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Evidence that D1 Processing Is Required for Manganese Binding and Extrinsic Protein Assembly into Photosystem II

2004; Elsevier BV; Volume: 279; Issue: 44 Linguagem: Inglês

10.1074/jbc.m408458200

ISSN

1083-351X

Autores

Johnna L. Roose, Himadri B. Pakrasi,

Tópico(s)

Enzyme Structure and Function

Resumo

Photosystem II (PSII) is a large membrane protein complex that catalyzes oxidation of water to molecular oxygen. During its normal function, PSII is damaged and frequently turned over. The maturation of the D1 protein, a key component in PSII, is a critical step in PSII biogenesis. The precursor form of D1 (pD1) contains a C-terminal extension, which is removed by the protease CtpA to yield PSII complexes with oxygen evolution activity. To determine the temporal position of D1 processing in the PSII assembly pathway, PSII complexes containing only pD1 were isolated from a CtpA-deficient strain of the cyanobacterium Synechocystis 6803. Although membranes from the mutant cell had nearly 50% manganese, no manganese was detected in isolated ΔctpAHT3 PSII, indicating a severely decreased manganese affinity. However, chlorophyll fluorescence decay kinetics after a single saturating flash suggested that the donor YZ was accessible to exogenous Mn2+ ions. Furthermore, the extrinsic proteins PsbO, PsbU, and PsbV were not present in PSII isolated from this mutant. However, PsbO and PsbV were present in mutant membranes, but the amount of PsbV protein was consistently less in the mutant membranes compared with the control membranes. We conclude that D1 processing precedes manganese binding and assembly of the extrinsic proteins into PSII. Interestingly, the Psb27 protein was found to be more abundant in ΔctpAHT3 PSII than in HT3 PSII, suggesting a possible role of Psb27 as an assembly factor during PSII biogenesis. Photosystem II (PSII) is a large membrane protein complex that catalyzes oxidation of water to molecular oxygen. During its normal function, PSII is damaged and frequently turned over. The maturation of the D1 protein, a key component in PSII, is a critical step in PSII biogenesis. The precursor form of D1 (pD1) contains a C-terminal extension, which is removed by the protease CtpA to yield PSII complexes with oxygen evolution activity. To determine the temporal position of D1 processing in the PSII assembly pathway, PSII complexes containing only pD1 were isolated from a CtpA-deficient strain of the cyanobacterium Synechocystis 6803. Although membranes from the mutant cell had nearly 50% manganese, no manganese was detected in isolated ΔctpAHT3 PSII, indicating a severely decreased manganese affinity. However, chlorophyll fluorescence decay kinetics after a single saturating flash suggested that the donor YZ was accessible to exogenous Mn2+ ions. Furthermore, the extrinsic proteins PsbO, PsbU, and PsbV were not present in PSII isolated from this mutant. However, PsbO and PsbV were present in mutant membranes, but the amount of PsbV protein was consistently less in the mutant membranes compared with the control membranes. We conclude that D1 processing precedes manganese binding and assembly of the extrinsic proteins into PSII. Interestingly, the Psb27 protein was found to be more abundant in ΔctpAHT3 PSII than in HT3 PSII, suggesting a possible role of Psb27 as an assembly factor during PSII biogenesis. Photosystem II (PSII), 1The abbreviations used are: PSII, photosystem II; pD1, precursor D1; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; F, fluorescence; Chl, chlorophyll; MES, 4-morpholineethanesulfonic acid; MALDI, matrix-assisted laser desorption ionization; MALDI-MS, MALDI mass spectrometry.1The abbreviations used are: PSII, photosystem II; pD1, precursor D1; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; F, fluorescence; Chl, chlorophyll; MES, 4-morpholineethanesulfonic acid; MALDI, matrix-assisted laser desorption ionization; MALDI-MS, MALDI mass spectrometry. a multisubunit protein complex localized to the thylakoid membranes of cyanobacteria and chloroplasts, performs a light-driven electron transfer from water to plastoquinones, generating molecular oxygen as a byproduct (1Goussias C. Boussac A. Rutherford A.W. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2002; 357 (1419–1320): 1369-1381Crossref PubMed Scopus (145) Google Scholar). Cyanobacterial PSII consists of more than 20 subunits including both membrane-integral and extrinsically associated proteins (2Kashino Y. Lauber W.M. Carroll J.A. Wang Q. Whitmarsh J. Satoh K. Pakrasi H.B. Biochemistry. 2002; 41: 8004-8012Crossref PubMed Scopus (278) Google Scholar, 3Pakrasi H.B. Annu. Rev. Genet. 1995; 29: 755-776Crossref PubMed Scopus (72) Google Scholar). In addition to its protein components, PSII also has many associated cofactors including chlorophylls, pheophytins, plastoquinones, manganese, non-heme iron, calcium, and chloride atoms as well as two heme groups (2Kashino Y. Lauber W.M. Carroll J.A. Wang Q. Whitmarsh J. Satoh K. Pakrasi H.B. Biochemistry. 2002; 41: 8004-8012Crossref PubMed Scopus (278) Google Scholar, 3Pakrasi H.B. Annu. Rev. Genet. 1995; 29: 755-776Crossref PubMed Scopus (72) Google Scholar). Recent structural studies (4Zouni A. Witt H.T. Kern J. Fromme P. Krauss N. Saenger W. Orth P. Nature. 2001; 409: 739-743Crossref PubMed Scopus (1754) Google Scholar, 5Kamiya N. Shen J.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 98-103Crossref PubMed Scopus (991) Google Scholar, 6Ferreira K.N. Iverson T.M. Maghlaoui K. Barber J. Iwata S. Science. 2004; 303: 1831-1838Crossref PubMed Scopus (2823) Google Scholar) have greatly advanced our knowledge of the arrangement of the components within the functional complex. In particular, new details on the structure of the tetra-manganese-calcium cluster of the oxygen-evolving complex have given key insights into the mechanism of the water oxidation reaction (6Ferreira K.N. Iverson T.M. Maghlaoui K. Barber J. Iwata S. Science. 2004; 303: 1831-1838Crossref PubMed Scopus (2823) Google Scholar).Despite these advances, these static structures are not adequate to understand the dynamic nature of the assembly and turn-over of the PSII complex. The structural complexity of PSII requires precise and regulated assembly, yet the PSII biogenesis pathway is poorly understood and many questions still remain as to how the components are assembled into a functional PSII complex. Furthermore, PSII assembly occurs frequently, because the PSII complex is rapidly turned over even under normal conditions (7Keren N. Berg A. van Kan P.J. Levanon H. Ohad I.I. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1579-1584Crossref PubMed Scopus (244) Google Scholar). As a consequence of the electron transfer reactions, the D1 protein is irreversibly damaged, removed from the PSII complex, and replaced with a newly synthesized D1 protein (8Andersson B. Aro E.M. Andersson B. Regulation of Photosynthesis. Kluwer Academic Publishers, The Netherlands2001: 377-393Google Scholar). Thus, PSII complexes can be generated from either newly synthesized components or from partially disassembled complexes.During PSII biogenesis, several events define the assembly of the oxygen-evolving complex, namely D1 processing, manganese cluster assembly, and extrinsic protein association. The D1 protein is synthesized in a precursor form (pD1) containing a C-terminal extension of 16 amino acids in cyanobacteria (9Nixon P.J. Trost J.T. Diner B.A. Biochemistry. 