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J. Phys. Chem. 1996, 100, 2762-2765
Homogeneous Line Width of the Different Vibronic Bands of Retinal Absorption in Bacteriorhodopsin by the Hole-Burning Technique Valey F. Kamalov, Tina M. Masciangioli, and Mostafa A. El-Sayed* School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0400 ReceiVed: October 6, 1995; In Final Form: January 3, 1996X
Using the hole-burning technique, resolved vibrational structure was observed in the retinal absorption spectrum of bacteriorhodopsin (bR) in poly(vinyl alcohol) (PVA) film at 10 K with 556 and 632 nm irradiation. The homogeneous line widths of V ) 1 and V ) 2 vibronic bands are estimated from the deconvolution of the observed spectrum. The absorption maximum is found to shift by 100-200 cm-1 by using the two excitation wavelengths; resulting from partial site selection due to the contribution of inhomogeneous broadening. The hole width produced by excitation near the zero-phonon band is found to be ∼1250 cm-1, which corresponds to a homogeneous width of ∼600 cm-1, and the low limit of dephasing time can be estimated as 20 fs. This width is found to be independent of the vibronic band observed.
Introduction Bacteriorhodopsin (bR), the other natural photosynthetic system besides chlorophyll, is a retinal protein membrane which absorbs light and undergoes the following photocycle:1-3 hν
0.2 ps
500 fs
3 ps
2 µs
bR568 Df (bR)* 98 I460 98 J625 98 K610 98 70 µs
ms
-H
+H
L550 9 8 M412 9 8 N f O f bR568 + + As a result of absorption, protons are pumped from inside the cell membrane to the extracellular surface.1-4. This leads to conversion of solar energy into electric energy which is further converted into chemical energy by converting ADP into ATP.5,6 The first step following absorption is the rapid photoisomerization7,8 process occurring in 450 fs. The detailed understanding of the dynamics of this process remains incomplete. This process has been previously discussed by Warshel,9 Birge,10 and Mathies.11 Unfortunately, the absorption spectrum is broad and structureless and gives little information regarding the potential surface or the electronic structure of retinal in its excited state along which the photoisomerization process takes place. Hole burning is one of the spectroscopic techniques used to help reduce the contribution of inhomogeneous broadening to the spectrum. This leads to sharper and thus more resolved spectra. Two groups have recently carried out hole-burning experiments on bR at low temperatures. Lee et al.12 observed a broad structureless hole with a bandwidth of 1600 cm-1 (fwhm) at 550 nm. Loppnow et al.13 observed photochemical hole burning with a hole width of 2500 cm-1 (fwhm). Broad holes in their study were only weakly dependent on photolysis wavelength. Both groups used glycerol/water mixtures for sample preparation. In this study, bR samples were prepared in a pH-buffered PVA film which we found maintained the photochemical cycle of bR at room temperature. These polymer films have been previously used in various optical applications (Kamalov et al.,14 Chen et al.15). This polymer is less polar than the glasses used previously and gave more resolvable spectra. Vibronic structure due to the CdC stretching vibration in the excited state was observed in hole-burning spectra at 10 K. From the observed X
Abstract published in AdVance ACS Abstracts, February 1, 1996.
0022-3654/96/20100-2762$12.00/0
spectrum, we were able to determine the change in the retinal electronic structure upon excitation and the rate of dephasing processes in each vibronic level observed. The valence bond method is used to qualitatively discuss the observed results. Experimental Section Purple membrane was isolated from Halobacterium halobium according to Oesterhelt and Stoeckenius16 and Becher and Cassim.17 The PVA film, 50% (wt/vol), was prepared (a modified procedure of Chen et al.15) in a 50 mM N-(2hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) buffer by heating to 98 °C. Approximately 1 mL of the PVA solution was mixed with 1 mL of concentrated bR (ca. 0.15 mM) in DDW. Quartz plates of 1 mm thickness were used as the substrate for films. After the bR-PVA solution was degassed under vacuum, the solution was spread on the substrate under a constant stream of nitrogen gas. Samples were mounted in a Janis CCS-150 closed-cycle refrigerator system (Janis Research Inc.) and was subsequently irradiated with yellow filter light to transform bR to the lightadapted form. The sample was protected from additional light and was cooled to 10 K. Absorption spectra were measured with a Beckman DU-650 UV-visible spectrophotometer. Samples were irradiated with a beam of a halogen lamp passed through a monochromator at various wavelengths. A He-Ne laser (632 nm) was also used for sample irradiation. Results Absorption spectra of bR/PVA film are shown in Figure 1 measured at 300 K (curve a) and 10 K (curve b). The absorption maximum of the light adapted bR/PVA sample at room temperature was at 560 nm. After decreasing the temperature down to 10 K the absorption maximum was red shifted to 567 nm. The low-energy tail of the absorption spectrum was gradually reduced due to Boltzman redistribution with decreasing the temperature. Small narrowing of absorption at the highenergy side of absorption band was also observed at 10 K. The small bands in the UV region (λ ) 350-450 nm) are due to transitions to higher electronic excited states. Figure 2 shows the difference in the absorption spectra for bR in PVA at 10 K with and without excitation light at 556 nm (curve a) and 632 nm (curve b). The irradiation dose was 10-2 J/cm2 for 556 nm excitation and 102 J/cm2 for 632 nm excitation. © 1996 American Chemical Society
Letters
Figure 1. Absorption spectrum of bR in PVA film at 300 K (curve a) and 10 K (curve b).
J. Phys. Chem., Vol. 100, No. 8, 1996 2763
Figure 3. Difference spectra measured immediately and 5 h after excitation at 632 nm.
absorption spectrum of the sample after cooling, irradiation, and subsequent heating was almost unchanged (within 5%) compared with the initial absorption spectrum (Figure 1, curve a). Discussion
Figure 2. Difference absorption spectra of bR in PVA at 10 K with excitation at 556 nm (a, solid line) and 632 nm (b, dotted line).
Irradiation time was 50 min for 556 nm. Absorption at 632 nm excitation wavelength was extremely low so that long irradiation time (540 min) was needed to create observable changes in the absorption spectrum while the low-power (4 mW) HeNe laser was used. Irradiation time dependence different spectra was measured to ensure that both spectra were taken in the low dose limit (
