Interaction of CPa-1 with the manganese-stabilizing protein of photosystem II: identification of domains on CPa-1 which are shielded from N-hydroxysuccinimide biotinylation by the manganese-stabilizing protein.

The structural organization of photosystem II proteins has been investigated by use of the amino group-labeling reagent N-hydroxysuccinimidobiotin (NHS-biotin) and calcium chloride-washed photosystem II membranes. We have previously shown that the presence of the extrinsic, manganese-stabilizing protein on photosystem II membranes prevents the modification of lysyl residues located on the chlorophyll protein CPa-1 (CP-47) by NHS-biotin [Bricker, T. M., Odom, W. R., & Queirolo, C. B. (1988) FEBS Lett. 231, 111-117]. Upon removal of the manganese-stabilizing protein by calcium chloride-washing, CPa-1 can be specifically modified by treatment with NHS-biotin. Preparative quantities of biotinylated CPa-1 were subjected to chemical cleavage with cyanogen bromide. Two major biotinylated peptides were identified with apparent molecular masses of 11.8 and 15.7 kDa. N-terminal sequence analysis of these peptides indicated that the 11.8-kDa peptide was 232G-330M and that the 15.7-kDa peptide was 360P-508V. The 15.7-kDa CNBr peptide was subjected to limited tryptic digestion. The two smallest tryptic fragments identified migrated at apparent molecular masses of 9.1 (nonbiotinylated) and 7.5 kDa (biotinylated). N-terminal sequence analysis and examination of the predicted amino acid sequences of these peptides suggest that the 9.1-kDa fragment was 422R-508V and that the 7.5-kDa fragment was 360P-421A. These results strongly suggest that two NHS-biotinylated domains, 304K-321K and 389K-419K, become exposed on CPa-1 when the manganese-stabilizing protein is removed by CaCl2 treatment. Both of these domains lie in the large extrinsic loop E of CPa-1.     

Extrinsic proteins of photosystem II: an intermediate member of PsbQ protein family in red algal PS II.

The oxygen-evolving photosystem II (PS II) complex of red algae contains four extrinsic proteins of 12 kDa, 20 kDa, 33 kDa and cyt c-550, among which the 20 kDa protein is unique in that it is not found in other organisms. We cloned the gene for the 20-kDa protein from a red alga Cyanidium caldarium. The gene consists of a leader sequence which can be divided into two parts: one for transfer across the plastid envelope and the other for transfer into thylakoid lumen, indicating that the gene is encoded by the nuclear genome. The sequence of the mature 20-kDa protein has low but significant homology with the extrinsic 17-kDa (PsbQ) protein of PS II from green algae Volvox Carteri and Chlamydomonas reinhardtii, as well as the PsbQ protein of higher plants and PsbQ-like protein from cyanobacteria. Cross-reconstitution experiments with combinations of the extrinsic proteins and PS IIs from the red alga Cy. caldarium and green alga Ch. reinhardtii showed that the extrinsic 20-kDa protein was functional in place of the green algal 17-kDa protein on binding to the green algal PS II and restoration of oxygen evolution. From these results, we conclude that the 20-kDa protein is the ancestral form of the extrinsic 17-kDa protein in green algal and higher plant PS IIs. This provides an important clue to the evolution of the oxygen-evolving complex from prokaryotic cyanobacteria to eukaryotic higher plants. The gene coding for the extrinsic 20-kDa protein was named psbQ' (prime).     

Structural and functional modulation of the manganese cluster in Ca(2+)-depleted photosystem II induced by binding of the 24-kilodalton extrinsic protein.

Depletion of functional Ca2+ from photosystem (PS) II membranes impairs O2 evolution. Redox properties of the Mn cluster as probed by thermoluminescence were modified differently in Ca(2+)-depleted PSII depending on the procedure for Ca2+ extraction. Ca2+ depletion by low-pH treatment gave rise to an abnormally modified S2 state exhibiting a thermoluminescence band with elevated peak temperature accompanied by a marked upshift in threshold temperature for its formation, whereas Ca2+ depletion by NaCl washing in the light followed by the addition of EDTA could generate a similarly modified S2 state only when the Ca(2+)-depleted PSII was reconstituted with the 24-kDa extrinsic proteins. These results indicated that manifestation of the abnormal properties of the Ca(2+)-depleted S2 state is significantly contributed by the association of the 24-kDa extrinsic protein to PSII. It was inferred that the 24-kDa extrinsic protein regulates the structure and function of the Mn cluster in the absence of functional Ca2+ through a conformational modulation of the intrinsic protein(s) that bind(s) both Mn and Ca. Features of the extrinsic protein-dependent modulation of the Mn cluster were discussed in relation to the function of Ca2+ in O2 evolution.     

