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bR----L, and bR----M photoreactions in bacteriorhodopsin mutants in which Asp residues 85, 96, 115, and 212 have been replaced by Asn and by Glu.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

Bacteriorhodopsin (bR) is a light-driven proton pump whose function includes two key membrane-based processes, active transport and energy transduction. Despite extensive research on bR and other membrane proteins, these processes are not fully understood on the molecular level. In the past ten years, the introduction of Fourier transform infrared (FTIR) difference spectroscopy along with related techniques including time-resolved FTIR difference spectroscopy, polarized FTIR, and attenuated total reflection FTIR has provided a new approach for studying these processes. A key step has been the utilization of site-directed mutagenesis to assign bands in the FTIR difference spectrum to the vibrations of individual amino acid residues. On this basis, detailed information has been obtained about structural changes involving the retinylidene chromophore and protein during the bR photocycle. This includes a determination of the protonation state of the four membrane-embedded Asp residues, identification of specific structurally active amino acid residues, and the detection of protein secondary structural changes. This information is being used to develop an increasingly detailed picture of the bR proton pump mechanism.

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 ...

Polarized Fourier transform infrared spectroscopy has been used to study the structure of purple membrane from Halobacterium halobium. Membranes were oriented by drying a suspension of membrane fragments onto Irtran-4 slides. Dichroism measurements of the amide I, II and A peaks were used to find the average spatial orientation of the bacteriorhodopsin alpha-helices. By deriving a function that relates the observed dichroism to the orientational order parameters for the peptide groups, helical axis distribution, and mosaic spread of the membranes, the average orientation of the alpha-helices was found to lie in a range of less than 26 degrees away from the membrane normal, agreeing with electron microscopic measurements. The frequency of the amide I and A peaks is at least 10 cm-1 higher than values found for most alpha-helical polypeptides and proteins. This may indicate that bacteriorhodopsin contains distorted alpha-helical conformations.

Isotopically labeled tyrosines have been selectively incorporated into bacteriorhodopsin (bR). A comparison of the low-temperature bR570 to K Fourier transform infrared-difference spectra of these samples and normal bR provides information about the role of tyrosine in the primary phototransition. Several tyrosine contributions to the difference spectrum are found. These results and comparison with the spectra of model compounds suggest that a tyrosinate group protonates during the bR570 to K transition. This conclusion is strongly supported by the results of UV difference spectroscopy.Elucidation of the mechanism by which bacteriorhodopsin (bR), a light-driven proton pump in the purple membrane of Halobacterium halobium functions remains an important problem in biology (1). Two molecular events have been implicated in the bR primary phototransition. (i) An all-trans to 13-cis isomerization of the retinal chromophore has been deduced from resonance Raman measurements (2). (ii) Movement of a proton has been surmised from picosecond visible absorption data (3). It is not known, however, which bR groups are involved in proton transfer or how this event is coupled to retinal chromophore isomerization.Recently, our group and others (4-12) have begun to study the molecular alterations occurring during the bR photocycle with Fourier transform infrared (FTIR) spectroscopy. It has been demonstrated by this work that FTIR is sufficiently sensitive to detect changes occurring in single chemical groups in both the bR chromophore and the protein component. It is important for further progress that contributions to the FTIR-difference spectrum from specific amino acid residues be identified. This can be accomplished by selectively incorporating in bR isotopically labeled amino acids such as [E-15N]lysine (13). Such an approach was recently used by Englehard et al., who were able to identify the 1760 cm-1 carboxyl vibration (4, 7) as due to an aspartic acid residue (11).We report here the results of FTIR measurements on bR samples containing L-ring-deuterated tyrosine and L-[ring-4-13C]tyrosine (carbon label nearest hydroxyl group). We compare these difference spectra with FTIR measurements on model tyrosine compounds at high and low pH and p2H. Our results indicate that one or more tyrosines change during the bR570 to K phototransition and suggest that a tyrosinate group in bR570 becomes protonated by K. These conclusions are strongly supported by UV difference measurements, which also suggest the possible involvement of tryptophan at this stage of the photocycle. (14) and was purified by recrystallization. All isotope substitutions were verified by NMR spectroscopy. MATERIALS AND METHODSHalobacterium halobium R1 was grown in a synthetic medium like that of Gochnauer and Kushner (15) Purple membrane was isolated by the method of Oesterhelt and Stoeckenius (16). Specific activity measurements indicated that 50-80% of the tyrosine residues were labeled in various preparations. Amino acid analysis showed that <10% of t...