Structural Changes of D1 C-terminal (cid:1) -Carboxylate during S-state Cycling in Photosynthetic Oxygen Evolution*

Changes in the chemical structure of (cid:1) -carboxylate of the D1 C-terminal Ala-344 during S-state cycling of photosynthetic oxygen-evolving complex were selectively measured using light-induced Fourier transform infrared (FTIR) difference spectroscopy in combination with specific [ 13 C]alanine labeling and site-directed mutagenesis in photosystem II core particles from Synechocystis sp. PCC 6803. Several bands for carboxylate symmetric stretching modes in an S 2 /S 1 FTIR difference spectrum were affected by selective 13 C labeling of the (cid:1) -carboxylate of Ala with L -[1- 13 C]alanine, whereas most of the isotopic effects failed to be induced in a site-directed mutant in which Ala-344 was replaced with Gly. Labeling of the (cid:1) -methyl of Ala with L -[3- 13 C]alanine had much smaller effects on the spectrum to induce isotopic bands due to a symmetric CH 3 deformation coupled with the (cid:1) -carboxylate. The isotopic bands for the (cid:1) -carbox-ylate of Ala-344 showed characteristic changes during S-state cycling. The bands appeared prominently upon the S 1 -to-S 2 transition and to a lesser extent upon the S 2 -to-S 3 transition but reappeared at slightly upshifted frequencies with the opposite sign upon the S 3 -to-S 0 transition. No obvious isotopic band appeared upon the S 0 -to-S 1 transition. These results indicate that the (cid:1) -car- boxylate of C-terminal Ala-344 is structurally associated with a manganese ion that becomes oxidized upon the S 1 -to-S 2 transition and reduced reversely upon the S 3 - to-S 0 transition but is not associated with manganese ion(s) oxidized during the S 0 -to-S 1 (and S 2 -to-S 3 ) transition(s). Consistently, L -[1- 13 C]alanine labeling also induced spectral changes in the low frequency (670–350 cm (cid:2) 1 ) S 2 /S 1 FTIR difference spectrum.

Photosynthetic water oxidation takes place in an oxygenevolving complex (OEC) 1 in which the catalytic metal cluster located on the lumenal side of the D1 protein is composed of four manganese ions and one Ca 2ϩ ion. Most of the potential ligands of the manganese/Ca 2ϩ cluster appear to be located on the D1 protein based on site-directed mutagenesis studies using the cyanobacterium Synechocystis sp. PCC 6803 (reviewed in Refs. [1][2][3]. These include Asp-170, Glu-189, His-190, His-332, Glu-333, His-337, Asp-342, and C-terminal Ala-344 (4 -9), which are arranged in close proximity to the cluster according to the x-ray structural model of S 1 state OEC (10 -13).
The D1 protein is synthesized and assembled into the PS II complex with a short C-terminal extension except for the protein in Euglena (14). Light-dependent assembly of the manganese/Ca 2ϩ cluster requires a free ␣-carboxylate of the C-terminal Ala-344, which occurs via cleavage of the C-terminal extension by the D1 C-terminal processing protease (CtpA) (15). None of the C-terminal truncated Synechocystis mutants in which Asn-335, Asp-342, Leu-343, and Ala-344 were replaced with a stop codon grew photoautotrophically and evolved oxygen (5). Site-directed replacement of D1-Ala-344 with Gly, Met, Ser, Val, Glu, or Gln in the D1-Ala-344-stop strain did not eliminate the capability for photoautotrophic growth and oxygen evolution (5). Lightinduced FTIR difference spectroscopy showed the isotopeinduced changes of carboxylate symmetric stretching bands from the ␣-carboxylate of Ala-344 upon the S 1 -to-S 2 transition by incorporating L-[1-13 C]alanine isotope (16). Replacement of the C-terminal Ala with Gly, which induced marked changes in the skeletal structure of the manganese/Ca 2ϩ cluster detected by light-induced FTIR difference spectroscopy in the low frequency region (17), indicated the close structural association of Ala-344 with the manganese/Ca 2ϩ cluster. These observations suggest that the ␣-carboxylate of D1-Ala-344 associates with manganese ion(s) as a direct ligand. Some x-ray structural models of PS II suggested possible ligation of the ␣-carboxylate of Ala-344 to the manganese ion(s) (10 -12), but a recent 3.5 Å x-ray model proposed the closest location of the ␣-carboxylate to the Ca 2ϩ ion that composes a cubane-like cluster core with three manganese ions (13). Notably, however, no electron density for the ␣-carboxylate of Ala-344 has been resolved in any x-ray models because of limited resolution and x-ray damages during data collection.
