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Fluorescence from the primary products of the bacteriorhodopsin photocycle
410
Journal of Luminescence 48 & 49 (1991) 410 414 North Holland
Fluorescence from the primary products of the bacteriorhodopsin photocycle Picosecond time-resolved fluorescence spectroscopy G.H. Atkinson1, D. Blanchard and T.L. Brack
Department of Chemistry and Optical Science Center, University of Arizona, Tucson, AZ 85721, USA
The fluorescence spectrum of the K-590 intermediate, formed several picoseconds after the optical initiation of the bacteriorhodopsin (BR) photocycle, is observed at room temperature using a two laser, pump-probe technique. The BR photocycle is initiated with a 565 nm pump laser pulse (6 ps) while the observed fluorescence is generated by the optically delayed 590 nm probe laser pulse (4 ps). The distinct emission from K-590, while unstructured and occurring in the same general spectral region as the emission from BR-570 itself, is distinguished by its approximately twofold greater emission quantum yield and a shift of the position of maximum emission intensity to higher energy (corresponding to a blue shift of 17 nm). The assignment of this emission to the K 590 is based on its time dependence. The K-590 fluorescence spectrum described here is obtained using a 40 ps pump-probe delay. At this time in the photocycle, the only transient species which can be excited by the picosecond probe laser is K 590. The changes in the ground and excited state properties of the retinal chromophore of BR during the initial 100 ps of the photocycle can he quantitatively described by a rate equation model in which the instantaneous populations of each of the photocycle species is calculated These results are used to fit the picosecond transient absorption (PTA) and time resolved fluorescence (PTRF) data. Differences in the time-dependence of the PTA and PTRF data require the inclusion into the rate-equation model of vibrationally excited, ground state species, BR’ and K’, which are formed during the optical pumping and by decay of J 625, respectively.
1. Introduction
The photocycle of bacteriorhodopsin (BR), the protein contained in the purple membrane (PM) of Halobacterium halobium has been studied extensively using several experimental techniques to elucidate the molecular mechanism(s) underlying the energy storage in the trans-membrane protein, This energy is utilized for proton pumping which separately is considered as a model for visual processes in rhodopsin [1,2]. Of particular interest are the events which follow the optical excitation on the picosecond time scale associated with configurational and conformational changes in the retinal chromophore [1,2]. Much of the understanding of the early kinetics has been based on transient absorption experiments which monitor changes in Senior Fulbright
Professor,
Germany, and
Professor, Hebrew University, Israel. 0022-2311/91 $03.50 c 1991
Lady Davis
the electronic properties of ground state species. These absorption results have led to several rateequation models which attempt to describe the picosecond events in the photocycle. Recently, picosecond time-resolved fluorescence (PTRF) measurements have provided an opportunity to monitor changes in the excited electronic states during the initial part of the photocycle [3]. By extending the rate equation models to describe the PTRF data, quantitative correlations between ground and excited state populations can be made. In fig. 1 the general scheme used in the rate equation model is presented on a schematic potential surface representing the isomerization of the retinal chromophore during the initial part of the photocycle. Recent attention has been directed at understanding the excited-state relaxation processes which occur less than 500 fs after optical excitation [5,6]. For example, femtosecond transient absorption studies have distinguished the Franck
Elsevier Science Publishers By. (North Holland)
OH. Atkinson eta!. / Fluorescence from primary products of the BR photocycle
3
\~4~J
411
ticularly interesting since their contributions in optical excitation and during the photocycle have
Purple membrane is isolated from Halobacterium halohium strain Ri using the procedure e
t)~
r te
Fig. 1. Schematic representation of the potential energy surfaces and the initial processes which occur in the BR photocycle. The various species present in the course of reaction are represented as letters followed by a number indicating the wavelength (in nm) of the visible absorption maximum for each species. Absorption is represented by solid, vertical arrows pointing up whereas fluorescence are represented by dashed, vertical arrows pointing down. Nonradiative relaxation, includ ing vibrational relaxation, is represented by wavy arrows. The common excited-state shared by BR and J is labeled CES while Franck Condon states populated by absorption are labeled FC. The remaining notation is defined in the text.
