Fourier transform infrared (FTIR) difference spectra have been obtained for the bR----K, bR----L, and bR----M photoreactions in bacteriorhodopsin mutants in which Asp residues 85, 96, 115, and 212 have been replaced by Asn and by Glu. Difference peaks that had previously been attributed to Asp COOH groups on the basis of isotopic labeling were absent or shifted in these mutants. In general, each COOH peak was affected strongly by mutation at only one of the four residues. Thus, it was possible to assign each peak tentatively to a particular Asp. From these assignments, a model for the proton-pumping mechanism of bR is derived, which features proton transfers among Asp-85, -96, and -212, the chromophore Schiff base, and other ionizable groups within the protein. The model can explain the observed COOH peaks in the FTIR difference spectra of bR photointermediates and could also account for other recent results on site-directed mutants of bR.

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...

The usefulness of stroboscopic time-resolved Fourier transform IR spectroscopy for studying the dynamics of biological systems is demonstrated. By using this technique, we have obtained broadband JR absorbance difference spectra after photolysis of bacteriorhodopsin with a time resolution of z50 ps, spectral resolution of 4 cm.1, and a detection limit of AA -10-4. These capabilities permit observation of detailed structural changes in individual residues as bacteriorhodopsin passes through its L, M, and N intermediate states near physiological temperatures. When combined with band assignments based on isotope labeling and site-directed mutagenesis, the stroboscopic Fourier transform IR difference spectra show that on the time scale of the L intermediate, has an altered environment that may be accompanied by change in its protonation state. On The recent publication of a high-resolution structure for bacteriorhodopsin (bR) based on EM (1) has focused attention on. relating the bR structure to its mechanism of lightdriven proton transport. Visible absorption spectroscopy (2, 3) originally established the cycle of transitions that occurs after light absorption by the retinal chromophore and showed that at room temperature the time scales of these reactions are in the range of 10 ps for bR -* K, 1 ,us for K -i L, 50 Us for L -+ M, and 5-10 ms for the M -+ N -O 0 -i bR steps. However, most of what is known about the actual structural changes corresponding to these transitions has come from vibrational spectroscopy. Resonance Raman spectroscopy has provided information selectively about the retinal chromophore (4-7) and more recently about aromatic residues (8). IR spectroscopy, on the other hand, is sensitive to changes throughout the protein. By trapping bR photoproducts through partial dehydration (9) or cooling (10)(11)(12), it has been possible to obtain very precise Fourier transform IR (FTIR) difference spectra corresponding to the bR -* K, bR -* L, and bR -* M transitions.With spectral assignments from isotope labeling (13-15) and site-directed mutagenesis (16, 17), FTIR difference spectra were used previously to develop a model for the protonpumping mechanism that involved proton transfers among the retinal Schiff base and residues Asp-96, Tyr-185, Asp-212, and . Along with a specific sequence of these proton transfers, this model included a detailed 3-dimensional structure for the retinal binding pocket and helices C, F, and G. This structural model took into account existing low-resolution information from both EM (18) and neutron diffraction (19). As it turns out, the detailed structural model deduced from FTIR spectroscopy (20) is very similar to that recently proposed (1) on the basis of electron cryomicroscopy with improved resolution. The EM results thus lend support to the general features of the mechanism previously proposed from FTIR spectroscopy.The mechanism proposed earlier (17) is thus a useful starting point for further investigations, although it is probably incorrect in a number of ...

We have obtained the resonance Raman spectrum of bacteriorhodopsin's primary photoproduct K with a novel low-temperature spinning sample technique. Purple membrane at 77 K is illuminated with spatially separated actinic (pump) and probe laser beams. The 514-nm pump beam produces a photostationary steady-state mixture of bacteriorhodopsin and K. This mixture is then rotated through the red (676 nm) probe beam, which selectively enhances the Raman scattering from K. The essential advantage of our successive pump-and-probe technique is that it prevents the fluorescence excited by the pump beam from masldng the red probe Raman scattering. K exhibits strong Raman lines at 1516, 1294, 1194, 1012, 957, and 811 cm-'. The effects of C15 deuteration on K's fingerprint lines correlate well with those seen in 13-cis model compounds, indicating that K has a 13-cis chromophore. However, the presence of unusually strong "lowwavenumber" lines at 811 and 957 cm-, attributable to hydrogen out-of-plane wags, indicates that the protein holds the chromophore in a distorted conformation after trans-*cis isomerization. Bacteriorhodopsin (BR), the major component of the purple membrane found in Halobacterium halobium, is a retinal-containing protein that acts as a solar energy converter (1, 2). Absorption of light by retinal in light-adapted BR drives the pigment through a proton-pumping photocycle that stores energy for ATP synthesis as a trans-membrane proton gradient (3,4). In order to understand the mechanism of this light-driven proton pump, we have been studying the structure of the parent BR molecule and its photoproducts ( Fig. 1) with resonance Raman spectroscopy.Raman spectra provide detailed vibrational information about chromophore structure (5-8) that is particularly useful when aided by selective isotopic modification ofretinal because these substitutions permit unambiguous characterization of the molecular vibrations. For example, we have recently shown that the 15-deuterio-induced changes in the 1100-1300 cm-' fingerprint vibrations ofthe chromophore provide a clear criterion for distinguishing between the 13-cis and all-trans configurations even in the presence ofprotein perturbations (9). We have used this method to show that the M412 intermediate contains a 13-cis retinal chromophore, whereas the parent BR chromophore is all-trans, in agreement with the most recent chromophore extraction results (10,11).This in situ demonstration of a trans--cis isomerization during proton pumping has focused our attention on K, the primary photoproduct in the proton-pumping cycle. Arguments based on analogies between the photochemical behavior of rhodopsin and BR (12), when coupled with the Raman and extraction results on M412 (7-11), suggest that the primary photochemical

