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Determination of the Primary Charge Separation Rate in Isolated Proc. Nati. Acad. Sci. USA Vol. 86, pp. 524-528, January 1989 Biophysics Determination of the primary charge separation rate in isolated photosystem II reaction centers with 500-fs time resolution (electron transfer/ultrafast spectroscopy/photosynthesis/Spinacia oleracea) MICHAEL R. WASIELEWSKIt, DOUGLAS G. JOHNSONt, MICHAEL SEIBERTt, AND GOVINDJEE§ tChemistry Division, Argonne National Laboratory, Argonne, IL 60439; tPhotoconversion Research Branch, Solar Energy Research Institute, Golden, CO 80401; and §Departments of Physiology and Biophysics, and Plant Biology, University of Illinois, Urbana, IL 61801 Communicated by Joseph J. Katz, October 3, 1988 ABSTRACT We have measured directly the rate of for- Pheoa - to a subsequent quinone electron acceptor takes mation of the oxidized chlorophyll a electron donor (P680') place in 510 ps. and the reduced electron acceptor pheophytin a- (Pheoa-) Until now it has proven very difficult to measure the following excitation ofisolated spinach photosystem II reaction primary charge separation rate in PSII RCs because isolation centers at 40C. The reaction-center complex consists of DI, D2, of a purified, stable RC preparation was not possible. Nanba and cytochrome b-559 proteins and was prepared by a proce- and Satoh (14) succeeded in isolating a complex that con- dure that stabilizes the protein complex. Transient absorption tained only D1, D2, and cytochrome b-559 proteins (see also difference spectra were measured from 440 to 850 nm as a refs. 15 and 16). Since this preparation bound four or five function of time with 500-fs resolution following 610-nm laser Chla molecules, two Pheoa molecules, and a single ,8- excitation. The formation of P680'-Pheoa- is indicated by the carotene molecule, and since Pheoa in the preparation was appearance of a band due to P680' at 820 nm and correspond- reduced following illumination, these authors suggested that ing absorbance changes at 505 and 540 nm due to formation of the complex was the RC ofPSII analogous to the RC ofpurple PheoaC. The appearance of the 820-nm band is monoexponen- photosynthetic bacteria. The bacterial RC also contains four tial with T = 3.0 - 0.6 ps. The time constant for decay of bacteriochlorophyll and two bacteriopheophytin molecules l*P680, the lowest excited singlet state of P680, monitored at (17-22). However, unlike RC preparations from Rhodopseu- 650 nm, is T = 2.6 _ 0.6 ps and agrees with that of the domonas viridis and Rhodobacter sphaeroides, the PSII RC appearance of P680+ within experimental error. Treatment of preparation does not contain bound quinone acceptors and the photosystem H reaction centers with sodium dithionite and has not been crystallized for structural studies. Nevertheless, methyl viologen followed by exposure to laser excitation, Takahashi et al. (23) and Danielius et al. (24) measured a conditions known to result in accumulation of Pheoa-, results 32-ns lifetime for P680+-Pheoa- in this material. Hansson et in formation of a transient absorption spectrum due to '*P680. al. (25) reported that the P680+-Pheoa- lifetime depends on We find no evidence for an electron acceptor that precedes the the number of antenna Chla molecules attached to the PSII formation of Pheoa-. RC. Okamura et al. (26) showed that recombination of the P680+-Pheoa- radical pair in the PSII RC complex leads to The primary processes of photosynthesis commence with formation of the triplet state of P680. This triplet possesses light absorption by antenna pigment-protein complexes (1), the spin polarization characteristic of a radical-pair precur- followed by exciton migration among the pigments (2). sor. Unfortunately, the PSII RC as isolated by Nanba and Trapping of the excitons by chlorophyll a (Chla) or bacterio- Satoh is rather unstable (27) and thus no data on the kinetics chlorophyll molecules in the reaction center (RC) is followed of the formation of P680+-Pheoa- have appeared as yet. by primary charge separation, which converts the excitation In this paper we report the direct measurement of the energy into chemical potential (3). In isolated RC prepara- kinetics ofthe primary charge separation in stabilized (27, 28) tions from purple photosynthetic bacteria, primary charge PSII RCs. separation occurs in about 3 ps (4-8). However, the situation is more complex with cyanobacteria and plants because they contain two photosystems. Photosystem I (PSI) oxidizes MATERIALS AND METHODS plastoquinone and reduces nicotinamide-adenine dinucleo- PSII RC complex was prepared from spinach (Spinacia tide phosphate (NADP+), while photosystem II (PSII) oxi- oleracea) PSII appressed membrane fragments (29, 30) by a dizes water to molecular oxygen and reduces plastoquinone. modification (27, 28) of the original Nanba and Satoh proce- The primary electron donor of PSI consists of one or two dure (14). All steps were carried out at 4°C in the dark. PSII Chla molecules and is called P700 because its long- membranes (30 mg ofChl at a concentration of 1 mg/ml) were wavelength absorption maximum is at 700 nm; the primary solubilized in 4% (wt/vol) Triton X-100/50 mM Tris HCI, pH electron donor of PSII is called P680 for similar reasons (3). 