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Femtosecond absorption spectroscopy of light-adapted and dark-adapted bacteriorhodopsin
Volume 137, number 4
CHEMICAL PHYSICS LETTERS
19 June 1987
FEMTOSECOND ABSORPTION SPECTROSCOPY OF LIGHT-ADAPTED AND DARK-ADAPTED BACTERIORHODOPSIN J.W. PETRICH ‘, J. BRETON b, J.L. MARTIN a,’ and A. ANTONETTI a aLaboratoired’optique AppliquPe,Ecole Polytechnique,ENSTA, INSERM U275. 91128 PalaiseauCedex, France bService de Biophysique,CEN Saclay, 91191 G&w-Yvette Cedex, France Received 9 February 1987; in final form 1 April 1987
Using tunable femtosecond laser pulses, we report the first measurements of the kinetics of formation of the initial photoproducts of dark-adapted bacteriorhodopsin (approximately 50% all-trans and 50% 13-cis, 15-cis retinal) and compare these results with those obtained for light-adapted bacteriorhodopsin (all-trans retinal). We find that for both light-adapted and dark-adapted bacteriorhodopsin, the initial photoproducts are formed with the same time constants. These results are discussed in terms of the photophysics of bacteriorhodopsin and the mechanism of formation of its photoproducts.
1. Introduction The protein bacteriorhodopsin (BR) is the major constituent of the purple membrane of the bacterium Halobacterium halobium. The role of BR is to convert light into chemical energy by pumping protons across the bacterial membrane [ 11. In the membrane, BR forms trimers which comprise a twodimensional hexagonal lattice [ 21. The BR monomer is a hydrophobic protein ( MWZ 26000) linked from its lysine 216 to a retinal chromophore by means of a protonated Schiff base. Prolonged illumination of BR prepares the retinal in an all-trans configuration. Early subpicosecond studies of BR were performed by Ippen et al. [ 31 and indicated that a bathochromic intermediate appeared in 1.Of 0.5 ps. Recent femtosecond absorption studies of the light-adapted BR have shown that subsequent to absorption of a photon, a species denoted as J (whose structure has not yet been determined) is formed in approximately 0.5 ps from a proposed excited state of BR [ 4-61. J has been determined to decay in 3-5 ps to the relatively long-lived (ps) bathochromic intermediate, K [ 4-61, whose structure and spectra have been studied by a variety of techniques [ 11. In a few milliseconds, the all-trans retinal is regener’ To whom correspondence should be addressed.
ated via a pathway involving intermediates denoted as L, M, and 0 which have also been characterized by their absorption spectra, kinetics, and involvement in proton pumping [ 11. In the dark, a thermodynamic equilibrium consisting of approximately 50% of the all-trans retinal and 50% of 13-cis, 15-cis retinal is established [ 7,8 1. These structures are displayed in fig. la. It has been shown that upon excitation the 13-cis, 15-cis retinal also forms a bathochromic intermediate whose absorption spectrum is similar to that of K in the alltrans or light cycle. This bathochromic intermediate formed from 13-cis, 15-cis retinal decays in the dark with a half-time of 37 ms at 20°C to both the 13-cis, 15-cis retinal and to the all-trans retinal, where the pathway to the 13-cis, 15-cis retinal predominates [ 15 1. Thus, the dark cycle, in itself, is ineffective in the pumping of protons and can only be considered to do so if there is a subsequent light source to excite the photogenerated all-trans retinal. The physiological significance of the dark cycle is not yet understood. It is now believed that the primary photophysical event in the light cycle involves a trans-to-cis isomerization [ 1 ] ; and it has been shown by resonance Raman studies at 77 K that the bathochromic intermediate of the light cycle, K, has undergone an almost complete trans-to&s isomerization about its C3=C4
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al Rebnal
( Light-adapted bacteriorhodopsrn1
Lys 216
i)
Retinal
(Dark-adaptedbacteriorhodopsinI
iii 1 Lys 216
Ii
all - trans 13-cis
ii 1
. l5-cis
iv1 k
Lys 216
? ( “bathochromrc
Intermediate *
1
( K. . bathochromic intermediate m1
W bans
cycle
cis cycle . A;
c=o 1: k--H
--
. A’:
hu AZ .,, 0 @
A;
Fig. 1. (a) Structures of retinal in (i) light-adapted and in (iii) dark-adapted bacteriorhodopsin before light absorption. The structures presented for K in the light cycle (ii) are controversial. While there is general agreement [9-l 1 ] that the C13=C,4bond is cis or “cisoid”, Schulten and Tavan [ 111have proposed an additional cis isomerization about the C,4-C,s single bond, which Gerwert and Siebert have suggested is supported by their FTIR results [ 111. As noted, no structure has yet been proposed for the analog of K in the dark cycle. (b) The mechanism proposed by Trissl and GHrtner for proton pumping in the trans (light) cycle and for its absence in the cis (dark) cycle. While light absorption effects a motion of the same magnitude and direction for both the all-trans and the 13-cis, 15-cis retinals, the protonation states of the aspartic acid residues (A, to A.,) suggested to be implicated in proton pumping [ 12,131 prevent proton translocation in the cis cycle. Figure adapted from H.-W. Trissl and W. Giirtner [ 141 with permission of the authors.