Condon population (FC1 in fig. 1) created by the optical excitation from other excited state populations formed by vibrational relaxation [6,7]. In this paper, the non-radiative and photochemical events in the BR photocycle occurring more than 1 ps after excitation are of primary interest. Specifically, PTA and PTRF data measured with a pump (6 ps) and probe (4 ps) laser configuration under experimental conditions which facilitate direct, quantitative comparisons are presented. Both types of data are analyzed using a rate-equation model which includes the transient populations of ground-state photochemical species (i.e. BR-570, J-625 and K-590), excited electronic states (BR*, J~and K*) formed during optical pumping, and vibrationally-excited species (BR’ and K’) generated during optical pumping and within the photocycle, respectively, Some of these species are known to be present from previous absorption studies [13] while others have not been explicitly treated in previous models (e.g. J’1’ and K*) or are reported here for the first time (e.g. BR’ and K’). The presence of vibrationally-excited ground-states (BR’ and K’) is par-
of Oesterhelt and Stoeckenius [8] with little modification [3]. The BR sample is prepared for use by suspending purple membrane in distilled water to obtain an optical density at 565 nm of 4.0. The pH of this suspension lies between 6 and 7 although no buffer is used. The BR sample is pumped through a 380 p.m diameter glass nozzle .
to form a jet stream so that the sample volume illuminated by the focused laser beams is exchanged within I p.s [3]. The laser system and detection apparatus used to acquire PTRF and PTA data have been described in detail elsewhere [3,9]. A mode-locked frequency doubled Nd:YAG laser synchronously pumps two independently tunable cavity dumped (1 MHz) dye lasers providing pairs of pump-probe picosecond pulses (4-6 Ps FWHM). After the 590 nm probe beam exits the adjustable optical delay line, it is made collinear with the 573 nm pump beam and directed to the flowing BR sample. Fluorescence is collected at right angles to the beam and flowing jet stream and is directed into a 1 m monochromator. The dispersed fluorescence is detected with a cooled photomultiplier tube (PMT) (Hamamatsu model R943/02). The PMT signal is sent to a lock-in amplifier referenced to a mechanical chopper placed in the path of the probe laser beam. This ensures that only emission induced by the probe laser is detected. Picosecond transient absorption (PTA) measurements are obtained by directing the probe laser light trans mitted through the BR sample to a photodiode (EG&G HUV-2000B) whose output is sent to the lock-in amplifier. To measure the transmitted probe intensity, I~,the lock-in is referenced to a mechanical chopper placed in the probe beam.
G.H. Atkinson cia!.
412
/
Fluorescence from primary products of the BR phorocycle
~ the change in the transmitted probe intensity induced by the pump beam’s effect on the sample, is measured with the chopper placed in the pump laser beam. The change in absorbance is given by K~I~/I1where K (In 10) [10]. In an —
—
analogous manner the PTRF signal is defined as ~If/1f [Ir(td) I~]/I,.where I~is the fluorescence signal obtained when the pump laser beam is prevented from reaching the sample and f(td) refers to the fluorescence intensity obtained at delay time, td, using both beams. Two types of PTRF data are reported here. The spectral profile of the fluorescence emission is obtained by positioning the delay line to the desired pump-probe delay interval and scanning the monochromator. The time evolution of the total probe laser induced fluorescence intensity is obtained by scanning the delay line while detecting the total nondispersed emission (the monochromator grating is replaced by a plane mirror for this purpose).
3. Results
The relative change in the total probe laser (590 nm) induced fluorescence intensity, M1/11, is
displayed in fig. 2 as a function of the pump-probe delay l.solid trace). These data are characterized by an initial decrease in the emission intensity during the cross-correlation period when the two laser pulses are overlapped in time followed by a rise in intensity returning to and then exceeding the level obtained when the emission is measured using the probe laser alone. This rise is complete by approximately 40 Ps and the higher level is maintained for at least 300 ps. The analogous PTA results are presented in fig. 3. The same general behavior is seen in these data although a more rapid rise is seen here. The spectrum of the fluorescence obtained using the low power (2 mW) probe laser alone is presented as the solid trace in fig. 4. This emission recorded over the spectral range of 650-900 nm is similar to emission assigned previously to BR-570 [3,11]. The fluorescence spectrum obtained when higher power (10 mW) pump excitation and a probe laser at a 40 ps delay is used is more intense and has approximately the same shape as that of the BR570 emission. The maximum of the fluorescence emission, however, is shifted toward shorter wavelengths. The dashed trace in fig. 4 is obtained when the contribution of the BR-570 emission is removed from the two laser, pump/probe data by subtracting from it the BR-570 fluorescence spectrum after it has been scaled by a factor of
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Fig. 2. Relative variation of fluorescence, ~ produced by the 590 nm, 4 ps probe laser pulse and induced by a 6 ps pump laser pulse at 573 nm as a function of time delay. t~.for a room temperature BR sample. The simulation based on the mechan ism with BR’ and K’ are presented as dashed lines. A poor fit is obtained with the simulation which does not include BR’ and K’ (the dotted trace) and also with the simulation where K” is permitted to relax within S1 to K” (~( K ~*) 0.5q51( BR”)) (dashed-dotted line) (see text).
