J. Phys. Chem. 1992, 96,6066-6071
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and Humanities (I.W.) and by the Office of Basic Energy Sciences, Division of Chemical Science, US.Department of Energy, under Contract DE-AC02-83CH 10093 (A.J.F.).
References and Notes (1) (a) Usui, Y.; Misawa, H.; Sakuragi, H.; Tokumaru, K. Bull. Chem. Soc. Jpn. 1987,60, 1573. (b) Usui, Y.; Sasaki, Y.; Ishii, Y.; Tokumaru, K. Bull. Chem. Sot. Jpn. 1988, 61, 3335. (2) (a) Willner, I.; Eichen, Y.; Man, S.;Doron, A. Isr. J . Chem., in press.
(b) Willner, I.; Marx, S.;Eichen, Y. Angew. Chem., In?. Ed. Engl., in press. (3) Willner, I.; Eichen, Y.; Rabinovitz, M.; Hoffman, R.; Cohen, S.J . Am. Chem. Sot. 1992, 114,637. (4) Willner, I.; Eichen, Y.;Joselevich. E. J. Phys. Chem. 1990, 94, 3092. (5) Benesi, H. A.; Hildebrand, J. H. J. Am. Chem. Sot. 1949, 71,2703. (6) (a) Hunter, C. A.; Sanders, J. K. M. J . Am. Chem. Soc. 1990, 112, 5525. (b) Price, S.L.; Stone, A. J. J . Chem. Phys. 1987,86,2859. (c) Rigby, M.; Smith, E. B.;Wakeham, W. A.; Maitland, G. C. The Forces Between Molecules; Clarendon: Oxford, 1986. (d) Intermolecular Interactions: From Diatomics ro Biopolymers; Pullman, B.,Ed.; Wiley: Chichester, 1978. (e) Fersht, A. R. Enzyme Structure and Mechanism; Freeman: New York, 1985.
(7) (a) Foster, L. Organic Charge-Transfer Complexes; Academic Press: New York, 1969; Chapters 2 and 8. (b) Nakamura, K.; Kai, Y.; Yasuoka, N.; Kasai, N. Bull. Chem. Soc. Jpn. 1981,61,3300. (c) Kisch, H.; Fernandez, A.; Wakatssuki, Y.; Yamazaki, H.Z . Naturforsch. 1985,406,292. (d) Bent, H. A. Chem. Rev. 1968, 68, 587. (e) Megehee, E. G.; Johnson, C. E.; Eisenberg, R. Inorg. Chem. 1989,28, 2423. (0 Reddington, M.V.; Slawin, A. M. Z.; Spencer, N.; Stoddart, F. J.; Vicent, V.; Williams, D. J. J. Chem. Soc., Chem. Commun. 1991,630. (9) Ashton, P. R.; Brown, C. L.; Chrystal, J. T.; Goodnow, T. T.; Kaifer, A. E.; Parry, K. P.; Philip, D.; Slawin, A. M. Z.; Spencer, N.; Stoddart, F. J.; Vicent, V.; Williams, D. J. J . Chem. Sot., Chem. Commun. 1991, 634. (8) The Chemistry of Silica; Iler, R. K., Ed.; Wiley: New York, 1979. (9) (a) Willner, I.; Degani, Y. J . Chem. Soc.. Chem. Commun. 1982, 761. (b) Willner, I.. Degani, Y. J. Am. Chem. SOC.1983, f05,6228. (c) Furlong, D. N.; Johansen, 0.;Launikonis, A.; M e r , J. W.; Mav, A. W. H.; Sasse, W. H. F. Aust. J . Chem. 1985, 38, 363. (IO) (a) Willner, I.; Otvos, J. W.; Calvin, M. J . Am. Chem. Soc. 1981,103, 3202. (b) Laane, C.; Willner, I.; Otvos, J. W.; Calvin, M. Proc. Narl. Acad. Sci. U.S.A. 1981, 78, 5928. (c) Willner, I.; Yang, J.-M.; Otvos, J. W.; Calvin, M. J . Phys. Chem. 1981,85, 3277. (1 1) Valdes-Aguilera, 0.;Neckers, D. C. Acc. Chem. Res. 1989, 22, 171.