During the process of photosynthetic oxygen evolution, two water molecules are oxidized to yield an oxygen molecule through five intermediates labeled S n (n ϭ 0 -4), where n denotes the number of oxidizing equivalents stored. Each S n state advances to the S nϩ1 state by absorbing a photon until reaching the highest oxidation state, S 4 , which spontaneously reverts to the lowest oxidation state, S 0 , concomitant with the release of an oxygen molecule (18,19). Because the thermally stable S 1 state predominates after prolonged dark incubation, S-state cycling starts from the S 1 state in the dark adapted OEC. However, the mechanism through which substrate water molecules are oxidized to an oxygen molecule remains still largely unknown. To understand the mechanism of water oxidation, it is indispensable to know the details of changes in the chemical structure of the OEC during S-state cycling. Infrared spectroscopy is a powerful technique for detecting subtle changes in molecular structure and chemical bonds accompanying chemical processes directly (20). Mid-frequency FTIR difference spectroscopy has been applied to studies on oxygen evolution for elucidating S-state-dependent changes in protein backbone and amino acid side groups, which structurally associate with the manganese/Ca 2ϩ cluster in a direct or indirect manner (21)(22)(23)(24)(25). However, the spectra obtained were a composite of changes of a large number of bands, and no band was exactly assigned to a specific amino acid residue in a protein, with the only exception being the bands for the ␣-carboxylate of the C-terminal Ala-344 of the D1 protein found in the S 2 /S 1 FTIR difference spectrum (16).
In the present study, we characterized the bands of C-terminal Ala-344 of the D1 protein in the mid-frequency S 2 /S 1 FTIR difference spectrum by labeling the ␣-carboxylate group with L-[1-13 C]alanine and the ␣-methyl group with L-[3-13 C]alanine in combination with site-directed mutation of Ala-344. We further report the changes in the mid-frequency bands for the ␣-carboxylate of C-terminal Ala-344 during S-state cycling in PS II core particles from Synechocystis sp. PCC 6803. This is the first report on detection of the S-state-dependent structural changes in a single amino acid ligand for the manganese/Ca 2ϩ cluster in OEC. It was also shown that bands in the low frequency (670 -350 cm Ϫ1 ) S 2 /S 1 FTIR difference spectrum exhibited characteristic changes due to L-[1-13 C]alanine labeling.

EXPERIMENTAL PROCEDURES
Sample Materials-A mutant with Gly instead of Ala at the D1 C-terminal (A344G-stop) was constructed based on the control strain of Synechocystis sp. PCC 6803, which lacks the D1 C-terminal extension and bears a His tag on the C-terminal of CP47 (Ala-344-stop) (17,26). For specific 13 C labeling of the ␣-carboxylate (1-C) or ␣-methyl carbon (3-C) of alanine, cells were grown photoautotrophically in BG11 medium supplemented with 0.5 mM L-[1-13 C]alanine or L-[3-13 C]alanine (99% 13 C enrichment, Cambridge Isotope Laboratories Inc.). Total incorporation of 13 C into 1-C or 3-C of Ala was evaluated with liquid chromatography/mass spectrometry analysis and found to be ϳ70% in thylakoid membranes. Thylakoid membranes obtained by disrupting cells using a Bead-Beater (Bio-Spec Products) followed by centrifugation were solubilized with n-dodecyl-␤-D-maltoside, and PS II core particles were chromatographically purified using a nickel column (17,26). The sample medium was replaced with 40 mM sucrose, 5 mM NaCl, 5 mM CaCl 2 , 10 mM Mes/NaOH, pH 6.0 (medium A) by precipitation of PS II core particles using 10% (w/v) polyethylene glycol 6000 and extensive washes with medium A for S 2 /S 1 FTIR difference spectra (17) or re-peated ultrafiltration in the presence of 0.06% (w/v) n-dodecyl-␤-Dmaltoside for S-state cycling (25). The O 2 -evolving activity of the core particles obtained was ϳ2500 mol of O 2 (mg of chlorophyll) Ϫ1 h Ϫ1 at 25°C using 4 mM potassium ferricyanide as an electron acceptor.