bond [ 9, lo]. The possibility of an accompanying isomerization about the C,&Z15 bond remains controversial at this writing [ 111 (see fig. 1a). The isomerization is accompanied by separation of the protonated Schiff base from a negatively charged counterion which is most likely a carboxylate group 370
of one of four aspartic acid residues [ 121 or a tyrosinate group [ 131. Trissl and Ggirtner [ 141 have recently shown that the magnitude and the sign of the transmembrane voltage accompanying this photogenerated charge separation are the same for both light- and dark-adapted BR and for bacteria-opsins
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combined with different retinals with various equilibrium populations of cis and trans conformations. In all cases the photovoltage appeared in less than 70 ps (the time resolution of their apparatus). These authors concluded from their study that the bathochromic intermediates derived from the 13-cis or the all-trans retinals resemble each other but are formed along different pathways. Here we present the first data obtained by transient absorption spectroscopy for the initial photoproducts of the dark cycle of BR and compare them with data obtained for the light cycle of BR. We discuss our results in terms of the BR photophysics and the mechanism of the formation of the bathochromic intermediates.
2. Experimental Transient absorption measurements were performed at room temperature with the apparatus described in detail elsewhere [ 16,171. Purple membrane from Halobacterium halobium was prepared by standard procedures [ 181 and suspended in distilled water containing 0.0 1% NaN3 (approximately 2 mg protein/ml). Dark-adaptation was effected by storage in the dark overnight. At our excitation wavelength of 6 12 nm, the optical density in 1 mm of the light-adapted and the dark-adapted samples was 0.44 and 0.33, respectively. Transient absorption measurements were performed by translating the sample in a 1 mm cuvette through the pump and the probe beams with a velocity of 12 mm/s. To further avoid sample damage (and, for the experiments involving the dark cycle, to ensure that we were always studying dark-adapted samples), the solution was also flowed through the translating cell with a peristaltic pump. The sample reservoir typically contained lo- 15 ml of the BR preparation and was either shielded from light (for the dark-adapted preparation) or illuminated with 50 mW of broadband 560 nm light (for the light-adapted preparation). These conditions were sufficient to produce and maintain complete dark or light adaptation of the samples, which were frequently monitored with a Cary 15 spectrophotometer (fig. 2). Sample damage induced by the time-resolved experiment was measured by the decrease in the maxima of the steady-state
wavelength (nm) Fig. 2. Absorption spectra of light-adapted (-) and of darkadapted (...) purple membrane taken at room temperature with a 1 mm cuvette. The procedure for light- and dark-adaptation is described in the text.
absorption spectra of the light- and dark-adapted samples and was ;5 6%.
3. Results Upon excitation of the light-adapted sample with 120 fs pulses at 612 nm, three distinct phenomena were observed. The BR was bleached in less than 50 fs, and simultaneously there appeared a species with an absorption maximum at 460 nm. This absorption decayed with a time constant of 500 f 40 fs to a species which has previously been designated as J. Our result for the time of formation of J is in good agreement with that of Nuss et al. (430 + 50 fs) [ 41. The decay of J to K is found to be 3.2 + 0.2 ps as measured by the absorption recovery at 580 nm. This result is in good agreement with that of Sharkov et al. (3.0f0.5 ps) [ 51 but is less than the 5 ps decay time obtained by Polland et al. [ 61. We obtain consistent time constants for the formation of J and K over the entire spectral range we investigated, 420 to 675 nm. Performing identical experiments on the darkadapted BR, we observed nearly identical kinetics of formation for the counterparts of J and K in the lightadapted preparation. The only noticeable difference is the amplitude of the absorption at its asymptotic value (figs. 3 and 4). The magnitude of this absorp371
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I/lo
111, LIGHT-ADAPTED
Fig. 3. Transient absorption due to the formation of an excited state of bacteriorhodopsin, BR*, and its subsequent decay to the ground-state species, J. &.,,=612 nm, A,,,=460 nm. BR* is produced in less than 50 fs. Satisfactory double-exponential fits were (a) for the dark-adapted sample: I(t)/I,=l-0.97 xexp( -t/490 fs)-0.03 exp( -t/3200 fs), (b) for the lightadapted sample: I(t)/&= l-O.97 exp( -t/530 fs) -0.03 exp( -t/3200 fs).