Primary Photochemical Events in Haiorhodopsin Studied by Subpicosecond Time-Resolved Spectroscopy Hideki Kandori, Keitaro Yoshihara,* Institute for Molecular Science, Myodaiji, Okazaki 444, Japan
Hiroaki Tomioka, and Hiroyuki Sasabe Frontier Research Program, RIKEN (The Institute of Physical and Chemical Research), Hirosawa, Wako, 351-01, Japan (Received: December 2, 1991; In Final Form: March 6, 1992)
Primary photochemical events of the light-driven chloride-pump halorhodopsin (hR) are studied at room temperature by subpicosecond transient absorption measurements. On excitation of hR with a 600-nm, 0.6-ps pulse, excited-state absorption and stimulated emission appear immediately in the 420-530-nm and 650-770-nm wavelength regions, respectively, and both decay with a time constant of 2.3 ps. The calculated absorption spectrum of the excited state of hR (hR*) has a peak at 516 nm and a shoulder at about 460 nm. Accompanied by the decay of hR*, the primary ground-state product appears at around 645 nm. The quantum yield of the product formation is determined to be 0.27. The detailed analysis of the kinetics at 645 nm provided the faster rise time of the product ($1.0 ps) than the decay of the excited state (2.3 ps), as well as the possible presence of the J-intermediate (hRJ). Instead of the simple sequential kinetic model considering three states of hR*, hRJ, and hR, which has been applied to the primary process of bacteriorhodopsin, a parallel channel model is suggested for the primary process of hR. After Franck-Condon excitation, the cis-trans isomerization to hRJ and the relaxation to hR* take place simultaneously. The latter decays only to hR via radiative and nonradiative processes. The present results suggest that the excited state having a reaction channel to cis-trans isomerization is not located at the potential minimum of hR* and that the relaxation process in the excited state is a process in competition with isomerization.
Introduction Halobacterium halobium contains the four retinal proteins: bacteriorhodopsin (bR), halorhodopsin (hR), sensoryrhodopsin (sR), and phoborhodopsin (pR). bR and hR work for energy generation by light-driven proton and chloride pumps, respectively,l**while sR and pR work for positive and negative phototaxis, re~pectively.~.~ Structural varieties and their relationship to the respective functions have been of much interest. Since a photon triggers reactions in each pigment, photochemical reactions of the pigments must be closely related to the functions of the pigments. Flash photolysis experiments on these retinal proteins have mainly focused on bR. In particular, recent developments in ultrashort pulse generation have enabled us to look directly at the relaxation processes in the excited state of the retinal proteins. A number of time-resolved studies on bR have given pictures on cis-trans isomerization in protein.s-ll According to these studies, the excited state of bR (bR*) depopulates and the first ground-state product J appears with the same rate constant of about (0.5 ps)-’. Since the J-intermediate has a 13-cis-likeconfig~ration,~*-’~ it has been concluded that cis-trans isomerization occurs along the reaction coordinates in the excited state. 0022-3654/92/2096-6066$03,00/0
Because it is less convenient to prepare samples, few studies by time-resolved spectroscopy have been made on either hR, sR, or pR. One may tend to consider that the primary photochemical events of these three pigments are same as that of bR. In 1985, however, Polland et al. reported that the lifetime of the excited state of hR (hR*) is 10-times longer ( 5 ps) than that of bR*.IS Although both hR and bR have the same chromophore and similar protein environments,I6 the primary reaction rates are 1-order of magnitude different. Thus we have started to investigate the primary photochemical events of hR by use of subpicosecond transient absorption spectnwcopy. In a previous paper, we reported the absorption spectrum of hR* which displayed a unique profile.” In the present article, we measure the transient absorption spectra of hR and investigate its primary photochemical events. The results obtained strongly imply that the primary process in hR cannot be described by the simple scheme which has been applied to bR.
