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J. Phys. Chem. 1996, 100, 5562-5569
Level Mixing and Energy Redistribution in Bacterial Photosynthetic Reaction Centers Gilad Haran,† Klaas Wynne,† Chris C. Moser,‡ P. Leslie Dutton,‡ and Robin M. Hochstrasser*,† Department of Chemistry and the Johnson Research Foundation, UniVersity of PennsylVania, Philadelphia, PennsylVania 19104-6323 ReceiVed: October 2, 1995; In Final Form: December 19, 1995X
We performed ultrafast polarized light experiments in which we pumped the 800 nm band and probed several near- and mid-IR transitions of photosynthetic reaction centers from Rhodobacter sphaeroides. Absorption into the upper excitonic level of the special pair (PY+) is part of this band, but it is not known whether PY+ behaves as a localized state or if it mixes with accessory bacteriochlorophyll (BChl) states. A calculation of the anisotropy of pump-probe signals fails to reproduce the experimental results if the localized picture is used. In fact, the transition into PY+ has to be 4-fold intensified over a simple exciton model prediction in order to give rise to anisotropies which are consistent with the experiment. This substantial intensification is inconsistent with previous experimental results. Agreement between theory and experiment can be achieved if the PY+ state is mixed with excited states of accessory BChl. Stimulated emission from the lower excitonic level of the special pair (PY-), probed at 950 nm after pumping at 800 nm, does not appear instantaneously, but rises with a time constant of 110 fs. A novel excited state absorption of the accessory BChls at 1200 nm, assigned as a monomer transition by comparison with a pump-probe experiment on free BChl dissolved in acetone, also decays with a ∼100 fs time constant. Although Fo¨rster energy transfer from accessory Bchl states to PY- can account for the fast transients, under the delocalized state picture suggested in this paper they would rather correspond to an internal conversion process from the mixed states to the PY- state.
1. Introduction Bacterial photosynthetic reaction centers (RCs) contain eight cofactors, arranged in a well defined, close to C2-symmetric structure within a protein matrix.1-3 The RC from Rhodobacter (Rb.) sphaeroides, which is the subject of the present study, contains four bacteriochlorophyll-a (BChl) molecules, two bacteriopheophytin-a (H) molecules, and two quinones (Q).1,3 The proximity of these cofactors led to “supermolecule” descriptions of the excited states of the RC, in which the interchromophoric coupling leads to mixed states.4-9 The strong coupling of the two bacteriochlorophylls (known as PM and PL) forming the “special pair” through excitonic and charge resonance interactions is well documented.4,6 However, the extent of coupling to the other cofactors is not established. The only published electronic structure calculation for the RC of Rb. sphaeroides8 assigns the absorption band at 860 nm to a transition into the lowest energy excitonic state of the special pair, which is an antisymmetric combination of the first excited states of the BChls, and is therefore designated PY-. It is well documented8,10-12 that this state is mixed with charge transfer states. These states should not contribute to the absorption strength of the 860 nm band, since the ground state is not considered to contain any charge transfer component. Mixing of the higher energy excitonic component (the symmetric combination of the first excited states of the BChls, PY+) with accessory BChl states8 is believed to lead to several transitions forming the band around 800 nm. To obtain more information on the composition of the 800 nm band and the ensuing dynamics, we performed ultrafast pump-probe spectroscopy on Rb. sphaeroides RC samples. Exciting the RC at 810 nm with a 40-60 fs laser pulse, we probed the dynamics using polarized laser pulses at 950 nm †
Department of Chemistry. Johnson Research Foundation. X Abstract published in AdVance ACS Abstracts, March 1, 1996. ‡
0022-3654/96/20100-5562$12.00/0
