Tyrosine and carboxyl protonation changes in the bacteriorhodopsin photocycle. 2. Tyrosines-26 and -64

Low-temperature Fourier transform infrared (FTIR) and UV difference spectroscopies combined with selective tyrosine nitration and tyrosine isotopic labeling have been used to investigate the participation of tyrosines-26 and -64 in the bacteriorhodopsin (bR) photocycle. Nitration of Tyr-26 has no detectable effect on the FTIR or UV difference spectra of the BR570----K630 or BR570----M412 transitions. In contrast, nitration of Tyr-64 causes changes in both the FTIR and UV spectra of these transitions. However, this nitration does not alter tyrosine peaks in the FTIR difference spectra which have previously been associated with the protonation of a tyrosinate by K630 and the deprotonation of a tyrosine by M412 [Roepe, P., Ahl, P. L., Das Gupta, S. K., Herzfeld, J., & Rothschild, K. J. (1987) Biochemistry (preceding paper in this issue)]. Instead, Tyr-64 nitration appears to affect other tyrosine peaks. These results and changes in UV difference spectra upon Tyr-64 nitration are consistent with the deprotonation of Tyr-64 by M412 as concluded previously [Scherrer, P., & Stoeckenius, W. (1985) Biochemistry 24, 7733-7740]. Effects on chromophore vibrations caused by Tyr-64 nitration are unaltered upon reducing the nitrotyrosine to aminotyrosine with sodium dithionite. Finally, nitro-Tyr-64 causes a shift in the frequency of a positive peak at 1739 cm-1 in the BR570----M412 FTIR difference spectrum which reflects the protonation of a carboxyl-containing residue [Engelhard, M., Gerwert, K., Hess, B., Kreutz, W., & Siebert, F. (1985) Biochemistry 24, 400-407; Roepe, P., Ahl, P. L., Das Gupta, S. K., Herzfeld, J., & Rothschild, K. J. (1987) Biochemistry (preceding paper in this issue)]. The shift does not occur for samples containing amino-Tyr-64. These data suggest that Tyr-64 may interact with this carboxyl group.     

Tyrosine protonation changes in bacteriorhodopsin. A Fourier transform infrared study of BR548 and its primary photoproduct

The structural alterations which occur in bacteriorhodopsin (bR) during dark adaptation (BR570----BR548) and the primary phototransition of the dark photocycle (BR548----KD610) have been investigated by Fourier transform infrared and UV difference spectroscopy. Possible contributions of tyrosine to the Fourier transform infrared difference spectra of these transitions were assigned by incorporating ring per-deuterated tyrosine into bR. Based on these data and UV difference measurements, we conclude that a stable tyrosinate exists in BR570 at physiological temperature and that it protonates during formation of BR548. A tyrosinate protonation has also been observed at low temperature during the primary phototransition of BR570 to the red-shifted photoproduct K630 (1). However, we now find that no tyrosine protonation change occurs during the primary phototransition of BR548 to the red-shifted intermediate KD610. Through analysis of bR containing isotopically labeled retinals, it was also determined that the chromophore of KD610 exits in a 13-trans, 15-cis configuration. On the basis of this evidence and previous studies on the structure of the chromophore in BR570, BR548, and K630, it appears that only the 13-trans,15-trans configuration of the protonated chromophore leads to a stable tyrosinate group. It is proposed that a tyrosinate residue is stabilized due to its interaction with the Schiff base positive charge in the BR570 chromophore. Isomerization of the chromophore about either the C13 = C14 or C = N bond disrupts this interaction causing a protonation of the tyrosinate.     

Fourier transform infrared study of the halorhodopsin chloride pump

Halorhodopsin (hR) is a light-driven chloride pump located in the cell membrane of Halobacterium halobium. Fourier transform infrared difference spectroscopy has been used to study structural alterations occurring during the hR photocycle. The frequencies of peaks attributed to the retinylidene chromophore are similar to those observed in the spectra of the related protein bacteriorhodopsin (bR), indicating that in hR as in bR an all-trans----13-cis isomerization occurs during formation of the early bathoproduct. Spectral features due to protein structural alterations are also similar for the bR and hR photocycles. For example, formation of the red-shifted primary photoproducts of both hR and bR results in similar carboxyl peaks in the 1730-1745-cm-1 region. However, in contrast to bR, no further changes are observed in the carboxyl region during subsequent steps in the hR photocycle, indicating that additional carboxyl groups are not directly involved in chloride translocation. Overall, the close similarity of vibrations in hR and bR photoproduct difference spectra supports the existence of some common elements in the molecular mechanisms of energy transduction and active transport by these two proteins.     

