriorhodopsin photocycle.

The first step of the bacteriorhodopsin (bR) photocycle involves the formation of a red-shifted product, K. Fourier transform infrared difference spectra of the bR570 to K630 transition at 81 K has been measured for bR containing different isotopic substitutions at the retinal Schiff base. In the case of bacteriorhodopsin containing a deuterium substitution at the Schiff base nitrogen, carbon 15, or both, we find spectral changes in the 1600-1610- and 1570-1580-cm-1 region consistent with the hypothesis that the K630 C=N stretching mode of a protonated Schiff base is located near 1609 cm-1. A similar set of Schiff base deuterium substitutions for retinal containing a 13C at the carbon 10 position strongly supports this conclusion. This assignment of the K630 C=N stretching vibration provides evidence that the bR Schiff base proton undergoes a substantial environmental change most likely due to separation from a counterion. In addition, a correlation is found between the C=N stretching frequency and the maximum wavelength of visible absorption, suggesting that movement of a counterion relative to the Schiff base proton is the main source of absorption changes in the early stages of the photocycle. Such a movement is a key prediction of several models of proton transport and energy transduction. Evidence is also presented that one or more COOH groups are involved in the formation of the K intermediate.

Solid-state 13C NMR studies of retinal in bacteriorhodopsin.

Solid-state 13C magic-angle sample spinning (MASS) NMR has been used to study lyophilized dark-adapted purple membrane containing 13C-labeled retinals. C-10-, C-11-, and C-12-labeled derivatives each showed two lines, assigned to the coexisting 13-cis and all-trans isomers. The isotropic chemical shifts, particularly of C-11, indicate that the Schiff base is protonated. Shift anisotropies are also similar to those of model compounds, indicating that this part of the chromophore is rigid and immobile and possesses the same degree of in-plane bending as crystalline retinal derivatives. Purple membrane samples labeled on the C-19- and C-20-methyl groups both give single lines from the retinal, upfield shifted by 2.1 and 1.0 ppm, respectively, from model compounds. In all cases, high-quality spectra were obtained from approximately 50-mg samples in modest signal-averaging times. These results suggest that it is now practical to exploit the enormous potential of MASS NMR for structural studies of 13C-labeled membrane proteins.

Bacteriorhodopsins with chromophores modified at the beta-ionone site. Formation and light-driven action of the proton pump.

The binding to bacterioopsin of the all-trans isomers of retinal analogues lacking the six-membered ring and differing in length of the conjugated chain, as well as the light-driven action of the proton pump of the resulting bacteriorhodopsin analogues, were studied. The 'opsin shifts' in these modified bacteriorhodopsins are all around 2700 cm-1 and do not depend on the number of double bonds in the chromophore. These experimental results suggest that the 4800 cm-1 'opsin shift' in unmodified bacteriorhodopsin consists of a contribution of about 2700 cm-1 due to the interaction of the protonated Schiff-base with the counterion. The extra 2100 cm-1 shift in bacteriorhodopsin is due to the specific interaction of the cyclohexene ring and the protein. Only the bacteriorhodopsin analogue with the same number of conjugated double bonds in the chromophore as bacteriorhodopsin itself shows light-driven proton pump action.

Determination of retinal Schiff base configuration in bacteriorhodopsin.

Resonance Raman spectra of the BR(568), BR(548), K(625), and L(550) intermediates of the bacteriorhodopsin photocycle have been obtained in (1)H(2)O and (2)H(2)O by using native purple membrane as well as purple membrane regenerated with 14,15-(13)C(2) and 12,14-(2)H(2) isotopic derivatives of retinal. These derivatives were selected to determine the contribution of the C(14)-C(15) stretch to the normal modes in the 1100- to 1400-cm(-1) fingerprint region and to characterize the coupling of the C(14)-C(15) stretch with the NH rock. Normal mode calculations demonstrate that when the retinal Schiff base is in the C[unk]N cis configuration the C(14)-C(15) stretch and the NH rock are strongly coupled, resulting in a large ( approximately 50-cm(-1)) upshift of the C(14)-C(15) stretch upon deuteration of the Schiff base nitrogen. In the C[unk]N trans geometry these vibrations are weakly coupled and only a slight (<5-cm(-1)) upshift of the C(14)-C(15) stretch is predicted upon N-deuteration. In BR(568), the insensitivity of the 1201-cm(-1) C(14)-C(15) stretch to N-deuteration demonstrates that its retinal C[unk]N configuration is trans. The C(14)-C(15) stretch in BR(548), however, shifts up from 1167 cm(-1) in (1)H(2)O to 1208 cm(-1) in (2)H(2)O, indicating that BR(548) contains a C[unk]N cis chromophore. Thus, the conversion of BR(568) to BR(548) (dark adaptation) involves isomerization about the C[unk]N bond in addition to isomerization about the C(13)[unk]C(14) bond. The insensitivity of the native, [14,15-(13)C(2)]-, and [12,14-(2)H(2)]K(625) and L(550) spectra to N-deuteration argues that these intermediates have a C[unk]N trans configuration. Thus, the primary photochemical step in bacteriorhodopsin (BR(568) --> K(625)) involves isomerization about the C(13)[unk]C(14) bond alone. The significance of these results for the mechanism of proton-pumping by bacteriorhodopsin is discussed.

Dark-adapted bacteriorhodopsin contains 13-cis, 15-syn and all-trans, 15-anti retinal Schiff bases.

13C NMR spectra of lyophilized dark-adapted [14-13C]retinyl-labeled bacteriorhodopsin show a large anomalous upfield shift for the 13C-14 resonance assigned to the 13-cis isomer, relative to both the all-trans isomer and model compounds. We attribute this to the so-called gamma effect, which results from a steric interaction between the C-14 retinal proton and the protons on the epsilon CH2 of the lysine. As a consequence of this observation, we infer that dark-adapted bacteriorhodopsin is composed of a mixture of all-trans, 15-anti (trans or E) and 13-cis, 15-syn (cis or Z) isomers. These occur in an approximate 4:6 ratio and are commonly identified as bR568 and bR548. This conclusion is based on an examination of the isotropic and anisotropic chemical shifts and a comparison with 13C shifts of the carbons adjacent to the C = N linkage in protonated ketimines. Other possible origins for the anomalous shift are examined and shown to be insufficient to account for either the size of the shift or the nature of the shift tensor. We discuss the consequences of this finding for the structure and photochemistry of bacteriorhodopsin.