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.

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...

ABSTRACT13C NMR spectra of Iyophilized 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 y effect, which results from a steric interaction between the C-14 retinal proton and the protons on the e 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 bRm and bR54. 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 ketinines. 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.Bacteriorhodopsin (bR), the single protein of the purple membrane (PM) of Halobacterium halobium (1), has been the subject of considerable experimental scrutiny for some time. Like rhodopsin (2), it contains as its chromophore the polyene aldehyde retinal, connected via a Schiff base linkage to the E-amino group of a lysine side chain (3). Although bR appears to resemble visual pigments (4) (14), the configuration of the C=-N Schiff base linkage in bR has not been considered. In addition, the interesting possibility that isomerization about this bond might occur during the photocycle has been largely overlooked. The inattention to this important question can be attributed in part to the absence of an experimental means to discriminate definitively between syn and anti isomers in bR. In a recent paper (15) we demonstrated that high-resolution solid-state 3C NMR is a potent means of establishing configuration about C=C bonds in bR. Here, we present evidence that it is equally effective in determining the C=N bond configuration and evinces that dark-adapted bR contains alltrans,15-anti and 13-cis,15-syn isomers in an approximately 4:6 ratio. The implications of this finding for both the structure of the chromophore and the bR photocycle will also be discussed. MATERIALS AND METHODS13C-14-labeled retinal was prepared by the method of ref. 16 and incorporated into white membrane as described (8, 15). The reconstituted PM was then lyophilized at 0.1 mm Hg (1 mm Hg = 133 Pa) and packed into a Kel-F rotor of the Andrew-Beams design (17). 13C magic-angle sample spinning (MASS) spectra were obtained at various spinning frequencies between 1.9 and 3.2 kHz, with a 13C frequency of 79.9 MHz. Typically, (,)1/21r) = 50 kHz was used for cross-polarization. Subsequently, the magnetization was sampled in the presence of 1H decoupling fields of 125 kHz. Usually, 15,000 transients were accumulated, with a recycle de...

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.