1992; 31: 10859-10871Crossref PubMed Scopus (194) Google Scholar) and 8–9 amino acids in eukaryotes (10Takahashi Y. Nakane H. Kojima H. Satoh K. Plant Cell Physiol. 1990; 31: 273-280Google Scholar, 11Takahashi M. Shiraishi T. Asada K. FEBS Lett. 1988; 240: 6-8Crossref PubMed Scopus (66) Google Scholar). The pD1 protein is co-translationally inserted into the thylakoid membrane, it associates with other PSII membrane components, and is cleaved after Ala-344 by CtpA, a C-terminal processing protease, to yield the mature D1 protein (12Diner B.A. Ries D.F. Cohen B.N. Metz J.G. J. Biol. Chem. 1988; 263: 8972-8980Abstract Full Text PDF PubMed Google Scholar, 13Anbudurai P.R. Mor T.S. Ohad I. Shestakov S.V. Pakrasi H.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8082-8086Crossref PubMed Scopus (137) Google Scholar). In addition, the inorganic cluster of manganese and calcium is assembled and several extrinsic proteins (PsbO, PsbU, and PsbV) associate with the luminal side of the complex. However, the temporal order of these events during PSII assembly remains poorly understood.Despite numerous studies with D1 processing mutants, it has been difficult to characterize PSII complexes from these mutants with respect to their manganese content and polypeptide compositions. Studies of D1 processing mutants have revealed that processing of pD1 is essential for the assembly of a functional manganese cluster. However, the exact number of manganese atoms associated with pD1-containing PSII complexes has been the subject of debate (12Diner B.A. Ries D.F. Cohen B.N. Metz J.G. J. Biol. Chem. 1988; 263: 8972-8980Abstract Full Text PDF PubMed Google Scholar, 13Anbudurai P.R. Mor T.S. Ohad I. Shestakov S.V. Pakrasi H.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8082-8086Crossref PubMed Scopus (137) Google Scholar, 14Trost J.T. Chisholm D.A. Jordan D.B. Diner B.A. J. Biol. Chem. 1997; 272: 20348-20356Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 15Metz J.G. Pakrasi H.B. Seibert M. Arntzen C.J. FEBS Letters. 1986; 205: 269-274Crossref Scopus (102) Google Scholar). Manganese measurements of membranes of the Scenedesmus obliquus LF1 mutant suggests that pD1-containing PSII complexes have only 1–2 manganese/PSII (16Metz J. Bishop N.I. Biochem. Biophys. Res. Commun. 1980; 94: 560-566Crossref PubMed Scopus (56) Google Scholar). EPR studies of the LF1 mutant revealed the lack of the S2 multiline signal suggesting that the LF1 mutant lacks the redox active manganese cluster of the oxygen-evolving complex (17Rutherford A. Seibert M. Metz J. Biochim. Biophys. Acta. 1988; 932: 171-176Crossref Scopus (25) Google Scholar). Analysis of thylakoid membranes from this mutant showed reduced binding of the 23-kDa protein, PsbP, and loss of the 17-kDa protein, PsbQ, suggesting that the C-terminal extension in pD1 interferes with the association of these extrinsic proteins (18Metz J.G. Bricker T.M. Seibert M. FEBS Lett. 1985; 185: 191-196Crossref Scopus (31) Google Scholar). Additionally, it has been suggested that the extrinsic PsbO protein binds with lower affinity to PSII in the D1 mutant S345P in Synechocystis 6803 in which pD1 is not processed (19Chu H.A. Nguyen A.P. Debus R.J. Biochemistry. 1994; 33: 6150-6157Crossref PubMed Scopus (43) Google Scholar).Recent structural studies also emphasize close spatial relationship of the D1 C terminus, the manganese cluster, and the extrinsic proteins. Based on the 3.7-Å structure PSII crystal structure from Thermosynechococcus vulcanus, the C-terminal oxygen in the carboxylate of the mature D1 protein at Ala-344 can be a direct ligand to one of the manganese atoms (5Kamiya N. Shen J.