Absence of the PsbQ Protein Results in Destabilization of the PsbV Protein and Decreased Oxygen Evolution Activity in Cyanobacterial Photosystem II*

We have previously reported that cyanobacterial photosystem II (PS II) contains a protein homologous to PsbQ, the extrinsic 17-kDa protein found in higher plant and green algal PS II (Kashino, Y., Lauber, W. M., Carroll, J. A., Wang, Q., Whitmarsh, J., Satoh, K., and Pakrasi, H. B. (2002) Biochemistry 41, 8004–8012) and that it has regulatory role(s) on the water oxidation machinery (Thornton, L. E., Ohkawa, H., Roose, J. L., Kashino, Y., Keren, N., and Pakrasi, H. B. (2004) Plant Cell 16, 2164–2175). In this work, the localization and the function of PsbQ were assessed using the cyanobacterium Synechocystis sp. PCC 6803. From the predicted sequence, cyanobacterial PsbQ is expected to be a lipoprotein on the luminal side of the thylakoid membrane. Indeed, experiments in this work show that upon Triton X-114 fractionation of thylakoid membranes, PsbQ partitioned in the hydrophobic phase, and trypsin digestion revealed that PsbQ was highly exposed to the luminal space of thylakoid membranes. Detailed functional assays were conducted on the psbQ deletion mutant (ΔpsbQ) to analyze its water oxidation machinery. PS II complexes purified from ΔpsbQ mutant cells had impaired oxygen evolution activity and were remarkably sensitive to NH2OH, which indicates destabilization of the water oxidation machinery. Additionally, the cytochrome c550 (PsbV) protein partially dissociated from purified ΔpsbQ PS II complexes, suggesting that PsbQ contributes to the stability of PsbV in cyanobacterial PS II. Therefore, we conclude that the major function of PsbQ is to stabilize the PsbV protein, thereby contributing to the protection of the catalytic Mn4-Ca1-Clx cluster of the water oxidation machinery.

Ca 2ϩ and Cl Ϫ . Deletion of psbV resulted in significant defects in PS II function; ⌬psbV cells exhibited a slower photoautotrophic growth rate and 40% oxygen evolution activity compared with wild type (15). From the biochemical and genetic studies, Shen and co-workers (4,16,17) proposed that PsbU and PsbV in cyanobacteria were similar in function to PsbQ and PsbP in chloroplasts. Accordingly, an evolutionary model was described in which PsbQ and PsbP replaced PsbU and PsbV at some point during evolution from the ancestral cyanobacterium to chloroplasts (2,17,18). However, this model is somewhat unsatisfactory, because no similarity in the predicted primary sequences or the crystallographic structures was found between these sets of proteins (PsbU/PsbV versus PsbP/PsbQ) (2, 19 -24).
Subsequent to these studies, proteomic analysis of highly purified PS II complexes isolated from a HT3 strain of Synechocystis 6803 identified a novel protein homologous to chloroplast PsbQ (5). Further analysis of the Synechocystis 6803 genome identified a protein homologous to PsbP (6). Initial functional analyses showed that both PsbP and PsbQ have regulatory role(s) in the oxygen evolution machinery, since the deletion of the corresponding genes results in retarded growth under Ca 2ϩ -or Cl Ϫ -limited conditions (6). The PsbQ protein was found to be associated with PS II complexes stoichiometrically, whereas the PsbP protein is substoichiometric in PS II (6). Therefore, PS II complexes from the cyanobacterium Synechocystis 6803 contain five associated extrinsic proteins: PsbO, PsbP, PsbQ, PsbU, and PsbV.