FTIR Measurements-Mid-frequency (1800 -1200 cm Ϫ1 ) and low frequency (670 -350 cm Ϫ1 ) FTIR spectra were recorded on a Bruker IFS-66v/S spectrophotometer equipped with a mercury cadmium telluride detector (EG&G Optoelectronics D316/6) and on a Bomen MB102 spectrophotometer equipped with a silicon bolometer (infrared, HDL-5), respectively, at 0°C (Ϯ0.03°C) as described previously (25,26). The PS II core particles (ϳ40 g of chlorophyll) suspended in medium A were mixed with 1 l of sodium ferricyanide solution (100 mM stock) as an electron acceptor. The PS II core suspension was deposited on a BaF 2 (mid-frequency) or an AgCl (low frequency) disk, partially dried under a stream of cool N 2 gas, and then covered with another BaF 2 or AgCl disk with a greased Teflon spacer after placing a droplet of 20% (v/v) glycerol/water solution adjacent to the sample for rehydration (25). After dark incubation at 0°C for 1 h, the sample was preflashed to reduce the oxidized non-heme iron on the acceptor side and to enrich the S 1 population. The sample was subjected to one flash for the S 1 -to-S 2 transition or four successive flashes at 10-s intervals for respective S-state transitions provided from a frequency-doubled Nd 3ϩ :YAG laser (Spectra Physics, INDI-50) as described previously (25,26). Singlebeam spectra in the mid-frequency (20 scans) or low frequency (10 scans) region were measured before and after the first flash for S 2 /S 1 difference or before the first flash and after each flash for the measurement of the flash-induced difference spectrum of each transition during the S-state cycling. To obtain difference spectra for the respective Sstate transitions, the single-beam spectrum before each flash was subtracted from that after the flash. To obtain higher quality spectra of S 2 /S 1 difference for close inspection of the isotopic bands than the spectra for S-state cycling, 80 -170 and 70 -89 mid-frequency spectra were accumulated for S 2 /S 1 difference and S-state cycling, respectively. 261-273 spectra were averaged for the low frequency region.
D1 C-terminal ␣-Carboxylate during S-state Cycling well as the amide I (1700 -1600 cm Ϫ1 ) and II (1600 -1500 cm Ϫ1 ) modes from polypeptide backbones (16, 24 -26). Labeling by L-[1-13 C]alanine induced changes of S 2 /S 1 spectra in the carboxylate symmetric stretching region below 1400 cm Ϫ1 in particles from the control Ala-344-stop cells (a) but minimal changes in particles from the mutant A344G-stop cells (c). These results were in good agreement with a previous report (16) and indicated that changes in the ␣-carboxylate group of D1 C-terminal Ala-344 are mainly responsible for these isotopic bands. Consistent with this view, labeling of the ␣-methyl of alanine by L-[3-13 C]alanine had little influence on spectra in the carboxylate symmetric stretching region of the control Ala-344-stop particles (b) other than the slight difference around 1354 cm Ϫ1 . Although these isotope labeling experiments also induced changes in the spectra at 1700 -1500 cm Ϫ1 , the region that includes asymmetric stretching bands of carboxylates, the effects of the isotope labeling on the spectra in this frequency region were ambiguous at present due to extensive overlap of intense amide bands. Fig. 1B shows double difference FTIR spectra for the S 2 /S 1 difference in the carboxylate symmetric stretching region (1500 -1200 cm Ϫ1 ), in which relatively small changes induced by the isotope labeling were clearly observable due to the absence of overlapping by intensive amide I and II