© Copyright 1998 by the American Chemical Society
VOLUME 102, NUMBER 13, MARCH 26, 1998
LETTERS Low-Temperature Retinal Photoisomerization Dynamics in Bacteriorhodopsin S. L. Logunov, T. M. Masciangioli, V. F. Kamalov, and M. A. El-Sayed* Laser Dynamics Laboratory, School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0400 ReceiVed: September 5, 1997; In Final Form: February 6, 1998
Retinal photoisomerization dynamics are studied at both room temperature and 20 K in wild-type bacteriorhodopsin using femtosecond pulses. We were able to resolve the decay at 20 K into two components with the dominant component having a similar lifetime to that observed at room temperature. This strongly suggests that the retinal lifetime at physiological temperature is barrierless. The minor, low-temperature long-lived component is discussed in terms of previous results obtained for fluorescence and transient absorption with lower time resolution, and the origin of this component is discussed in terms of low-temperature glass heterogeneity.
Introduction Since the early observation of light emission from wild-type bacteriorhodopsin (bR) in the mid-70s1,2 the relaxation behavior of the excited state of bR continues to be of great interest. Through the use of picosecond and femtosecond spectroscopic techniques, absorption by excited-state bR at 480 nm is shown to decay in 0.5 ps3-7 at room temperature forming J- and K-intermediates with a quantum yield of 0.66.8-10 The ground state of light-adapted bR is composed of all-trans-retinal and has an absorption maximum at 568 nm. The temperature dependence of the excited-state properties of bR was studied previously by several groups. Shapiro et al.2 observed a very strong temperature dependence of the quantum yield of bR emission at low temperature with a sharp rise below 100 K, suggesting the presence of two excited electronic states with an activation barrier separating them. The early low-temperature fluorescence data are somewhat controversial and vary substantially from group to group, depending on the experimental conditions used.1,2,8,11,12 Transient absorption spectroscopy was used in the 1980s to study the relaxation behavior of the excited state of bR at low temperatures with 30-ps temporal resolution.7 It was found that dynamics of the
excited state persist for longer times at 13 K than at room temperature (80 ps7 vs 0.5 ps3-5). This report is the first experimental investigation of bR at low temperature using pulses of femtosecond duration. We measured the decay of the excited state at room temperature and at 20 K. At room temperature the decay is exponential with a 0.5-ps lifetime, in agreement with previous results.3-7 At 20 K, the decay is nonexponential but can be resolved into two components, the dominant one with a 0.5 ps lifetime and a minor component with a 0.9-1.2 ps lifetime. The time scale for photoisomerization for bR at 20 K appears to be dependent on the sample studied, with the main variation being in the amplitude of the slow component (time scale longer than 50 ps). The temperature dependence obtained is analyzed and discussed in terms of sample heterogeneity in which there is a distribution of activation barriers in the excited state. Experimental Section The bR sample was isolated from Halobacterium salinarium according to Oesterhelt and Stoeckenius.13 A 2-mm quartz cell of bR in a mixture of glycerol and water (3:1, v:v) was mounted in a Janis CCS-150 closed cycled refrigerator system (CTI Cryogenics). The ground-state absorption spectrum was mea-
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2304 J. Phys. Chem. B, Vol. 102, No. 13, 1998
Figure 1. Transient absorption spectra of bR in a glycerol/water glass at 20 K, measured at different time delays (0, 1.2, 4, and 8 ps) after the 600-nm excitation pulse.
sured using a UV-vis spectrophotometer (Beckman). A PTI Inc. fluorimeter was used for the fluorescence steady-state measurements. During all steady-state measurements, special care was taken to ensure that there was minimal irradiation of the sample to avoid accumulation of the K-intermediate. Between measurements, the sample was irradiated with red light (>650 nm) to convert any accumulated K-intermediate back to the ground state.6,8 The transient spectrophotometric experimental setup was described previously.10,14 Briefly, subpicosecond pulses from a dye laser (Satori, Coherent) were amplified by a regenerative amplifier with a repetition rate of 10 Hz. The detection system was either a liquid nitrogen cooled CCD (Princeton Instruments, EV-1024 UV) for transient spectra or a couple of photodiodes for changes in absorption at a single wavelength in the kinetics measurements. Amplified pulses of the dye laser (600-610 nm), with a duration of about 350 fs, were used as a pump source, while white light continuum (420-800 nm) generated in a water cell provided probe pulses. To convert any accumulated K-intermediate of bR back to the all-trans-configuration, the sample was irradiated continuously with a pulsed laser that produced 10-ns pulses with 0.2 mJ/pulse at 630 nm. This beam was obtained via stimulated Raman scattering of the second harmonic of a 10-Hz Nd:YAG laser in a 10-cm cell of ethanol. Spectra were corrected by taking into account group-velocity dispersion of the white light continuum. The kinetic data were fit by a least-squares method followed by a deconvolution procedure. To estimate the quantum yield of K-intermediate formation, the ratio of the number of bR photoconverted to the total number of molecules excited at zero time10,14 is needed. The total number of bR molecules excited was obtained from the deconvolution of the transient absorption spectrum at time zero from the ground-state bleach. The number of bR photoconverted was obtained from the deconvolution of the transient spectrum at 8 ps from the ground-state bleach and K-intermediate absorption spectrum8 (assuming that the shape of the absorption spectrum does not change from 77 to 20 K). Results Figure 1 shows transient absorption spectra of bR at 20 K measured at different delay times (0, 1.2, 4, and 8 ps) after excitation by a 600-nm pulse. Three spectral regions are observed corresponding to transient absorption of the excited electronic state of bR at 450-530 nm,3-5,7 a bleaching corre-
Letters
Figure 2. Comparison of time-resolved spectrum of wild-type bR (at time zero and 20 K, solid line) with the weighted inverted absorption spectrum of the sample (at 20 K, dashed line). This shows that the photoproduct (K-intermediate) contribution to the transient spectrum is negligible.
