10648
J. Phys. Chem. 1995,99, 10648-10657
Probing Bacteriorhodopsin Photochemistry with Nonlinear Optics: Comparing the Second Harmonic Generation of bR and the Photochemically Induced Intermediate K Oleg Bouevitch; Aaron Lewis,*,? and Mordechai Sheves*,* Division of Applied Physics, The Hebrew University of Jerusalem, Jerusalem 91904, Israel, and Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel Received: October 13, 1994; In Final Form: February 10, 1 9 9 9
The nonlinear optical properties of the bacteriorhodopsin chromophore in the bR568 and K states are investigated by second harmonic generation. The comparison of amplitudes and phases of the second-order nonlinear optical polarizabilities of the retinal chromophore in the two states has revealed a noticeable increase of the induced dipole of the retinal as a result of the bR568 K transition. The results have been explained in terms of recent theoretical understandings of the nonlinear optical properties of polyenes. Within the context of these understandings we have discussed the molecular origins of the light-induced color changes and the possible mechanism of photon energy storage observed in this protein.
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Introduction
Cryostat
Second harmonic generation (SHG) has been employed as a unique method to characterize the structure and function of biological systems.’-3 In this report, we extend our previous applications of this probe in order to understand the mechanism of the photochemistry of the light-induced proton pump bacteriorhodopsin (bR) which is found in the purple membrane (PM) of Halobacterium halobium. Numerous ~ t u d i e s ~have - ~ been aimed at trying to resolve the fundamental photochemical interactions in bR,7 but this problem remains one of the outstanding questions in biophysics. In terms of the primary photochemical event it is known that within 450 fs the initial pigment state traverses the excited state and reaches the ground state photoproduct, called J.8-9 During this process, which occurs with 70% quantum efficiency,IO*’ >23% of the photon energy is stored.I2 The crucial question is what are the molecular processes involved in this photochemically based transformation. The first intermediate that can be stabilized after photon absorption is not J but K. K is produced from J in 3 ps and lives for microseconds. K, however, can be stabilized at liquid nitrogen temperatures indefinitely, and this has led to numerous studies being performed with a variety of spectroscopic technique~.~?~~’ In terms of the optical spectroscopies available, nonlinear optical interactions, provided by SHG, provide a set of unique advantages both experimentally and from the point of view of the information that can be extracted on the structure and the function of bR. The obtainable fundamental information includes details on the absolute structural orientation of the chromophore 2 and the induced dipole, dpex, of the chromophore in the protein 3. This information is obtained with an experimental technique that is relatively simple to implement, is nonbleaching using infrared probing beams, and has a large signal to noise ratio due to the gigantic nonlinearities exhibited by bR. It is important to note that the near resonant bR hyperpolarizability3>I4 measured at 1.064 pm, that is related to the induced dipole dpex of the bR chromophore, is 1.15 times bigger than the largest resonant hyperpolarizability measured in specially optimized synthetic organic compound^.'^
@
The Hebrew University of Jerusalem. The Weizmann Institute of Science. Abstract published in Advance ACS Abstracts, April 1, 1995.
0022-365419512099-10648$09.00/0
He-Ne
Figure 1. Experimental arrangement: QS:ML Nd:YAG, Coherent “Antares” Q-switched mode-locked Nd:YAG laser; P, Glan-Thompson polarizer; ;2/2, phase plate; F, filter; x-cut, quartz plate; RGP, rotatable glass plate; L, lens; IF, 532 nm interference filter; M, 20 cm monochromator; PMT, cooled RCA C3 1034 photomultiplier.