A truncated mutant of the extrinsic 23-kDa protein that absolutely requires the extrinsic 17-kDa protein for Ca2+ retention in photosystem II

One function of the extrinsic 23-kDa protein in photosystem II (OEC23) is to retain Ca(2+ )and Cl(-), two essential cofactors for photosynthetic oxygen evolution. A truncated mutant of OEC23 (OEC23 Delta19) revealed that 19 residues of the N-terminus of OEC23 were necessary for Ca(2+ )retention but not for its proper interaction with OEC17, the extrinsic 17-kDa protein in photosystem II. The lost ability of OEC23 Delta19 to reconstitute the oxygen-evolving activity was partially restored by OEC17 binding, suggesting the involvement of OEC17 in Ca(2+ )retention in photosystem II.     

Positive Charges on Lysine Residues of the Extrinsic 18 kDa Protein Are Important to Its Electrostatic Interaction with Spinach Photosystem Ⅱ Membranes

To determine the contribution of charged amino acids to binding with the photosystem II complex （PSII）, the amino or carboxyl groups of the extrinsic 18 kDa protein were modified with N- succinimidyl propionate （NSP） or glycine methyl ester （GME） in the presence of a water-soluble carbodiimide, respectively. Based on isoelectric point shift, 4-10 and 10-14 amino groups were modified in the presence of 2 and 4 mM NSP, respectively. Similarly, 3-4 carboxyl groups were modified by reaction with 100 mM GME. Neutralization of negatively charged carboxyl groups with GME did not alter the binding activity of the extrinsic 18 kDa protein. However, the NSP-modified 18 kDa protein, in which the positively charged amino groups had been modified to uncharged methyl esters, failed to bind with the PSII membrane in the presence of the extrinsic 23 kDa protein. This defect can not be attributed to structural or conformational alterations imposed by chemical modification, as the fluorescence and circular dichroism spectra among native, GME- and NSP-modified extrinsic 18 kDa proteins were similar. Thus, we have concluded that the positive charges of lysyl residues in the extrinsic 18 kDa protein are important for its interaction with PSII membranes in the presence of the extrinsic 23 kDa protein. Furthermore, it was found that the negative charges of carboxyl groups of this protein did not participate in binding with the extrinsic 23 kDa protein associated with PSII membranes.     

Isolation and characterization of the 10-kDa and 22-kDa polypeptides of higher plant photosystem 2

Two polypeptides of 10 kDa and 22 kDa, shown to be components of the higher plant photosystem 2, were purified and examined. A NaCl/Triton X-100 treatment was designed, which released these two polypeptides from the thylakoid membrane, in concert with the extrinsic 16-kDa and 23-kDa proteins, concomitant with a loss in oxygen-evolution activity. After this treatment the oxygen-evolving activity of the photosystem 2 membranes devoid of the 10-kDa and the 22-kDa polypeptides could be restored with CaCl2, but not by readdition of the purified 23-kDa protein. This deficiency was caused by an inability of the 23-kDa protein to rebind to the photosystem 2 membranes. In analogy, the oxygen-evolution activity of a highly purified photosystem 2 core preparation, devoid of the 10-kDa and 22-kDa polypeptides, was stimulated by CaCl2, but not by the 23-kDa protein. We, therefore, suggest that the 10-kDa or the 22-kDa polypeptides provide a binding-site for the extrinsic 23-kDa protein to the thylakoid membrane. The 10-kDa and 22-kDa polypeptides were isolated through ion-exchange chromatography in the presence of detergents. They both displayed hydrophobic properties, verified by their low proportion of polar amino acid residues and their partition to the hydrophobic phase during Triton X-114 fractionation. The purified polypeptides did not contain metallic cofactors or substances with absorption in the visible region of the spectrum.     

