Manganese-stabilizing Protein Research Articles

Inorganic Cofactor Stabilization and Retention: The Unique Functions of the Two PsbO Subunits of Eukaryotic Photosystem II

Eukaryotic PsbO, the photosystem II (PSII) manganese-stabilizing protein, has two N-terminal sequences that are required for binding of two copies of the protein to PSII [Popelkova, H., et al. (2002) Biochemistry 41, 10038-10045; Popelkova, H., et al. (2003) Biochemistry 42, 6193-6200]. In the work reported here, a set of selected N-terminal truncation mutants of PsbO that affect subunit binding to PSII were used to determine the effects of PsbO stoichiometry on the Mn, Ca(2+), and Cl(-) cofactors and to characterize the roles of each of the PsbO subunits in PSII function. Results of the experiments with the PsbO-depleted PSII membranes reconstituted with the PsbO deletion mutants showed that the presence of PsbO does not affect Ca(2+) retention by PSII in steady-state assays of activity, nor is it required for Ca(2+) to protect the Mn cluster against reductive inhibition in darkness. In contrast to the results with Ca(2+), PsbO increases the affinity of Cl(-) for the active site of the O(2)-evolving complex (OEC) as expected. These results together with other data on activity retention suggest that PsbO can stabilize the Mn cluster by facilitating retention of Cl(-) in the OEC. The data presented here indicate that each of two copies of PsbO has a distinctive function in PSII. Binding of the first PsbO subunit fully stabilizes the Mn cluster and enhances Cl(-) retention, while binding of the second subunit optimizes Cl(-) retention, which in turn maximizes O(2) evolution activity. Nonspecific binding of some PsbO truncation mutants to PSII has no functional significance.

Structural and functional aspects of the MSP (PsbO) and study of its differences in thermophilic versus mesophilic organisms

The Manganese Stabilizing Protein (MSP) of Photosystem II (PSII) is a so-called extrinsic subunit, which reversibly associates with the other membrane-bound PSII subunits. The MSP is essential for maximum rates of O(2) production under physiological conditions as stabilizes the catalytic [Mn(4)Ca] cluster, which is the site of water oxidation. The function of the MSP subunit in the PSII complex has been extensively studied in higher plants, and the structure of non-PSII associated MSP has been studied by low-resolution biophysical techniques. Recently, crystal structures of PSII from the thermophilic cyanobacterium Thermosynechococcus elongatus have resolved the MSP subunit in its PSII-associated state. However, neither any crystal structure is available yet for MSP from mesophilic organisms, higher plants or algae nor has the non-PSII associated form of MSP been crystallized. This article reviews the current understanding of the structure, dynamics, and function of MSP, with a particular focus on properties of the MSP from T. elongatus that may be attributable to the thermophilic ecology of this organism rather than being general features of MSP.

Site-Directed Mutagenesis of Conserved C-Terminal Tyrosine and Tryptophan Residues of PsbO, the Photosystem II Manganese-Stabilizing Protein, Alters Its Activity and Fluorescence Properties

The extrinsic photosystem II PsbO subunit (manganese-stabilizing protein) contains near-UV CD signals from its complement of aromatic amino acid residues (one Trp, eight Tyr, and 13 Phe residues). Acidification, N-bromosuccinimide modification of Trp, reduction or elimination of a disulfide bond, or deletion of C-terminal amino acids abolishes these signals. Site-directed mutations that substitute Phe for Trp241 and Tyr242, near the C-terminus of PsbO, were used to examine the contribution of these residues to the activity and spectral properties of the protein. Although this substitution is, in theory, conservative, neither mutant binds efficiently to PSII, even though these proteins appear to retain wild-type solution structures. Removal of six residues from the N-terminus of the W241F mutant restores activity to near-wild-type levels. The near-UV CD spectra of the mutants are modified; well-defined Tyr and Trp peaks are lost. Characterizations of the fluorescence spectra of the full-length WF and YF mutants indicate that Y242 contributes significantly to PsbO's Tyr fluorescence emission and that an excited-state tyrosinate could be present in PsbO. Deletion of W241 shows that this residue is a major contributor to PsbO's fluorescence emission. Loss of function is consistent with the proposal that a native C-terminal domain is required for PsbO binding and activity, and restoration of activity by deletion of N-terminal amino acids may provide some insights into the evolution of this important photosynthetic protein.