Characterization of the conformational change in the M1 and M2 substates of bacteriorhodopsin by the combined use of visible and infrared spectroscopy.

A combination of visible and Fourier transform infrared (FTIR) spectroscopies is used to characterize the formation of the M1 and M2 substates of the bacteriorhodopsin photocycle in glucose-embedded, hydrated thin films. Difference FTIR bands in the amide I region verify the previously reported existence of a significant peptide backbone conformational change in the transition from M1 to M2. The visible absorption spectra demonstrate that contamination of the M-intermediate samples by L, N, or other non-M species should contribute negligibly to the observed changes in the amide I region, and this conclusion is supported by comparison of specific carboxyl group peaks with corresponding bands in published L and N FTIR difference spectra. Based upon spectroscopic results, an extension of the C-T Model (Fodor, S., Ames, J., Gebhard, R., van den Berg, E., Stoeckenius, W., Lugtenberg, J., and Mathies, R. (1988) Biochemistry 27, 7097-7101) is presented. The results of this work suggest that protein structural changes should be clearly visible in M-bR, difference Fourier density maps and that these structural changes may in turn elucidate how bacteriorhodopsin actively pumps ions across the purple membrane of Halobacterium halobium.     

Orientation of the bacteriorhodopsin chromophore probed by polarized Fourier transform infrared difference spectroscopy

Polarized, low-temperature Fourier transform infrared (FTIR) difference spectroscopy has been used to investigate the structure of bacteriorhodopsin (bR) as it undergoes phototransitions from the light-adapted state, bR570, to the K630 and M412 intermediates. The orientations of specific retinal chromophore and protein groups relative to the membrane plane were calculated from the linear dichroism of the infrared bands, which correspond to the vibrational modes of those groups. The linear dichroism of the chromophore C=C and C-C stretching modes indicates that the long axis of the polyene chain is oriented at 20-25 degrees from the membrane plane at 250 K and that it orients more in-plane when the temperature is reduced to 81 K. The polyene plane is found to be approximately perpendicular to the membrane plane from the linear dichroism calculations of the HOOP (hydrogen out-of-plane) wags. The orientation of the transition dipole moments of chromophore vibrations in the K630 and M412 intermediates has been probed, and the dipole moment direction of the C=O bond of an aspartic acid that is protonated in the bR570----M412 transition has been measured.     

Effects of amino acid substitutions in the F helix of bacteriorhodopsin. Low temperature ultraviolet/visible difference spectroscopy

Site-specific mutagenesis in combination with low temperature UV/visible difference spectroscopy has been used to investigate the role of individual amino acids in the structure and function of bacteriorhodopsin (bR). We examined the effects of eight single amino acid substitutions, all in the putative F helix, on the absorption of bR as well as formation of the K and M intermediates. Both the absorbance spectra and the photocycle difference spectra of Escherichia coli expressed bR as well as the mutants S183A, P186G, and E194Q all closely resembled the corresponding purple membrane spectra. In contrast the Pro-186----Leu substitution resulted in the loss of the normal photocycle and a large blue shift in the bR state lambda max. Thus, Pro-186 appears to play a critical role in maintaining the normal protein-chromophore interactions, although the pyrrolidine ring is not essential since proline could be replaced by glycine at this position. The mutants W182F, W189F, and S193A did not appear to be directly involved in the bathochromic shift of bR since they all had lambda max's close to that of purple membrane and produced intermediates similar to K and M. However, alterations in the UV and visible difference spectra as well as the appearance of some irreversibility in the photoreactions indicate that these mutants have altered protein-chromophore interactions during the photocycle. Unlike the other mutants examined, Y185F exhibited a red-shifted form of bR and K raising the possibility that Tyr-185 is directly involved in color regulation. In addition, UV difference peaks previously associated with a tyrosine deprotonation were absent in Y185F indicating that Tyr-185 undergoes protonation changes during the photocycle in agreement with recent Fourier transform infrared difference measurements (Braiman, M.S., Mogi, T., Stern, L. J., Hackett, N., Chao, B. H., Khorana, H.G., and Rothschild, K. J. (1988) Proteins: Structure, Function, and Genetics 3, 219-229). Our results suggest that Trp-182, Tyr-185, Pro-186, Trp-189, and Ser-193, all of which are within a 100 degrees segment of the F helix, are part of a retinal-binding pocket.     