tion is consistent with that predicted from the equilibrium spectra (fig. 2) and serves as a criterion for the integrity of the dark-adapted samples. Because dark-adapted BR is a mixture of 50% alltrans retinal and 5OW 13-cis, 15cis retinal, and because the absorption cross section of the cis isomer at the 6 12 nm excitation wavelength is about half that of the all-trans isomer [ 151 (fig. 2)) there is a possibility that the time constants for the formation of the photoproducts of the 13-cis, 15-cis retinal are different from those of the all-trans retinal but cannot be adequately resolved in our absorption measurements. To investigate this possibility, we simulated absorption recovery curves in which the time constants of the photoproducts of the 13-cis, 1S-cis retinal were varied and compared these curves with our data. For the signal-to-noise ratio provided by our experiment, the counterparts of J and K in the dark cycle must be formed approximately 1.5 times more rapidly or more slowly than J and K in 372
Fig. 4. Transient bleaching of dark- and light-adapted bacteriorhodopsin on two different time scales. Collecting the data on a longer time scale allows a more accurate determination of the contribution of the long-lived decay component. A,,,,,, = 6 12 nm, I pmbe=580 nm. The fast recovery of the bleaching represents the formation of the first ground-state intermediate, which is designated J in the light cycle. The slower recovery of the bleaching represents the formation of the second ground-state intermediate, designated Kin the light cycle. Double-exponential fits to the data from the dark-adapted samples yielded: (a) I(t)lfo=0.88 Xexp( -t/490 fs) +0.12 exp( - t/3200 fs): 4 ps full scale; (c) I(t)/Z,=0.75 exp( - t/500 fs) +0.25 exp( - t/3200 fs): = 30 ps full scale. Double-exponential tits to the data from the light-adapted samples yielded: (b) I( t)&=0.70 exp( -t/530 fs) +0.30 xexp( -t/3200 fs): 4 ps full scale; (d) I( t)/1,=0.70 exp( -t/500 fs) +0.30 exp( -t/3200 fs): x 30 ps full scale.
the light cycle to produce a significant difference in our absorption data. We therefore conclude that the initial photoproducts of the light and the dark cycles are formed with nearly identical time constants.
4. Discussion 4.1. Photophysics of the retinal chromophore in bacteriorhodopsin
The quantum yield for the bleaching of lightadapted BR has been measured to be 0.33 [ 19-211 and 0.60 [22]. The independence of the quantum yield on both excitation wavelength and temperature has been interpreted in terms of a trans-to-cis isomerization which takes place after complete thermal relaxation and which requires no activation energy
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[ 20,231. An interesting explanation for a quantum yield of 0.33 arises if it is postulated that light excitation creates a precursor which forms BR* in less than 50 fs (the time resolution of our apparatus). Such a precursor has been suggested by Nuss et al. [4] to be a delocalized excited state of the bacteriorhodopsin trimer. In this case the quantum yield of 0.33 could be interpreted as the efficiency of exciton trapping by a monomer of the trimeric unit. While this explanation is appealing, the existence of efficient exciton coupling [ 241 in the trimer has been called into question. Notably, the large absorption polarization of K has been cited by El-Sayed et al. [25] as evidence that exciton transfer is not competitive with BR photochemistry since rapid exciton transfer is expected to produce depolarized intermediates. Godfrey [26] has discussed the problem of exciton coupling in bacteriorhodopsin trimers in terms of the limits of strong and weak coupling. In the strong coupling limit, it is assumed that the exciton coupling is much larger than the electronic absorption bandwidth [ 27,281 and that all vibronic levels of one monomer are in resonance with those of another. In the weak coupling limit, however, the exciton coupling is much less than the electronic bandwidth and resonance interaction only occurs among individual vibronic levels. Godfrey points out that using Hemenger’s value of 64 cm- ’ [ 291 for the exciton coupling (as opposed to larger estimates for the coupling strength [24,30]), an exciton transfer time of x 90 fs is obtained in the strong coupling limit. In the weak coupling limit, however, the exciton transfer time may easily be increased by an order of magnitude [ 261. Since J is formed in 500 fs, such a weak exciton coupling could produce a situation where the exciton transfer time is significantly slower than the formation time of the first ground-state intermediate of BR. 4.2. The light and the dark cycles: photochemistry Based on the similarity of the absorption spectra of J and K and the fact that K already has its retinal in a cis, or at least partly cis, conformation [ 9, lo], Nuss et al. [ 41 suggested that photoisomerization has already been accomplished with the formation of J. Polland et al. [6] studied the kinetics of bacterioopsin combined with a retinal analog modified to
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prohibit transformation to a 13-cis configuration. They found that such an analog has an excited-state lifetime of 10 ps, a 12-fold enhancement of its fluorescence quantum yield compared with that of native BR [ 3 11, and formed no photoproduct. This was cited as further evidence that the primary photophysical event in light-adapted BR is a rapid transto-cis isomerization about the CL3=C4 bond. For light-adapted and dark-adapted BR and for bacteria-opsins containing retinal analogs whose equilibrium populations ranged from 30 to 85% 13cis, Trissl and Giirtner [ 141 found that, within their time resolution of 70 ps, charge separation was effected with the same rate and in a direction opposite to that of proton translocation [32]. Based on these observations, they suggested that the inability of the dark cycle to pump protons could not be explained in terms of a photoisomerization of the 13cis, 15-cis retinal in a direction opposite so that of the all-trans retinal. They concluded that the bathochromic intermediates derived from the 13-cis and the all-trans retinals resemble each other in their conformation and approach their respective conformations by two different routes. Finally, they suggested that the ability of BR to pump protons in the light cycle is more a consequence of different protonation states of the amino acid residues of the protein rather than of different geometries of the parent retinal moieties (see fig. 1b). Our transient absorption data are consistent with the conclusions and the model presented by Trissl and Gartner [ 141. A bathochromic intermediate that appears with the same time constant in both the darkand the light-adapted preparations need not have the same structure in both of the preparations. For example, it has been shown that a bathochromic shift in the retinal absorption spectrum can be produced by either separation of the protonated Schiff base from its counterion or by protonation of the counterion [ 331: in either instance the net positive charge of the reninal polyene chain increases [ 341. We thus suggest that the similarities in the time constants for the formation of the bathochromic intermediates in the dark and the light cycles are not due to the acquisition of similar retinal structures but to similar displacements from their counter ions. Although it may be argued that the simplest mechanism for such a displacement would involve an all-trans to 13-cis 373
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isomerization in the light cycle and a cis-to-trans isomerization about the C&N bond in the dark cycle (fig. 1) which would, in fact, yield identical intermediates, it is probably more accurate, as Trissl and GIrtner [ 141 suggest, to speak in terms of “transoid” and “cisoid” transformations involving simultaneous distortions of other bonds in the polyene chain instead of isolated and complete trans-to-cis and cis-to-trans isomerizations when discussing the formation of the bathochromic intermediates. This is borne out by the observation of Braiman and Mathies [ 9, lo] : at 77 K, the resonance Raman spectrum of K in the light cycle exhibits intense out-ofplane hydrogen stretching frequencies which are suggestive of an incomplete, or at least an unrelaxed, cis configuration about the &=C,., bond.