Morphological and molecular characterization of a spontaneously tuberizing potato mutant: an insight into the regulatory mechanisms of tuber induction

BackgroundTuberization in potato (Solanum tuberosum L.) represents a morphogenetic transition of stolon growth to tuber formation, which is under complex environmental and endogenous regulation. In the present work, we studied the regulatory mechanisms and the role of different morphogenetic factors in a newly isolated potato mutant, which exhibited spontaneous tuberization (ST). The ST mutant was characterized in detail at morphological, physiological and biochemical levels.ResultsTuberization of the ST mutant grown in the soil was photoperiod-insensitive; predominantly sessile tubers formed directly from axillary buds even under continuous light. Single-node cuttings of the ST mutant cultured in vitro frequently formed tubers or basal tuber-like swellings instead of normal shoots under conditions routinely used for shoot propagation. The tuberization response of ST cuttings under light was dependent on sucrose, the concentration of which had to exceed certain threshold that inversely correlated with irradiance. Gibberellic acid prevented tuberization of ST cuttings, but failed to restore normal shoot phenotype and caused severe malformations. Carbohydrate analysis showed increased levels of both soluble sugars and starch in ST plants, with altered carbohydrate partitioning and metabolism. Comparative proteomic analysis revealed only a few differences between ST- and wild-type plants, primary amongst which seemed to be the absence of an isoform of manganese-stabilizing protein, a key subunit of photosystem II.ConclusionST mutant exhibits complex developmental and phenotypic modifications, with features that are typical for plants strongly induced to tuberize. These changes are likely to be related to altered regulation of photosynthesis and carbohydrate metabolism rather than impaired transduction of inhibitory gibberellin or photoperiod-based signals. The effect of gibberellins on tuberization of ST mutant suggests that gibberellins inhibit tuberization downstream of the inductive effects of sucrose and other positive factors.

Expression, assembly and auxiliary functions of photosystem II oxygen-evolving proteins in higher plants

The oxygen-evolving complex (OEC) of higher plant photosystem II (PSII) consists of an inorganic Mn(4)Ca cluster and three nuclear-encoded proteins, PsbO, PsbP and PsbQ. In this review, we focus on the assembly of these OEC proteins, and especially on the role of the small intrinsic PSII proteins and recently found "novel" PSII proteins in the assembly process. The numerous auxiliary functions suggested during the past few years for the OEC proteins will likewise be discussed. For example, besides being a manganese-stabilizing protein, PsbO has been found to bind calcium and GTP and possess a carbonic anhydrase activity. In addition, specific roles have been suggested for the two isoforms of the PsbO protein in Arabidopsis thaliana. PsbP and PsbQ seem to play an additional role in the formation of PSII supercomplexes and in grana stacking, besides their originally recognized role in providing a proper calcium and chloride ion concentration for water splitting.

Six Specific Lysine Residues are Crucial in Maintaining the Structure and Function of Soluble Manganese Stabilizing Protein

When manganese stabilizing protein (MSP) was treated with 0.5 mM N-succinimidyl propionate (NSP), the rebinding ability and oxygen-releasing capabilities of the modified MSP were not altered, in spite of changes of MSP surface Lys residues. Furthermore, far-ultraviolet circular dichroism and intrinsic fluorescence spectra analysis revealed that 0.5 mM NSP-modified MSP retained most of its native secondary and tertiary structure. Mapping of the sites of NSP modification by Staphylococcus V(8) protease digestion of the modified protein, as well as analysis by matrix-assisted laser desorption ionization-time of flight mass spectrometry, indicated that seven Lys residues were modified. The results suggested that these residues are not absolutely essential to the structure and function of MSP. However, when the NSP concentration was increased to 4 mM, the modified MSP was unable to bind photosystem II and completely lost its reactivating capability. Both far-ultraviolet circular dichroism and intrinsic fluorescence spectra analysis revealed a clear conformational change in MSP after 4 mM NSP treatment, suggesting that some Lys residues are involved in maintaining the structure and function of MSP. Analysis by matrix-assisted laser desorption ionization-time of flight mass spectrometry indicated that another six Lys residues, namely Lys20, Lys101, Lys196, Lys207, Lys130 (or Lys137) and Lys66 (or Lys76), were modified by 4 mM NSP. Therefore, these six Lys residues are crucial in maintaining the structure and function of soluble MSP.