Stoichiometric association of extrinsic cytochrome c550 and 12 kDa protein with a highly purified oxygen-evolving photosystem II core complex from Synechococcus vulcanus.

A highly purified, native photosystem II (PS II) core complex was isolated from thylakoids of Synechococcus vulcanus, a thermophilic cyanobacterium by lauryldimethylamine N-oxide (LDAO) and dodecyl beta-D-maltoside solubilization. This native PS II core complex contained, in addition to the proteins that have been well characterized in the core complex previously purified by LDAO and Triton X-100, two more extrinsic proteins with apparent molecular weights of 17 and 12 kDa. These two proteins were associated with the core complex in stoichiometric amounts and could be released by treatment with 1 M CaCl2 or 1 M alkaline Tris but not by 2 M NaCl or low-glycerol treatment, indicating that they are the real components of PS II of this cyanobacterium. N-Terminal sequencing revealed that the 17 and 12 kDa proteins correspond to the apoprotein of cytochrome c550, a low potential c-type cytochrome, and the 9 kDa extrinsic protein previously found in a partially purified PS II preparation from Phormidium laminosum, respectively. In spite of retention of these two extrinsic proteins, no homologues of higher plant 23 and 17 kDa extrinsic proteins could be detected in this cyanobacterial PS II core complex.     

Structural organization of proteins on the oxidizing side of photosystem II. Two molecules of the 33-kDa manganese-stabilizing proteins per reaction center.

The 33-kDa manganese-stabilizing protein stabilizes the manganese cluster in the oxygen-evolving complex. There has been, however, a considerable amount of controversy concerning the stoichiometry of this photosystem II (PS II) component. In this paper, we have verified the extinction coefficient of the manganese-stabilizing protein by amino acid analysis, determined the manganese content of oxygen-evolving photosystem II membranes and reaction center complex using inductively coupled plasma spectrometry, and determined immunologically the amount of the manganese-stabilizing protein associated with photosystem II. Oxygen-evolving photosystem II membranes and reaction center complex preparations contained 258 +/- 11 and 67 +/- 3 chlorophyll, respectively, per tetranuclear manganese cluster. Immunoquantification of the manganese-stabilizing protein using mouse polyclonal antibodies on "Western blots" demonstrated the presence of 2.1 +/- 0.2 and 2.0 +/- 0.3 molecules of the manganese-stabilizing protein/tetranuclear manganese cluster in oxygen-evolving PS II membranes and highly purified PS II reaction center complex, respectively. Since the manganese-stabilizing protein co-migrated with the D2 protein in our electrophoretic system, accurate immunoquantification required the inclusion of CaCl2-washed PS II membrane proteins or reaction center complex proteins in the manganese-stabilizing protein standards to compensate for the possible masking effect of the D2 protein on the binding of the manganese-stabilizing protein to Immobilon-P membranes. Failure to include these additional protein components in the manganese-stabilizing protein standards leads to a marked underestimation of the amount of the manganese-stabilizing protein associated with these photosystem II preparations.     

Identification of domains on the extrinsic 23 kDa protein possibly involved in electrostatic interaction with the extrinsic 33 kDa protein in spinach photosystem II.

To elucidate the domains on the extrinsic 23 kDa protein involved in electrostatic interaction with the extrinsic 33 kDa protein in spinach photosystem II, we modified amino or carboxyl groups of the 23 kDa protein to uncharged methyl ester groups with N-succinimidyl propionate or glycine methyl ester in the presence of a water-soluble carbodiimide, respectively. The N-succinimidyl propionate-modified 23 kDa protein did not bind to the 33 kDa protein associated with PSII membranes, whereas the glycine methyl ester-modified 23 kDa protein completely bound. This indicates that positive charges on the 23 kDa protein are important for electrostatic interaction with the 33 kDa protein associated with the PSII membranes. Mapping of the N-succinimidyl propionate-modified sites of the 23 kDa protein was performed using Staphylococcus V8 protease digestion of the modified protein followed by determination of the mass of the resultant peptide fragments with MALDI-TOF MS. The results showed that six domains (Lys11-Lys14, Lys27-Lys38, Lys40, Lys90-Lys96, Lys143-Lys152, Lys166-Lys174) were modified with N-succinimidyl propionate. In these domains, Lys11, Lys13, Lys33, Lys38, Lys143, Lys166, Lys170 and Lys174 were wholly conserved in the 23 kDa protein from 12 species of higher plants. These positively charged lysyl residues on the 23 kDa protein may be involved in electrostatic interactions with the negatively charged carboxyl groups on the 33 kDa protein, the latter has been suggested to be important for the 23 kDa binding [Bricker, T.M. & Frankel, L.K. (2003) Biochemistry42, 2056-2061].     