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Using a dual-beam flow technique, we have obtained resonance Raman spectra of the M412 photointermediates of both native purple membrane (15H M412) and purple membrane regenerated with 15-deuterioretinal (15D M412). For comparison, we have also obtained Raman spectra of the n-butylamine Schiff bases of the 13-cis and all-trans isomers of 15H and 15D retinal. The 15D model compound spectra, when compared to the 15H spectra, show isotopically induced spectral changes that are markedly different for the two isomers. There is a very close agreement between the frequency and intensity changes which occur upon deuteration of M412 and those which occur upon deuteration of the 13-cis model compound, but not even a qualitative correspondence exists when M412 and the all-trans model compound are similarly compared. These data demonstrate that the chromophore of M412 is an unprotonated Schiff base of 13-cis-retinal rather than all-trans-retinal. An analogous spectral comparison of 15H and 15D light-adapted bacteriorhodopsin (bRLA) with the 15H and 15D protonated Schiff bases of 13-cis- and all-trans-retinal demonstrates that bRLA contains an all-trans chromophore, in agreement with previous extraction experiments. Thus, a trans leads to cis isomerization occurs in the proton-pumping photocycle of Halobacterium halobium.

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.

We have obtained room-temperature transient infrared difference spectra of the M412 photoproduct of bacteriorhodopsin (bR) by using a "rapid-sweep" Fouriertransform infrared (FT-IR) technique that permits the collection of an entire spectrum (extending from 1000 to 2000 cm-' with 8-cm'1 resolution) in 5 ms. These spectra exhibit <10-4 absorbance unit of noise, even utilizing wet samples containing -40 pmol of bR in the measuring beam. The bR --M transient difference spectrum is similar to FT-IR difference spectra previously obtained under conditions where M decay was blocked (low temperature or low humidity). In particular, the transient spectrum exhibits a set of vibrational difference bands that were previously attributed to protonation changes of several tyrosine residues on the basis of isotopic derivative spectra of M at low temperature. Our rapid-sweep FT-IR spectra demonstrate that these tyrosine/tyrosinate bands are also present under more physiological conditions. Despite the overall similarity to the low-temperature and low-humidity spectra, the room-temperature bR --M transient difference spectrum shows significant additional features in the amide I and amide II regions. These features' presence suggests that a small alteration of the protein backbone accompanies M formation under physiological conditions and that this conformational change is inhibited in the absence of liquid water.Infrared difference spectroscopy is a useful technique for measuring protein structural changes. Every residue has infrared-active group vibrations that are potentially sensitive to changes in covalent bonding (e.g., conformation, protonation state) and in noncovalent interactions with the surrounding environment (e.g., hydrogen bonding, steric hindrance). Although the presence of many IR-active groups in a large protein leads to a very complex IR spectrum, careful null measurements make it possible to observe only the small subset of vibrations that change during a biochemical transformation.The photoreactive proteins bacteriorhodopsin (bR) and rhodopsin are ideally suited for observing such difference spectra. By photolyzing these proteins inside a spectrometer, it has been possible to make very precise measurements of the resulting IR absorbance changes (1-12). These IR difference spectra have provided a wealth of information. For example, it has been shown that during the photoreaction of bR (Xmax = 568 nm) to M (Xmax = 412 nm), an aspartate residue becomes protonated (1, 7); additional protonation changes of carboxylic acid residues occur at other steps in the bR photocycle (7, 9, 10). More recently, IR difference spectra (along with UV difference spectra) have detected changes in protonation of several tyrosines in the photointermediates between bR and M (8-10). Such spectra provide important experimental tests of proposed proton-translocation mechanisms for bR.Early IR difference spectra of bR and rhodopsin photoproducts were obtained by using flash photolysis techniques and single-wavelength transient m...