Materials and Methods The hR sample was prepared as described previ0us1y.l~ A bacteriorhodopsin-deficient strain, OD2, was grown in a 20-L 0 1992 American Chemical Society
Primary Processes of Halorhodopsin medium. Culture condition and membrane preparation were described earlier.'* hR was isolated according to the method described previously.l9 In the present experiment, nonaoyl-Nmethylglucamide (MEGA-9) was used instead of n-octyl-8-Dglucopyranoside (octylglucoside), because hR is more stable in MEGA-9 than in octylglucoside. hR was purified and concentrated on a concentrator resin (Bio-Gel Concentrator Resin, Bio-Rad). The apparatus for obtaining transient absorption spectra is described previously.20 Briefly, the subpicosecond light source is an amplified, synchronously pumped, hybridly mode-locked dye laser. The laser system produces a 600-nm, 0.6-ps, and 500 pJ pulse at a 10-Hz repetition rate. The amplified laser pulse is split with a dichroic beam splitter. One part of the 600-nm subpicosecond pulse is used to excite the samples after adequate attenuation. Since the absorbance change due to hR* (490 nm) displays a linear increase up to 50-60 pJ, a 40-pJ pulse is used for the experiments. The diameter of the pulse is 2.2 mm at the sample position. The other part is used to generate a subpicosecond continuum probe pulse. The continuum is obtained by focusing the laser beam into a 1-cm cell containing a D20/H20mixture (1:2). To eliminate the remaining 600-nm light, we placed either a blue glass filter (Melles Griot, BG28) or a cut-off filter (Schott, RG630 or RG645) in the measurement at shorter or longer wavelengths than 600 nm, respectively. The continuum probe energy is less than 1% that of the pump pulse. The continuum was split into two parts by a half mirror and then focused into two independent 25-cm spectrographs. One of the beams is adjusted to overlap with the excitation beam at the sample cell, to monitor the absorbance change due to the excitation, and the other beam is used as a reference. The light path of the sample cell is 2 mm, and the sample (total volume 2 mL) is flowed by a peristeric pump. The continuum light intensities are dispersed via the spectrograph and directed onto 512 multichannel photodiodes (MCPD, Hamamatsu Photonics). The signals on the MCPD are scanned, digitized, accumulated, and transferred to a computer for calculation of the absorption difference spectrum. To improve the signal-to-noise ratio, 200 accumulations of the excitation and the reference spectra are obtained to calculate the difference spectrum at each delay setting. Merence spectra shown in this article is a sum of 3-4 independent experiments (namely 600-800 accumulations). During measurements a part of the pump pulse is monitored with a photodiode for each pulse. The signal is measured with a sample-and-hold circuit, digitized, and transferred to a computer for normalization of the pumping intensity. It is noted that the arrival times of a probe pulse are different in respective wavelengths, Le., as the probing wavelength is shorter the arrival is delayed more as suggested previ~usly.~We are determining the delay time as described previously;20that is, obtained spectra are corrected according to the absorbance change of a standard dye (Nile Blue in methanol). Every measurement was carried out at room temperature (20 "C). ReSult.9 Figure 1 shows the time-resolved difference absorption spectra in the blue region (370-540 nm). Positive absorption appears immediately after excitation and decays on the early picosecond time scale without giving any spectral changes. The observed spectra display a broad peak in the wavelength region of 460-5 10 nm. This is assigned to the absorption spectrum of hR*.I7 The broad profile of the hR* spectrum implies an overlap of more than one absorption band. Kinetic measurements, however, show that the decay time courses are identical at three wavelengths: 470, 490, and 510 nm (Figure 2). At present, single exponential fittings are attempted in considering the pulse duration (0.6 ps). Each figure in Figure 2 can be fitted with the same time constant of 2.3 ps. Thus it is shown that hR* depopulates with a time constant of 2.3 ps from the absorption measurement. Figure 3 shows the difference absorption spectra in the red region (620-780 nm). In these spectra, three species are observed.