A rare protein fluorescence behavior where the emission is dominated by tyrosine: case of the 33-kDa protein from spinach photosystem II

An abnormal fluorescence emission of protein was observed in the 33-kDa protein which is one component of the three extrinsic proteins in spinach photosystem II particle (PS II). This protein contains one tryptophan and eight tyrosine residues, belonging to a "B type protein". It was found that the 33-kDa protein fluorescence is very different from most B type proteins containing both tryptophan and tyrosine residues. For most B type proteins studied so far, the fluorescence emission is dominated by the tryptophan emission, with the tyrosine emission hardly being detected when excited at 280 nm. However, for the present 33-kDa protein, both tyrosine and tryptophan fluorescence emissions were observed, the fluorescence emission being dominated by the tyrosine residue emission upon a 280 nm excitation. The maximum emission wavelength of the 33-kDa protein tryptophan fluorescence was at 317 nm, indicating that the single tryptophan residue is buried in a very strong hydrophobic region. Such a strong hydrophobic environment is rarely observed in proteins when using tryptophan fluorescence experiments. All parameters of the protein tryptophan fluorescence such as quantum yield, fluorescence decay, and absorption spectrum including the fourth derivative spectrum were explored both in the native and pressure-denatured forms.     

Cooperation of Photosynthetic Reaction Center of Photosystem II under Weak Light as Shown by Light Intensity-dependence of the Half-effective Dose of Atrazine

The binding constant (K) and number of binding sites (N) of atrazine to isolated photosystem (PS) II membranes were measured with an apparent correlation between N and the activity of oxygen evolution. Upon the addition of an electron acceptor, N became equal to the total number of the population of PS II reaction centers irrespective of having oxygen-evolving activity, about 4 mmol per mole of chlorophyll, with a concomitant decline of K from 1.32 (±0.34) × 10⁷ M^–1 to 4.09 (±0.40) × 10⁶ M^–1 . NH₂OH and NaCl treatments, which inactivate oxygen evolution, affected neither the binding to PS II membranes of the extrinsic 33-kDa protein or of atrazine. The atrazine binding sites that are latent in CaCl₂-treated PS II membranes was partially restored by the reconstitution of the membranes with isolated extrinsic 33-kDa protein. An oxidizing system involving the 33-kDa protein may provide a suitable structure of PS II reaction center complex for atrazine binding. The level of inhibition of oxygen-evolving activity by atrazine under the saturating intensity of light parallels the fraction of the photosystem (PS) II reaction center with the quinone-binding site blocked by atrazine. In contrast, under a rate-limiting intensity of light, percents of remaining oxygen-evolving activity after the addition of atrazine correlated with the 1.33th power of the fraction of atrazine-free binding sites. Inhibition of PS II complexes more than one that bound with atrazine suggests a cooperation between PS II complexes to evolve oxygen under weak light intensity.     

Interaction of CPa-1 with the manganese-stabilizing protein of photosystem II: identification of domains cross-linked by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide.