bands. Each double difference spectrum was obtained by subtracting the  (27), and the coupled bands were affected by the 1-13 C (16) or 3-13 C labeling 2 in the 12 C/ 13 C difference spectrum of alanine solution. In contrast, the asymmetric CH 3 deformation mode was hardly affected by both types of labeling, and the CH bending mode sensitive to the 1-13 C labeling was little changed upon the 3-13 C labeling. Therefore, the 1356(Ϫ)/1349(ϩ) cm Ϫ1 bands in the [3-12 C]/[3-13 C]alanine spectrum (b) may be ascribed to the symmetric CH 3 deformation modes of the ␣-methyl of D1 Ala-344 coupled with the ␣-carboxylate. The [1-12 C]/[1-13 C]alanine spectrum for the A344G-stop particles contained bands at 1305(Ϫ)/1291(ϩ)/ 1278(Ϫ) cm Ϫ1 . Since D1 Ala-344 was replaced with Gly in the A344G-stop particles, it is likely that these bands are caused by Ala residue(s) separate from the C-terminal residue. There are several alanine residues that reside possibly close to the manganese/Ca 2ϩ cluster in the luminal side of the D1 protein. One of these residues may be responsible for the 1309 -1277 cm Ϫ1 bands, although it is not clear which vibrational modes are responsible for the bands. The coupled ␣-methyl band of D1 Ala-344 and the bands from the other Ala residue(s) are likely to at least in part contribute to the bands in the [1-12 C]/[1-13 C]alanine spectrum for the control Ala-344-stop particles. Other minor bands at 1381(Ϫ) and 1373(ϩ) cm Ϫ1 cannot be assigned at present, although apparent S-state dependence indicated that some species concerning S-state cycling are responsible for the bands.
Structural Changes of D1 C-terminal Ala-344 Carboxylate during S-state Cycling- Fig. 2 shows the FTIR difference spectra at 1800 -1200 cm Ϫ1 obtained during S-state cycling of PS II core particles from the unlabeled control (black lines) and L-[1-13 C]alanine-labeled (red lines) cells. The difference spectra induced by the first (a), second (b), third (c), and fourth (d) flashes correspond to the S 2 /S 1 , S 3 /S 2 , S 0 /S 3 , and S 1 /S 0 difference spectra, respectively. Spectra showed characteristic changes that were dependent on the number of flashes. Each spectrum for the S-state cycling of unlabeled control (Ala-344-stop) particles was nearly identical to that of particles isolated from a histidine-tagged wild-type Synechocystis strain retaining the C-terminal extension (25) and very similar to PS II core particles from Thermosynechococcus elongatus (21,23,24) and spinach (22,25). Overall features of the L-[1-13 C]alanine-labeled spectra for S-state cycling were similar to those for the unlabeled control, but distinct changes were clearly observed in the carboxylate symmetric stretching regions below 1400 cm Ϫ1 in spectra induced by the first (a) and third (c) flashes but not in those induced by the second (b) and fourth (d) flashes. Spectral features in the 1700 -1500 cm Ϫ1 region showed some differences in band intensity between the unlabeled controls and L-[1-13 C]alanine-labeled spectra. This frequency region is expected to involve asymmetric carboxylate stretching vibrations that are the counterpart of the labeling-sensitive symmetric modes, and the observed differences may include the effects of the labeling on the C-terminal Ala-344 carboxylate during the S-state cycling. However, we cannot analyze the details of the labeling effects on the spectral features at 1700 -1500 2 Y. Kimura cm Ϫ1 because the band intensity tends to fluctuate easily due to the presence of the large background from the water and amide absorptions.