5. Conclusion We have studied the transient absorption spectra of the initial photoproducts of the light and the dark cycles of BR. BR*, J, and K in the light cycle, and their counterparts in the dark cycle, are formed with similar time constants. This observation is consistent with the photovoltage measurements of Trissl and Giirtner [ 141. We suggest that the similarity of the time constants in both instances is due to equivalent separations between the protonated Schiff base of the retinal and the counterion furnished by the protein rather than to adoption of equivalent structures of the retinal. Resonance Raman techniques will provide more detailed structural information on the nature of the early-time photoproducts at room temperature; and such experiments with techniques providing subpicosecond resolution [ 351 should prove useful in better understanding the nature of the bathochromic intermediates in the light and the dark cycles. Also, one might determine whether the species J is structurally distinct from K or is a vibrationally hot form of K.
Acknowledgement We thank H.-W. Trissl for very stimulating discussions and J.-L. Rigaud, M. Seigneuret, and A. Bluzat for their generous gift of purple membrane. 374
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JWP was supported by an NSF Industrialized Countries postdoctoral fellowship, an INSERM poste orange, and a fellowship from La Fondation pour la Recherche Medicale. Parts of this work were funded by INSERM, ENSTA, and Le Minis&e de la Recherche et de la Technologie.
References
[ 11W. Stoeckenius and R.A. Bogomolni, Ann. Rev. Biochem. 52 (1982) 587. [2] R. Henderson and P.N.T. Unwin, Nature 257 (1975) 28. [ 3 ] E.P. Ippen, C.V. Shank, A. Lewis and M.A. Marcus, Science 200 (1978) 1279. [4] M.C. Nuss, W. Zinth, W. Kaiser, E. Kijlling and D. Oesterhelt, Chem. Phys. Letters 117 (1985) 1. [5]A.V. Sharkov, A.V. Pakulev, S.V. Chekalin and Y.A. Matveetz, Biochim. Biophys. Acta 808 (1985) 94. [ 61 H.-J. Polland, M.A. Franz, W. Zinth, W. Kaiser, E. Kijlling and D. Oesterhelt, Biophys. J. 49 (1986) 65 1. [7] M.J. Pettei, A.P. Yudil, K. Nakanishi, R. Henselman and W. Stoeckenius, Biochemistry 16 (1977) 1955. [8] G.S. Harbison, SO. Smith, J.A. Pardoen, C. Winkel, J. Lugtenburg, J. Herzfeld, R.A. Mathies and R.G. Griffin, Proc. Natl. Acad. Sci. US 8 1 (1984) 1706. [ 91 M.S. Braiman and R.A. Mathies, Proc. Natl. Acad. Sci. US 79 (1982) 403. [ lo] S.O. Smith, J. Lugtenburg and R.A. Mathies, J. Membrane Biol. 85 (1985) 95. [ 111 K. Schulten and P. Tavan, Nature 272 (1978) 85; P. Tavan and K. Schulten, Biophys. J. 50 (1986) 8 1; K. Genvert and F. Siebert, EMBO J. 5 (1986) 805; S.O. Smith, I. Homung, R. van der Steen, J.A. Pardoen, M.S. Braiman, J. Lugtenburg and R.A. Mathies, Proc. Natl. Acad. Sci. US 83 (1986) 967. [ 121 M. Engelhard, K. Gerwert, B. Hess, W. Kreutz and F. Siebert, Biochemistry 24 (1985) 400. [ 131 G. Dollinger, L. Eisenstein, S.-L. Lin, K. Nakanishi and I. Termini, Biochemistry 25 (1986) 6524. [ 141 H.-W. Trissl and W. G%rtner, Biochemistry, to be published. [ 151 W. Sperling, P. Carl. C.N. Rafferty and N. Dencher, Biophys. Struct. Mech. 3 (1977) 79. [ 161 J.L. Martin, A. Migus, C. Poyart, Y. Lecarpentier, R. Astier and A. Antonetti, Proc. Natl. Acad. Sci. US 80 (1983) 173. [ 171 J.L. Martin, A. Migus, C. Poyart, Y. Lecarpentier, R. Astier and A. Antonetti, in: Ultrafast phenomena, Vol. 4, eds. D.H. Auston and K.B. Eisenthal (Springer, Berlin, 1984) p. 447. [ 181 D. Oesterhelt and W. Stoeckenius, Meth. Enzymol. 31A (1984) 667. [ 191 C.R. Goldschmidt, M. Ottolenghi and R. Korenstein, Biophys. J. 16 (1976) 839; C.R.Goldschmidt, 0. Kalisky, T. Rosenfeld and M. Ottolenghi, Biophys. J. 17 ( 1977) 179.