Conformational changes in bacteriorhodopsin studied by infrared attenuated total reflection.

We report on a new method based on Fourier transform infrared (FTIR)-difference spectroscopy for studying the conformational changes occurring during the photocycle of bacteriorhodopsin. Previous studies have been made by measuring the absorbance of an infrared (IR) beam transmitted through a thin hydrated purple membrane film. In contrast, the present study utilizes the technique of attenuated total reflection (ATR). Purple membrane is fixed on the surface of a germanium internal reflection crystal and immersed in a buffer whose pH and ionic composition can be varied. Measurements of the amide I and II absorbance with light polarized parallel and at 45 degrees to the crystal surface reveals that the membrane is highly oriented. An ATR-FTIR-difference spectrum of the light to dark (bR570 to bR548) transition is similar but not identical to the transmittance FTIR-difference spectrum. This disagreement between the two methods is shown to be due in the ATR case to the absorption of transition moments oriented predominantly out of the membrane plane. Raising the pH of La3+ substituted purple membrane films from 6.8 to 8.0 slows the M-decay rate sufficiently so that a bR570 to M412 difference spectrum can be obtained with steady state illumination at room temperature. A comparison of this difference spectrum with that obtained at -23 degrees C using the transmittance method reveals several changes that cannot be attributed to out-of-plane transition moments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Time-resolved Fourier transform infrared spectroscopy of the polarizable proton continua and the proton pump mechanism of bacteriorhodopsin

Nanosecond-to-microsecond time-resolved Fourier transform infrared (FTIR) spectroscopy in the 3000-1000-cm(-1) region has been used to examine the polarizable proton continua observed in bacteriorhodopsin (bR) during its photocycle. The difference in the transient FTIR spectra in the time domain between 20 ns and 1 ms shows a broad absorption continuum band in the 2100-1800-cm(-1) region, a bleach continuum band in the 2500-2150-cm(-1) region, and a bleach continuum band above 2700 cm(-1). According to Zundel (G., J. Mol. Struct. 322:33-42), these continua appear in systems capable of forming polarizable hydrogen bonds. The formation of a bleach continuum suggests the presence of a polarizable proton in the ground state that changes during the photocycle. The appearance of a transient absorption continuum suggests a change in the polarizable proton or the appearance of new ones. It is found that each continuum has a rise time of less than 80 ns and a decay time component of approximately 300 micros. In addition, it is found that the absorption continuum in the 2100-1800-cm(-1) region has a slow rise component of 190 ns and a fast decay component of approximately 60 micros. Using these results and those of the recent x-ray structural studies of bR(570) and M(412) (H. Luecke, B. Schobert, H.T. Richter, J.-P. Cartailler, and J. K., Science 286:255-260), together with the already known spectroscopic properties of the different intermediates in the photocycle, the possible origins of the polarizable protons giving rise to these continua during the bR photocycle are proposed. Models of the proton pump are discussed in terms of the changes in these polarizable protons and the hydrogen-bonded chains and in terms of previously known results such as the simultaneous deprotonation of the protonated Schiff base (PSB) and Tyr185 and the disappearance of water molecules in the proton release channel during the proton pump process.     

Photoacoustic photocalorimetry and spectroscopy of Halobacterium halobium purple membranes.