S-state dependence of the carboxylate symmetric stretching bands of the D1 C-terminal Ala-344 was revealed more clearly in the L-[1-12 C]alanine/L-[1-13 C]alanine double difference spectra shown in Fig. 3, which were obtained by subtracting L-[1-13 C]alanine-labeled S-state difference spectra from the unlabeled S-state difference spectra. The double difference spectra obtained are predominantly ascribed to S-state-dependent structural changes of the C-terminal Ala-344 carboxylate. The bands clearly and prominently observed in the double difference spectrum for S 2 /S 1 difference (a) largely disappeared in the double difference spectrum for S 3 /S 2 difference (b). Note that the spectral features of the double difference spectrum for the S 3 /S 2 difference somewhat resembled those for the S 2 /S 1 difference, suggesting some contribution of the isotopic bands for S 2 /S 1 difference to the double difference spectrum for the second flash due to the miss-hit (ϳ18%) after the first flash (25). The double difference spectrum for S 0 /S 3 (c) showed prominent bands, overall features of which were similar to those in the double difference spectrum for S 2 /S 1 but with the opposite sign. Interestingly, the peak position for each band in spectrum c was slightly upshifted as compared with that for the corresponding band in spectrum a. No obvious band was apparent in the double difference spectrum for S 1 /S 0 (d), indicating that few structural changes in the C-terminal Ala-344 carboxylate group were induced during the S 0 -to-S 1 transition. The periodic changes in the spectral features clearly demonstrated that the ␣-carboxylate group of the D1 C-terminal Ala-344 characteristically changes structure and/or chemical natures during Sstate cycling.
Effects of L- [1-13 C]Alanine Labeling on Low Frequency S 2 /S 1 Spectrum- Fig. 4 shows the low frequency (670 -350 cm Ϫ1 ) S 2 /S 1 FTIR difference spectra of PS II core particles from the unlabeled control (black line) and L-[1-13 C]alanine-labeled (red line) cells. The control spectrum agreed well with that reported previously (17) and showed bands at 652(Ϫ), 642(ϩ), 629(ϩ), 617(Ϫ), 606(ϩ), 590(ϩ), and 577(Ϫ) cm Ϫ1 as well as many medium and low intensity bands. The bands at ϳ400(Ϫ) cm Ϫ1 and 590(ϩ) cm Ϫ1 were ascribed to the ferricyanide added to the sample suspension as an electron acceptor and ferrocyanide formed by photoreduction of ferricyanide, respectively (26). Although the spectrum of L-[1-13 C]alanine-labeled particles was basically similar to the unlabeled control spectrum, small but distinctive differences between the two spectra were observed reproducibly at Ͼ590 cm Ϫ1 . The intensity of the 617(Ϫ) cm Ϫ1 band changed considerably, and the bands at 642(ϩ), 629(ϩ), and 617(Ϫ) cm Ϫ1 were downshifted slightly upon the L-[1-13 C]alanine labeling. Most of the bands for S 2 /S 1 in the low frequency difference spectrum were significantly affected upon universal 13 C labeling with the exception of the 577(Ϫ) cm Ϫ1 band for the putative skeletal vibration of the manganese cluster (26). Therefore, it is conceivable that the ␣-carboxylate in the C-terminal Ala-344 is responsible for low frequency bands affected by L-[1-13 C]alanine labeling. The low frequency result is compatible with the view that the ␣-carboxylate of the D1 C-terminal Ala-344 associates with metal ion(s) as a direct ligand. The C-terminal carboxylate ligates a manganese ion in an unidentate manner throughout S-state cycling. This manganese ion is oxidized from Mn III to Mn IV during the S 1 -to-S 2 transition and reduced from Mn IV to Mn III during the S 3 -to-S 0 transition, but shows no valence change during the S 2 -to-S 3 or S 0 -to-S 1 transition. An alternative interpretation was presented in the parentheses. The C-terminal carboxylate ligates a Ca 2ϩ ion in an unidentate manner in the S 0 and S 1 states but unidentately ligates a manganese ion, which has been oxidized to Mn IV upon the S 2 state formation, instead of Ca 2ϩ in the S 2 and S 3 states. See "Discussion" for details. D1 C-terminal ␣-Carboxylate during S-state Cycling