Determination of the primary charge separation rate in isolated photosystem II reaction centers with 500-fs time resolution

Abstract

We have measured directly the rate of formation of the oxidized chlorophyll a electron donor (P680') and the reduced electron acceptor pheophytin a-(Pheoa-) following excitation of isolated spinach photosystem II reaction centers at 40C. The reaction-center complex consists of DI, D2, and cytochrome b-559 proteins and was prepared by a procedure that stabilizes the protein complex. Transient absorption difference spectra were measured from 440 to 850 nm as a function of time with 500-fs resolution following 610-nm laser excitation. The formation of P680'-Pheoa-is indicated by the appearance of a band due to P680' at 820 nm and corresponding absorbance changes at 505 and 540 nm due to formation of PheoaC. The appearance of the 820-nm band is monoexponential with T = 3.0 -0.6 ps. The time constant for decay of l*P680, the lowest excited singlet state of P680, monitored at 650 nm, is T = 2.6 _ 0.6 ps and agrees with that of the appearance of P680+ within experimental error. Treatment of the photosystem H reaction centers with sodium dithionite and methyl viologen followed by exposure to laser excitation, conditions known to result in accumulation of Pheoa-, results in formation of a transient absorption spectrum due to '*P680. We find no evidence for an electron acceptor that precedes the formation of Pheoa-.The primary processes of photosynthesis commence with light absorption by antenna pigment-protein complexes (1), followed by exciton migration among the pigments (2). Trapping of the excitons by chlorophyll a (Chla) or bacteriochlorophyll molecules in the reaction center (RC) is followed by primary charge separation, which converts the excitation energy into chemical potential (3). In isolated RC preparations from purple photosynthetic bacteria, primary charge separation occurs in about 3 ps (4-8 (25) reported that the P680+-Pheoa-lifetime depends on the number of antenna Chla molecules attached to the PSII RC. Okamura et al. (26) showed that recombination of the P680+-Pheoa-radical pair in the PSII RC complex leads to formation of the triplet state of P680. This triplet possesses the spin polarization characteristic of a radical-pair precursor. Unfortunately, the PSII RC as isolated by Nanba and Satoh is rather unstable (27) and thus no data on the kinetics of the formation of P680+-Pheoa-have appeared as yet.In this paper we report the direct measurement of the kinetics of the primary charge separation in stabilized (27, (27,28) of the original Nanba and Satoh procedure (14). All steps were carried out at 4°C in the dark. PSII membranes (30 mg of Chl at a concentration of 1 mg/ml) were solubilized in 4% (wt/vol) Triton X-100/50 mM Tris HCI, pH 7.2, for 1 hr with stirring. The material was then centrifuged for 1 hr at 100,000 x g, and the resultant supernatant was loaded onto a 15 x 1.6-cm column of Fractogel TSK DEAE-650S (EM Science, Gibbstown, NJ, but now sold by Supelco, Bellefonte, PA, under the name TSK-GEL Toyopearl DEAE-650S) preequilibrated with 50 mM Tris HCI, pH 7.2/30 mM NaCI/0.05% ...

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.…”

“…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.…”

Similarity of primary radical pair recombination in photosystem II and bacterial reaction centers

“…An estimate of dG('P680*-P680'Pheo-) can be obtained from the following argument: the free energy of P680'Pheo-necessarily is below that of 'P680*, as can be concluded from the fast charge separation at high and low temperatures [4,5,36] which only is possible for an electron transfer rate near the activationless case, implying a negative free energy gap dG('P*-PCH-). Therefore, the backward electron transfer P68O+Pheo--+ 'P680* with the rate k_, is activated and decreases upon lowering the temperature.…”

“…These D,D,-preparations contain 4-6 chlorophylls a (Chl), 2 pheophytins a (Pheo), 1 p-carotene, but no quinones [3]. Electron transfer from the primary donor P680 to a Pheo was reported to occur in a few picoseconds even at low temperatures [4,5], although measurements with better time resolution point to a slightly slower charge separation preceded by energy transfer processes [6]. P680'Pheo-decays on the nanosecond timescale [7-91 by recombination via the radical pair mechanism, as can be concluded from the magnetic field dependence of the recombination [9] and from the spin polarization of the triplet state 3P680* pointing to a radical pair precursor [lo].…”