Biochemistry 1985, 24, 3035-3043
3035
Active and Resting States of the 02-Evolving Complex of Photosystem IIt Warren F. Beck, Julio C. de Paula, and Gary W. Brudvig* Department of Chemistry, Yale University, New Haven, Connecticut 0651 1 Received May 1 1 , 1984; Revised Manuscript Received December 28, 1984
ABSTRACT: During dark adaptation, a change in the 02-evolving complex (OEC) of spinach photosystem
I1 (PSII) occurs that affects both the structure of the M n site and the chemical properties of the OEC, as determined from low-temperature electron paramagnetic resonance (EPR) spectroscopy and O2 measurements. The S2-state multiline E P R signal, arising from a Mn-containing species in the OEC, exhibits different properties in long-term (4 h a t 0 "C) and short-term (6 min at 0 "C) dark-adapted PSII membranes or thylakoids. The optimal temperature for producing this E P R signal in long-term dark-adapted samples is 200 K compared to 170 K for short-term dark-adapted samples. However, in short-term dark-adapted samples, illumination a t 170 K produces an E P R signal with a different hyperfine structure and a wider field range than does illumination a t 160 K or below. In contrast, the line shape of the Sz-state EPR signal produced in long-term dark-adapted samples is independent of the illumination temperature. The EPRdetected change in the M n site of the O E C that occurs during dark adaptation is correlated with a change in O2consumption activity of PSII or thylakoid membranes. PSII membranes and thylakoid membranes slowly consume O2following illumination, but only when a functional OEC and excess reductant are present. W e assign this slow consumption of O2to a catalytic reduction of O2 by the O E C in the dark. The rate of O2consumption decreases during dark adaptation; long-term dark-adapted PSII or thylakoid membranes do not consume O2despite the presence of excess reductant. The EPR-detected change in the M n site of the O E C and the decline of the O2consumption activity observed in PSII or thylakoid membranes occur with the same time constant. It is proposed that a structural change in the Mn site of the OEC occurs during dark adaptation, changing the electron-transport properties of the donor side of PSII and causing a conversion from an active, 02-consuming state to a resting state incapable of O2 consumption.
x e 0,-evolving complex (OEC)' of photosystem I1 (PSII) catalyzes the four-electron oxidation of water to 02.In the S-state model of Kok and co-workers (Kok et al., 1970; Forbush et al., 1971), successive light-induced charge separations in the PSII reaction center advance the OEC through five oxidation states, Si, i = 0-4, with the release of an O2 molecule being accompanied by the rapid conversion of the S4 state to the lowest oxidation state, So. Dark adaptation causes the S2and S3states to decay to the S, state, producing a distribution of 75% SI and 25% So shortly after continuous illumination. In the S-state model of Kok et al., it is assumed that the So and SI states are stable in order to explain the characteristic oscillation with a period of four of the 0, yields from a series of short flashes of light (Joliot & Kok, 1975). Velthuys & Visser (1975) and, more recently, Vermaas et al. (1984) have shown that the So/S1 ratio depends on the length of dark adaptation; the calculated So concentration is nearly zero in extensively dark-adapted samples. Apparently, the PSII OEC undergoes conversions during the dark-adaptation process that stabilize the S1 state relative to the other S states. However, the mechanism of this conversion is not understood. There have been numerous reports of changes in the properties of the PSII OEC related to the extent of dark adaptation. More double hits (advances of two S states during a single flash of light) are observed in flash-induced 0, yield experiments employing long-term dark-adapted samples than in Supported by the U.S. Department of Agriculture (83-CRCR-11318), the National Institutes of Health (1-ROl-GM32715-01), Research Corporation, the donors of the Petroleum Research Fund, administered by the American Chemical Society, the Camille and Henry Dreyfus Foundation, the Chicago Community Trust/Searle Scholars Program, and a NSF fellowship to W.F.B.
0006-2960/85/0424-3035$01.50/0
experiments using briefly dark-adapted samples [see Wydryzynski (1982) and references cited therein' Also, extensive dark adaptation decreases the number of protL.,s released from the OEC after the initial flash of light (Forster et al., 1981). Further, the amplitude of the 50 "C thermoluminescence band in chloroplasts, which is related to charge recombination from the So and S1 states, is strongly dependent on the length of dark adaptation, decreasing exponentially with a tl12of 10 min at 25 "C (Demeter et al., 1984). Each of these results could be accounted for if the PSII OEC undergoes a structural reorganization during dark adaptation, affecting the properties of the So and S1 states. However, the molecular details of the reorganization are not revealed by any of the techniques discussed above. In this work, we consider the nature of the change in the PSII OEC that occurs during dark adaptation. To probe the structural aspects of the change, we use low-temperature electron paramagnetic resonance (EPR) spectroscopy of the Mn site. A variety of studies [reviewed by Amesz (1983)l strongly suggest that Mn ions play a crucial role in the mechanism of photosynthetic O2 evolution. Dismukes & Siderer (1981) showed that a multiline EPR signal from a metal ion cluster containing Mn can be produced in spinach thylakoids frozen quickly following a single laser flash and
'
Abbreviations: chl, chlorophyll; DCBQ, 2,s-dichloro-p-benzoquinone: DCMU, 3-(3,4-dichlorophenyl)-I,I-dimethylurea; EDTA, ethylenediaminetetraacetic acid; EPR, electron paramagnetic resonance; HEPES, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid: OEC, 02-evolving complex: PSI, photosystem I; PSII, photosystem 11; P680, primary electron donor in PSII; QA. primary electron acceptor in PSII; Qe, secondary electron acceptor in PSII; TPB, sodium tetraphenylborate: Tris, tris(hydroxymethy1)aminomethane.
