Resonance Raman spectra of light-adapted bacteriorhodopsin (BRS68) have been obtained using purple membrane regenerated with isotopic retinal derivatives. The chromophore was labeled with "C at positions 5 , 6, 7, 8,9, 10, 11, 12, 13, 14, and 15, while deuterium substitutions were made at positions 7, 8, 10, 11, 12, 14, and 15 and on the Schiff base nitrogen.On the basis of the observed isotopic shifts, empirical assignments have been made for the vibrations observed between 700 and 1700 cm-I. A modified Urey-Bradley force field has been refined to satisfactorily reproduce the vibrational frequencies and isotopic shifts. Of particular importance is the assignment of the normal modes in the structurally sensitive 1100-1300 cm-' "fingerprint region" to specific combinations of C-C stretching and CCH rocking motions. The methyl-substituted "C8-C9" and "C12-C13n stretches are highest in frequency at 1214 and 1248-1255 cm-I, respectively, as a result of coupling with their associated C-methyl stretches. The C8-C9 and CI2-Cl3 stretches also couple strongly with the CloH and C14H rocks, respectively. The 1169-cm-' mode is assigned as a relatively localized CIo-CII stretch, and the 1201-cm-' mode is a localized CI4-Cl5 stretch. The frequency ordering and spacing of the C-C stretches in BR568 is the same as that observed in the all-trans-retinal protonated Schiff base. However, each vibration is -10 cm-I higher in the pigment as a result of increased r-electron delocalization.The frequencies and Raman intensities of the normal modes are compared with the predictions of theoretical models for the ground-and excited-state structure of the retinal chromophore in bacteriorhodopsin.Chemical reactions that occur in the active sites of biological macromolecules such as enzymes, photosynthetic pigments, and heme proteins often involve rapid changes in the structure of transiently bound substrate molecules or covalently bound prosthetic groups. Vibrational spectroscopy is a powerful method for studying the molecular changes involved in these reactions since the frequencies and intensities of the vibrational normal modes of an enzyme substrate or prosthetic group are sensitive to both molecular structure and environment. Resonance Raman spectroscopy is a useful technique for obtaining vibrational spectra of specific chromophoric groups within proteins. By selecting a laser excitation wavelength within the absorption band of retinal pigments or heme proteins, it is possible to selectively enhance the chromophore resonances over the more numerous protein Furthermore, the use of pulsed laser techniques can provide picosecond time-resolution, sufficient to monitor very fast biochemical reaction^.^ Fourier transform infrared (FTIR) difference spectroscopy offers a second approach for obtaining spectra of reactive groups in macrom~lecules.~ In both the Raman and FTIR techniques, interpreting the changes in vibrational spectra in terms of molecular structure or environment requires the assignment of the vibrational lines to specific norm...

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.

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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)

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

Resonance Raman spectra of the BR568, BR54, K625, and L5so intermediates of the bacteriorhodopsin photocycle have been obtained in 1H20 and 2H20 by using native purple membrane as well as purple membrane regenerated with 14,15-13C2 and 12,14-2H2 isotopic derivatives of retinal. These derivatives were selected to determine the contribution of the C14-C15 stretch to the normal modes in the 1100-to 1400-cm-' fingerprint region and to characterize the coupling of the C14-C15 stretch with the NH rock. Normal mode calculations demonstrate that when the retinal Schiff base is in the C=N cis configuration the C14-C15 stretch and the NH rock are strongly coupled, resulting in a large (-50-cm'1) [14,and [12, and L550 spectra to N-deuteration argues that these intermediates have a C=N trans configuration. Thus, the primary photochemical step in bacteriorhodopsin (BR5" -K625) involves isomerization about the ClY=Cl4 bond alone. The significance of these results for the mechanism of proton-pumping by bacteriorhodopsin is discussed.Bacteriorhodopsin functions as a photochemical proton pump in the purple membrane of Halobacterium halobium (1). Absorption of light by bacteriorhodopsin's retinal prosthetic group converts the light-adapted pigment, BR568, to the red-absorbing intermediate K625, which thermally decays back to BR568 through the intermediates L550, M412, and 0Ow(2). The initial photochemical step involves a trans --cis isomerization about the C13=C14 bond of retinal (3-6), which is followed by deprotonation of the Schiff base nitrogen in the L550 --M412 transition (7). Recently it has been shown that reprotonation of the Schiff base and thermal reisomerization of the C13=C14 bond occur in the conversion of M412 to O64o (8). In the dark, BR568 converts to darkadapted bactenorhodopsin, which contains a 60:40 mixture of 13-cis and all-trans protonated Schiff base chromophores denoted BR548 and BR568, respectively. It is generally accepted that chromophore isomerization and Schiff base protonation/deprotonation play an active role in the mechanism of this proton pump. An important element in establish-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.ing the orientation and molecular motion of the Schiff base proton in bacteriorhodopsin's photocycle is the configuration of the retinal-lysine Schiff base bond. However, no experimental determination of the C=N configuration has yet been made.Resonance Raman spectroscopy can be used to examine the structure of the protein-bound retinal chromophore in bacteriorhodopsin (9). To interpret these spectra, it is necessary to assign the vibrational lines to specific normal modes and to establish how the vibrations are affected by changes in chromophore geometry. Selective isotopic substitution of the retinal chromophore facilitates the vibrational assignments, while model compounds and normal mode calculations ca...