A 20-kDa protein homologous to plant PsbQ was found in PS II complexes purified from a primitive red alga, Cyanidium caldarium, which also contains the PsbU and PsbV proteins (18). Careful genome analysis of cyanobacteria showed that most cyanobacterial genomes contain genes homologous to psbP and psbQ (25), consistent with our findings for Synechocystis 6803 (5,6). These new findings challenge the proposed model that PsbP and PsbQ in plants have functionally replaced PsbU and PsbV from cyanobacteria. Indeed, recent studies of the PS II extrinsic proteins have focused on more rigorously assigning functions to each of the five extrinsic proteins in cyanobacterial PS II (26,27). The function of PsbU was reexamined, and this new analysis of ⌬psbU indicates that PsbU in cyanobacterial PS II functions to support a stable structural architecture of the water-splitting system (26). Summerfield et al. (27) extended our findings on PsbQ, showing stringent requirements for PsbQ in vivo. They reported that the ⌬psbQ mutant had impaired growth at elevated temperatures. They also combined the ⌬psbQ mutation with other PS II extrinsic protein mutations in Synechocystis 6803 (27). The double deletion mutant ⌬psbQ⌬psbV was not able to grow photoautotrophically, whereas the ⌬psbQ⌬psbO and ⌬psbQ⌬psbU mutants showed exacerbated growth defects relative to the single mutants, especially in medium lacking Ca 2ϩ and Cl Ϫ . Accordingly, they concluded that PsbQ has a role in optimizing PS II activity in Synechocystis 6803 cells, which is absolutely required under specific physiological conditions. Although these recent studies highlight a role of PsbQ in cyanobacterial PS II, the reported structural models of cyanobacterial PS II complexes from Thermosynechococcus elon-gatus and Thermosynechococcus vulcanus do not contain PsbQ (19,23,24). Thus, it is not known how PsbQ interacts with the other extrinsic proteins (PsbO, PsbU, and PsbV) nor how this may contribute to the water oxidation reaction. In this report, we specifically focus on the localization and the function of PsbQ within the water oxidation machinery of cyanobacterial PS II. Our results demonstrate that PsbQ associates with the luminal surface of PS II despite differences in its biochemical properties relative to the other extrinsic proteins, PsbO, PsbU, and PsbV. Functional analyses of the water oxidation machinery in the psbQ deletion mutant (⌬psbQ) show significant effects on the donor side of PS II, including destabilization of PsbV in the complex and a more exposed catalytic center. From these data, we propose that the major function of PsbQ is to stabilize the PsbV protein on the luminal surface of PS II and protect the catalytic Mn 4 -Ca 1 -Cl x cluster from exogenous reductants.
Isolation of Thylakoid Membranes and Purification of PS II Complexes-Thylakoid membrane and PS II complexes were isolated as described in Ref. 5 and resuspended in MMCG solution, containing 50 mM MES-NaOH (pH 6.0), 10 mM MgCl 2 , 5 mM CaCl 2 , and 25% glycerol.
Isolation of Right Side-out and Inside-out Vesicles-To isolate thylakoid membranes containing predominantly right side-out vesicles (30), cells in MMCG solution were broken as described in Ref. 5 and collected at 36,000 ϫ g after the removal of unbroken cells. For the isolation of thylakoid membranes containing mostly inside-out vesicles (30), cells suspended in 50 mM MES-NaOH (pH 6.0), 10 mM MgCl 2 , 5 mM CaCl 2 were passed through a French pressure cell twice at 100 megapascals. After removal of unbroken cells by centrifugation at 3,000 ϫ g, the resulting vesicles were collected at 160,000 ϫ g. Both types of vesicles were suspended in MMCG solution in the final step of preparation. No protease inhibitors were added during these isolations. The lysozyme treatment was omitted to avoid possible proteolytic degradation. Instead, the samples were thoroughly kept cool and processed quickly.
Fractionation of Thylakoid Membrane-Triton X-114 phase partitioning of thylakoid membrane was performed essentially as described in Ref. 31 at 100 g of Chl/ml. Triton X-114 was purchased from Sigma.