The structural organization of photosystem II proteins has been investigated by use of the zero-length protein cross-linking reagent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and monoclonal and polyclonal antibody reagents. Photosystem II membranes were treated with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide which cross-links amino groups to carboxyl groups which are in van der Waals contact. This treatment did not affect the oxygen evolution rates of these membranes and increased the retention of oxygen evolution after CaCl2 washing. Analysis of the proteins cross-linked by this treatment indicated that two cross-linked species with apparent molecular masses of 95 and 110 kDa were formed which cross-reacted with antibodies against both the 33-kDa manganese-stabilizing protein and the chlorophyll protein CPa-1. Cleavage of the 110-kDa cross-linked species with cyanogen bromide followed by N-terminal sequence analysis was used to identify the peptide fragments of CPa-1 and the manganese-stabilizing protein which were cross-linked. Two cyanogen bromide fragments were identified with apparent molecular masses of 50 and 25 kDa. N-Terminal sequence analysis of the 50-kDa cyanogen bromide fragment indicates that this consists of the C-terminal 16.7-kDa fragment of CPa-1 and the intact manganese-stabilizing protein. This strongly suggests that the manganese-stabilizing protein is cross-linked to the large extrinsic loop domain of CPa-1. N-Terminal analysis of the 25-kDa cyanogen bromide fragment indicates that this consists of the C-terminal 16.7-kDa peptide of CPa-1 and the N-terminal 8-kDa peptide of the manganese-stabilizing protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photoactivation of Oxygen-Evolving Enzyme in Dark-Grown Pine Cotyledons: Relationship between Assembly of Photosystem II Proteins and Integration of Manganese and Calcium

Dark-grown cotyledons of pine (Pinus thunbergit) did not exhibitO₂ evolution, but this capability was rapidly activated by illuminationfor a short period (photoactivation). To examine the biochemicalchanges which accompany the process of photoactivation in gymnosperms,a method enabling the preparation of highly active O₂-evolvingphotosystem II (PS II) membranes was applied to light-grown,dark-grown, and photoactivated cotyledons. PS II membranes preparedfrom light-grown cotyledons exhibited high O₂-evolving activity,and contained all the intrinsic proteins as well as the threeextrinsic proteins (32, 23 and 17 kDa) associated with PS II.These membranes were also found to contain 4.4 Mn and 0.83 Ca/PSII reaction center. PS II membranes from dark-grown cotyledonscontained all the intrinsic proteins, but preserved only 32kDa extrinsic protein, and zero Mn and 0.85 Ca/PS II reactioncenter. The two extrinsic proteins (23 and 17 kDa) absent inthe PS II membranes from dark-grown cotyledons were, however,present as mature forms in whole thylakoid membranes from thecorresponding sample. The PS II membranes isolated from photoactivatedcotyledons showed a high activity of O₂ evolution and retainedthe three extrinsic proteins, 5.3 Mn and 1.1 Ca/PS II reactioncenter, respectively. The results indicated that Mn and thetwo extrinsic proteins were tightly integrated in the O₂-evolvingapparatusduring the process of photoactivation but integration of Capreceded the integration of Mn by photoactivation. (Received December 9, 1991; Accepted February 1, 1992)     

Carboxylate groups on the manganese-stabilizing protein are required for efficient binding of the 24 kDa extrinsic protein to photosystem II

The effects of the modification of carboxylate groups on the manganese-stabilizing protein on the binding of the 24 kDa extrinsic protein to Photosystem II were investigated. Carboxylate groups on the manganese-stabilizing protein were modified with glycine methyl ester in a reaction facilitated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The manganese-stabilizing protein which was modified while associated with NaCl-washed membranes could bind to calcium chloride-washed PS II membranes and reconstitute oxygen evolution in a manner similar to that observed for unmodified manganese-stabilizing protein (Frankel, L.K, Cruz, J. C. and Bricker, T. M. (1999) Biochemistry 38, 14271-14278). However, PS II membranes reconstituted with this modified protein were defective in their ability to bind the extrinsic 24 kDa protein of Photosystem II. Mapping of the sites of modification was carried out by trypsin and Staphylococcus V8 protease digestion of the modified protein and analysis by MALDI mass spectrometry. These studies indicated that the domains (1)E-(71)D, (97)D-(144)D, and (180)D-(187)E are labeled when the manganese-stabilizing protein is bound to NaCl-washed Photosystem II membranes. We hypothesize that modified carboxylates, possibly residues (1)E, (32)E, (139)E, and/or (187)E, in these domains are responsible for the altered binding affinity of the 24 kDa protein observed.     