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 98-103Crossref PubMed Scopus (991) Google Scholar). However, a more recent crystal structure from Thermosynechococcus elongatus at a 3.5 Å resolution has placed the D1 C terminus near the Ca2+ ion of the manganese-calcium cluster but did not include it as a ligand (6Ferreira K.N. Iverson T.M. Maghlaoui K. Barber J. Iwata S. Science. 2004; 303: 1831-1838Crossref PubMed Scopus (2823) Google Scholar). Furthermore, the D1 C terminus makes numerous contacts with the three extrinsic proteins, PsbO, PsbU, and PsbV (6Ferreira K.N. Iverson T.M. Maghlaoui K. Barber J. Iwata S. Science. 2004; 303: 1831-1838Crossref PubMed Scopus (2823) Google Scholar). However, it is difficult to extrapolate a temporal sequence of events from these structural details.To investigate the temporal position of D1 processing in the PSII assembly pathway, we have isolated PSII complexes containing only pD1 from a CtpA-deficient strain of Synechocystis 6803. We were unable to detect any manganese atoms associated with these PSII complexes, indicating that D1 processing is required for any stable manganese interaction. The extrinsic polypeptides of PSII, PsbO, PsbU, and PsbV were also absent, suggesting that the C-terminal extension of pD1 precludes their binding. Finally, one protein, Psb27, was found to be significantly more abundant in such PSII complexes.MATERIALS AND METHODSBacterial Strains and Culture Conditions—Synechocystis 6803 cultures were grown at 30 °C under 30 μmol of photons m–2 s–1 of white light in TES-buffered BG-11 medium (20Allen M.M. J. Phycol. 1968; 4: 1-4Crossref PubMed Scopus (1002) Google Scholar) with air bubbling. The HT3 strain of Synechocystis 6803, which has a hexahistidine tag at the C-terminal end of the CP47 protein (21Bricker T.M. Morvant J. Masri N. Sutton H.M. Frankel L.K. Biochim. Biophys. Acta. 1998; 1409: 50-57Crossref PubMed Scopus (97) Google Scholar) was a generous gift from Prof. T. M. Bricker (Louisiana State University, Baton Rouge, LA). Growth medium for the ΔctpA cells was supplemented with 5 mm glucose, 10 μm 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and 3 μg/ml erythromycin, whereas growth medium for the ΔctpAHT3 cells was supplemented with 5 mm glucose, 10 μm DCMU, 5 μg/ml kanamycin, and 3 μg/ml erythromycin. Because the ΔctpA and ΔctpAHT3 cells are light-sensitive, they were cultured at a lower light intensity by wrapping culture bottles in Kimwipes.Mutant Construction—The ΔctpA deletion construct was prepared as follows. A 6-kb BamHI/HindIII fragment of Synechocystis 6803 DNA containing the ctpA locus was cloned into a pUC19 vector lacking an EcoRI site (pSL794). The ctpA locus within pSL794 (1.4 kb), which is flanked on either side by EcoRI sites, was replaced with a 1.5-kb EcoRI fragment containing an erythromycin resistance marker (pSL795). The double mutant ΔctpAHT3 was constructed by transforming HT3 cells with the ΔctpA construct. Complete segregation of the ΔctpA mutation was determined by PCR using the following primers, 5′-AAGTCCATGCTGTGGAAGC-3′ and 5′-GGATGCCTTTACTTATGGC-3′.Fluorescence Measurements—Fluorescence measurements were conducted to test the segregation of the ΔctpA mutation by verifying the low fluorescent ΔctpA phenotype. Briefly, HT3, ΔctpA, and ΔctpAHT3 cells were grown in BG11 medium supplemented with 5 mm glucose in 12-well microtiter plates. Fo and Kautsky fluorescence induction were measured on a FluorCam 690M (Photon Systems Instruments, Brno, Czech Republic). The average Fo was calculated from a 3-s measurement before actinic illumination. Samples were then subjected to continuous actinic light (300 μmol of photons m–2 s–1) at 30% intensity for 10 s and fluorescence (F) was monitored by 33-μs measuring flashes every 40 ms. Fluorescence traces were normalized to Fo to compare the signals from different