Protease Treatment-Stock solution of trypsin (6,750 units/ ml; Sigma; pancreatic type II crude) was added to 1 ml of thylakoid membrane suspension (100 g of Chl/ml) to 0, 34, 68, and 135 units/100 g of Chl (equivalent to around 4 mg of protein). The mixtures were incubated at 30°C for 10 min at pH 6.0. Note that because the pH was not optimal for trypsin activity, the digestion was performed at 30°C. The reactions were stopped by adding one-third volume of denaturing solution (20 mM EDTA, 5% lithium dodecyl sulfate, 40 mM dithiothreitol, 172 mM Tris, pH 6.8, and 0.5 M sucrose) and cooling to 0°C.
Oxygen Evolution Assay-Steady state oxygen evolution was measured on a Clark-type electrode in the presence of 1 mM potassium ferricyanide and 0.5 mM 2,6-dichloro-p-benzoquinone as electron acceptors at 2 g of Chl/ml in MMCS buffer containing 50 mM MES-NaOH, (pH 6.0), 10 mM MgCl 2 , 5 or 20 mM CaCl 2 and 0.5 M sucrose. To assess the effect of NH 2 OH, oxygen evolution was measured after PS II complexes (2.0 g of Chl/ml in MMCG buffer, 20 mM CaCl 2 ) were incubated for 1 h on ice in the dark in the presence of variable concentrations of NH 2 OH (34). Flash oxygen yield measurements were measured using a bare platinum electrode as described in Ref. 26.
Fluorescence Kinetics-Q A reoxidation kinetics was measured at room temperature using a double-modulation fluorometer, FL-3320 (Photon System Instruments, Brno, Czech Republic) with FluorWin software (version 3.6.3.3). The cell concentration for each sample was adjusted to OD 730 ϭ 0.08 (ϳ2 g of Chl/ml) measured on a DW2000 spectrophotometer (SLM-Aminco, Urbana, IL). The samples were dark-adapted for 3 min prior to measuring.
Optical Measurements-Chl a concentration was determined by the method of Porra et al. (35).

Cyanobacterial PsbQ Is a Luminal Protein with a Putative
Lipid Anchor-We have previously postulated that PsbQ in cyanobacteria is a luminally targeted lipoprotein (6). Indeed, cyanobacterial PsbQ was not removed from PS II by biochemical treatments (1 M CaCl 2 or 1 M Tris-HCl, pH 8.0) that removed the other extrinsic proteins (PsbO, PsbU, and PsbV) (5). This result was quite different from the characteristics of chloroplast PsbQ, which is removed by high salt treatment (36).
Triton X-114 partitioning, a well established method to separate hydrophobic and hydrophilic proteins (37), was used to assess the hydrophobicity of the mature cyanobacterial PsbQ protein. Thylakoid membranes were subjected to Triton X-114 partitioning, and the proteins were fractionated into a hydrophobic fraction (Triton X-114 fraction; Fig. 1, lane 3), an aqueous fraction (lane 4), and an insoluble fraction (very hydrophilic; lane 5). Upon Coomassie staining, the separate fractions showed different polypeptide profiles. As expected, the hydrophilic phycobiliproteins partitioned into the aqueous and insoluble fractions (Fig. 1, Coomassie staining and no staining, lanes 4 and 5). Heme staining was used to detect PsbV (cytochrome c 550 , apparent molecular mass of 22 kDa), which fractionated into the aqueous phase ( Fig. 1, heme staining, lane 4). This result is in agreement with previous reports that PsbV is removed by high salt treatment (4,5). Unlike these hydrophilic proteins, PsbQ partitioned into the hydrophobic fraction ( Fig.  1, PsbQ, lane 3).
Although PsbQ partitioned into the hydrophobic phase ( Fig.  1), this protein has no predicted hydrophobic domain and is expected to be exposed on one side of the thylakoid membrane. Right side-out and inside-out thylakoid membranes were prepared and subjected to limited digestion by trypsin to assess the topology of PsbQ (Fig. 2). PsbQ was not digested in the right side-out membrane samples but was digested in the inside-out membrane samples, similar to the digestion pattern for the luminal protein PsbV (cytochrome c 550 ). This result demonstrates that PsbQ is located on the luminal side of thylakoid membrane. Altogether, the data from the Triton X-114 partitioning and the trypsin digestion experiments are consistent with the previous prediction that PsbQ is targeted to the thylakoid lumen and cleaved by signal peptidase II to yield an N-terminal cysteine, which is modified with a lipid anchor (6).