0 1985 American Chemical Society
3036
BIOCHEMISTRY
proposed that this EPR signal arises from the S2state. This proposal was confirmed by Brudvig et al. (1983a), who also showed that the S2-state EPR signal can be produced by continuous illumination at 200 K of dark-adapted spinach thylakoids. We show in this paper that the intensity and hyperfine structure of the Sz-state EPR signals produced by low-temperature illumination of PSII membranes or thylakoids are strongly dependent on the length of dark adaptation, indicating that the environment of the Mn center of the OEC changes during dark adaptation. We have also examined the effect of dark adaptation on the 0, consumption behavior of PSII membranes and thylakoids. Sayre & Homann (1979) have reported that chloroplasts consume 0,immediately after illumination for a period of a few minutes. We report that following this rapid phase of O2 consumtion a slower, exponentially decaying phase of Oz consumption occurs during dark adaptation. The decay of the slow phase of O2 consumption and the change in the EPR properties of the Mn site occur synchronously,suggesting that these two phenomena are related. On the basis of these results, it is suggested that during dark adaptation the PSII OEC undergoes a change from an active, O,-consuming state to a resting state incapable of 0, consumption and that this process involves a structural change in the Mn site of the OEC. EXPERIMENTAL PROCEDURES Thylakoids were isolated from 2-h dark-adapted market spinach leaves by the high-salt/EDTA procedure of Yocum et al. (1981) and were suspended for storage at 77 K at approximately 5 mg of chl/mL in the storage buffer, containing 20 mM MES-NaOH, pH 6.0, 15 mM NaCl, and 30% (v/v) ethylene glycol. PSII membranes were prepared by the procedure of Berthold et al. (1981), as modified by Sandusky et al. (1983), except that 5 mM EDTA and 1 g/L bovine serum albumin (Sigme fraction V powder) were added to all buffers used. All steps in the procedure were done in darkness. PSII membranes were suspended for storage at 77 K at approximately 5 mg of chl/mL in the storage buffer. Tris-washed PSII membranes were made by incubating PSII membranes (0.5 mg of chl/mL) in 0.85 M Tris-HC1 (Sigma), pH 8.0, on ice and in room light for 30 min. The Tris-washed PSII membranes were resuspended after centrifugation in the storage buffer. Chlorophyll concentrations were determined spectrophotometrically by the method of Arnon (1949). The EPR signal I1 spin-quantitation procedure of Babcock et al. (1983) using potassium nitrosodisulfonate as the spin standard was employed to determine the ratio of chlorophyll to PSII reaction center in PSII membrane suspensions. In the preparations used in this study, the ratio varied between 175 and 200 chl/PSIl. The low-temperature illumination method of Brudvig et al. (1983a,b) was used to generate the S2 state for EPR study. Before dark adaptation, the samples were illuminated in a 0 or 25 "C water bath for 5 min by unfiltered, low-intensity incandescent light (100 W/m2). After the light source was turned off, the samples were held in the water bath for a specified period of time in complete darkness. At the end of the dark-adaptation period, samples were frozen in liquid nitrogen. EPR scans of the dark-adapted samples were taken and stored on diskette by a DEC MINC-23 computer. Then, samples were removed from the EPR cavity and were placed in a nitrogen gas flow temperature-control apparatus. The sample temperature was monitored with a gold-chrome1 thermocouple (Air Products) calibrated with g carbon-glass resistor (Lake Shore Cryotronics) held in a sample tube. The
BECK, D E PAULA,
AND BRUDVIG
samples were allowed to equilibrate at the chosen temperature (140-230 K) for 3 min before illumination (1 20 s) with white light from a 100-W quartz-halogen lamp filtered with 10 cm of water (700 W/m2). The samples were then quickly cooled with liquid nitrogen. EPR scans of the illuminated samples were recorded in the same manner employed for the darkadapted samples. EPR scans were performed at 7 K on a JEOL ME-3X X-band EPR spectrometer equipped with an Oxford ESR-900 liquid helium cryostat. Sample temperatures were determined with a carbon-glass resistor (Lake Shore Cryotronics) contained in a sample tube in the sample position. O2evolution and consumption measurements were made on a YSI Model 53 O2 monitor equipped with a Teflon-membrane-covered Clark-type O2electrode. A Neslab RTE-9DD circulator bath was used to maintain the sample cell's water jacket at 25.0 OC and to equilibrate the temperature of the electrode