strains.Chlorophyll fluorescence decay after a single saturating flash was measured using an FL100 fluorometer (Photon Systems Instruments, Brno, Czech Republic). PSII samples were diluted to 2 μg of Chl/ml in resuspension buffer (50 mm MES-NaOH, pH 6.0, 10 mm MgCl2, 5 mm CaCl2, and 25% glycerol). Samples were incubated under ambient light for 10 min with and without the addition of 1 mm MnCl2 followed by an incubation in darkness for 10 min. DCMU was added to a final concentration of 10 μm just prior to measurement. The average Fo was calculated as the average signal after four measuring flashes from the dark-adapted sample. Samples were subjected to a 30-μs saturating actinic flash to give Fm followed by a series of 3-μs measuring flashes to follow F over a 1-s measurement period. The fluorescence signals were normalized as (F – Fo)/(Fm – Fo).Isolation of PSII—PSII preparations from the HT3 and ΔctpAHT3 strains were isolated according to Ref. 2Kashino Y. Lauber W.M. Carroll J.A. Wang Q. Whitmarsh J. Satoh K. Pakrasi H.B. Biochemistry. 2002; 41: 8004-8012Crossref PubMed Scopus (278) Google Scholar.Determination of Manganese Content—The concentrations of manganese were measured on an AA600 atomic absorption spectrophotometer (PerkinElmer Life Sciences). Membranes samples were prepared as follows. Cells were harvested and washed with 20 mm HEPES, pH 7.8, 5 mm EDTA buffer according to Ref. 22Keren N. Kidd M.J. Penner-Hahn J.E. Pakrasi H.B. Biochemistry. 2002; 41: 15085-15092Crossref PubMed Scopus (72) Google Scholar, resuspended in HCMS buffer (50 mm HEPES-NaOH, 5 mm CaCl2, 10 mm MgCl2, and 1 m sucrose, pH 7.8) to a concentration of 500 μg of Chl/ml, and broken with 0.17-mm glass beads. The cell lysate was centrifuged at 3000 × g for 3 min to remove the glass beads and unbroken cells. The resulting supernatant was then centrifuged at 40,000 × g for 20 min to pellet the cell membranes. The membranes were resuspended in HCMS buffer to ∼800 μg of Chl/ml. The membrane samples were diluted to 10 μg of Chl/ml in 80% nitric acid and allowed to digest overnight. The digested membranes were then diluted 1:2 in deionized water for metal analysis. PSII samples were diluted to 5 μg of Chl/ml in deionized water. The manganese:PSII ratio was calculated based on 41 molecules of Chl/PSII (2Kashino Y. Lauber W.M. Carroll J.A. Wang Q. Whitmarsh J. Satoh K. Pakrasi H.B. Biochemistry. 2002; 41: 8004-8012Crossref PubMed Scopus (278) Google Scholar).Protein Detection—PSII samples (7 μg of Chl/lane) were fractionated using the SDS-PAGE system described in Ref. 2Kashino Y. Lauber W.M. Carroll J.A. Wang Q. Whitmarsh J. Satoh K. Pakrasi H.B. Biochemistry. 2002; 41: 8004-8012Crossref PubMed Scopus (278) Google Scholar and visualized with Coomassie Blue staining (Sigma). Membrane samples (5 μg of Chl/lane) were fractionated using a 16% acrylamide, 6 m urea SDS-PAGE system. For immunodetection, fractionated proteins were blotted onto nitrocellulose membranes, and the CP47, D1, D2, and PsbO proteins were identified using the corresponding specific antibodies. The hybridization signals were detected using chemiluminescence reagents (Pierce) and then visualized on a Fujifilm LAS-1000 plus imager (Fujifilm, Stamford, CT). PsbV (cytochrome c550) was detected based on heme staining using chemiluminescence reagents. The identity of the Psb27 band was confirmed by MALDI-MS analysis (see below).MALDI Mass Spectrometry—The protein band corresponding to Psb27 was excised from a Coomassie-stained gel, reduced with 10 mm dithiothreitol for 30 min, alkylated with 55 mm iodoacetamide for 30 min, and digested with trypsin at 37 °C for 12 h. The resulting peptide fragments were analyzed on a MALDI time-of-flight Voyager DE STR instrument (Applied Biosystems, Foster City). The Protein Prospector program was used to identify hits in the Synechocystis 6803 translated open reading frames data base (46Clauser K.R. Baker P.R. Burlingame A.L. Anal. Chem. 1999; 71: 2871-2882Crossref PubMed Scopus (975) Google Scholar).Membrane Association of PSII Proteins—Membranes were partially solubilized as described in Ref. 23Qian M. Al-Khaldi S.F. Putnam-Evans C. Bricker T.M. Burnap R.L. Biochemistry. 