Mutagenesis of basic residues R151 and R161 in manganese-stabilizing protein of photosystem II causes inefficient binding of chloride to the oxygen-evolving complex.

Manganese-stabilizing protein of photosystem II, an intrinsically disordered polypeptide, contains a high ratio of charged to hydrophobic amino acid residues. Arg151 and Arg161 are conserved in all known MSP sequences. To examine the role of these basic residues in MSP structure and function, three mutants of spinach MSP, R151G, R151D, and R161G, were produced. Here, we present evidence that replacement of Arg151 or Arg161 yields proteins that have lower PSII binding affinity, and are functionally deficient even though about 2 mol of mutant MSP/mol PSII can be rebound to MSP depleted PSII membranes. R161G reconstitutes O(2) evolution activity to 40% of the control, while R151G and R151D reconstitute only 20% of the control activity. Spectroscopic and biochemical techniques fail to detect significant changes in solution structure. More extensive O(2) evolution assays revealed that the Mn cluster is stable in samples reconstituted with each mutated MSP, and that all three Arg mutants have the same ability to retain Ca(2+) as the wild-type protein. Activity assays exploring the effect of these mutations on retention of Cl(-), however, showed that the R151G, R151D, and R161G MSPs are defective in Cl(-) binding to the OEC. The mutants have Cl(-) K(M) values that are about four (R161G) or six times (R151G and R151D) higher than the value for the wild-type protein. The results reported here suggest that conserved positive charges on the manganese-stabilizing protein play a role in proper functional assembly of the protein into PSII, and, consequently, in retention of Cl(-) by the O(2)-evolving complex.

Structural Dynamics of the Manganese-Stabilizing ProteinEffect of pH, Calcium, and Manganese

The photosystem-II-associated 33-kDa extrinsic manganese-stabilizing protein is found in all oxygen-evolving organisms. In this paper, we show that this protein undergoes pH-induced conformational changes in the physiological pH range. At a neutral pH of 7.2, the hydrophobic amino acid residues that are most likely located inside the beta barrel are "closed" and the protein binds neither Mn2+ nor Ca2+ ions. When the protein is transferred to a solution with a slightly acidic pH of 5.7, hydrophobic amino acid residues become exposed to the surrounding medium, enabling them to bind the fluorescent probe 8,1-ANS. At this pH-induced open state, Mn2+ and Ca2+ bind to the manganese-stabilizing protein. The pH values used in this study, 7.2 and 5.7, are typical of the pH found in the thylakoid lumen in the dark and light, respectively. A model is presented in which the manganese-stabilizing protein undergoes a pH-dependent conformational change that in turn influences its capacity to bind calcium and manganese. In this model, the proton-dependent conformational changes of the tertiary structure of the manganese-stabilizing protein are of functional relevance for the regulation of substrate (water) delivery to and product (proton) release from the water-oxidizing complex by forming a proton-sensing proton-transport pathway.

Structure and Activity of the Photosystem II Manganese-Stabilizing Protein: Role of the Conserved Disulfide Bond

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. 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. 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. This mutant was further characterized by incubation at 90 degrees C to determine the effect of loss of the disulfide bridge on MSP thermostability and solution structure. After heating at 90 degrees 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.