Enthalpy changes associated with intermediates of the photocycle of bacteriorhodopsin (bR) in light-adapted Halobacterium halobium purple membranes, and decay times of these intermediates, are obtained from photoacoustic measurements on purple membrane fragments. Our results, mainly derived from modulation frequency spectra, show changes in the amount of energy stored in the intermediates and in their decay times as a function of pH and/or salt concentration. Especially affected are the slowest step (endothermic) and a spectroscopically unidentified intermediate (both at pH 7). This effect is interpreted in terms of cation binding to the protein, conformational changes of which are thought to be connected with the endothermic process. Wavelength spectra are used to obtain heat dissipation spectra, which allow identification of wavelength regions with varying photoactivity, and estimation of the amounts of enthalpy stored in the photointermediates. Because of bleaching and accumulation of intermediates, however, and because of the small fraction of light energy stored during photocycle, quantitative information cannot be obtained. From photoacoustic wavelength spectra of purple membrane fragments equilibrated at 63% relative humidity, rise and decay times of the bR570 and M412 intermediates are calculated.     

Time-resolved ultraviolet resonance Raman studies of protein structure: application to bacteriorhodopsin.

Time-resolved ultraviolet resonance Raman spectra of bacteriorhodopsin are used to study protein structural changes on the nanosecond and millisecond time scales. Excitation at 240 nm is used to selectively enhance vibrational scattering from tyrosine so that changes in its hydrogen bonding and protonation state can be examined. Both nanosecond and millisecond UV Raman difference spectra indicate that none of the tyrosine residues change ionization state during the BR----K and BR----M transitions. However, intensity changes are observed at 1172 and 1615 cm-1 in the BR----M UV Raman difference spectra. The 1615-cm-1 feature shifts down 25 cm-1 in tyrosine-d4-labeled BR, consistent with its assignment as a tyrosine vibration. The intensity changes in the BR----M UV Raman difference spectra most likely reflect an increase in resonance enhancement that occurs when one or more tyrosine residues interact more strongly with a hydrogen-bond acceptor in M412. The frequency of the v7a feature (1172 cm-1) in the BR----M UV Raman difference spectra supports this interpretation. The proximity of Tyr-185 and Asp-212 in the retinal binding pocket suggests that deprotonation of the Schiff base in M412 causes Tyr-185 to stabilize ionized Asp-212 by forming a stronger hydrogen bond.     

Fourier transform infrared evidence for Schiff base alteration in the first step of the bacteriorhodopsin photocycle

The first step of the bacteriorhodopsin (bR) photocycle involves the formation of a red-shifted product, K. Fourier transform infrared difference spectra of the bR570 to K630 transition at 81 K has been measured for bR containing different isotopic substitutions at the retinal Schiff base. In the case of bacteriorhodopsin containing a deuterium substitution at the Schiff base nitrogen, carbon 15, or both, we find spectral changes in the 1600-1610- and 1570-1580-cm-1 region consistent with the hypothesis that the K630 C=N stretching mode of a protonated Schiff base is located near 1609 cm-1. A similar set of Schiff base deuterium substitutions for retinal containing a 13C at the carbon 10 position strongly supports this conclusion. This assignment of the K630 C=N stretching vibration provides evidence that the bR Schiff base proton undergoes a substantial environmental change most likely due to separation from a counterion. In addition, a correlation is found between the C=N stretching frequency and the maximum wavelength of visible absorption, suggesting that movement of a counterion relative to the Schiff base proton is the main source of absorption changes in the early stages of the photocycle. Such a movement is a key prediction of several models of proton transport and energy transduction. Evidence is also presented that one or more COOH groups are involved in the formation of the K intermediate.     