1997; 36: 15244-15252Crossref PubMed Scopus (40) Google Scholar with some modifications. Briefly, cells were washed with 20 mm HEPES, pH 7.8, 5 mm EDTA and broken with glass beads. The resulting cell lysate (∼300 μg of Chl/ml) was treated with 0.04% (w/v) of the detergent β-dodecyl maltoside (Amresco) and immediately centrifuged at 60,000 × g for 40 min to pellet the membranes. The supernatant fraction was carefully removed, and the membranes were suspended in HCMS buffer to 800 μg of Chl/ml. Supernatant and membranes samples were analyzed for PSII protein content. The ImageJ program was used to quantify the relative amounts of different proteins in the samples.RESULTSΔctpAHT3 Mutant Construction—To characterize the PSII complexes containing only the pD1 protein, we generated the double mutant ΔctpAHT3 in Synechocystis 6803, in which (a) the CP47 protein has a C-terminal histidine tag to facilitate PSII isolation (21Bricker T.M. Morvant J. Masri N. Sutton H.M. Frankel L.K. Biochim. Biophys. Acta. 1998; 1409: 50-57Crossref PubMed Scopus (97) Google Scholar), and (b) the ctpA gene is deleted resulting in the presence of pD1 in the PSII complex (13Anbudurai P.R. Mor T.S. Ohad I. Shestakov S.V. Pakrasi H.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8082-8086Crossref PubMed Scopus (137) Google Scholar). The ΔctpA construct used for this study differs from that described previously (13Anbudurai P.R. Mor T.S. Ohad I. Shestakov S.V. Pakrasi H.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8082-8086Crossref PubMed Scopus (137) Google Scholar) to be compatible with the kanamycin-resistant HT3 mutation. The ΔctpA and ΔctpAHT3 mutants described in the present work contain a 1.5-kb erythromycin resistance marker at the ctpA locus (Fig. 1A). Complete segregation of the ΔctpA mutation was confirmed by PCR analysis of the ctpA locus (Fig. 1B). The presence of the C-terminal hexahistidine tag at the psbB locus was confirmed by nucleotide sequencing (Fig. 1C). Because a low fluorescence phenotype has been observed for other D1 processing protease mutants (12Diner B.A. Ries D.F. Cohen B.N. Metz J.G. J. Biol. Chem. 1988; 263: 8972-8980Abstract Full Text PDF PubMed Google Scholar, 16Metz J. Bishop N.I. Biochem. Biophys. Res. Commun. 1980; 94: 560-566Crossref PubMed Scopus (56) Google Scholar, 24Shestakov S.V. Anbudurai P.R. Stanbekova G.E. Gadzhiev A. Lind L.K. Pakrasi H.B. J. Biol. Chem. 1994; 269: 19354-19359Abstract Full Text PDF PubMed Google Scholar), the fluorescence properties of the ΔctpA and ΔctpAHT3 mutants were also examined to verify segregation. Fig. 1D shows that the ΔctpA and ΔctpAHT3 mutants have negligible Kautsky fluorescence induction curves compared with that of HT3 cells.Manganese Content of ΔctpAHT3 Membranes and Isolated PSII—Mutants containing a defective D1 processing protease have PSII complexes with no oxygen evolution activity specifically because of the absence of an active manganese cluster (12Diner B.A. Ries D.F. Cohen B.N. Metz J.G. J. Biol. Chem. 1988; 263: 8972-8980Abstract Full Text PDF PubMed Google Scholar, 13Anbudurai P.R. Mor T.S. Ohad I. Shestakov S.V. Pakrasi H.