The PsbV Protein Partially Dissociates from ⌬psbQHT3 PS II Complexes-The polypeptide profiles of HT3 and ⌬psbQHT3 PS II complexes are shown in Fig. 3. As expected, the PsbQ protein is absent from the ⌬psbQHT3 PS II complexes. Also, PsbV was partially lost in ⌬psbQHT3 PS II, indicating that, in the absence of PsbQ, the structure of the water oxidation machinery becomes labile. The fact that PsbV, a luminal PS II protein, is affected by the absence of PsbQ is also consistent with the luminal localization of PsbQ determined during the preceding experiments.
The ⌬psbQ Mutant Has Defects in PS II Water Oxidation-To address the contribution of PsbQ to the water oxidation process, fluorescence kinetics measurements were performed using cells grown in normal BG11. Because PsbV became labile in PS II upon the absence of PsbQ, the ⌬psbV mutant was also assayed for comparison. For samples at the same chlorophyll concentration, the F o value for ⌬psbQ cells was essentially the same as that of wild type cells (0.735 Ϯ 0.0802 versus 0.715 Ϯ 0.0805; S.D., n ϭ 5-6). This is consistent with previous data showing no significant difference in the relative amounts of PS II between wild type and ⌬psbQ cells (6). Whereas the F o value of ⌬psbV cells is significantly increased relative to wild type (1.08 Ϯ 0.136; S.D., n ϭ 5-6), indicating an increase in impaired PS II complexes. The normalized variable fluorescence yield in ⌬psbQ was also comparable with that in wild type (0.742 Ϯ 0.0864 versus 0.694 Ϯ 0.0379; S.D., n ϭ 5-6), whereas that of ⌬psbV was much lower (0.307 Ϯ 0.121; S.D., n ϭ 5-6). The fluorescence decay kinetics for cells after a single saturating flash, which represents the reoxidation kinetics of Q A Ϫ in PS II, are shown in Fig. 4. Qualitatively, the ⌬psbQ mutant has a decay curve intermediate to that of wild type and the ⌬psbV mutant.
To quantitatively compare the fluorescence decay curves for all strains, they were fit with three components of different decay half-times ( Table 1). The first phase reports on the electron transfer from Q A to Q B (38 -40). The second phase represents the turnover of plastoquinone molecules at the Q B site (41). The last component with the longest half-time is the oxidation of Q A by PS I (42). It is noteworthy that the decay half-time of the  fastest component in ⌬psbQ was 20% faster than that in wild type; furthermore, the relative amounts of each component in ⌬psbQ differed from those in wild type. This result is consistent with that observed in the ⌬psbV mutant in which the water oxidation process is highly modified. Flash oxygen yield measurements were also conducted to assay the function of the water oxidation machinery in the absence of PsbQ. ⌬psbQ and ⌬psbV cells showed a characteristic but somewhat modified period four oscillation. The relative oxygen yields after first and second flashes were slightly larger in ⌬psbQ cells than in wild type cells but smaller than in ⌬psbV (data not shown).