0
4
( Fig. 3. Variation of absorbance (~A)at 590 nm induced by a 2 3 ps laser pulse at 573 nm as a function of time delay, r~,for a room temperature BRsample. A fit to the data is superimposed as a dashed line.
OH. Atkinson et a!. / Fluorescence from primary products of the BR photocvcle 2 731
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tion of BR-570. The solution of these rate equations yields the time dependent populations of the reacting species which are then used to simulate the PTA and PTRF observations. In order to fit both PTA and PTRF data using the same molecular parameters, it is necessary to include the vibrationally-excited species BR’ and K’ explicitly in the model and to assign to these species absorption coefficients, emission quantum yields and relaxation lifetimes which differ from their respective relaxed forms.
I 850
Wavelength Inr,iI
Fig. 4. Fluorescence spectrum of the K-590 intermediate in the BR photocycle obtained at room temperature and with 590 nm probe excitation. The fluorescence spectrum of BR-570 at room temperature recorded with pulsed (2.4 mW) excitation at 590 nm is shown as a dashed line for comparison. The K-590 spectrum is obtained by subtracting the scaled (0.6) BR-590 fluorescence spectrum from the 40 ps PTRF spectrum. This analysis is based on the simulation’s prediction that 40% of the initial concentration of BR-570 is converted into K-590. From ref. [3].
0.6. The scaling factor is determined from the kinetic simulation model used to analyze the time dependent data (see below). Varying this scaling factor between 0.7 and 0.5 has very little effect on the shape of the residual emission,
4. Discussion The assignment of the additional emission intensity observed at pump-probe delays of 40 ps or longer to the K-590 intermediate is based on the generally accepted reaction scheme for the early portion ofthe photocycle (see fig. 1). At these times in the photocycle the only species present are BR-570 and K-590 and therefore, after removing the BR-570 contribution, only contributions from K-590 remain. The analysis of the PTA and PTRF data (including the scaling factor) is based on a model of the early photocycle describing the changes in the populations of the known photocycle intermediates using rate equations which take into account all of the photochemical and photophysical processes initiated by the photo-excita-
5. Kinetic model The differential equations needed to treat the early photocycle are described in detail elsewhere [9,10]. The PTA and PTRF data are simulated by convoluting the pump-induced populations with the probe laser’s Gaussian temporal profile. While .
.
.
