Photoassembly of the Photosystem II (Mn) 4 Cluster in Site-Directed Mutants Impaired in the Binding of the Manganese-Stabilizing Protein † (original) (raw)
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Biochemistry, 1998
The extrinsic manganese stabilizing protein of photosystem II is required for Mn retention by the O 2 -evolving complex, accelerates the rate of O 2 evolution, and protects photosytem II against photoinhibition. We report results from studies of the in vitro reconstitution of spinach photosytem II with recombinant manganese stabilizing protein with C-terminal deletions of two, three, and four amino acids. The deletions were the result of amber mutations introduced by site-directed mutagenesis. Removal of the C-terminal dipeptide (Glu-Gln) did not diminish the ability of the manganese stabilizing protein either to rebind to or to restore high rates of O 2 evolution to photosystem II preparations depleted of the native protein. Deletion of the C-terminal tripeptide (Leu-Glu-Gln) resulted in weakened but specific binding of manganese stabilizing protein to photosystem II and minimal recovery of O 2 evolution activity.
Biochemistry, 2011
In the current X-ray crystallographic structural models of photosystem II, Glu354 of the CP43 polypeptide is the only amino acid ligand of the oxygen-evolving Mn 4 Ca cluster that is not provided by the D1 polypeptide. To further explore the influence of this structurally unique residue on the properties of the Mn 4 Ca cluster, the CP43-E354Q mutant of the cyanobacterium Synechocystis sp. PCC 6803 was characterized with a variety of biophysical and spectroscopic methods, including polarography, EPR, X-ray absorption, FTIR, and mass spectrometry. The kinetics of oxygen release in the mutant were essentially unchanged from those in wild type. In addition, the oxygen flash yields exhibited normal period four oscillations having normal S state parameters, although the yields were lower, correlating with the mutant's lower steady-state rate (approximately 20% compared to wild type). Experiments conducted with H 2 18 O showed that the fast and slow phases of substrate water exchange in CP43-E354Q thylakoid membranes were accelerated 8.5-and 1.8-fold, respectively, in the S 3 state compared to wild type. Purified oxygen-evolving CP43-E354Q PSII core complexes exhibited a slightly altered S 1 state Mn-EXAFS spectrum, a slightly altered S 2 state multiline EPR signal, a substantially altered S 2-minus-S 1 FTIR difference spectrum, and an unusually long lifetime for the S 2 state (>10 h) in a substantial fraction of reaction centers. In contrast, the S 2 state Mn-EXAFS spectrum was nearly indistinguishable from that of wild type. The S 2-minus-S 1 FTIR difference spectrum showed alterations throughout the amide and carboxylate stretching regions. Global labeling with 15 N and specific labeling with L-[1-13 C]alanine revealed that the mutation perturbs both amide II and carboxylate stretching modes and shifts the symmetric carboxylate stretching modes of the R-COOgroup of D1-Ala344 (the C-terminus of the D1 polypeptide) to higher frequencies by 3-4 cm-1 in both the S 1 and S 2 states. The EPR and FTIR data implied that 76-82% of CP43-E354Q PSII centers can achieve the S 2 state and that most of these can achieve the S 3 state, but no evidence for advancement beyond the S 3 state was observed in the FTIR data, at least not in a majority of PSII centers. Although the X-ray absorption and EPR data showed that the CP43-E354Q mutation only subtly perturbs the structure and spin state of the Mn 4 Ca cluster in the S 2 state, the FTIR and H 2 18 O exchange data show that the mutation strongly influences other properties of the Mn 4 Ca cluster, altering the response of numerous carboxylate and amide groups to the increased positive charge that develops on the cluster during the S 1 to S 2 transition and weakening the binding of both substrate water molecules (or water-derived ligands), especially the one that exchanges rapidly in the S 3 state. The FTIR data provide evidence that CP43-Glu354 coordinates to the Mn 4 Ca cluster in the S 1 state as a bridging ligand between two metal ions but provide no compelling evidence that this residue changes its coordination mode during the S 1 to S 2 transition. The H 2 18 O exchange data provide evidence that CP43-Glu354 interacts with the Mn ion that ligates the substrate water molecule (or water-derived ligand) that is in rapid exchange in the S 3 state. The light-driven oxidation of water in photosystem II (PSII) 1 produces nearly all of the O 2 on Earth and drives the production of nearly all of its biomass. Photosystem II is an integral membrane protein complex that is located in the thylakoid membranes of plants, algae, and cyanobacteria. It is a homodimer in vivo, having a total molecular mass of over 700 kDa. Each monomer consists of at least 20 different subunits and contains over 60 organic and inorganic cofactors including 35 Chl a and 12 carotenoid molecules. Each monomer's primary subunits include the membrane spanning polypeptides CP47 (56 kDa), CP43 (52 kDa), D2 (39 kDa), and D1 (38 kDa) and the extrinsic polypeptide PsbO (26.8 kDa). The D1 and D2 polypeptides are homologous and together form a heterodimer at