The distribution of S-states after 10 min of dark adaptation was somewhat perturbed in ⌬psbQ cells relative to wild type cells. ⌬psbQ cells exhibited a higher fraction of the S 3 -state (4.5 Ϯ 0.18 versus 2.3 Ϯ 0.18%; S.E., n ϭ 4 -8) and a lower fraction of the S 0 -state (32 Ϯ 0.83 versus 37 Ϯ 0.81%; S.E., n ϭ 4 -8) The distribution in ⌬psbV cells was more perturbed relative to that in wild type cells with an increased population of S 2 -and S 3 -states and a lower fraction of S 0 -and S 1 -states (data not shown). The yield itself was also affected, in that the averaged amplitude after damping (flash number [13][14][15][16] in ⌬psbQ cells was typically 90% of that in wild type cells (n ϭ 5) but still larger than that in ⌬psbV cells (24% of that in wild type; n ϭ 6).  5 shows the kinetics of oxygen release from cells poised in the S 3 -state, as detected using a bare platinum electrode for wild type, ⌬psbQ, and ⌬psbV cells (14,43). The rise kinetics for wild type and ⌬psbQ cells were identical, giving the same peak time (ϳ17 ms following the third flash), whereas ⌬psbV cells showed a slower rise time and ϳ7-ms slower peak time than the other strains (Fig. 5). Interestingly, the decay half-time of the oxygen release curve was different for wild type and ⌬psbQ cells (29.5 and 39 ms, respectively). In the ⌬psbQ cells, the decay was decelerated, but not to the extent of that in ⌬psbV cells (62.8 ms). Here again, the ⌬psbQ mutant displays a phenotype intermediate to that of wild type and ⌬psbV. Although this signal has been used previously as an indirect measurement of S 3 -to S 0 -state advancement (14,43), other factors, including long oxygen diffusion pathways to the electrode and oxygen consumption by the cells, also contribute to its lifetime.
The Structural Integrity of the PS II Water Oxidation Complex Is Compromised in the Absence of PsbQ-It has been reported that the removal of PsbP and PsbQ from higher plant PS II membrane preparations causes a remarkable decrease in the oxygen evolution rate and an increase in the Ca 2ϩ and Cl Ϫ requirement for activity and exposes the oxidizing side of PS II to exogenous reductants, such as NH 2 OH (12,44). Table 2 shows the steady state oxygen evolution activities of isolated PS II complexes from HT3 and ⌬psbQHT3. The activity of ⌬psbQHT3 PS II complexes was lower than that of HT3 even in the presence of 20 mM CaCl 2 (60% of HT3 PS II). Whereas HT3 PS II evolved oxygen at the same rate in the presence of 5 and 20 mM CaCl 2 , the rate of oxygen evolution by ⌬psbQHT3 PS II

TABLE 1 Exponential decay components in Q A ؊ reoxidation kinetics
Fluorescence decay kinetics was measured in wild type, ⌬psbQ, and ⌬psbV as in Fig.  4 and was deconvoluted to three exponential decay components. Curve fitting was performed using KaleidaGraph software (version 3.6 for Macintosh; Synergy Software, Reading, PA), with a Levenberg-Marquardt regression algorithm. The curves were better fit with three exponential decay components rather than two exponential decay components. Numbers in parentheses are S.E. (n ϭ 6 or 7). To further analyze the structural integrity of the isolated PS II complexes, the effect of hydroxylamine on steady state oxygen evolution activity was measured (Fig. 6). In the presence of NH 2 OH, the rate of oxygen evolution in HT3 PS II decreased by around 20% independent of the NH 2 OH concentration tested. In contrast, the oxygen evolution activity of ⌬psbQHT3 PS II decreased in a clearly NH 2 OH concentration-dependent manner, with a steep decrease up to 20 M NH 2 OH and a gradual decrease above 20 M NH 2 OH. These results show that the water oxidation machinery in HT3 PS II is largely protected from the small reductant NH 2 OH, whereas that of ⌬psbQHT3 PS II is highly exposed and therefore easily accessed and damaged by NH 2 OH.

DISCUSSION
The current models for PS II extrinsic proteins include five proteins in cyanobacteria (PsbO, PsbU, PsbV, PsbP, and PsbQ) and three proteins in higher plants (PsbO, PsbP, and PsbQ) (6). The PsbO protein, which is shared by both systems, may have a common function in cyanobacteria and plants. However, the PsbU and PsbV proteins have a specialized function in cyanobacteria, whereas PsbP and PsbQ have a modified function between the two systems. Therefore, it is necessary to reevaluate the roles of each of these proteins in the water oxidation reaction. The current study has focused on the localization and function of cyanobacterial PsbQ.