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[ 201 J.B. Hurley, T.G. Ebrey, B. Honig and M. Ottolenghi, Nature
[29] R.P. Hemenger, J. Chem. Phys. 69 (1978) 2279.
270 (1977) 540. [ 211 J.B. Hurley and T.G. Ebrey, Biophys. J. 22 (1978) 49. [22] D. Oesterhelt and B. Hess, European J. Biochem. 37 (1973) 316; D. Oesterhelt, P. Hegemann and J. Tittor, EMBO J. 4 (1985) 2351. 123] T.G. Ebrey and B. Honig, in: Biological events probed by ultrafast laser spectroscopy, ed. R.R. Alfano (Academic Press, New York, 1982) pp. 271,281. [ 241 T.C. Ebrey, B. Becher, B. Mao, P. Kilbride and B. Honig, J. Mol. Biol. 112 (1977) 377. 1251 M.A. El-Sayed, B. Karvaly and J. Fukumoto, Proc. Natl. Acad. Sci. US 78 (1981) 7512. 1261 R.E. Godfrey, Biophys. J. 38 (1982) 1. 127) Th. Wrster, in: Modem quantum chemistry, ed. 0. Sinanoglu (Academic Press, New York, 1966) p. 93. [28] W.T. Simpson and D.L. Peterson, J. Chem. Phys. 26 (1957) 588.
[ 301 A.N. Kriebel and A.C. Albrecht, J. Chem. Phys. 65 (1976) 4575.
[ 3 1 ] H.-J. Polland, M.A. Franz, W. Zinth, W. Kaiser, E. Kijlling and D. Oesterhelt, Biochim. Biophys. Acta 767 (1984) 635. [32] H.-W. Trissl, Biochim. Biophys. Acta 723 (1983) 327; L. Keszthelyi, Biochim. Biophys. Acta 598 (1980) 429; A. Fahr, P. tiuger and E. Bamberg, J. Membrane Biol. 60 (1981) 51. [ 331 P.E. Blatz, J.H. Mohler and H.V. Navangard, Biochemistry 11 (1972) 848; U.C. Fischer and D. Oesterhelt, Biophys. J. 31 (1980) 139. [34] P. Tavan, K. Schulten and D. Oesterhelt, Biophys. J. 47 (1985) 415. [ 351 J.W. Petrich, J.L. Martin, D. Houde, C. Poyart and A. Orszag, Biochemistry, submitted for publication.
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FEMTOSECOND ABSORPTION SPECTROSCOPY OF LIGHT-ADAPTED AND DARK-ADAPTED BACTERIORHODOPSIN J.W. PETRICH ‘, J. BRETON b, J.L. MARTIN a,’ and A. ANTONETTI a aLaboratoired’optique AppliquPe,Ecole Polytechnique,ENSTA, INSERM U275. 91128 PalaiseauCedex, France bService de Biophysique,CEN Saclay, 91191 G&w-Yvette Cedex, France Received 9 February 1987; in final form 1 April 1987
Using tunable femtosecond laser pulses, we report the first measurements of the kinetics of formation of the initial photoproducts of dark-adapted bacteriorhodopsin (approximately 50% all-trans and 50% 13-cis, 15-cis retinal) and compare these results with those obtained for light-adapted bacteriorhodopsin (all-trans retinal). We find that for both light-adapted and dark-adapted bacteriorhodopsin, the initial photoproducts are formed with the same time constants. These results are discussed in terms of the photophysics of bacteriorhodopsin and the mechanism of formation of its photoproducts.
1. Introduction The protein bacteriorhodopsin (BR) is the major constituent of the purple membrane of the bacterium Halobacterium halobium. The role of BR is to convert light into chemical energy by pumping protons across the bacterial membrane [ 11. In the membrane, BR forms trimers which comprise a twodimensional hexagonal lattice [ 21. The BR monomer is a hydrophobic protein ( MWZ 26000) linked from its lysine 216 to a retinal chromophore by means of a protonated Schiff base. Prolonged illumination of BR prepares the retinal in an all-trans configuration. Early subpicosecond studies of BR were performed by Ippen et al. [ 31 and indicated that a bathochromic intermediate appeared in 1.Of 0.5 ps. Recent femtosecond absorption studies of the light-adapted BR have shown that subsequent to absorption of a photon, a species denoted as J (whose structure has not yet been determined) is formed in approximately 0.5 ps from a proposed excited state of BR [ 4-61. J has been determined to decay in 3-5 ps to the relatively long-lived (ps) bathochromic intermediate, K [ 4-61, whose structure and spectra have been studied by a variety of techniques [ 11. In a few milliseconds, the all-trans retinal is regener’ To whom correspondence should be addressed.