is possible to use the PTA simulation to obtain reasonable fits for the absorption data, it is not possible to fit the PTRF data without including the vibrationally-excited species, BR’ and K’. This is illustrated in fig. 2 where the dotted trace represents the best fit obtainable using the PTRF simulation without K’. It is clear that the rise of the fluorescence intensity after the pump pulse is slower than that predicted by the simulation without K’. The dash-dotted trace shown in fig. 2 represents the simulation’s prediction when K’ is included in the model and is given the same quantum yield of fluorescence as K-590. Only when the K’ quantum yield of emission is set to zero is it possible to fit the PTRF data and the PTA data with a consistent set of molecular parameters. The dashed trace in fig. 2 represents the simulated PTRF signal predicted using both BR’ and K’ with a K’ quantum yield for emission set to zero. The parameters used in these simulations are presented and discussed in detail elsewhere [9,10]. Several other approaches for modifying the simulation model have been explored such as models which include various back-reactions from J-625 and K-590, but none of these alone produce satisfactory simulations of the PTRF data. The inclusion of the vibrationally-excited species in the model is justified by the independent observation it
414
OH. Atkinson eta!. / Fluorescence from primary products of the BR photocycle
of substantial anti-Stokes resonance Raman scattering from BR that has been optically excited [12]. These observations indicate the presence of BR’ produced by the relaxation of the optically excited S1 BR-570 [12]. Simulations that include BR’ as the only vibrationally-excited species can be used to fit the PTRF data only if very long relaxation times (“=20 ps) for the BR’ species are employed, Such slow vibrational relaxation is not consistent with the observed decay of the anti-Stokes signals attributed to BR’ in the resonance Raman studies [12]. The ground-state vibrational relaxation properties assigned to K’ are similar to those attributed to BR’ both in the simulation and in the analysis of anti-Stokes resonance Raman results [12]. The “’8 ps relaxation times found for BR’ and K’ are similar to intermolecular vibrational energy transfer times between polyatomic molecules and their solvents temperatures [13,14]. It is likely then that attheroom vibrational relaxation measured here reflects the transfer of energy from the retinal chromophore to its protein environment. In these processes BR’ and K’ act similarly. Vibrational relaxation within the excited electronic states of BR’ and K’, however, are distinct. The rapid (0.2 ps) vibrational relaxation in S~ dominates the decay of BRI* while there is no support for an analogous process in K’. The PTRF data can only be simulated accurately if the initially populated levels of Kn* relax directly to K’ without any significant vibrational relaxation in the excited electronic state. These differences in excited state relaxation properties may reflect differences in the degree to which the retinal chromophore interacts with its protein environment with K1* being more strongly perturbed and thus having more efficient nonradiative decay channels directly to the ground state. The successful simulation of the PTRF data also provides values for the relative populations of BR-
570 and K-590 at delays of 50 p5 to 500 ns. At such delays, these are the only two species present. The shift in the intensity maximum of the emission observed between the BR-570 and K-590 spectra (fig. 4) can be viewed as a reduction in the Stokes shift of the fluorescence spectrum relative to the absorption spectrum (141 c.f. 178 nm). This reduced Stokes shift indicates a smaller change in geometry between the ground and excited electronic states of K-590. Changes in these PTRF signals in this time regime again may reflect vanations in excited state retinal interactions with its protein environment. References [1] W. Stoeckenius, R.H. Lozier and R.A. Bogomolni, Bio-
chim. Biophys. Acta 505 (1979) 215. [2] M. Ottolenghi, in: Advances in Photochemistry, eds. J.N. Pitts, G.S. Hammond, K. Gollnick 97. and D. Grossjean
Interscience, NY, 1980)H.12Lemaire, p. [3] (Wiley OH. Atkinson, D. Blanchard, T.L. Brack and H. Hayashi, Biophys. J. 55 (1989) 263. [4] W. Stoeckenius and R.A. Bogomolni, Ann. Rev. Biochem. 51(1982) 587.
[5] H-i. Polland, MA. Franz, W. Zinth, W. Kaiser, E. Kolling and D. Oesterhelt, Biophys. J. 49 (1986) 651. [6] R.A. Mathies, C.H. Brito Cruz, W.T. Polland and CV. Shank, Science 240 (1988) 777. [7] i. Dobler, W. Zinth and W. Kaiser, Chem. Phys. Lett. 144 (1988) 215. [8] D. Oesterhelt and W. Stoeckenius, Methods Enzymol. 31 (1974) 667. [9] D. Blanchard, D. Gilmore, T. Brack, H. Lemaire, D. Hughes and G.H. Atkinson, Chem. Phys., submitted. [10] 1) Blanchard, Ph 1) Dissertation, Ijniversité de loceph Fourier, Grenoble (1990). [11] R. Govindjee, T.G. Ebrey and AR. Crofts, Biophys. J 30 (1980) 231. [12] T.L. Brack and G.H. Atkinson, J. Phys. Chem., in press. [13] W. Kaiserand A. Seilmeier, in: Time-Resolved Vibrational Spectroscopy, eds. Stockburger and Laubereau (Springer, Berlin, 1985) p. 42. [14] A. Seilmeier and W. Kaiser, in: Ultrashort Laser Pulses and Applications, ed. W. Kaiser (Springer, Berlin, 1988) p. 279.