Determination of the localization of PsbQ within the cyanobacterial PS II complex is critical for understanding the roles of all of the associated extrinsic proteins. Structural studies have not yet resolved the PsbQ protein in PS II (19,23,45,46). Cyanobacterial PsbQ associates with PS II complexes but shows different biochemical properties than higher plant PsbQ, since it is not removed by 1 M CaCl 2 or 1 M Tris (5,6). In this work, the physical properties of cyanobacterial PsbQ were investigated using Triton X-114 partitioning. Our results show that PsbQ fractionated into the hydrophobic Triton X-114 phase (Fig. 1). This result is consistent with our previous prediction from sequence analysis that PsbQ is a lipoprotein (6). This kind of modification has been reported previously in Synechocystis 6803 for the periplasmic protein NrtA (47). Thus, it is reasonable to assume that the N-terminal lipid modification causes PsbQ to partition into the hydrophobic Triton X-114 phase and anchors PsbQ to the membrane such that it is not removed by 1 M CaCl 2 or 1 M Tris (5).
The fact that cyanobacterial PsbQ is a lipoprotein may explain why PsbQ is present in HT3 PS II but not in the current crystallographic model (19,23,24). HT3 PS II was purified under mild detergent conditions (33), and it is expected that the lipid bound to the N terminus of PsbQ should remain anchored to the hydrophobic domain of PS II. However, the prevailing procedure to purify membrane protein complexes suitable for crystallization includes a step(s) to remove excess lipids (33, 48 -50). Thus, when the lipids around the PS II complex were removed during purification, it is conceivable that PsbQ was also removed or could no longer anchor to the lipid-depleted PS II complex. Tll2057, the PsbQ homologue in T. elongatus used for PS II crystallographic studies, (available on the World Wide Web at www.kazusa.or.jp/cyano/Thermo/index.html), is also predicted to contain a lipoprotein signal peptide. The functional significance of the N-terminal lipid anchor in cyanobacterial PsbQ is unclear, since PsbQ in plants lacks this modification.
To confirm the luminal localization of cyanobacterial PsbQ, thylakoid membrane vesicles of opposite orientations were subjected to trypsin digestion. PsbQ was digested by trypsin in the inside-out membrane samples, whereas it was protected from digestion in the right side-out membrane samples, as was the bona fide luminal protein PsbV (cytochrome c 550 ). We have reported previously that upon trypsin digestion of right sideout membranes prepared in this way, the luminal PsbO protein is protected from digestion, whereas the cytoplasmically exposed PsaD protein is degraded. The opposite digestion profiles were observed for the inside-out prepared membranes (30). Our current results clearly indicate that PsbQ localizes to the luminal side of thylakoid membrane and excludes the possibility of the presence of a transmembrane domain in cyanobacterial PsbQ. Altogether, these data confirm that PsbQ closely associates to the luminal side of cyanobacterial PS II complexes (Fig. 2) (5), and it can be concluded that PsbQ is a component of the water oxidation complex in cyanobacterial PS II.
Based on the above mentioned localization of PsbQ, the role of PsbQ in relation to the water oxidation reaction was assessed. The variable yield of the fluorescence ((F m Ϫ F o )/F o ) in ⌬psbQ was comparable with that of wild type, whereas the yield in ⌬psbV was much smaller than that of wild type (typically less than 45%). The rate of Q A reoxidation was accelerated in ⌬psbQ as well as ⌬psbV with the decay half-time of the major, fastest component 20% faster in the mutant cells compared with that of wild type cells ( Fig. 4 and Table 1). Although Q A is localized on the reducing side of PS II, defects on the oxidizing side of PS II directly cause an acceleration of Q A reoxidation (51). This result indicates that the absence of PsbQ affects the stability of the water oxidation complex.