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8082-8086Crossref PubMed Scopus (137) Google Scholar, 25Metz J.G. Wong J. Bishop N.I. FEBS Lett. 1980; 114: 61-66Crossref Scopus (89) Google Scholar). It was previously reported that membranes from the Scenedesmus LF1 mutant had a significant decrease in manganese content. Therefore, it was concluded that the assembly of a functional manganese cluster was not possible in D1 processing mutants, because only 2 of the 4 manganese atoms involved in oxygen evolution activity could bind to PSII (25Metz J.G. Wong J. Bishop N.I. FEBS Lett. 1980; 114: 61-66Crossref Scopus (89) Google Scholar). However, it was unclear whether the manganese content of membranes was representative of the manganese bound to PSII complexes.To resolve the issue of the number of manganese atoms associated with pD1-containing PSII, the manganese content of membranes and isolated PSII samples from HT3 and ΔctpAHT3 cells were measured using atomic absorption spectroscopy. Table I shows that the manganese content of ΔctpAHT3 membranes is indeed ∼50% that of HT3 membranes on a per chlorophyll basis. However, manganese measurements of isolated PSII complexes revealed a different interpretation of the ability of manganese to bind to pD1-containing PSII complexes. As expected, HT3 PSII complexes had a manganese:PSII of 4, but PSII complexes from the ΔctpAHT3 mutant were devoid of any manganese.Table IManganese content of membranes and purified PSII samplesMn/ChlMn/PSIInmol/molHT3 membranes32.3 ± 1.2 (n = 3) (100%)NDΔctpAHT3 membranes16.3 ± 2.5 (n = 3) (50.5%)NDHT3 PSII98.0 ± 4.8 (n = 4) (100%)4.0 ± 0.2 (n = 4)ΔctpAHT3 PSII1.0 ± 0.1 (n = 4) (1.1%)0.04 ± 0.01 (n = 4) Open table in a new tab Another widely used technique to examine the presence of a functional manganese cluster in PSII is to measure the decay of chlorophyll fluorescence after a single saturating flash in the presence of DCMU. Upon illumination, charge separation gives the high fluorescent state (Y +Z P680 Q –A), which decays over time as charge recombination occurs between Q –A and other electron acceptors within PSII. The presence of DCMU prevents normal Q –A to QB electron transfer. Thus, chlorophyll fluorescence decay in the presence of DCMU reflects charge recombination between Q –A and the donor side of PSII. In PSII complexes without a functional manganese cluster, charge recombination occurs between Q –A and Y +Z with a t½ ≥ 1 ms (26Diner B.A. Methods Enzymol. 1998; 297: 337-360Crossref Scopus (23) Google Scholar). However, the addition of exogenous Mn2+ ions can block charge recombination between Q –A and Y +Z by donating electrons directly to Y +Z. The site at which this Mn2+ is bound and oxidized is predicted to be the first binding site during manganese cluster assembly, also referred to as the high affinity manganese binding site (9Nixon P.J. Trost J.T. Diner B.A. Biochemistry. 1992; 31: 10859-10871Crossref PubMed Scopus (194) Google Scholar, 27Nixon P.J. Diner B.A. Biochemistry. 1992; 31: 942-948Crossref PubMed Scopus (224) Google Scholar).Exogenous Mn2+ ions block charge recombination between Q –A and Y +Z over a timescale of 1 s (27Nixon P.J. Diner B.A. Biochemistry. 1992; 31: 942-948Crossref PubMed Scopus (224) Google Scholar). This property has been previously used to monitor the integrity of the high affinity manganese binding site in various D1 mutants (9Nixon P.J. Trost J.T. Diner B.A. Biochemistry. 1992; 31: 10859-10871Crossref PubMed Scopus (194) Google Scholar, 27Nixon P.J. Diner B.A. Biochemistry. 1992; 31: 942-948Crossref PubMed Scopus (224) Google Scholar, 28Boerner R.J. Nguyen A.P. Barry B.A. Debus R.J. Biochemistry. 1992; 31: 6660-6672Crossref PubMed Scopus (106) Google Scholar). Fig. 2 shows the charge recombination kinetics between Q –A and the donor side of ΔctpAHT3 PSII in the presence and absence of 1 mm MnCl2. In the absence of added Mn2+, the decay curve is characteristic of charge recombination between Q –A and Y +Z with a t½ of 20 ms. Similar decay curves and t½ values (10–20 ms) have been reported for the Scenedesmus LF-1 mutant and the Synechocystis D1 S345P and A344Stop mutants (9Nixon P.J. Trost J.T. Diner B.A. Biochemistry. 