7.2, for 1 hr with stirring. The material was then cen- Using 6-ps and 1.5-ps time resolution, respectively, Fenton et trifuged for 1 hr at 100,000 x g, and the resultant superna- al. (9) and Wasielewski et al. (10) have shown that PSI tant was loaded onto a 15 x 1.6-cm column of Fractogel TSK preparations enriched in P700 (1 P700 per 40 Chl molecules) DEAE-650S (EM Science, Gibbstown, NJ, but now sold by undergo primary charge separation in about 14 ps. Nuijs et al. Supelco, Bellefonte, PA, under the name TSK-GEL Toyo- (11, 12) have suggested that electron transfer from P680 to pearl DEAE-650S) preequilibrated with 50 mM Tris HCI, pH pheophytin a (Pheoa) in PSII preparations enriched in P680 7.2/30 mM NaCI/0.05% Triton X-100. The column was then (1 P680 per 80 Chl) occurs in <35 ps. Schatz et al. (13), using washed with the same buffer until the eluent was colorless, a preparation containing 1 P680 per 80 Chl from Synecho- and the RC fraction was eluted with a 30-200 mM NaCl coccus, have estimated that the electron transfer from gradient in 50 mM Tris HCI, pH 7.2/0.05% Triton X-100. At The publication costs of this article were defrayed in part by page charge Abbreviations: Chl, chlorophyll; PS11, photosystem II; RC, reaction payment. This article must therefore be hereby marked "advertisement" center; P680; primary electron donor of PSII; *P680, lowest excited in accordance with 18 U.S.C. §1734 solely to indicate this fact. singlet state of P680; Pheoa, pheophytin a. 524 Downloaded by guest on September 25, 2021 Biophysics: Wasielewski et al. Proc. Natl. Acad. Sci. USA 86 (1989) 525 this point the RC fraction was concentrated by precipitation 1.2- with polyethylene glycol, which also serves to stabilize the material (27, 28). Polyethylene glycol (32.5% wt/vol; Mr = 3,350) was added slowly and dispersed with a soft paintbrush. 1.0 After 90 min of incubation, the suspension was centrifuged at 31,300 x g for 15 min. The pellet was resuspended in 50 mM 0.8- Tris HCl (pH 7.2) with no detergent and then centrifuged at 1100 x g for 90 sec to pellet mostly colorless material E- 0.6- containing polyethylene glycol aggregates. The supernatant C RC material was stored at -80'C until use. Photochemical competence of the RCs was assayed by photoinduced elec- 0.4- tron transport from diphenylcarbazide to silicomolybdate at 40C (28) and by observation ofthe wavelength ofthe red-most optical absorption band at 674 nm. The photochemical 0.2- activity of this preparation ranges from 1350 to 2250 ,umol of silicomolybdate reduced per mg of Chl per hr. As the RCs 0.0 degrade, the 674-nm band shifts to 669 nm (27). Measure- 350 450 550 650 750 ments made before and after laser excitation showed that the Wavelength, nm absorption maximum of the sample was always between 672 nm and 674 nm. FIG. 1. Ground-state absorption spectrum of PSH1 RCs used in RC samples for picosecond spectroscopy (117 ,tg of Chl the transient absorption experiments. per ml) were thawed in dim light and 0.04% Triton X-100 The transient absorption difference spectrum obtained 10 (final concentration) was added immediately to keep the ps following a 500-fs laser flash at 610 nm [Fig. 2, spectrum material from aggregating (28). The samples were then A (solid trace)] shows a strong bleaching at 674 nm and a transferred to a nitrogen atmosphere and the following were positive absorption starting near 740 nm and extending past added (final concentration) in the indicated order: glucose (20 850 nm. Since P680 absorbs near 674 nm in detergent-treated mM), catalase (0.039 mg/ml), and glucose oxidase (0.1 mg/ PSII membranes (32) and Pheoa absorbs close to 682 nm in ml) (28). After mixing, the samples were transferred anaer- PSII RCs (14), the observed red bleach should be a convo- obically or aerobically to a sealed cuvette for spectroscopy at lution of the bleaches due to P680 and Pheoa. The fact that 4°C. Under these conditions the samples did not aggregate the observed bleach occurs at 674 nm could be due to a larger and remained active for the duration of the experiment. change in molar extinction coefficient (Ae) for P680 -- P680' Subpicosecond time-resolved transient absorption mea- compared to Pheoa -* Pheoa-, electrochromic effects, and/ surements were obtained with the laser apparatus described or the presence of 0.04% Triton X-100 required to disaggre- elsewhere (10).
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