The Journal of Physical Chemistry, Vol. 96, No. 14, 1992 6067
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At 0.7 ps, two negative signals can be observed. One is at shorter than 650 nm and the other is at 650-780 nm. Since hR has an absorption shorter than 650 nm, the former is assigned to the
6068 The Journal of Physical Chemistry, Vol. 96, No. 14, 1992
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ground-state depletion, while the latter should be assigned to the stimulated emission from hR*. The emission maximum is located at 715 nm. As time proceeds, a third feature appears toward the positive side at 620-680 nm. This is due to the transient absorption of the primary intermediate of hR, namely hRK, which was reported previously.ls Two points should be noted here: (i) The amplitude of the transient positive absorption is small (about 0.015 in absorbance unit). The difference absorbance signal due to the photoproduct is notably smaller (about 6 times) than that due to hR* (Figure 1). (ii) Even when the generation of the product is completed, the stimulated emission signal remained. The second point is clearly illustrated in the kinetic observations (Figure 4). As discussed above, the negative signal at 645 nm (Figure 4a) is due to both ground-state depletion of hR and stimulated emission from hR*, while the positive signal is due to the generation of a primary intermediate. The fitting curve in Figure 4a shows that the time constant of the overall process is 1.0 ps. The negative signal at 720 nm (Figure 4b), on the other hand, is due only to stimulated emission from hR*. The obtained decay time constant (2.3 ps) is in good agreement with that of excited-state absorption (Figure 2). The detailed analysis of 645-runkinetics to determine the product formation rate is camed out in the Discussion. Although we are measuring difference absorption spectra between hR* and hR in Figure l, the absolute absorption spectrum of hR* can provide more information on configuration and/or conformation. In order to determine the absolute absorption spectrum of an intermediate, we have to know the percentage of the bleach of the pigmente2*Different from bR or rhodopsin, a long-lived excited state of hR makes it possible to observe the bleaching directly. Since difference absorbance at 615-650 nm (Figure 3) is due to the ground-state depletion, we can estimate the percentage of depletion by comparison between the difference spectra and absolute spectrum of hR. We choose the difference spectrum at 0.3 p for the estimation (Figure 5) because it displays the greatest depletion so that the contribution of the photoproduct is negligible. In Figure Sa the stimulated emission maximum is at about 700 nm, which is located at a 10-15 nm shorter wavelength than those at later times (Figure 3). indicating that the spectra in Figure 3 is reflected by the appearance of the product.
Figure 4. Kinetics of the hR absorption changes at two wavelengths: 645 (a) and 720 nm (b). (a) The negative signal just after excitation is due
to both ground-statedepletion of hR and stimulated emission from hR*, while the pwitive signal at a longer time is due to the generation of a primary intermediate of hR. The calculated curve (solid l i e ) shows a time constant for the overall process of 1.0 p. (b) The negative signal is due to stimulated emission from hR*. The calculated w e (solid lie) shows a time constant of 2.3 p, which is in good agreement to those obtained from the decay of the excited state absorption.