ated via a pathway involving intermediates denoted as L, M, and 0 which have also been characterized by their absorption spectra, kinetics, and involvement in proton pumping [ 11. In the dark, a thermodynamic equilibrium consisting of approximately 50% of the all-trans retinal and 50% of 13-cis, 15-cis retinal is established [ 7,8 1. These structures are displayed in fig. la. It has been shown that upon excitation the 13-cis, 15-cis retinal also forms a bathochromic intermediate whose absorption spectrum is similar to that of K in the alltrans or light cycle. This bathochromic intermediate formed from 13-cis, 15-cis retinal decays in the dark with a half-time of 37 ms at 20°C to both the 13-cis, 15-cis retinal and to the all-trans retinal, where the pathway to the 13-cis, 15-cis retinal predominates [ 15 1. Thus, the dark cycle, in itself, is ineffective in the pumping of protons and can only be considered to do so if there is a subsequent light source to excite the photogenerated all-trans retinal. The physiological significance of the dark cycle is not yet understood. It is now believed that the primary photophysical event in the light cycle involves a trans-to-cis isomerization [ 1 ] ; and it has been shown by resonance Raman studies at 77 K that the bathochromic intermediate of the light cycle, K, has undergone an almost complete trans-to&s isomerization about its C3=C4
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Volume 137, number 4
al Rebnal
( Light-adapted bacteriorhodopsrn1
Lys 216
i)
Retinal
(Dark-adaptedbacteriorhodopsinI
iii 1 Lys 216
Ii
all - trans 13-cis
ii 1
. l5-cis
iv1 k
Lys 216
? ( “bathochromrc
Intermediate *
1
( K. . bathochromic intermediate m1
W bans
cycle
cis cycle . A;
c=o 1: k--H
--
. A’:
hu AZ .,, 0 @
A;
Fig. 1. (a) Structures of retinal in (i) light-adapted and in (iii) dark-adapted bacteriorhodopsin before light absorption. The structures presented for K in the light cycle (ii) are controversial. While there is general agreement [9-l 1 ] that the C13=C,4bond is cis or “cisoid”, Schulten and Tavan [ 111have proposed an additional cis isomerization about the C,4-C,s single bond, which Gerwert and Siebert have suggested is supported by their FTIR results [ 111. As noted, no structure has yet been proposed for the analog of K in the dark cycle. (b) The mechanism proposed by Trissl and GHrtner for proton pumping in the trans (light) cycle and for its absence in the cis (dark) cycle. While light absorption effects a motion of the same magnitude and direction for both the all-trans and the 13-cis, 15-cis retinals, the protonation states of the aspartic acid residues (A, to A.,) suggested to be implicated in proton pumping [ 12,131 prevent proton translocation in the cis cycle. Figure adapted from H.-W. Trissl and W. Giirtner [ 141 with permission of the authors.
bond [ 9, lo]. The possibility of an accompanying isomerization about the C,&Z15 bond remains controversial at this writing [ 111 (see fig. 1a). The isomerization is accompanied by separation of the protonated Schiff base from a negatively charged counterion which is most likely a carboxylate group 370
of one of four aspartic acid residues [ 121 or a tyrosinate group [ 131. Trissl and Ggirtner [ 141 have recently shown that the magnitude and the sign of the transmembrane voltage accompanying this photogenerated charge separation are the same for both light- and dark-adapted BR and for bacteria-opsins
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combined with different retinals with various equilibrium populations of cis and trans conformations. In all cases the photovoltage appeared in less than 70 ps (the time resolution of their apparatus). These authors concluded from their study that the bathochromic intermediates derived from the 13-cis or the all-trans retinals resemble each other but are formed along different pathways. Here we present the first data obtained by transient absorption spectroscopy for the initial photoproducts of the dark cycle of BR and compare them with data obtained for the light cycle of BR. We discuss our results in terms of the BR photophysics and the mechanism of the formation of the bathochromic intermediates.
2. Experimental Transient absorption measurements were performed at room temperature with the apparatus described in detail elsewhere [ 16,171. Purple membrane from Halobacterium halobium was prepared by standard procedures [ 181 and suspended in distilled water containing 0.0 1% NaN3 (approximately 2 mg protein/ml). Dark-adaptation was effected by storage in the dark overnight. At our excitation wavelength of 6 12 nm, the optical density in 1 mm of the light-adapted and the dark-adapted samples was 0.44 and 0.33, respectively. Transient absorption measurements were performed by translating the sample in a 1 mm cuvette through the pump and the probe beams with a velocity of 12 mm/s. To further avoid sample damage (and, for the experiments involving the dark cycle, to ensure that we were always studying dark-adapted samples), the solution was also flowed through the translating cell with a peristaltic pump. The sample reservoir typically contained lo- 15 ml of the BR preparation and was either shielded from light (for the dark-adapted preparation) or illuminated with 50 mW of broadband 560 nm light (for the light-adapted preparation). These conditions were sufficient to produce and maintain complete dark or light adaptation of the samples, which were frequently monitored with a Cary 15 spectrophotometer (fig. 2). Sample damage induced by the time-resolved experiment was measured by the decrease in the maxima of the steady-state
wavelength (nm) Fig. 2. Absorption spectra of light-adapted (-) and of darkadapted (...) purple membrane taken at room temperature with a 1 mm cuvette. The procedure for light- and dark-adaptation is described in the text.
absorption spectra of the light- and dark-adapted samples and was ;5 6%.