Transient photovoltages in purple membrane multilayers. Charge displacement in bacteriorhodopsin and its photointermediates

The photovoltaic properties of bacteriorhodopsin molecules and their photochemical intermediates have been investigated in an experimental cell consisting of multilayered films of highly oriented, dry fragments of purple membrane and lipid sandwiched between two metal (Pd) electrodes. The electrical time constant of these sandwich cells containing between 5 and 30 layers is less than 10(-5) S. Bright illumination of these cells with actinic flashes of approximately 1 ms duration generates transient photovoltages. These photovoltages, which make the extracellular surface of purple membrane positive with respect to the intracellular surface, follow the time course of the flash with no detectable latency. The amplitude of the photovoltages increases linearly with light intensity and their action spectrum matches the absorption spectrum of the light-adapted state of bacteriorhodopsin, BR570. In these dry multilayer cells, the slow photointermediates of bacteriorhodopsin, M412, N520 and O640 are long lived. Illumination of the sandwich cells with long duration (200 ms) pulses of light results, therefore, in the formation of photomixtures containing all these slow photointermediates. Flash illumination of the sandwich cells immediately following the conditioning pulse produces photovoltages whose action spectra match the absorption spectra of the M412 and N520 photointermediates. The M412 photovoltages, like the BR570 photovoltages, follow the time course of the actinic flash with no detectable latency and increase in amplitude linearly with light intensity. But, unlike the BR570 photovoltage, the M412, N520 and O640 photovoltages make the extracellular surface of purple membrane negative with respect to the intracellular surface. Through the of their specific photovoltaic signals, M412 and N520 are shown to be kinetically distinct photointermediates of bacteriorhodopsin. Detection of fast photovoltages with these characteristics in the absence of any ionic solution, and in parallel with spectrophotometric changes, suggest that they arise from charge displacements in the bacteriorhodopsin molecules and their photointermediates as they undergo photochemical conversion in response to the absorption of photons.     

Vibrational spectroscopy of bacteriorhodopsin mutants: light-driven proton transport involves protonation changes of aspartic acid residues 85, 96, and 212

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.     

Electric response of a back photoreaction in the bacteriorhodopsin photocycle.

The electric response of a back photoreaction in the bacteriorhodopsin photocycle was investigated. The proton pumping activity of green flash excited bacteriorhodopsin stops if the M412 form is illuminated by blue light (Karvaly and Dancsházy, 1977). In the present work a fast negative displacement current signal was measured in an oriented membrane suspension system, indicative of back movement of protons from M412 to BR570. Quantitative evaluation of the data shows that there are at least two steps in the back reaction, with different rate constants. The temperature dependence of the rate constants show simple linear Arrhenius behavior between 5 degree and 40 degree C. The rate constants were slower by a factor of 1.8 in D2O suspension. The relevance of the protein electric response signals (PERS) observed in this paper to the early receptor potential is discussed.     

Ultrafast infrared spectroscopy of bacteriorhodopsin.

Picosecond infrared spectroscopy is developed and used for the first time to study the dynamics of photoexcited bacteriorhodopsin (BR). Both spectral and time-resolved data are obtained. The results open an entirely new approach to investigations of the BR photocycle. The infrared difference spectrum (K minus BR570) recorded at ambient temperature between 1,560 and 1,700 cm-1 is not identical with the spectrum reported for a frozen sample. Three bands of the K state at 1,622, 1,610, and 1,580 cm-1 and the bleaching at 1,637 cm-1 (C = NH stretch) are seen. These new spectral lines appear in less than 10 ps.     

Mechanism of Nonlinear Photoinduced Anisotropy in Bacteriorhodopsin and its Derivatives

Polymer films made with photosensitive chromophore protein bacteriorhodopsin (BR) from the extreme halophile Halobacterium salinarium as well as films made with BR derivatives exhibit a nonlinear photoinduced anisotropy. Two different methods can be used to induce anisotropy in polymer BR films. The first method is based on the anisotropic properties of the initial form of the photocycle, BR570 (B-type anisotropy). Another method is based on the anisotropic properties of the longest-lived photocycle intermediate M412 (M-type anisotropy). CW gas lasers were employed to induce a reversible anisotropy in polymer BR films. Nonlinear photoinduced anisotropy is discussed in the context of a model for the anisotropic photoselection of BR molecules under linearly polarized light. A comparison of the experimental dependencies of nonlinear photoinduced anisotropy on laser intensity with similar calculated dependencies enables one to determine the molecular dichroism of BR and its derivatives not only for the initial form of the photocycle, B but also for the longest-lived intermediate M. Here we present the data showing the correlation between the laser induced nonlinear anisotropic properties and chromophore/protein interactions in BR. The effect of polymer binder on the nonlinear photoanisotropic properties of polymer BR films is also described.     