(where stimulated emission from PY- could be detected), at 1200 nm (where a novel transient absorption signal was observed), and at 3840 nm (where absorption into the band described by Walker et al.13 and Wynne et al.31 was detected). Using the anisotropy values of the transients measured at these wavelengths, we deduce below that PY+ has a significant role in processes initiated by absorption in the 800 nm region. A 100 fs rise in the stimulated emission signal at 950 nm and an equivalent decay of the transient absorption at 1200 nm were detected. We discuss here the origins of these ultrafast transients and suggest that they can arise from an internal conversion process between a mixed accessory BChl- PY+ state and the PY- state. 2. Materials and Methods Preparation of Reaction Center Protein. RCs were isolated from photosynthetically grown Rb. sphaeroides strain R-26 by the method of Clayton and Wang,14 using the detergent lauryldimethylamine oxide to solubilize the photosynthetic membranes. RCs where further purified by FPLC (Pharmacia) to an absorbance ratio between 280 and 802 nm of 1.2-1.4. The final RC solution (at a concentration of 0.1 mM) was prepared in 10 mM Tris buffer (pH 8), 0.04% lauryldimethylamine oxide, and 1 mM DTT. The latter was added in order to reduce quinone A (QA) of the RC, thus decreasing the “recycling time” into the nanosecond regime. RC samples were placed between calcium fluoride windows in a 250 µm thick cell, which was spun and translated vertically fast enough to enable replacement of the sample volume exposed to the laser beam between consecutive laser shots. An absorption spectrum of the Rb. sphaeroides RC sample is shown in Figure 1. Bacteriochlorophyll-a (BChl-a) was extracted with methanol and acetone from the lyophilized cells of the carotenoid-free mutant R-26 of Rb. sphaeroides and purified chromatographically on DEAE-Sepharose CL-6B. This method followed © 1996 American Chemical Society
Level Mixing in Bacterial Photosynthetic RCs
J. Phys. Chem., Vol. 100, No. 13, 1996 5563 TABLE 1: Anisotropy Values Obtained from Fits to Experimental Data with Pump Wavelength of 810 nm probe wavelength (nm)
probed transition
anisotropy
PYP+HPYP+HPYP+H-
0.11 ( 0.01 0.03 ( 0.01 0.10 ( 0.01 0.12 ( 0.01 0.05 ( 0.01 0.02 ( 0.04
950 1215 3840 Figure 1. Near-IR absorption spectrum of the photosynthetic reaction center from Rb. sphaeroides. Dashed line: spectrum of the 50 fs pulse used to excite the reaction centers in the pump/probe experiments. The pulse almost completely overlaps the 800 nm band of the reaction center, but also excites a fraction of the 860 nm band.
Scherz and Parson15 using Rb. sphaeroides R-26 rather than Rhodospirillum rubrum. The procedure was performed quickly in the dark, with intermediate and final material held under an argon atmosphere to minimize the potential for irreversible lightinduced oxidation of the BChl. Spectra confirmed the purity of the bacteriochlorophyll before storage in liquid nitrogen. BChl-a was dissolved in acetone for the time-resolved measurements. Femtosecond Spectroscopy. Pulses (40-60 fs, 100 µJ) at 810 nm (at a repetition rate of 4.3 kHz) were produced by a home-built chirped-pulse Ti-sapphire amplifier similar to that described by Wynne et al.16 The amplifier was seeded by 20 fs pulses from a Ti-sapphire oscillator17 and pumped by a Quantronix intracavity-doubled Q-switched Nd:YLF laser. Half of the amplified beam was sent through a variable delay line, attenuated to 100 nJ using a polarizer, and then used to pump the sample. A half-wave plate in the pump beam was used to change its polarization relative to the probe beam. To generate probe wavelengths of 950 and 1200 nm, the other half of the amplified beam was focused on a 2 mm sapphire window to generate white light. A portion of the white light was selected with a band-pass interference filter (Spectrogon or Corion), sent through a polarizer, and used as a probe. To generate probe wavelengths around 3.8 µm, an optical parametric amplifier was used. In this device, which will be described in detail in a forthcoming publication,18 single-filament white light generated in a 2 mm sapphire window was focused collinearly with a strong (30-40 µJ) 810 nm beam on a potassium titanylphosphate (KTP) crystal. Using type II phase matching, we were able to produce 50-100 nJ of mid-IR light from this device, tunable from 1.5 to 4.2 µm. The pulse width of the pump and probe pulses was determined from the rise time of the signal produced by free-carrier absorption in a thin silicon wafer. For both near-IR probe wavelengths used (see the Results section) the pulse widths were