3. Results Upon excitation of the light-adapted sample with 120 fs pulses at 612 nm, three distinct phenomena were observed. The BR was bleached in less than 50 fs, and simultaneously there appeared a species with an absorption maximum at 460 nm. This absorption decayed with a time constant of 500 f 40 fs to a species which has previously been designated as J. Our result for the time of formation of J is in good agreement with that of Nuss et al. (430 + 50 fs) [ 41. The decay of J to K is found to be 3.2 + 0.2 ps as measured by the absorption recovery at 580 nm. This result is in good agreement with that of Sharkov et al. (3.0f0.5 ps) [ 51 but is less than the 5 ps decay time obtained by Polland et al. [ 61. We obtain consistent time constants for the formation of J and K over the entire spectral range we investigated, 420 to 675 nm. Performing identical experiments on the darkadapted BR, we observed nearly identical kinetics of formation for the counterparts of J and K in the lightadapted preparation. The only noticeable difference is the amplitude of the absorption at its asymptotic value (figs. 3 and 4). The magnitude of this absorp371
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I/lo
111, LIGHT-ADAPTED
Fig. 3. Transient absorption due to the formation of an excited state of bacteriorhodopsin, BR*, and its subsequent decay to the ground-state species, J. &.,,=612 nm, A,,,=460 nm. BR* is produced in less than 50 fs. Satisfactory double-exponential fits were (a) for the dark-adapted sample: I(t)/I,=l-0.97 xexp( -t/490 fs)-0.03 exp( -t/3200 fs), (b) for the lightadapted sample: I(t)/&= l-O.97 exp( -t/530 fs) -0.03 exp( -t/3200 fs).
tion is consistent with that predicted from the equilibrium spectra (fig. 2) and serves as a criterion for the integrity of the dark-adapted samples. Because dark-adapted BR is a mixture of 50% alltrans retinal and 5OW 13-cis, 15cis retinal, and because the absorption cross section of the cis isomer at the 6 12 nm excitation wavelength is about half that of the all-trans isomer [ 151 (fig. 2)) there is a possibility that the time constants for the formation of the photoproducts of the 13-cis, 15-cis retinal are different from those of the all-trans retinal but cannot be adequately resolved in our absorption measurements. To investigate this possibility, we simulated absorption recovery curves in which the time constants of the photoproducts of the 13-cis, 1S-cis retinal were varied and compared these curves with our data. For the signal-to-noise ratio provided by our experiment, the counterparts of J and K in the dark cycle must be formed approximately 1.5 times more rapidly or more slowly than J and K in 372
Fig. 4. Transient bleaching of dark- and light-adapted bacteriorhodopsin on two different time scales. Collecting the data on a longer time scale allows a more accurate determination of the contribution of the long-lived decay component. A,,,,,, = 6 12 nm, I pmbe=580 nm. The fast recovery of the bleaching represents the formation of the first ground-state intermediate, which is designated J in the light cycle. The slower recovery of the bleaching represents the formation of the second ground-state intermediate, designated Kin the light cycle. Double-exponential fits to the data from the dark-adapted samples yielded: (a) I(t)lfo=0.88 Xexp( -t/490 fs) +0.12 exp( - t/3200 fs): 4 ps full scale; (c) I(t)/Z,=0.75 exp( - t/500 fs) +0.25 exp( - t/3200 fs): = 30 ps full scale. Double-exponential tits to the data from the light-adapted samples yielded: (b) I( t)&=0.70 exp( -t/530 fs) +0.30 xexp( -t/3200 fs): 4 ps full scale; (d) I( t)/1,=0.70 exp( -t/500 fs) +0.30 exp( -t/3200 fs): x 30 ps full scale.
the light cycle to produce a significant difference in our absorption data. We therefore conclude that the initial photoproducts of the light and the dark cycles are formed with nearly identical time constants.