Isolation of the D1 Protein and a 28-kDa Fragment of the D2 Protein from Spinach Photosystem II Membranes

The 32-kDa D1 protein, which contained no lysine in spinachchloroplasts, as deduced from its DNA code, was isolated byhigh-performance gel permeation chromatography in the presenceof 0.1% SDS and 4 M urea. Three proteins of the photosystemII reaction center complex have a molecular mass of 30–35kDa, and two, the D2 protein and the peripheral 33-kDa protein,were severed into peptide fragments by Achromobacter lysyl endopeptidase(EC 3.4.14.50 [EC] ) before the chromatography. The isolated D1 proteindid not contain chlorophylls and pheophytins but had an absorptionmaximum at 265 nm probably due to bound plastoquinone. A peptidefragment of 28 kDa from the D2 protein was also isolated fromspinach photosystem II membranes and the wheat photosystem IIreaction center. Antibodies raised against the 28-kDa peptidefrom wheat bound to the 34-kDa D2 protein, which suggested thatthis peptide was the largest sequence of Aspl4-Lys265. The fragmentof wheat D2 protein showed absorption maxima at 413 and 682nm attributable to bound pheophytin that probably had been convertedfrom chlorophyll a during the isolation process. (Received June 29, 1987; Accepted October 21, 1987)     

Low pH-Induced Dissociation of Three Extrinsic Proteins from O2-Evolving Photosystem II

Three extrinsic proteins involved in oxygen evolution reversiblydissociated from Photo-system II (PS II) membranes at acidicpHs showing distinctly different pH dependencies. The pHs forhalf dissociation of 17, 23 and 33 kDa extrinsic proteins weredetermined to be 5.0, 4.1 and 3.6, respectively. The half dissociationpHs of 17 and 23 kDa proteins were much lower than their respectiveisoelectric points, while that for 33 kDa protein was closeto its isoelectric point. It was suggested that protonationof the negatively charged binding domain on membrane proteinscauses dissociation of the former two extrinsic proteins, whereasprotonation of the extrinsic protein itself is responsible forthe dissociation of 33 kDa protein. Based on these, featuresof low pH-induced dissociation of extrinsic proteins and Mnfrom PS II were discussed. (Received September 25, 1990; Accepted February 13, 1991)     

The manganese-stabilizing protein is required for photosystem II assembly/stability and photoautotrophy in higher plants

Interfering RNA was used to suppress the expression of two genes that encode the manganese-stabilizing protein of photosystem II in Arabidopsis thaliana, MSP-1 (encoded by psbO-1, At5g66570), and MSP-2 (encoded by psbO-2, At3g50820). A phenotypic series of transgenic plants was recovered that expressed high, intermediate, and low amounts of these two manganese-stabilizing proteins. Chlorophyll fluorescence induction and decay analyses were performed. Decreasing amounts of expressed protein led to the progressive loss of variable fluorescence and a marked decrease in the fluorescence quantum yield (F(v)/F(m)) in both the absence and the presence of dichloromethylurea. This result indicated that the amount of functional photosystem II reaction centers was compromised in the plants that exhibited intermediate and low amounts of the manganese-stabilizing proteins. An analysis of the decay of the variable fluorescence in the presence of dichlorophenyldimethylurea indicated that charge recombination between Q ((A-)) and the S(2) state of the oxygen-evolving complex was seriously retarded in the plants that expressed low amounts of the manganese stabilizing proteins. This may have indicated a stabilization of the S(2) state in the absence of the extrinsic component. Immunological analysis of the photosystem II protein complement indicated that significant losses of the CP47, CP43, and D1 proteins occurred upon the loss of the manganese-stabilizing proteins. This indicated that these extrinsic proteins were required for photosystem II core assembly/stability. Additionally, although the quantity of the 24-kDa extrinsic protein was only modestly affected by the loss of the manganese-stabilizing proteins, the 17-kDa extrinsic protein dramatically decreased. The control proteins ribulose bisphosphate carboxylase and cytochrome f were not affected by the loss of the manganese-stabilizing proteins; the photosystem I PsaB protein, however, was significantly reduced in the low expressing transgenic plants. Finally, it was determined that the transgenic plants that expressed low amounts of the manganese-stabilizing proteins could not grow photoautotrophically.     