Journal of Luminescence 48 & 49 (1991) 410 414 North Holland
Fluorescence from the primary products of the bacteriorhodopsin photocycle Picosecond time-resolved fluorescence spectroscopy G.H. Atkinson1, D. Blanchard and T.L. Brack
Department of Chemistry and Optical Science Center, University of Arizona, Tucson, AZ 85721, USA
The fluorescence spectrum of the K-590 intermediate, formed several picoseconds after the optical initiation of the bacteriorhodopsin (BR) photocycle, is observed at room temperature using a two laser, pump-probe technique. The BR photocycle is initiated with a 565 nm pump laser pulse (6 ps) while the observed fluorescence is generated by the optically delayed 590 nm probe laser pulse (4 ps). The distinct emission from K-590, while unstructured and occurring in the same general spectral region as the emission from BR-570 itself, is distinguished by its approximately twofold greater emission quantum yield and a shift of the position of maximum emission intensity to higher energy (corresponding to a blue shift of 17 nm). The assignment of this emission to the K 590 is based on its time dependence. The K-590 fluorescence spectrum described here is obtained using a 40 ps pump-probe delay. At this time in the photocycle, the only transient species which can be excited by the picosecond probe laser is K 590. The changes in the ground and excited state properties of the retinal chromophore of BR during the initial 100 ps of the photocycle can he quantitatively described by a rate equation model in which the instantaneous populations of each of the photocycle species is calculated These results are used to fit the picosecond transient absorption (PTA) and time resolved fluorescence (PTRF) data. Differences in the time-dependence of the PTA and PTRF data require the inclusion into the rate-equation model of vibrationally excited, ground state species, BR’ and K’, which are formed during the optical pumping and by decay of J 625, respectively.
1. Introduction
The photocycle of bacteriorhodopsin (BR), the protein contained in the purple membrane (PM) of Halobacterium halobium has been studied extensively using several experimental techniques to elucidate the molecular mechanism(s) underlying the energy storage in the trans-membrane protein, This energy is utilized for proton pumping which separately is considered as a model for visual processes in rhodopsin [1,2]. Of particular interest are the events which follow the optical excitation on the picosecond time scale associated with configurational and conformational changes in the retinal chromophore [1,2]. Much of the understanding of the early kinetics has been based on transient absorption experiments which monitor changes in Senior Fulbright
Professor,
Germany, and
Professor, Hebrew University, Israel. 0022-2311/91 $03.50 c 1991
Lady Davis
the electronic properties of ground state species. These absorption results have led to several rateequation models which attempt to describe the picosecond events in the photocycle. Recently, picosecond time-resolved fluorescence (PTRF) measurements have provided an opportunity to monitor changes in the excited electronic states during the initial part of the photocycle [3]. By extending the rate equation models to describe the PTRF data, quantitative correlations between ground and excited state populations can be made. In fig. 1 the general scheme used in the rate equation model is presented on a schematic potential surface representing the isomerization of the retinal chromophore during the initial part of the photocycle. Recent attention has been directed at understanding the excited-state relaxation processes which occur less than 500 fs after optical excitation [5,6]. For example, femtosecond transient absorption studies have distinguished the Franck
Elsevier Science Publishers By. (North Holland)
OH. Atkinson eta!. / Fluorescence from primary products of the BR photocycle
3
\~4~J
411
ticularly interesting since their contributions in optical excitation and during the photocycle have
Purple membrane is isolated from Halobacterium halohium strain Ri using the procedure e
t)~
r te
Fig. 1. Schematic representation of the potential energy surfaces and the initial processes which occur in the BR photocycle. The various species present in the course of reaction are represented as letters followed by a number indicating the wavelength (in nm) of the visible absorption maximum for each species. Absorption is represented by solid, vertical arrows pointing up whereas fluorescence are represented by dashed, vertical arrows pointing down. Nonradiative relaxation, includ ing vibrational relaxation, is represented by wavy arrows. The common excited-state shared by BR and J is labeled CES while Franck Condon states populated by absorption are labeled FC. The remaining notation is defined in the text.