4. Discussion 4.1. Photophysics of the retinal chromophore in bacteriorhodopsin
The quantum yield for the bleaching of lightadapted BR has been measured to be 0.33 [ 19-211 and 0.60 [22]. The independence of the quantum yield on both excitation wavelength and temperature has been interpreted in terms of a trans-to-cis isomerization which takes place after complete thermal relaxation and which requires no activation energy
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[ 20,231. An interesting explanation for a quantum yield of 0.33 arises if it is postulated that light excitation creates a precursor which forms BR* in less than 50 fs (the time resolution of our apparatus). Such a precursor has been suggested by Nuss et al. [4] to be a delocalized excited state of the bacteriorhodopsin trimer. In this case the quantum yield of 0.33 could be interpreted as the efficiency of exciton trapping by a monomer of the trimeric unit. While this explanation is appealing, the existence of efficient exciton coupling [ 241 in the trimer has been called into question. Notably, the large absorption polarization of K has been cited by El-Sayed et al. [25] as evidence that exciton transfer is not competitive with BR photochemistry since rapid exciton transfer is expected to produce depolarized intermediates. Godfrey [26] has discussed the problem of exciton coupling in bacteriorhodopsin trimers in terms of the limits of strong and weak coupling. In the strong coupling limit, it is assumed that the exciton coupling is much larger than the electronic absorption bandwidth [ 27,281 and that all vibronic levels of one monomer are in resonance with those of another. In the weak coupling limit, however, the exciton coupling is much less than the electronic bandwidth and resonance interaction only occurs among individual vibronic levels. Godfrey points out that using Hemenger’s value of 64 cm- ’ [ 291 for the exciton coupling (as opposed to larger estimates for the coupling strength [24,30]), an exciton transfer time of x 90 fs is obtained in the strong coupling limit. In the weak coupling limit, however, the exciton transfer time may easily be increased by an order of magnitude [ 261. Since J is formed in 500 fs, such a weak exciton coupling could produce a situation where the exciton transfer time is significantly slower than the formation time of the first ground-state intermediate of BR. 4.2. The light and the dark cycles: photochemistry Based on the similarity of the absorption spectra of J and K and the fact that K already has its retinal in a cis, or at least partly cis, conformation [ 9, lo], Nuss et al. [ 41 suggested that photoisomerization has already been accomplished with the formation of J. Polland et al. [6] studied the kinetics of bacterioopsin combined with a retinal analog modified to
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prohibit transformation to a 13-cis configuration. They found that such an analog has an excited-state lifetime of 10 ps, a 12-fold enhancement of its fluorescence quantum yield compared with that of native BR [ 3 11, and formed no photoproduct. This was cited as further evidence that the primary photophysical event in light-adapted BR is a rapid transto-cis isomerization about the CL3=C4 bond. For light-adapted and dark-adapted BR and for bacteria-opsins containing retinal analogs whose equilibrium populations ranged from 30 to 85% 13cis, Trissl and Giirtner [ 141 found that, within their time resolution of 70 ps, charge separation was effected with the same rate and in a direction opposite to that of proton translocation [32]. Based on these observations, they suggested that the inability of the dark cycle to pump protons could not be explained in terms of a photoisomerization of the 13cis, 15-cis retinal in a direction opposite so that of the all-trans retinal. They concluded that the bathochromic intermediates derived from the 13-cis and the all-trans retinals resemble each other in their conformation and approach their respective conformations by two different routes. Finally, they suggested that the ability of BR to pump protons in the light cycle is more a consequence of different protonation states of the amino acid residues of the protein rather than of different geometries of the parent retinal moieties (see fig. 1b). Our transient absorption data are consistent with the conclusions and the model presented by Trissl and Gartner [ 141. A bathochromic intermediate that appears with the same time constant in both the darkand the light-adapted preparations need not have the same structure in both of the preparations. For example, it has been shown that a bathochromic shift in the retinal absorption spectrum can be produced by either separation of the protonated Schiff base from its counterion or by protonation of the counterion [ 331: in either instance the net positive charge of the reninal polyene chain increases [ 341. We thus suggest that the similarities in the time constants for the formation of the bathochromic intermediates in the dark and the light cycles are not due to the acquisition of similar retinal structures but to similar displacements from their counter ions. Although it may be argued that the simplest mechanism for such a displacement would involve an all-trans to 13-cis 373
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isomerization in the light cycle and a cis-to-trans isomerization about the C&N bond in the dark cycle (fig. 1) which would, in fact, yield identical intermediates, it is probably more accurate, as Trissl and GIrtner [ 141 suggest, to speak in terms of “transoid” and “cisoid” transformations involving simultaneous distortions of other bonds in the polyene chain instead of isolated and complete trans-to-cis and cis-to-trans isomerizations when discussing the formation of the bathochromic intermediates. This is borne out by the observation of Braiman and Mathies [ 9, lo] : at 77 K, the resonance Raman spectrum of K in the light cycle exhibits intense out-ofplane hydrogen stretching frequencies which are suggestive of an incomplete, or at least an unrelaxed, cis configuration about the &=C,., bond.
5. Conclusion We have studied the transient absorption spectra of the initial photoproducts of the light and the dark cycles of BR. BR*, J, and K in the light cycle, and their counterparts in the dark cycle, are formed with similar time constants. This observation is consistent with the photovoltage measurements of Trissl and Giirtner [ 141. We suggest that the similarity of the time constants in both instances is due to equivalent separations between the protonated Schiff base of the retinal and the counterion furnished by the protein rather than to adoption of equivalent structures of the retinal. Resonance Raman techniques will provide more detailed structural information on the nature of the early-time photoproducts at room temperature; and such experiments with techniques providing subpicosecond resolution [ 351 should prove useful in better understanding the nature of the bathochromic intermediates in the light and the dark cycles. Also, one might determine whether the species J is structurally distinct from K or is a vibrationally hot form of K.
Acknowledgement We thank H.-W. Trissl for very stimulating discussions and J.-L. Rigaud, M. Seigneuret, and A. Bluzat for their generous gift of purple membrane. 374
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JWP was supported by an NSF Industrialized Countries postdoctoral fellowship, an INSERM poste orange, and a fellowship from La Fondation pour la Recherche Medicale. Parts of this work were funded by INSERM, ENSTA, and Le Minis&e de la Recherche et de la Technologie.
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