Figure 3. Excited-state kinetics measured at 480 nm and 20 K for three different samples of bR (closed diamonds, open circles, and closed circles). The solid line is a fit of the kinetic decay measured for sample 3 with the following decay parameters: (0.8 exp(-t/1.2 ps) + 0.2 exp(-t/80 ps)). The inset is the same kinetics on a shorter time scale. The kinetics of bR measured at 300 K is also shown (open diamonds).
sponding to the depletion of the ground-state molecules of alltrans-retinal at 530-610 nm, and transient absorption attributed to the J- and K-intermediates at 620-700 nm.5,7 The transient spectrum at time zero is compared to the weighted inverted ground-state absorption spectrum of bR at 20 K in Figure 2. This is done to verify that the K-intermediate does not contribute to the spectrum at this time. If there is a buildup of K-intermediate in these experiments, one should observe its absorption in the red wing of the ground-state bleach at zero time with a maximum at ∼650 nm. This is not observed in the 0-ps delay spectrum in Figure 2. The fact that the zero time delay groundstate bleach and the inverted ground-state absorption spectrum are similar in the 650-nm region strongly suggests that K does not accumulate under our experimental conditions with red light irradiation. Using this method, the maximum amount of K present per pump laser shot is estimated to be less than 20%. The possibility of the K-intermediate contributing to the kinetics of the excited state will be discussed later. Figure 3 shows excited-state decay data at 20 K monitored at 480 nm for three different samples prepared and measured in the same way (these samples were similar to each other at room temperature). As one can see, there is a variation in the
Letters amplitude of the slow component of the decay. The decay curves are resolved into two components. By eliminating the contributions of the slow component from the short-lived component in each decay curve, and fitting to ln I vs t, an average lifetime of 0.58 ( 0.1 ps is obtained for the short component. This is the same number determined for the lifetime at room temperature within experimental error. This strongly suggests that the photoisomerization process (or the process occurring with 0.5-ps lifetime) observed under physiological conditions is barrierless. The transient spectrum is found to be the same for all three samples, indicating that the long-lived intermediate has a similar spectrum to the short-lived one (Figure 1). At room-temperature both the spectral and temporal behaviors of the bR in the glycerol/water mixtures at 450-530 nm are similar to that in aqueous solution. This absorption band is assigned to absorption by the excited electronic state.5 Let us now consider the possibility that the K-intermediate accumulates and contributes to the excited-state kinetics observed at low temperature. It is possible that both the excited state of the K-intermediate trapped at low temperature and the ground state of all-trans-retinal in bR contribute to the observed double-exponential decay of excited-state bR. If this is correct, the fast component of the decay would have to correspond to the K-intermediate and the slow one to the excited state of alltrans-retinal because the quantum yield for fluorescence from the K-intermediate is much lower than that for ground-state bR at low temperature.15 The contribution of the K-intermediate, however, can be ruled out because the ratio of the two components of the excited-state decay is >4:1 (the fast-component amplitude to that of the slow component), but the absorption for both the K-intermediate and ground-state bR is about the same at the excitation wavelength (600 nm). Furthermore, the photostationary state between the K-intermediate and the bR ground state should correspond to a ratio of ∼0.4/0.6 (assuming the quantum yield of the K-intermediate formation is close to 0.66, and the quantum yield formation of all-trans-retinal from K-intermediate is 116). If this photostationary state is formed, the contribution of ground-state bR to the dynamics of the excited state should be at least 60%. In our excited-state kinetics, however, the contribution of the slow component is always less than 20%. This rules out the possibility that the fast component is due to the K-intermediate and the slow component is due to ground-state bR. The formation of both J- and K-intermediates can be seen in Figure 1 to be very similar to that observed at room temperature.3-6 The J-intermediate absorbs maximally around 650 nm and decays to the K-intermediate within 4-6 ps. The determination of the quantum yield of photoisomerization for the fast component (based on the amount of transient the K-intermediate formed) is equal to 0.6, which is similar to the value of 0.66 obtained at 77 K,8 and room temperature.9 The high value of the quantum yield of K-intermediate formation obtained in our measurements is another argument against the possible formation of a photostationary state between the K-intermediate and the ground-state bR. Discussion Excited-state relaxation of bR at room temperature is wellestablished owing to the contributions of several laboratories.3,4,6,17 Mathies et al.3 used femtosecond pulses (60-fs pump, 6-fs probe) to monitor the dynamics of retinal in bR. Kobayashi et al.4 showed that the stimulated emission and excited-state absorption both decayed with the same time constant of 0.5 ps.