Biochemistry, 2002
The importance of the N-terminal domain of manganese stabilizing protein in binding to photosystem II has been previously demonstrated [Eaton-Rye and Murata (1989) Biochim. Biophys. Acta 977, 219-226; Odom and Bricker (1992) Biochemistry 31, 5616-5620]. In this paper, we report results from a systematic study of functional and structural consequences of N-terminal elongation and truncation of manganese stabilizing protein. Precursor manganese stabilizing protein is the unprocessed wild-type protein, which carries an N-terminal extension of 84 amino acids in the form of its chloroplastic signal peptide. Despite its increased size, this protein is able to reconstitute O 2 evolution activity to levels observed with the mature, processed protein, but it also binds nonspecifically to PSII. Truncation of wild-type manganese stabilizing protein by site-directed mutagenesis to remove three N-terminal amino acids, resulting in a mutant called ∆G3M, causes no loss of activity reconstitution, but this protein also exhibits nonspecific binding. Further truncation of the wild-type protein by ten N-terminal amino acids, producing ∆E10M, limits binding of manganese stabilizing protein to 1 mol/mol of photosystem II and decreases activity reconstitution to about 65% of that obtained with the wild-type protein. Because two copies of wild type normally bind to photosystem II, amino acids in the domain 4 K-10 E must be involved in the binding of one copy of manganese stabilizing protein to photosystem II. Spectroscopic analysis (CD and UV spectra) reveals that N-terminal elongation and deletion of manganese stabilizing protein influence its overall conformation, even though secondary structure content is not perturbed. Our data suggest that the solution structure of manganese stabilizing protein attains a more compact solution structure upon removal of N-terminal amino acids.
Biochemistry, 1991
The photosystem I1 (PSII) reaction center complex coordinates a cluster of M n atoms that are involved in the accumulation of oxidizing equivalents generated by light-induced charge separations within the intrinsic portion of the PSII complex. A 33-kDa extrinsic protein, termed the Mn-stabilizing protein (MSP), has been implicated in the stabilization of two of the four Mn atoms of the cluster, yet the precise role of this protein in 0, evolution remains to be elucidated. Here we describe the construction of a mutant of the cyanobacterium Synechocystis sp. PCC6803 in which the entire gene encoding MSP has been deleted. Northern and immunoblot analyses indicate that other PSII proteins are expressed and accumulated, despite the absence of MSP. Fluorescence emission spectra at 77 K indicate PSII assembles in the mutant, but that the binding of M S P is required for the normal fluorescence characteristics of the PSII complex, and suggest a specific interaction between MSP and CP47. Fluorescence induction measurements indicate a reduced rate of forward electron transport to the primary electron donor, P680, in the mutant. It is concluded
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2001
Exposure of photosystem II membranes to trypsin that has been treated to inhibit chymotrypsin activity produces limited hydrolysis of manganese stabilizing protein. Exposure to chymotrypsin under the same conditions yields substantial digestion of the protein. Further probing of the unusual insensitivity of manganese stabilizing protein to trypsin hydrolysis reveals that increasing the temperature from 4 to 25³C will cause some acceleration in the rate of proteolysis. However, addition of low (100 WM) concentrations of NH 2 OH, that are sufficient to reduce, but not destroy, the photosystem II Mn cluster, causes a change in PS II-bound manganese stabilizing protein that causes it to be rapidly digested by trypsin. Immunoblot analyses with polyclonal antibodies directed against the N-terminus of the protein, or against the entire sequence show that trypsin cleavage produces two distinct peptide fragments estimated to be in the 17^20 kDa range, consistent with proposals that there are 2 mol of the protein/mol photosystem II. The correlation of trypsin sensitivity with Mn redox state(s) in photosystem II suggest that manganese stabilizing protein may interact either directly with Mn, or alternatively, that the polypeptide is bound to another protein of the photosystem II reaction center that is intimately involved in binding and redox activity of Mn. ß