Condon population (FC1 in fig. 1) created by the optical excitation from other excited state populations formed by vibrational relaxation [6,7]. In this paper, the non-radiative and photochemical events in the BR photocycle occurring more than 1 ps after excitation are of primary interest. Specifically, PTA and PTRF data measured with a pump (6 ps) and probe (4 ps) laser configuration under experimental conditions which facilitate direct, quantitative comparisons are presented. Both types of data are analyzed using a rate-equation model which includes the transient populations of ground-state photochemical species (i.e. BR-570, J-625 and K-590), excited electronic states (BR*, J~and K*) formed during optical pumping, and vibrationally-excited species (BR’ and K’) generated during optical pumping and within the photocycle, respectively, Some of these species are known to be present from previous absorption studies [13] while others have not been explicitly treated in previous models (e.g. J’1’ and K*) or are reported here for the first time (e.g. BR’ and K’). The presence of vibrationally-excited ground-states (BR’ and K’) is par-
of Oesterhelt and Stoeckenius [8] with little modification [3]. The BR sample is prepared for use by suspending purple membrane in distilled water to obtain an optical density at 565 nm of 4.0. The pH of this suspension lies between 6 and 7 although no buffer is used. The BR sample is pumped through a 380 p.m diameter glass nozzle .
to form a jet stream so that the sample volume illuminated by the focused laser beams is exchanged within I p.s [3]. The laser system and detection apparatus used to acquire PTRF and PTA data have been described in detail elsewhere [3,9]. A mode-locked frequency doubled Nd:YAG laser synchronously pumps two independently tunable cavity dumped (1 MHz) dye lasers providing pairs of pump-probe picosecond pulses (4-6 Ps FWHM). After the 590 nm probe beam exits the adjustable optical delay line, it is made collinear with the 573 nm pump beam and directed to the flowing BR sample. Fluorescence is collected at right angles to the beam and flowing jet stream and is directed into a 1 m monochromator. The dispersed fluorescence is detected with a cooled photomultiplier tube (PMT) (Hamamatsu model R943/02). The PMT signal is sent to a lock-in amplifier referenced to a mechanical chopper placed in the path of the probe laser beam. This ensures that only emission induced by the probe laser is detected. Picosecond transient absorption (PTA) measurements are obtained by directing the probe laser light trans mitted through the BR sample to a photodiode (EG&G HUV-2000B) whose output is sent to the lock-in amplifier. To measure the transmitted probe intensity, I~,the lock-in is referenced to a mechanical chopper placed in the probe beam.
G.H. Atkinson cia!.
412
/
Fluorescence from primary products of the BR phorocycle
~ the change in the transmitted probe intensity induced by the pump beam’s effect on the sample, is measured with the chopper placed in the pump laser beam. The change in absorbance is given by K~I~/I1where K (In 10) [10]. In an —
—
analogous manner the PTRF signal is defined as ~If/1f [Ir(td) I~]/I,.where I~is the fluorescence signal obtained when the pump laser beam is prevented from reaching the sample and f(td) refers to the fluorescence intensity obtained at delay time, td, using both beams. Two types of PTRF data are reported here. The spectral profile of the fluorescence emission is obtained by positioning the delay line to the desired pump-probe delay interval and scanning the monochromator. The time evolution of the total probe laser induced fluorescence intensity is obtained by scanning the delay line while detecting the total nondispersed emission (the monochromator grating is replaced by a plane mirror for this purpose).
3. Results
The relative change in the total probe laser (590 nm) induced fluorescence intensity, M1/11, is
displayed in fig. 2 as a function of the pump-probe delay l.solid trace). These data are characterized by an initial decrease in the emission intensity during the cross-correlation period when the two laser pulses are overlapped in time followed by a rise in intensity returning to and then exceeding the level obtained when the emission is measured using the probe laser alone. This rise is complete by approximately 40 Ps and the higher level is maintained for at least 300 ps. The analogous PTA results are presented in fig. 3. The same general behavior is seen in these data although a more rapid rise is seen here. The spectrum of the fluorescence obtained using the low power (2 mW) probe laser alone is presented as the solid trace in fig. 4. This emission recorded over the spectral range of 650-900 nm is similar to emission assigned previously to BR-570 [3,11]. The fluorescence spectrum obtained when higher power (10 mW) pump excitation and a probe laser at a 40 ps delay is used is more intense and has approximately the same shape as that of the BR570 emission. The maximum of the fluorescence emission, however, is shifted toward shorter wavelengths. The dashed trace in fig. 4 is obtained when the contribution of the BR-570 emission is removed from the two laser, pump/probe data by subtracting from it the BR-570 fluorescence spectrum after it has been scaled by a factor of
39() urn
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41
40
Fig. 2. Relative variation of fluorescence, ~ produced by the 590 nm, 4 ps probe laser pulse and induced by a 6 ps pump laser pulse at 573 nm as a function of time delay. t~.for a room temperature BR sample. The simulation based on the mechan ism with BR’ and K’ are presented as dashed lines. A poor fit is obtained with the simulation which does not include BR’ and K’ (the dotted trace) and also with the simulation where K” is permitted to relax within S1 to K” (~( K ~*) 0.5q51( BR”)) (dashed-dotted line) (see text).