J. Phys. Chem. B, Vol. 102, No. 13, 1998 2305 Within this time, the retinal molecule can either return to the all-trans (ground-state)-conformation (through internal conversion) or form the J-intermediate (through photoisomerization) followed by the formation of the other intermediates in the bR photocycle. Recently more accurate data with 100-fs laser excitation were obtained.17-19 The lack of spectral dynamics was found in both the excited-state absorption and stimulated emission. Moreover, the excited-state decay as well as the stimulated emission decay were found to be multiexponential with decay constants of 0.5 and 3 ps. To explain these data, another model was proposed in which the Ag and Bu states are involved in the torsional dynamics of the excited state.20 In this model a flat part was introduced to the torsional coordinate potential. It was suggested by Anfinrud et al.17,18 that the dynamics of the wave packet in this part of the potential surface dominates the spectral response of retinal. The multiexponential decay of the excited state, however, suggests a more complicated behavior. Previous low time resolution studies suggested the presence of a large activation barrier on the potential surface. It is still questionable, however, whether the relaxation pathway at low temperature is the same as that at room temperature. Nevertheless there are numerous data found in the literature for bR that support fast or slow torsional dynamics at low temperature. Steady-state fluorescence spectroscopy was used in the mid70s to obtain information on the excited-state dynamics of bR. Sineshchekov et al.,1 and independently Shapiro et al.,2 showed that the emission at 700 nm is due to all-trans-retinal in bR. Shapiro et al.2 measured the temperature dependence of the emission quantum yield and discovered that the quantum yield increased greatly when the temperature was decreased below 100 K. The sharp rise in the emission intensity can be associated with a decrease in nonradiative decay at low temperatures. This in turn leads to an increase in the excitedstate lifetime, which was observed by Alfano et al.11 (40 ps at 90 K) and Shapiro et al.2 (80 ps at 77 K). Sharkov et al.7 obtained a value of 80 ps for the formation of the K-intermediate at 13 K after excitation by 30-ps pulses at 532 nm in an ethylene glycol/water mixture. In the present paper, the lifetime of the dominant short component of the excited state of bR in the glycerol/water mixture does not change greatly between room temperature and 20 K. There is a slow component, however, which may be correlated with the 80-ps component found earlier by Sharkov et al.6 and other workers that used lasers with pulses of 30 ps. The amplitude of this component varies from sample to sample, though, and does not exceed 20% of the total signal of the excited-state absorption in our experiment. This biexponential behavior in our experiments cannot be explained by a photostationary mixture of the K-intermediate and ground-state bR, as was discussed previously. The biexponential decay of the excited state of bR at low temperature could instead indicate the existence of two states of bR. A model suggesting the existence of two states of bR with proton transfer between them was previously proposed.2,3,21,22 Proton-transfer occurrence after photoexcitation is questionable, but the possibility of different photostationary states between different states of bR in an inhomogeneous sample is highly possible. This could lead to parallel cycles. The inhomogeneity of bR samples was discussed in the literature, and there are numerous experimental data showing that this occurs on the basis of the multiexponential decays of the other photocycle intermediates,23 supported by the large contribution of the inhomogeneous line width to the absorption spectrum by use of hole-burning techniques.24,25 The
2306 J. Phys. Chem. B, Vol. 102, No. 13, 1998 large variation in the observed contribution of the slow to fast components could result from changes in the sample inhomogeneity resulting from differences of the quality of the glass formed at low temperature in the different experiments. Possible variation in local heating could also result in differences in sample inhomogeneity in these glasses in each experiment. The presence of a fast component in the excited-state decay of bR at 20 K presented here leads to an estimation of a very small barrier26 (