Flash yield oxygen measurements showed a slightly altered S-state distribution for ⌬psbQ relative to wild type cells, consistent with the lower oxygen evolution activity observed in ⌬psbQ cells (6,27). The rise kinetics of oxygen release from cells poised in the S 3 -state in ⌬psbQ cells was identical to that of wild type cells (Fig. 5). Although the oxygen signal after the third flash is not a direct measurement of oxygen release from the enzyme, this signal has been correlated to the kinetics of oxygen release from the S 3 -state, assuming that the diffusion rates of oxygen from different cells to the electrode are similar (14,43). Whereas ⌬psbQ cells behaved similarly to wild type cells for the rise kinetics of oxygen release, the decay kinetics of oxygen release in ⌬psbQ cells were slower compared with wild type but not to the extent of that in ⌬psbV cells (Fig. 5). The biological significance of the decay part of the oxygen release curve is not known, and additional processes (i.e. oxygen diffusion to the electrode and oxygen consumption by the cells) contribute to this part of the signal.
The absence of PsbQ had specific consequences on the extrinsic protein PsbV (cytochrome c 550 ). Whereas the Chl a-normalized amount of PsbV in ⌬psbQ cells was comparable with that in wild type cells (measured by heme staining; data not shown), there was a significant decrease in the amount of PsbV associated with PS II complexes purified from the ⌬psbQHT3 mutant (Fig. 3). The absence of PsbQ did not result in the complete removal of PsbV, and this is consistent with previous reports that the ⌬psbQ mutant grew faster than ⌬psbV mutant in both normal medium and Ca 2ϩ -or Cl Ϫ -depleted medium (6). From these results, we conclude that PsbQ is in close proximity to the PsbV protein within PS II and functions to stabilize the extrinsic proteins in the PS II water oxidation complex. These results suggest that a common role for PsbQ in cyanobacteria and plants is to stabilize other PS II extrinsic proteins and modulate the Cl Ϫ requirement for oxygen evolution activity. However, the specific effect on PsbV stability in cyanobacterial PS II upon the loss of PsbQ is somewhat surprising, because higher plant PS II complexes do not contain PsbV.
The absence of PsbQ leads to low water oxidation activity in isolated PS II and the higher requirement of Ca 2ϩ and Cl Ϫ for oxygen evolution (Table 2). This is consistent with the observed effect on PsbV in the ⌬psbQ mutant. To further probe the stability of the water oxidation machinery in PS II complexes lacking PsbQ, the effect of NH 2 OH was measured. Ghanotakis et al. (12) have shown that if PsbP and PsbQ are removed from intact PS II membranes from higher plants, the small reductant NH 2 OH causes extensive damage to the water-oxidizing side of PS II. Thus, PsbP and PsbQ, along with PsbO, shield the water oxidization machinery of plant PS II from the luminal space, and once these proteins are removed, the accessibility of NH 2 OH to the Mn 4 -Ca 1 -Cl x cluster increases significantly. The same effect was observed in isolated PS II complexes from the ⌬psbQHT3 mutant. ⌬psbQHT3 PS II was considerably more sensitive to NH 2 OH, whereas only a limited effect was observed for HT3 PS II (Fig. 6). These results highlight the role of PsbQ in stabilizing the components of the water oxidation machinery in cyanobacterial PS II.
In conclusion, PsbQ associates with the luminal side of cyanobacterial PS II complexes and participates in the water oxidation reaction. PsbQ is important for stabilizing PsbV within the PS II complex, and the majority of the defects described for ⌬psbQ can be explained by a partial loss of PsbV. However, PsbQ must have a role beyond that of stabilizing PsbV, because the double deletion mutant ⌬psbQ⌬psbV cannot grow photoautotrophically, whereas the respective single mutants can grow photoautotrophically (27). It is possible that PsbQ contributes to the stabilization of the other extrinsic proteins on the luminal side of PS II, and the absence of both PsbQ and PsbV results in such an instability of the water oxidation machinery that it can no longer support photoautotrophic growth. Thus, PsbQ is an important extrinsic protein in cyanobacterial PS II, which contributes to the protection of the catalytic Mn 4 -Ca 1 -Cl x cluster of the water oxidation machinery.