Biochemistry, 1998
Photosystem II catalyzes the light-driven oxidation of water and reduction of plastoquinone in oxygenic photosynthesis. The manganese stabilizing protein (MSP) of photosystem II is an extrinsic subunit that plays an important role in catalytic activity. This subunit can be extracted and rebound to the photosystem II reaction center. Extraction is associated with decreased stability of manganese binding by the enzyme and by loss in high rates of oxygen evolution activity; reconstitution reverses these phenomena. Since little is known about the assembly of complex membrane proteins, we have employed isotope editing and vibrational spectroscopy to obtain information about any changes in secondary structure that occur in MSP upon functional reconstitution to photosystem II. The spectroscopic data obtained are consistent with substantial changes in conformation when MSP binds to photosystem II; approximately 30-40% of the peptide backbone undergoes a change in secondary structure. These conclusions were reached by comparing different aliquots, before and after binding, of the same 13 [C]MSP sample. Analysis of amide I band line shapes through Fourier deconvolution and nonlinear regression suggests that binding of MSP to photosystem II is associated with a decrease in random structure and an increase in-sheet content. We conclude that binding of MSP to the reaction center can induce folding of MSP. Our results also indicate that, in solution, MSP can sample a variety of conformational states, which differ in hydrogen bonding of the peptide backbone.
Journal of Biological Chemistry, 1999
Photosystem II catalyzes photosynthetic water oxidation. The oxidation of water to molecular oxygen requires four sequential oxidations; the sequentially oxidized forms of the catalytic site are called the S states. An extrinsic subunit, the manganese-stabilizing protein (MSP), promotes the efficient turnover of the S states. MSP can be removed and rebound to the reaction center; removal and reconstitution is associated with a decrease in and then a restoration of enzymatic activity. We have isotopically edited MSP by uniform 13 C labeling of the Escherichia coli-expressed protein and have obtained the Fourier transform infrared spectrum associated with the S 1 to S 2 transition in the presence either of reconstituted 12 C or 13 C MSP. 13 C labeling of MSP is shown to cause 30-60 cm ؊1 shifts in a subset of vibrational lines. The derived, isotope-edited vibrational spectrum is consistent with a deprotonation of glutamic/ aspartic acid residues on MSP during the S 1 to S 2 transition; the base, which accepts this proton(s), is not located on MSP. This finding suggests that this subunit plays a role as a stabilizer of a charged transition state and, perhaps, as a general acid/base catalyst of oxygen evolution. These results provide a molecular explanation for known MSP effects on oxygen evolution.
Plant molecular biology, 2000
To investigate the interaction between the manganese-stabilizing protein (MSP) and cytochrome c-550 (cyt. c-550) of the photosystem II (PSII) complex in the cyanobacterium Synechocystis sp. PCC6803, three site-directed amino acid substitution mutants in MSP (MSP-D159N, MSP-R163L, MSP-D 159N/R 163L) were created by single and double amino acid substitution mutagenesis. The modified psbO genes encoding the mutants forms of MSP were used to transform a single-deletion mutant deltapsO strain lacking MSP as well as a double-deletion strain deltapsbO:deltapsbV lacking both MSP and cyt. c-550. The mutant forms of MSP were expressed in each case and all permitted autotrophic growth in strains expressing cyt. c-550. However, when the MSP mutations were introduced into a strain which lacks cyt. c-550 (deltapsbV), the two single amino acid substitution mutants (deltapsbV:MSP-D159N and deltapsbV:MSP-R 163L) failed to grow photoautotrophically. These strains exhibited coupled O2-evolving activity of 68-77% compared to the wild-type control using CO2 as an electron acceptor and maximal uncoupled O2-evolution rates of 42-57% using 2,6-dichloro-p-benzoquinone (DCBQ) as an artificial electron acceptor. Interestingly, when the two amino acid substitutions were together in the absence of cyt. c-550 (deltapsbV:MSP-D159N/R163L), the mutant grew photoautotrophically and the oxygen-evolving activities were higher than in the single mutants. This indicates that the MSP-D159N mutant suppresses the non-autotrophic phenotype of MSP-R163L (or vice versa) in the absence of cyt. c-550. The possibilities of a direct (ionic) or indirect interaction between D159 and R163 of MSP are discussed.