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Primary Processes of Halorhodopsin
The Journal of Physical Chemistry, Vol. 96, No. 14, 1992 6069
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Discussion The quantum yield of hRKformation is determined. Since the absolute absorption s p e c " of hRKis reported by Tittor et we can calculate the difference absorbance at a selected wavelength. When hR has an absorbance of 0.6 at the maximum and all hR molecules are converted to hRK,an absorbance change at 630 nm is estimated to be 0.16. Since the conversion rate of hR to hR* is 0.35and the observed difference absorbance at 630 nm is 0.015 (Figure 3), the quantum yield of hRKformation is calculated to be 0.27 f 0.05 (= 0.015/(0.16 X 0.35)). This value is similar to that of the formation of a deprotonated and long-lived species, h k l 0 (0.34).% These results suggest that no back-reaction takes place during the subsequent thermal process, and the efficiency of chloride pumping is determined directly by efficiency of the primary photochemical process, namely cis-trans isomerization. Kinetic results show that only a single exponential fitting is sufficient for the wavelengths at 470,490,510(hR* absorption; Figure 2), and 720 nm (stimulated emission; Figure 4b), and the common decay of 2.3 p gives the decay lifetime of hR*. On the other hand, kinetics at 645 nm is more complicated. As is easily observed from the spectral feature (Figure 3), there are three spectral components at 645 nm; the negative signals due to (1) ground-state depletion of hR (1680 nm) and (2) stimulated emission from hR* (620-780 nm) and the positive signal due to (3) generation of the ground-state product (620-680nm). Since there are no wavelengths regions in which only the product (hR,) is observed (Figure 3), we have to isolate the rise signal in order to obtain the formation time of the product. The absorbancechange at 645 nm, PODa5, should be denoted as follows AODus (-cI exp(-klt) - c2 exp(-kzr) + c3 (1- exp(-k,t)))@F(t) (1) here positive preexponential factors, c,, c,, and c3,are proportional to the respective absorbance changes in amplitude, and the kinetic change is convoluted with the instrumental function F(t). In F(t), a sech2 representation of the pump and probe pulses is used with 0.6 p FWHM. The first and second terms show the decaying process of the negative signal, correspondingto the ground-state recovery (rate constant, k,)and the decay of stimulated emission (rate constant, k2),respectively, while the third term represents the formation process of the product whose rate constant is kJ.
1
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Time / ps Figure 7. Simulations of kinetic change due to the appearance of the ground-state intermediate of hR at 645 nm. The filled circles and the fitted thin curves both in a and b indicate the experimental and fitting results, respectively, as described in Figure 4a. The absorbance change at 645 nm, AODM5,should be denoted as the sum of the three processts (eq 1): (1) ground-state depletion, (2) stimulated emission from hR*, and (3) formation of the ground-state product. In order to isolate the positive signal due to product formation, we subtracted the negative signals from the obtained kinetics (AODM5). All simulations are carried out taking into account the pulse duration of 0.6 ps. (a) The negative thin curve is a simulation curve of the first and the second terms in q 1, in which both k l (ground-staterecovery) and k2 (decay of the stimulated emission) are (2.3 ps)-l. It displays a single exponential decay in 2.3 ps. The thick curve indicates the subtraction result of the negative signal from the obtained kinetics, implying that the rise time of the product is 0.6 & 0.2 ps and there is a subsequent decay proccss of -3 ps. (b) The negative thin curve is a simulation curve of the first and the second terms in eq 1, in which kl (ground-state recovery) is (2.3ps)-l and k2 (decay of the stimulated emission) is chosen to (1.0 ps)-'. It displays a double exponential decay of 1.0 and 2.3 ps, and the contribution of the ground-statedepletion and the stimulated emission is taken to be equal (cI:c2= 1:l). The contributionsare taken from the intensity of transient absorption measurements at 645 nm (Figures 3 and 5). The thick curve indicates the subtraction result of the negative signal from the obtained kinetics, implying that the rise time of the product is 0.9 0.2 ps and there is a subsequent decay process of -3 ps.
