J. Phys. Chem. 1996, 100, 1945-1949
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Transient Absorption Studies of the Primary Charge Separation in Photosystem II Brent Donovan, Larry A. Walker II, Charles F. Yocum, and Roseanne J. Sension* Department of Chemistry, UniVersity of Michigan, Ann Arbor, Michigan 48109 ReceiVed: July 17, 1995; In Final Form: October 20, 1995X
Femtosecond transient absorption studies of the primary charge separation in photosystem II (PSII) are presented. A careful study of the dependence of the observed signal on laser intensity demonstrates that the multiple excitation of reaction centers produces additional fast components not observed at low excitation energy. In the regime where the observed signals are linear with excitation energy, a 20 ( 2 ps rise of the pheophytin anion absorption, bleach of the pheophytin Qx absorption, and appearance of the chlorophyll cation absorption are observed. Three different protocols, involving varying exposure of the PSII complex to the detergent Triton X-100, are used to prepare D1-D2-Cyt b559 complexes from spinach. The kinetic signals are independent of the method of sample preparation.
Introduction The initial charge transfer and energy transfer processes in photosynthetic systems have excited a great deal of experimental and theoretical effort in recent years. Most of the studies have been concerned with charge separation in the photosynthetic reaction center of purple bacteria, most notably Rhodobacter spheroides and Rhodopseudomonus Viridis. However, an increasing amount of this effort has been directed toward an investigation of primary charge separation in the related photosystem II (PSII) reaction center of green plants. Several picosecond1-4 and femtosecond5-13 transient absorption or fluorescence studies have been performed to directly investigate the primary charge separation event in photosystem II. As a result of these measurements and subsequent interpretations, a controversy has developed with regard to the time constant for the primary charge separation process. Klug and co-workers have published a series of papers in support of a ca. 20 ps effectiVe time constant for the appearance of the charge-separated state.7,8,10,11,13 Their conclusions are supported by the experiments of McCauley et al.9 and Freiberg et al.2 A ca. 20 ps effective time constant is consistent with a trap-limited model for charge separation in PSII. On the other hand, several groups have published data interpreted as supporting a ca. 3 ps time constant for the primary charge separation.1,3-6,12 A 3 ps time constant is appealing for several reasons: (1) it is consistent with low-temperature holeburning experiments;14-15 (2) it is consistent with early predictions based on kinetic models and picosecond fluorescence experiments;16 (3) it is in agreement with the well-established 2.5-3.5 ps time constant for primary charge separation in the reaction center of purple bacteria.17,18 An important aspect of this controversy is that the femtosecond spectroscopic measurements of the only two groups to have performed systematic subpicosecond studies of charge separation in PSII are in disagreement with each other.12,13 Both groups have observed signals with a wide range of time constants, although the fast and slow components have differing amplitudes in the two sets of measurments. A fast ca. 3 ps is dominant in one set, while a slower ca. 21 ps component is dominant in the other set. In light of this disagreement we have performed a series of subpicosecond transient absorption measurments on the reaction center of PSII. In these studies, X
Abstract published in AdVance ACS Abstracts, January 1, 1996.
0022-3654/96/20100-1945$12.00/0
we have concentrated on the effect of sample preparation and laser intensity on the observed transient absorption signals. The most interesting finding is the sensitivity of the observed transient absorption signals to laser intensity. A variety of fast components, unobserved at low laser intensities, become prominent at higher laser intensities. At low excitation energies the signals observed are most consistent with a 20 ( 2 ps effective time constant for charge separation. The method of sample preparation has little or no effect on the signals measured. Experimental Section Femtosecond transient absorption measurements have been performed as described elsewhere.19 Briefly, a regeneratively amplified Ti:sapphire laser system is used to produce ca. 150 fs pulses at 800 nm with a 1 kHz repetition rate. The beam is split, and half of the beam is frequency doubled in a β-barium borate nonlinear optical crystal. The intensity of this beam is attenuated using neutral density filters to provide the 400 nm excitation pulse. In experiments where the pump intensity is varied, 4% of the pump beam is split off and monitored with a photodiode to determine the relative intensities. The highest pump intensity used is 5 ns) and a component having a lifetime of ca. 20 ( 2 ps. For the data obtained with a probe wavelength of 690 nm it was also necessary to include a fast exponential decay of ca. 2-3 ps. For all other probe wavelengths the only time-varying component had a characteristic time constant of ca. 20 ( 2 ps. At all wavelengths, the data presented here are consistent with those reported by Klug and co-workers.7,8,10,11,13 The absence of any ultrafast ( 5 ns) and a component having a lifetime of ca. 20 ( 2 ps. Although we cannot rule out the presence of a 3 ps component of small amplitude, inclusion of such a component is not required by the data.