The 33 kDa protein can be removed without affecting the association of the 23 and 17 kDa proteins with the luminal side of PS II of spinach

An earlier study shows that a 30 min incubation of spinach PS II submembrane fragments at pH 6.3 in the presence of 10 microM HgCl(2) induces a 40% depletion of the 33 kDa protein without the apparent release of the 17 and 23 kDa proteins [Bernier, M., and Carpentier, R. (1995) FEBS Lett. 360, 251-254]. Here we report that the photosystem II 33 kDa extrinsic protein is fully removed by HgCl(2) added at micromolar and higher concentrations (0.25, 20, and 50 microM), with the 17 and 23 kDa extrinsic proteins and other intrinsic proteins remaining bound to the reaction center. The data presented here put in doubt the "regulatory cap" model of PS II, which follows the OEC-33 kDa-23 kDa-17 kDa binding order, as these results directly demonstrate that the 33 kDa protein can be removed without affecting the binding of the 23 and 17 kDa proteins to the intrinsic subunits of PS II. This suggests that each extrinsic protein may possess its own binding site on PS II. A possible mechanism for HgCl(2) upon the release of the 33 kDa protein is discussed.     

Light-dependent accumulation and localization of photosystem II proteins in maize

We have raised antibodies against several major components of photosystem II. These antisera, which are directed against the apoproteins of two chlorophyll-binding proteins (CPa-1 and CPa-2), the apoprotein of light-harvesting complex II and the 33-kDa extrinsic protein of the oxygen-evolving complex, were used to examine the light regulation of photosystem II assembly in maize. The principal findings of this study are as follows. The 33-kDa protein is present in dark-grown maize and the content increases 5-10-fold upon illumination. The level of the protein is mediated at least in part by phytochrome and is independent of the accumulation of chlorophyll. In contrast, none of the three chlorophyll-binding proteins examined was detectable in leaves of maize grown in darkness or under other light regimes where chlorophyll does not accumulate. Even in the absence of photosystem II assembly, the 33-kDa protein is properly transported across the thylakoid into the lumen. However, the protein does not attach in the normal way to the inner surface of the membrane under these conditions.     

Site-directed mutagenesis of glutamate residues in the large extrinsic loop of the photosystem II protein CP 43 affects oxygen-evolving activity and PS II assembly

The psbC gene encodes the intrinsic chlorophyll protein CP 43, a component of photosystem II in higher plants, green algae, and cyanobacteria. Oligonucleotide-directed mutagenesis was used to introduce mutations into the portion of psbC that encodes the large extrinsic loop E of CP 43 in the cyanobacterium Synechocystis 6803. Three mutations, E293Q, E339Q, and E352Q, each produced a strain with impaired photosystem II activity. The E293Q mutant strain grew photoautotrophically at rates comparable to the control strain. Immunological analyses of several PS II components indicated that this mutant accumulated normal quantities of PS II proteins. However, this mutant evolved oxygen to only 56% of control rates at saturating light intensities. Measurements of total variable fluorescence yield indicated that this mutant assembled approximately 60% of the fully functional PS II centers found in the control strain. The E339Q mutant grew photoautotrophically at a severely reduced rate. Both immunological analysis and variable fluorescence yield experiments indicated that E339Q assembled a normal complement of PS II centers. However, this mutant was capable of evolving oxygen to only 20% of control rates. Variable fluorescence yield experiments demonstrated that this mutant was inefficient at using water as an electron donor. Both E293Q and E339Q strains exhibited an increased (approximately 2-fold) sensitivity to photoinactivation. The E352Q mutant was the most severely affected. This mutant failed to grow photoautotrophically and exhibited essentially no capacity for oxygen evolution. Measurements of total variable fluorescence yield indicated that this mutant assembled no functional PS II centers. Immunological analysis of isolated thylakoid membranes from E352Q revealed a complete absence of CP 43 and reduced levels of both the D1 and manganese-stabilizing proteins. These results suggest that the mutations E293Q and E339Q each produce a defect associated with the oxygen-evolving complex of photosystem II. The E352Q mutation appears to affect the stability of the PS II complex. This is the first report showing that alteration of negatively charged residues in the CP 43 large extrinsic loop results in mutations affecting PS II assembly/function.