0
4
( Fig. 3. Variation of absorbance (~A)at 590 nm induced by a 2 3 ps laser pulse at 573 nm as a function of time delay, r~,for a room temperature BRsample. A fit to the data is superimposed as a dashed line.
OH. Atkinson et a!. / Fluorescence from primary products of the BR photocvcle 2 731
-
/ 1
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tion of BR-570. The solution of these rate equations yields the time dependent populations of the reacting species which are then used to simulate the PTA and PTRF observations. In order to fit both PTA and PTRF data using the same molecular parameters, it is necessary to include the vibrationally-excited species BR’ and K’ explicitly in the model and to assign to these species absorption coefficients, emission quantum yields and relaxation lifetimes which differ from their respective relaxed forms.
I 850
Wavelength Inr,iI
Fig. 4. Fluorescence spectrum of the K-590 intermediate in the BR photocycle obtained at room temperature and with 590 nm probe excitation. The fluorescence spectrum of BR-570 at room temperature recorded with pulsed (2.4 mW) excitation at 590 nm is shown as a dashed line for comparison. The K-590 spectrum is obtained by subtracting the scaled (0.6) BR-590 fluorescence spectrum from the 40 ps PTRF spectrum. This analysis is based on the simulation’s prediction that 40% of the initial concentration of BR-570 is converted into K-590. From ref. [3].
0.6. The scaling factor is determined from the kinetic simulation model used to analyze the time dependent data (see below). Varying this scaling factor between 0.7 and 0.5 has very little effect on the shape of the residual emission,
4. Discussion The assignment of the additional emission intensity observed at pump-probe delays of 40 ps or longer to the K-590 intermediate is based on the generally accepted reaction scheme for the early portion ofthe photocycle (see fig. 1). At these times in the photocycle the only species present are BR-570 and K-590 and therefore, after removing the BR-570 contribution, only contributions from K-590 remain. The analysis of the PTA and PTRF data (including the scaling factor) is based on a model of the early photocycle describing the changes in the populations of the known photocycle intermediates using rate equations which take into account all of the photochemical and photophysical processes initiated by the photo-excita-
5. Kinetic model The differential equations needed to treat the early photocycle are described in detail elsewhere [9,10]. The PTA and PTRF data are simulated by convoluting the pump-induced populations with the probe laser’s Gaussian temporal profile. While .
.
.