Photosynthesis Research, 2005
The 33-kDa manganese-stabilizing protein (MSP) of Photosystem II (PS II) maintains the functional stability of the Mn cluster in the enzyme's active site. This protein has been shown to possess characteristics similar to those of the intrinsically disordered, or natively unfolded proteins [Lydakis-Simantiris et al. (1999b) Biochemistry 38: 404-414]. Alternately it was proposed that MSP should be classified as a molten globule, based in part on the hypothesis that its lone disulfide bridge is necessary for structural stability and function in solution [Shutova et al. (2000) FEBS Lett. 467: 137-140]. A site-directed mutant MSP (C28A,C51A) that eliminates the disulfide bond reconstitutes O 2 evolution activity and binds to MSP-free PS II preparations at wild-type levels Biochim. Biophys. Acta 1274: 135-142]. This mutant was further characterized by incubation at 90°C to determine the effect of loss of the disulfide bridge on MSP thermostability and solution structure. After heating at 90°C for 20 min, C28A,C51A MSP was still able to bind to PS II preparations at molar stoichiometries similar to those of WT MSP and reconstitute O 2 evolution activity. A fraction of the protein aggregates upon heating, but after resolubilization, it regains the ability to bind to PS II and reconstitute O 2 evolution activity. Characterization of the solution structure of C28A,C51A MSP, using CD spectroscopy, UV absorption spectroscopy, and gel filtration chromatography, revealed that the mutant has a more disordered solution structure than WT MSP. The disulfide bond is therefore unnecessary for MSP function and the intrinsically disordered characteristics of MSP are not dependent on its presence. However, the disulfide bond does play a role in the solution structure of MSP in vivo, as evidenced by the lability of a C20S MSP mutation in Synechocystis 6803 [Burnap et al. (1994) Biochemistry 33: 13712-13718]. composed of sucrose (0.4 M), MES (50 mM, pH 6.0), NaCl (10 mM)); SW-PS II -Photosystem II preparation treated with 2 M NaCl to extract Ca 2+ and the 23 and 17 kDa extrinsic polypeptides; USW-PS II -Photosystem II preparation treated with 2 M NaCl to extract Ca 2+ and the 23 and 17 kDa extrinsic polypeptides, followed by incubation with 3.1 M urea and 240 mM NaCl to remove the manganese-stabilizing protein Photosynthesis Research (2005) 85: 359-372 Ó Springer 2005
Journal of Biological Chemistry
The 33-kDa manganese-stabilizingprotein stabilizes the manganese cluster in the oxygen-evolving complex. There has been, however, a considerable amount of controversy concerning the stoichiometry of this photosystem I1 (PS 11) component. In this paper, we have verified the extinction coefficient of the manganesestabilizing protein by amino acid analysis, determined the manganese content of oxygen-evolving photosystem I1 membranes and reaction center complex using inductively coupled plasma spectrometry, and determined immunologically the amount of the manganesestabilizing protein associated with photosystem 11. Oxygen-evolving photosystem I1 membranes and reaction center complex preparations contained 258 f 11 and 67 f 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 f 0.2 and 2.0 f 0.3 molecules of the manganese-stabilizing ProteinJtetranuclear manganese cluster in oxygen-evolving PS I1 membranes and highly purified PS I1 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 CaClz-washed PS I1 membrane proteins or reaction center complex proteins in the manganesestabilizing 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 I1 preparations.