In this paper we compare the SHG of the initial pigment state bR568 with the photochemically generated K intermediate at 77 K and derive from our data information on the induced dipole, dpex, of these two states. The data obtained are compared to a variety of bR states that can be produced with absorption maxima that mimic the red-shifted K state, and all these results are analyzed in terms of the current theoretical understandings of SHG in conjugated polyenes. Finally, we analyze the data in terms of prevalent models that could define this primary event in bR. Materials and Methods The experimental arrangement (Figure 1) included a Qswitched mode-locked Nd:YAG laser as the source of the fundamental exciting wavelength at 1064 nm. The laser was operated at a frequency of 80 Hz with an 80 mW average power and a 2 mm beam diameter. In addition, there was the required polarizing and directing optics together with an x-cut quartz crystal followed by a rotatable glass plate (RGP). The x-cut quartz crystal was employed as a source of defined secondharmonic (SH) emission. The sample was cooled in an optical cryostat, and the SH signal at 532 nm was detected with a cooled RCA C3 1034 photomultiplier. A set of appropriate spectral filters/monochromator was used to cut off the fundamental emission. The bR could be switched between photostationary states in which there were various concentrations of bR and K using a variety of light sources. These were a CW argon ion laser emitting light in the blue-green region, and an incandescent 0 1995 American Chemical Society
Probing Bacteriorhodopsin Photochemistry
J. Phys. Chem., Vol. 99, No. 26, 1995 10649
lamp with appropriate filters for wavelength selection that were used to produce the bR/K species. A HeNe laser and a diode laser emitting at 670 nm were also used in order to return the K species to bR. Two kinds of bR films were used in the experiments. Purple membrane -poly( vinyl alcohol) (PM -PVA) films were chosen for their excellent optical quality and long-term stability.I6 Electric-field-sedimentedPM filmsI7 possess a defined orientation of the PM fragments that stack onto a Sn02-covered glass in a highly oriented fashion that results in strong (p-polarized) SH signals. Both kinds of films are prepared according to established
procedure^.^,' 6,1 PM-PVA Films. A 5-g portion of PVA, Sigma, MW 25 000, was dissolved by boiling in 25 mL of 50 mM HEPES
buffer adjusted to pH 7. PM in the same buffer was carefully washed, and a 0.5 mL of an OD 10 suspension was used. A 3 -4 mL portion of the PVA solution at room temperature was gently mixed with 0.5 mL of the PM suspension and degassed. The resulting PM-PVA suspension was spread onto a fused quartz plate, 5 cm in diameter. A slow flow of filtered dried air was used to dry the film overnight. The thickness of the dried film was about 0.2 mm with an OD of between 0.1 and 0.2. The absorption spectrum did not differ from that of an aqueous PM suspension at pH 7. Electric-Field-SedimentedPM Films. PM was washed in double-distilled water several times. A 0.1-0.2 mL portion of the suspension was spread between a SnOz-covered glass plate and a flat silver electrode. The distance between electrodes was 2 mm. Both electrodes touched the suspension and an electric field of 20-30 V/cm was applied for not more than 1 min. Excess water was then removed and the sample dried overnight in a humid environment. A film of light-adapted bR containing 100% all-trans retinal (ATR) was cooled down in the dark to 77 K. The amplitude and phase of the second-order susceptiliblity tensor components describing SHG at a wavelength of 532 nm (fundamental laser wavelength of 1064 nm) were then determined by measuring SHG signals from a bR film illuminated with CW visible light at various wavelengths using different polarization angles of the linearly polarized fundamental laser beam. The illumination with visible light shifted photostationary concentrations of the bR568 and K photointennediates, and this resulted in a change of the SH intensity and phase. These changes of the magnitude and phase of the SHG, caused by changes in the relative bR/K composition of the film induced with the visible light, were recorded and processed to assess the change of the induced dipole of the retinal chromophore of bR upon the bR568 K transition. The measurement of the phase of SHG, or the phase of the second-order susceptibility of bR film, was performed in the following way. A 1.196 mm thick x-cut quartz plate was inserted into the path of the fundamental infrared beam (Figure 1). The plate itself generated a SH signal that had intensity equal to that of the bR film when the plate is tuned to one of the maxima of Maker fringes. The phases of SHG from both sources were, of course, fixed relative to the phase of the squared fundamental emission; they were determined by the phases of the corresponding second-order susceptibilities. The rotatable glass plate, marked as RGP in Figure 1, was placed between the two SHG sources (the x-cut plate and the bR film) to serve as the dispersive element modulating the relative phase of the squared fundamental wave and the wave of SH generated by the quartz plate. From the interference pattern from the two SHG sources, obtained by the slow (0.5 deg/min) rotation of