The action of lanthanum ions and formaldehyde on the proton-pumping function of bacteriorhodopsin

The photochemical cycle and the proton-pumping function of bacteriorhodopsin modified with lanthanum and formaldehyde has been studied. In both preparations, the M412 leads to BR570 transition time has been found to increase considerably. The deceleration of the photochemical cycle has been shown to be accompanied by inhibition of the millisecond phase of the photoelectrical response of bacteriorhodopsin membranes associated with phospholipid-impregnated collodion film. Photoelectrogenic activity measured with permeable ion probe in proteoliposomes was also inhibited. Effects of lanthanum were reversed by EDTA. Formation of M412 was slightly accelerated and the microsecond electrogenic phase was not affected by lanthanum and by formaldehyde. It is concluded that lanthanum, but not formaldehyde, can be used as a specific reversible inhibitor of the second half of the bacteriorhodopsin photocycle and of the associated H+ uptake on the cytoplasmic side of the halobacterial membrane. Possible mechanisms of these effects are discussed.     

O-H stretching vibration in Fourier transform difference infrared spectra of bacteriorhodopsin

FTIR difference spectroscopic studies of M intermediate and LA bacteriorhodopsin in the O-H stretching region show bands at 3671 and 3641 cm-1, respectively. The O-H stretching bands in this region may reflect protonation-deprotonation changes or environmental change in the tyrosine residues in bR.     

Active internal waters in the bacteriorhodopsin photocycle. A comparative study of the L and M intermediates at room and cryogenic temperatures by infrared spectroscopy

We present time-resolved room-temperature infrared difference spectra for the bacteriorhodopsin (bR) photocycle at 8 cm (-1) spectral and 5 micros temporal resolution, from 4000 to 800 cm (-1). An in situ hydration method allowed for a controlled and stable sample hydration (92% relative humidity), largely improving the quality of the data without affecting the functionality of bR. Experiments in both H 2 (16)O and H 2 (18)O were conducted to assign bands to internal water molecules. Room-temperature difference spectra of the L and M intermediates minus the bR ground state (L-BR and M-BR, respectively) were comprehensively compared with their low-temperature counterparts. The room-temperature M-BR spectrum was almost identical to that obtained at 230 K, except for a continuum band. The continuum band contains water vibrations from this spectral comparison between H 2 (16)O and H 2 (18)O, and no continuum band at 230 K suggests that the protein/solvent dynamics are insufficient for deprotonation of the water cluster. On the other hand, an intense positive broadband in the low-temperature L-BR spectrum (170 K) assigned to the formation of a water cavity in the cytoplasmic domain is absent at room temperature. This water cavity, proposed to be an essential feature for the formation of L, seems now to be a low-temperature artifact caused by restricted protein dynamics at 170 K. The observed differences between low- and room-temperature FTIR spectra are further discussed in light of previously reported dynamic transitions in bR. Finally, we show that the kinetics of the transient heat relaxation of bR after photoexcitation proceeds as a thermal diffusion process, uncorrelated with the photocycle itself.     

Transient spectroscopy of bacterial rhodopsins with an optical multichannel analyzer. 1. Comparison of the photocycles of bacteriorhodopsin and halorhodopsin

We used a gated optical multichannel analyzer to measure transient flash-induced absorption changes in bacteriorhodopsin (BR) and halorhodopsin (HR) and developed criteria for calculating the absorption spectra of the photocycle intermediates and the kinetics of their rise and decay. The results for BR agree with data reported by a large number of other authors. The results for HR in the presence of chloride are consistent with earlier data and reveal an additional intermediate, not previously seen, in the submicrosecond time scale. Although an M412-like intermediate is not in the HR photocycle, a one-by-one comparison of the rest of the intermediates observed for BR and HR indicates a striking similarity between the photocycles of the two bacterial rhodopsins. This was previously not apparent, perhaps because the experimental approaches to the spectroscopy of the two pigments were different and the data were thus more fragmented.