out the presence of a 3 ps component of small amplitude, inclusion of such a component is not required by the data. The dominant observed time constant in these key spectral regions is ca. 20 ps rather than ca. 3 ps, as reported in ref 13. The only positive evidence for a 3 ps charge separation in the present series of measurements is obtained at 690 nm. The fast 2-3 ps component is observed by several groups in this wavelength region.3,4,13 This fast component may be significant, as stimulated emission from P680 should make its largest contribution to the observed signal in this spectral region. However, this kinetic component can also be attributed to energy relaxation processes. If the dominant effective time constant for the formation of the charge-separated state was ca. 3 ps, this time constant would be expected to be well resolved at both 544 and 730 nm as well as 690 nm. It is postulated as a result of several femtosecond and picosecond studies, that rapid energy equilibration (τ < 300 fs) occurs between P680, two accessory chlorophyll molecules, and two pheophytin molecules.4,10 Much slower energy transfer is observed between the remaining two accessory chlorophyll molecules and P680 (ca. 20-30 ps).3,4 It is remotely possible that these two accessory chlorophyll molecules are selectively excited in our experiments, although the breadth and intensity of the chlorophyll and pheophytin absorption bands around 400 nm make any selective excitation extremely unlikely. In this
case, the dominance of a 20 ( 2 ps time constant could reflect slow energy transfer from the distant chlorophyll to the trap, rather than the inherent effective rate for primary charge separation. However, the spectral arguments above and the agreement between the transient absorption signals reported in this paper and those reported by Klug et al. for selective excitation of P680 at 694 nm make this hypothesis implausible.13 The Effect of Sample Preparation. Several different methods of sample preparation are currently used to produce the D1-D2-Cyt b559 complex commonly used for femtosecond and picosecond studies of charge separation in PSII. We have specifically compared three methods of sample preparation in this study as described above. Prep A is a modification of the initial method of Nanba and Satoh21 with Triton X-100 replaced by a milder detergent after the initial extraction and chromatography steps. Prep B also uses Triton X-100 to remove the antennae proteins. Prep C avoids the use of Triton X-100 in the extraction of the D1-D2-Cyt b559 complex. Data were obtained for all three of these preparations at 650, 680, and 690 nm. Data were also obtained for preps B and C at 544 nm and for preps A and C at 730 nm. No differences were observed for the three preparations at 650, 680, or 690 nm. The data obtained at 544 and 730 nm are shown in Figure 5. While the initial gain at 730 nm is not seen with prep C, the time constant for the appearance of the persistent ion-pair absorption band is ca. 21 ps for both samples. The signals observed at 544 nm are also similar. The relative magnitudes of the instantaneous absorption and the final bleach vary slightly, but, within experimental error, the time scale for the appearance of the bleach is unaffected by the sample preparation protocol. Although the kinetic signals are essentially unaffected by the sample preparation protocol, the samples are not identical. The protocol for prep C consistently provides samples of higher concentration and better activity. As shown in Figure 5, the signals obtained from these samples are consistently stronger for the same pump intensity and nominal sample concentration (as determined by the absorbance of the Qy band). The samples prepared by protocol A were consistently less stable, more prone to laser damage, and had to be replaced on average much more often than samples prepared according to the other two protocols.
Primary Charge Separation in Photosystem II Conclusions The transient absorption measurements presented in this paper are most consistent with the results presented by Klug and coworkers,13 rather than with those reported by Wiederrecht et al.12 The dependence of the observed signal on laser intensity indicates that the results in ref 12 were probably obtained with laser intensities resulting in multiple excitations in individual reaction centers. The data presented in this paper do not independently establish either a 3 or 21 ps effective time constant for primary charge separation in PSII. Taken in context with the more extensive results presented in ref 13, the present measurements do provide strong support for an effective 21 ps time constant for charge separation. This effective time constant is obtained from both the decay of the pheophytin Qx absorption band and the appearance of absorption characteristics of the ion-pair state. However, this 21 ps effective time constant should not be construed as a first-order time constant for electron transfer. The primary processes in PSII appear too complicated for such a simplistic interpretation of the data. Acknowledgment. Support for this research was provided by a grant from the National Science Foundation (MCB9418390) and the Cindy Yoder Research Award of the University of Michigan to R.J.S. C.F.Y. was supported by NSF (MCB-9314173) and USDA-NCGRIP-92-37306-7662. We would also like to acknowledge the assistance of the Center for Ultrafast Optical Science (NSF PHY-9319017) in the construction of our femtosecond laser system. References and Notes (1) Roelofs, T. A.; Kwa, S. L. S.; van Grondelle, R.; Dekker, J. P.; Holzwarth, A. R. Biochim. Biophys. Acta 1993, 1143, 147. (2) Freiberg, A.; Timpmann, K.; Moskalenko, A. A.; Kuznetsova, N. Y. Biochim. Biophys. Acta 1994, 1184, 45. (3) Schelvis, J. P. M.; van Noort, P. I.; Aartsma, T. J.; van Gorkom, H. J. Biochim. Biophys. Acta 1994, 1184, 242. (4) Holzwarth, A.; Mu¨ller, M. G.; Gatzen, G.; Hucke, G. M.; Griebenow, K. J. Lumin. 1994, 60&61, 497. (5) Wasielewski, M. R.; Johnson, D. G.; Seibert, M.; Govindjee. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 524. (6) Wasielewski, M. R.; Johnson, D. G.; Govindjee; Preston, C.; Seibert, M. Photosynth. Res. 1989, 22, 89. (7) Durrant, J. R.; Hastings, G. R.; Hong, Q.; Barber, J.; Porter, G.; Klug, D. R. Chem. Phys. Lett. 1992, 188, 54. (8) Hastings, G.; Durrant, J. R.; Barber, J.; Porter, G.; Klug, D. R.; Biochemistry 1992, 31, 7638.
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