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the glass plate, the phase of the nonlinear susceptibility of the bR sample could be determined.I8
Theoretical Considerations Since a main objective of this paper is to obtain information on the induced dipoles in the bR, K, and other states of bR, we first review the theoretical approach that leads from the observed macroscopic SHG intensities to the induced dipoles of the chromophore that are of interest to us. The intensity Zz0 of the SHG is related to the nonlinear susceptibility The nonlinear susceptibility is related to the molecular hyperpolarizability tensor p. Quantum-chemical calculation^'^ and Stark effect spectroscopy experiments20have demonstrated that the lowest energy electronic transition of the retinylidene chromophore includes a significant degree of charge transfer (CT) character along the long molecular axis 2'. Since the SH frequency is close to the frequency of this transition, the near-resonant hyperpolarizability tensor is dominated by only one component and this component, /3z,zy,is shown below2',22
x@).
where Wng is the transition frequency, w is the exciting laser frequency, f is oscillator strength of the transition, dpexis the induced dipole, and r is the damping constant. As can be seen in the above expression, the induced dipole, dp,,, is proportional to pz,z,z, and can be calculated from wng,f,r, and pZ,,,,,. The values wng and f are calculated from the known absorption spectra of bR568 and K.23 The contribution of the lowest electronic transition to the absorption spectra of bR568 and K is approximated with a log-normal function. This allows one to minimize possible incorporation from higher electronic transitions into calculated values of ungand$ The incorporation of other higher electronic states into p itself is thought to be negligible, for several reasons. First, there is ample experimental evidence (see for example Chemla and Z Y S S ~that ~ ) the two-level approximation of the second-order polarizability, p, works remarkably well for CT molecules. Second, there is a good correlation between the induced dipoles of retinal Schiff bases obtained, on the one hand, from SHG3,24 and, on the other, from the very different technique of Stark effect spectroscopy.20 Of significance is the fact that the induced dipoles calculated based on the SH results of these investigations were also based on this same two level model, and comparisons with Stark effect spectroscopy were within 5-6%. Third, the higher electronic states are not in resonance and should not change significantly in the bR-K transition. The absorption spectra of bR and K are shown in Figure 2. The value of r b R was taken from the literature.8 The value of r K was estimated experimentally by comparing phases of nearresonant complex x'2'bR and x ( ~ ) K . The value of molecular hyperpolarizabilitypz,z,z, of the retinal chromophore can be found from component(s) of the tensor of the nonlinear susceptibility x ( ~ )The . connection between the molecular hyperpolarizability /3z,z,z, and components of the macroscopic nonlinear susceptibility ~ ( can ~ be 1 written25as
Bouevitch et al.
10650 J. Phys. Chem., Vol. 99, No. 26, 1995
correction factors and (complex) Fresnel transmission coefficients. However, all these effects are insignificant as we show below. To estimate the order of magnitude of these effects, we 2 c9 calculated the incorporation of the absorption peak around Lo = 568 nm into the complex index of refraction n = n’ - in”, 0 F where n” = d/4nG, n’ - 1 = 2n”(wo - o ) / y , and a typical absorption coefficient of the electric-field-sedimented bR film rz Q, at 568 nm is a(Lo)= 2.3 x lo3 cm-’. For the value of y , the 0 0 full width at half maximum (fwhm) of the bR absorption contour C was taken. The estimate gives n’’ 5 0.94 x and n’ - 1 0 .< 0.005. The last value is in line with the experimental 40 estimations of coherence length of the near-resonant SHG in t .-c bR The calculations show that during the transition X W bR568 bR600 (or bR568 K), the local field factor changes by