*
Although the accurate rate constant of the ground-state recovery
(k,)could not be measured directly, it is most likely to be (2.3 ps)-' that is the lifetime of hR*. On the other hand, the decay rate constant of the stimulated emission probed at 645 nm (k2) may not be (2.3 ps)-', because the present probing wavelength (645 nm) is a blue-tail of the emission spectrum (Figure 3) and the shorter lifetime in bluer emission is possible due to emissions from the nonrelaxed state.1° In Figure 7,we made two different attempts to isolate the positive signal due to the product by subtracting the negative signals: the decay time of the stimulated emission is either 2.3 ps (a) or 1.0 ps (b). According to the simulation shown in Figure 7a,the first and second terms of eq 1 have t$e same rate constants (k,= kz = (2.3 ps)-') so that the negative signal decays in a single exponential manner (negative thin curve). The calculated signal due to the product (thick curve) increases and decays in an early picosecond time scale. This is well constructed by a single exponential rise of 0.6 f 0.2 ps with an instrumental response time of 0.6 ps, followed by decay to the other state in -3 ps. The simulation
Kandori et al.
6070 The Journal of Physical Chemistry, Vol. 96, No. 14, 1992
hR*(FC)
hRJ (1 3-cis)
hR (all-trans) Figure 8. Primary photochemical scheme in hR. Open, solid thin, and solid thick arrows indicate photon absorption, internal conversion, and both radiative and nonradiative processes, respectively. (a) In this scheme, excited hR molecule starts relaxation from the Franck-Condon state (hR*(FC)) to an excited equilibrium state (hR*(EQ)) along the C 1 3 C I Idouble bond rotational coordinate. Two channels are open in the equilibrium state: one is to hRJ by internal conversion, while the other is backward to hR. This simple scheme, which has been applied to the primary process of bR, cannot explain the present experimental results (see text). (b) In this scheme, hRJ is generated directly from hR*(FC) along the C13-C14 double bond rotational coordinate in 11.0 ps. hR* is generated as the result of relaxation process in the excited state. hR* is converted to hR by a radiative or a nonradiative process in 2.3 ps.
result shown in Figure 7b is essentially the same as that in Figure 7a. The calculated signal (thick curve in Figure 7b) is constructed by a single exponential rise of 0.9 f 0.2 ps with an instrumental response time of 0.6 ps, followed by a decay to the other state in -3 ps. It should be noted that the rise time of the primary intermediate of hR is 11.0 ps in any case (Figure 7), and it is faster than the decay of the excited state (2.3 ps). The present simulation also reveals another new aspect, Le., the presence of an extra intermediate. According to the present simulations,a primary intermediate is generated in 1 1.O ps and it is converted to the other state in -3 ps. The similar kinetic change is also observed in bR, and the former is called the J-intermediate (bR,) and the latter is called the K-intermediate (bRK)?+ bRI was assigned to be a vibrationally hot ground state having the 134s form in bR, indicating that the J to K conversion is the vibrational ccoling pr0ce~s.I~ Although we have no apparent spectroscopic observation of the J-intermediate, the kinetic simulation implies the presence of hR, as the precursor to hRKand the conversion rate to be similar to that of bR.7-9J4 With the spectral and kinetic results shown above, let us construct a scheme for the primary process of hR. A possible scheme for hR is shown in Figure 8a with the following states: hR, the original ground state; hR*(FC), excited Franck-Condon state; hR*(EQ), excited equilibrium state; and hRJ, primary intermediate. Although the scheme in Figure 8a is most commonly used for bR, it is not favorable to apply hR. The faster rise of hRJ (11.0 ps) than the lifetime of hR* (2.3 ps) cannot be explained by this simple scheme. We therefore ought t o reconsider the potential surface of the excited state of hR. The relaxation processes in the excited state probably proceed not one-dimensionally along the c 1 3 4 1 4 double bond rotational coordinate ( F i i 8a) but multidimensionally. In Figure 8b we are proposing another scheme, which is more appropriate for hR. In this scheme, hR, is formal diractly from the excited Franck+ndon state along the CI3+4 double bond rotational coordinate in 11.0 ps (an
intermediate state like hR*(EQ) in Figure 8a may be present on the reaction pathway). hR* is formed as the result of relaxation process in the excited state. The reaction coordinate should be different from the C13