and buffers before an assay. Output from the YSI O2 monitor was digitized by the MINC-23 computer. The 0,consumption assay media consisted of 4.0 mL of assay buffer [20 mM MES, 15 mM NaCl, 5 mM EDTA, and 30% (v/v) ethylene glycol, pH 6.01 and 250 pM DCBQ (Eastman Kodak, recrystallized twice from 95% ethanol), to which PSII membranes ranging in concentration from 1 to 150 pg of chl/mL had been added. EDTA was present in the assay buffer to avoid complication from free metal ion catalyzed O2consumption [see Miles (1976)]. Thylakoids were assayed in the same medium except that 2 mM ammonium chloride was added as an uncoupler. In some assays, as noted, 100 pM TPB (Aldrich gold label) was included. Illumination for O2 evolution/consumption assays was provided by a 1000-W quartz-halogen lamp filtered by Schott KG-5 heatabsorbing and GG-495 filters contained in a water bath (1200 W/m2). Two protocols were followed for illuminating samples prior to measurement of the 0, consumption. In some assays, the sample was added to the assay medium, the 0, electrode was positioned, and the medium was allowed to equilibrate for 5 min. Then the system was illuminated for a period of 0.5-2 min. The concentration of O2was measured prior to, during, and following the illumination. In other assays, the sample was added to the assay medium, allowed to equilibrate for 5 min, and then illuminated for 0.5-2 min while the electrode was kept in a temperature-regulated sample of the assay medium. Following illumination, the sample was allowed to equilibrate for a total of 4 min in the dark before positioning of the 0, electrode and measurement of the O2concentration; 30 pM DCMU, 1 mM sodium ascorbate, or 3.5 mM potassium ferricyanide was added to some assays 2 min after illumination. Following illumination of the sample, all steps were done in darkness. This second protocol was followed to allow the addition of reagents after illumination and dark reequilibration and also to determine if complications arose from illumination of the 0, electrode. When no additions were made, both protocols produced equivalent results. RESULTS EPR Studies of the S2 State. Figure 1 shows the effect of dark adaptation at 0 OC on the intensity of the S,-state EPR signal produced by illumination of PSII membranes and thylakoids at 200 K. The EPR signal intensity produced by illumination at 200 K after 6-min incubation in the dark at 0 O C was weak (Figure la). Samples illuminated at 200 K after longer periods of dark adaptation (Figure lb,c) produced much more intense S2-state EPR signals. The 4-h darkadapted sample produced an s,-state EPR signal very similar
VOL. 24, NO. 12, 1985
STATES OF THE 0 2 - E V O L V h G COMPLEX
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Magnetic Field (Gauss) Magnetic F i e l d (Gauss) FIGURE 1: Dark-adaptation dependence of the intensity of the S2-statemultiline EPR signal produced by 200 K illumination of dark-adapted PSII membranes (left) and thylakoids (right). The spectra are the difference between the spectrum after 200 K illumination (120 s) and the spectrum of the same dark-adapted sample. The g = 2.0 region of the spectrum is not shown. EPR spectrometer conditions: microwave frequency 8.81 GHz, microwave power 4 mW, modulation frequency 100 kHz, and modulation amplitude 20 G. PSII membranes and thylakoids ( 5 mg of chl/mL) were dark adapted at 0 O C for (a) 6 min, (b) 2 h, and (c) 4 h. to that observed by Dismukes & Siderer (1981), Brudvig et al. (1983a,b), and Hansson & Andreasson (1982). Further dark adaptation caused little change in either the intensity or the hyperfine structure of the S2-state EPR signal. The S,-state spectra from thylakoids showed the same dependence on dark adaptation as did PSII membranes (Figure 1). We can thus dismiss the possibility that the process occurring during dark adaptation was an abnormality induced by the Triton X-100 used in the PSII isolation procedure. We can also exclude the possibility that the dependence of the S2-stateEPR signals on the length of dark adaptation was due to decomposition of the samples. Control trials, in which samples were incubated at 0 OC in the dark for 4 h, illuminated at 0 "C to cycle the S states, and then dark adapted for 6 min at 0 OC, subsequently produced the S2-state EPR signal typical of short-term dark-adapted samples. The question arises as to whether 6 min of dark adaptation at 0 OC was sufficient to allow most of the S2 and S3 states present during the preillumination to decay to the SIstate. We find that the EPR spectra of samples dark adapted for 6 min at 0 OC show