1992; 31: 10859-10871Crossref PubMed Scopus (194) Google Scholar). As evident from the lack of decay after 600 ms, the addition of exogenous Mn2+ ions blocks charge recombination between Q –A and Y +Z in ΔctpAHT3 PSII. The structure of the high affinity binding site is not significantly altered in the ΔctpAHT3 mutant, because Mn2+ ions could still access and reduce Y +Z.Fig. 2Charge recombination kinetics between Q–A and the donor side ofΔctpAHT3 PSII in the presence and absence of Mn2+. The charge recombination kinetics of isolated ΔctpAHT3 PSII complexes (2 μg of Chl/ml) were measured in the presence (solid) and absence (dashed) of 1 mm MnCl2 as described under "Materials and Methods." The arrow indicates the saturating actinic flash.View Large Image Figure ViewerDownload (PPT)These results suggest that manganese affinity to PSII is severely decreased in the absence of D1 processing. Because the manganese content of membranes from the ΔctpAHT3 mutant is ∼50% of that of HT3, it is possible that the PSII-associated manganese does not survive the PSII isolation procedure or that there is another protein(s) that bind manganese in the membrane. These data from isolated PSII complexes suggest that D1 cleavage is a requirement for the stable interaction of any of the four manganese atoms with PSII.ΔctpAHT3 PSII Complexes Lack the Extrinsic Proteins PsbO, PsbV, and PsbU—It has been reported previously that pD1 is incorporated into PSII complexes containing the membrane components D2, CP47, CP43, and cytochrome b559 (12Diner B.A. Ries D.F. Cohen B.N. Metz J.G. J. Biol. Chem. 1988; 263: 8972-8980Abstract Full Text PDF PubMed Google Scholar, 13Anbudurai P.R. Mor T.S. Ohad I. Shestakov S.V. Pakrasi H.B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8082-8086Crossref PubMed Scopus (137) Google Scholar, 18Metz J.G. Bricker T.M. Seibert M. FEBS Lett. 1985; 185: 191-196Crossref Scopus (31) Google Scholar, 29Zhang L. Paakkarinen V. van Wijk K.J. Aro E.M. Plant Cell. 2000; 12: 1769-1782Crossref PubMed Scopus (127) Google Scholar). Metz et al. (18Metz J.G. Bricker T.M. Seibert M. FEBS Lett. 1985; 185: 191-196Crossref Scopus (31) Google Scholar) also observed a decrease in the affinity of the extrinsic PsbP and PsbQ proteins for the thylakoid membranes in the Scenedesmus LF-1 mutant, suggesting that the presence of the C-terminal extension on D1 interferes with the binding of these components. The ΔctpAHT3 mutant allowed a more detailed characterization of the protein components of pD1-containing PSII complexes.The polypeptide profiles of PSII isolated from HT3 and ΔctpAHT3 strains are shown in Fig. 3. The proteins were identified based on their migration in the SDS-PAGE system described in Ref. 2Kashino Y. Lauber W.M. Carroll J.A. Wang Q. Whitmarsh J. Satoh K. Pakrasi H.B. Biochemistry. 2002; 41: 8004-8012Crossref PubMed Scopus (278) Google Scholar or by immunodetection. As expected, ΔctpAHT3 PSII complexes contained only pD1 and no processed D1. Notably, among the extrinsic proteins, PsbO, PsbQ, and PsbV could not be detected on immunoblots of ΔctpAHT3 PSII (data not shown). Although the PsbQ protein has not been identified in PSII structural studies, it has recently been identified as a regulatory component of cyanobacterial PSII (30Thornton L.E. Ohkawa H. Roose J.L. Kashino Y. Keren N. Pakrasi H.B. Plant Cell. 2004; 16: 2164-2175Crossref PubMed Scopus (150) Google Scholar). PsbU could not be detected on Coomassie-stain

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