is possible to use the PTA simulation to obtain reasonable fits for the absorption data, it is not possible to fit the PTRF data without including the vibrationally-excited species, BR’ and K’. This is illustrated in fig. 2 where the dotted trace represents the best fit obtainable using the PTRF simulation without K’. It is clear that the rise of the fluorescence intensity after the pump pulse is slower than that predicted by the simulation without K’. The dash-dotted trace shown in fig. 2 represents the simulation’s prediction when K’ is included in the model and is given the same quantum yield of fluorescence as K-590. Only when the K’ quantum yield of emission is set to zero is it possible to fit the PTRF data and the PTA data with a consistent set of molecular parameters. The dashed trace in fig. 2 represents the simulated PTRF signal predicted using both BR’ and K’ with a K’ quantum yield for emission set to zero. The parameters used in these simulations are presented and discussed in detail elsewhere [9,10]. Several other approaches for modifying the simulation model have been explored such as models which include various back-reactions from J-625 and K-590, but none of these alone produce satisfactory simulations of the PTRF data. The inclusion of the vibrationally-excited species in the model is justified by the independent observation it
414
OH. Atkinson eta!. / Fluorescence from primary products of the BR photocycle
of substantial anti-Stokes resonance Raman scattering from BR that has been optically excited [12]. These observations indicate the presence of BR’ produced by the relaxation of the optically excited S1 BR-570 [12]. Simulations that include BR’ as the only vibrationally-excited species can be used to fit the PTRF data only if very long relaxation times (“=20 ps) for the BR’ species are employed, Such slow vibrational relaxation is not consistent with the observed decay of the anti-Stokes signals attributed to BR’ in the resonance Raman studies [12]. The ground-state vibrational relaxation properties assigned to K’ are similar to those attributed to BR’ both in the simulation and in the analysis of anti-Stokes resonance Raman results [12]. The “’8 ps relaxation times found for BR’ and K’ are similar to intermolecular vibrational energy transfer times between polyatomic molecules and their solvents temperatures [13,14]. It is likely then that attheroom vibrational relaxation measured here reflects the transfer of energy from the retinal chromophore to its protein environment. In these processes BR’ and K’ act similarly. Vibrational relaxation within the excited electronic states of BR’ and K’, however, are distinct. The rapid (0.2 ps) vibrational relaxation in S~ dominates the decay of BRI* while there is no support for an analogous process in K’. The PTRF data can only be simulated accurately if the initially populated levels of Kn* relax directly to K’ without any significant vibrational relaxation in the excited electronic state. These differences in excited state relaxation properties may reflect differences in the degree to which the retinal chromophore interacts with its protein environment with K1* being more strongly perturbed and thus having more efficient nonradiative decay channels directly to the ground state. The successful simulation of the PTRF data also provides values for the relative populations of BR-
570 and K-590 at delays of 50 p5 to 500 ns. At such delays, these are the only two species present. The shift in the intensity maximum of the emission observed between the BR-570 and K-590 spectra (fig. 4) can be viewed as a reduction in the Stokes shift of the fluorescence spectrum relative to the absorption spectrum (141 c.f. 178 nm). This reduced Stokes shift indicates a smaller change in geometry between the ground and excited electronic states of K-590. Changes in these PTRF signals in this time regime again may reflect vanations in excited state retinal interactions with its protein environment. References [1] W. Stoeckenius, R.H. Lozier and R.A. Bogomolni, Bio-
chim. Biophys. Acta 505 (1979) 215. [2] M. Ottolenghi, in: Advances in Photochemistry, eds. J.N. Pitts, G.S. Hammond, K. Gollnick 97. and D. Grossjean
Interscience, NY, 1980)H.12Lemaire, p. [3] (Wiley OH. Atkinson, D. Blanchard, T.L. Brack and H. Hayashi, Biophys. J. 55 (1989) 263. [4] W. Stoeckenius and R.A. Bogomolni, Ann. Rev. Biochem. 51(1982) 587.
[5] H-i. Polland, MA. Franz, W. Zinth, W. Kaiser, E. Kolling and D. Oesterhelt, Biophys. J. 49 (1986) 651. [6] R.A. Mathies, C.H. Brito Cruz, W.T. Polland and CV. Shank, Science 240 (1988) 777. [7] i. Dobler, W. Zinth and W. Kaiser, Chem. Phys. Lett. 144 (1988) 215. [8] D. Oesterhelt and W. Stoeckenius, Methods Enzymol. 31 (1974) 667. [9] D. Blanchard, D. Gilmore, T. Brack, H. Lemaire, D. Hughes and G.H. Atkinson, Chem. Phys., submitted. [10] 1) Blanchard, Ph 1) Dissertation, Ijniversité de loceph Fourier, Grenoble (1990). [11] R. Govindjee, T.G. Ebrey and AR. Crofts, Biophys. J 30 (1980) 231. [12] T.L. Brack and G.H. Atkinson, J. Phys. Chem., in press. [13] W. Kaiserand A. Seilmeier, in: Time-Resolved Vibrational Spectroscopy, eds. Stockburger and Laubereau (Springer, Berlin, 1985) p. 42. [14] A. Seilmeier and W. Kaiser, in: Ultrashort Laser Pulses and Applications, ed. W. Kaiser (Springer, Berlin, 1988) p. 279.