Femtosecond photodichroism studies of isolated photosystem II reaction centers.

Abstract

Photosynthetic conversion oflight energy into chemical potential begins in reaction center protein complexes, where rapid charge separation occurs with nearly unit quantum efficiency. Primary charge separation was studied in isolated photosystem H reaction centers from spinach containing 6 chlorophyll a, 2 pheophytin a (Pheo), 1 cytochrome b559, and 2 f-carotene molecules. Time-resolved pump-probe kinetic spectroscopy was carried out with 105-fs time resolution and with the pump laser polarized parallel, perpendicular, and at the magic angle (54.70) relative to the polarized probe beam. The time evolution of the transient absorption changes due to the formation of the oxidized primary electron donor P680+ and the reduced primary electron acceptor Pheo-were measured at 820 nm and 545 nm, respectively. In addition, kinetics were obtained at 680 nm, the wavelength ascribed to the Q transition of the primary electron donor P680 in the reaction center. At each measured probe wavelength the kinetics of the transient absorption changes can be fit to two major kinetic components. The relative amplitudes of these components are strongly dependent on the polarization of the pump beam relative to that of the probe. At the magic angle, where no photoselection occurs, the amplitude of the 3-ps component, which is indicative of the charge separation, dominates. When the primary electron acceptor Pheo is reduced prior to P680 excitation, the 3-ps component is eliminated.The detailed mechanism of primary charge separation in isolated reaction center (RC) complexes from photosynthetic organisms is an important topic of current interest (1-4). Femtosecond transient absorption spectroscopy has been used to probe a variety of issues regarding primary charge separation in the RCs of anoxygenic bacteria. It is of considerable interest to obtain the analogous data for photosystem II (PSII) from algae and higher plants because of the similarities between the bacterial and PSII RCs (5, 6). The first reported isolation of the PSII RC, the D1-D2-cytochrome b559 (cyt-b559) protein complex, from higher plants by Nanba and Satoh (5) yielded a complex containing 4-5 chlorophyll a (Chl) molecules (6-8). Subsequent work (9, 10) demonstrated that the RC complex, as originally isolated, was quite unstable, and thus of limited use in many spectroscopic studies. Fortunately, simple modifications to the Nanba and Satoh procedure, such as substituting the detergent dodecyl P-maltoside (DM) for Triton X-100 (9) and adding an active enzymatic 02-scrubbing system in situ (10), improve the stability of the PSII RC under prolonged illumination. The use of DM does not alter the spectroscopic properties of the RC as does Triton X-100 (11-13). Other changes in the isolation procedure have led to stable RCs that contain 6 Chl, 2 pheophytin a (Pheo), 1 cyt-b559, and 2 3-carotene molecules per RC (14-17).Carrying out detailed spectroscopic studies of PSII RCs is difficult because at room temperature the Q bands of all the chlorophylls and pheophytins wi...

Wavelength and intensity dependent primary photochemistry of isolated Photosystem II reaction centers at 5°C

“…The polarization purity of both the pump and probe beams was very high, as both beams pass through calcite polarizers before reaching the sample. Like all of the previous work on PSII RCs done in our laboratory [18,25,31,35], the probe beam was polarized at the magic angle (54.7 ° ) with respect to the pump beam. This polarization configuration, unlike having the two beams with parallel or perpendicular polarizations, allows direct observation of population kinetics without artifacts due to polarization phenomena.…”

“…Pheo a) and charge separation can be monitored at the Pheo a Qx band at ~ 544 nm [18,28,[30][31][32][33]. While the data from this feature are complicated by the presence of bleaching due to 1 • Pheo a (from both direct excitation of and excitation energy transfer to Pheo a), it is still probably the best indicator of charge separation that has been found so far.…”

“…Much of the work has been done probing the composite Q>. band itself [15][16][17][25][26][27][28][31][32][33][34]. Because of the spectral congestion of the six Chl a and two Pheo a pigments in this band, it is difficult to use bleaching as an indicator of ground state depopulation of a specific pigment or group thereof.…”

“…Near room temperature, the RC kinetics have been studied by both time resolved Chl a fluorescence [22][23][24] and transient absorption techniques [15][16][17][18][25][26][27][28][29][30][31][32][33]. While the time-resolved fluorescence measurements are hindered by the presence of processes occurring faster than the time resolution of the instrument, the transient absorption measurements are hampered by the lack of unambiguous indicators differentiating excitation energy transfer from charge separation.…”

Charge separation in the reaction center of photosystem II studied as a function of temperature

“…A straightforward interpretation of the results from pump-probe measurements in terms of pigment to pigment energy transfer and charge separation rates has proven difficult, mainly due to the congested absorption spectrum of the PSII RC. At room temperature (RT), most groups observed kinetics with lifetimes of 1-3 ps, 10-20 ps and Ͼ1 ns (14)(15)(16)(17)(18)(19)(20) as well as 100-600 fs (21)(22)(23)(24)(25)(26)(27). At very low temperature (4 K) the lifetime of singlet-excited P680 (P680*) was estimated to be 1.9 ps by transient hole-burning spectroscopy (28).…”