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bR----L, and bR----M photoreactions in bacteriorhodopsin mutants in which Asp residues 85, 96, 115, and 212 have been replaced by Asn and by Glu.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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