Acetonitrile‐induced unfolding of the photosystem II manganese‐stabilizing protein studied by electrospray mass spectrometry

In this paper an acetonitrile-induced unfolding of the manganese-stabilizing protein (MSP) of photosystem II was discovered. More distinct unfolding states of MSP were identified than previously by using mainly electrospray ionization mass spectrometry (ESI-MS), together with fluorescence spectra and far-UV circular dichroism (CD) at pH 2.0, 6.2 or 11.6, and with acetonitrile concentrations from 0 to 50%. At pH 6.2 with acetonitrile concentration changing from 0 to 10%, relatively broad charge-state distributions and poor intensity were observed in ESI-MS, indicating the presence of coexisting conformers. It was concluded that the structure of the MSP protein is unlikely to be a tightly folded form. When the concentration of acetonitrile was 20-40%, simulating the state in the biological membrane, changes in the state of unfolding of MSP were observed to a certain extent using ESI-MS, fluorescence and CD spectroscopy. The charge-state distribution in ESI-MS was found to move toward high states (from 13+ to 27+ to 15+ to 31+) with increasing acetonitrile concentration. At pH 2.0, the MSP structure is rearranged into an unfolded state, and at pH 11.6 the MSP structure is induced to assume another unordered state by deprotonation of appropriate residues. An interesting observation was that a second peak envelope emerged with 20-50% acetonitrile in the medium at pH 11.6.

Assembly and function of the Photosystem II manganese stabilizing protein: lessons from its natively unfolded behavior

The Photosystem II (PS II) manganese stabilizing protein (MSP) possesses characteristics, including thermostability, ascribed to the natively unfolded class of proteins (Lydakis-Simantiris et al. (1999) Biochemistry 38: 404-414). A site-directed mutant of MSP, C28A, C51A, which lacks the -S-S- bridge, also binds to PS II at wild-type levels and reconstitutes oxygen evolution activity [Betts et al. (1996) Biochim Biophys Acta 1274: 135-142], although the mutant protein is even more disordered in solution. Both WT and C28A, C51A MSP aggregate upon heating, but an examination of the effects of protein concentration and pH on heat-induced aggregation showed that each MSP species exhibited greater resistance to aggregation at a pH near their pI (5.2) than do either bovine serum albumin (BSA) or carbonic anhydrase, which were used as model water soluble proteins. Increases in pH above the pI of the MSPs and BSA enhanced their aggregation resistance, a behavior which can be predicted from their charge (MSP) or a combination of charge and stabilization by -S-S- bonds (BSA). In the case of aggregation resistance by MSP, this is likely to be an important factor in its ability to avoid unproductive self-association reactions in favor of formation of the protein-protein interactions that lead to formation of the functional oxygen evolving complex.

Influence of the 33 kDa manganese-stabilizing protein on the structure and substrate accessibility of the oxygen-evolving complex of photosystem II.

The 33 kDa manganese-stabilizing extrinsic protein binds to the lumenal side of photosystem II (PS II) close to the Mn(4)Ca cluster of the oxygen-evolving complex, where it limits access of small molecules to the metal site. Our previous finding that the removal of this protein did not alter the magnetic coupling regime within the manganese cluster, measured by electron spin-echo envelope modulation [Gregor, W., and Britt, R. D. (2000) Photosynth. Res. 65, 175-185], prompted us to examine whether this accessibility control is also true for substrate water, using the same pulsed EPR technique. Comparing the deuteron modulation of the S(2)-state multiline signal of PS II membranes, equilibrated with deuterated water (D(2)O) after removal or retention of the 33 kDa protein, we observed no change in the number and the distance of deuterons magnetically coupled to manganese, indicating that the number and distance of water molecules bound to the manganese cluster are independent of bound 33 kDa protein in the S(1) state, in which the sample was poised prior to cryogenic illumination. A simple modulation depth analysis revealed a distance of 2.5-2.6 A between the closest deuteron and manganese. These results are in agreement with our refined X-ray absorption analysis. The manganese K-edge positions, reflecting their oxidation states, and the extended X-ray absorption fine structure amplitudes and distances between the manganese ions and their oxygen and nitrogen ligands (1.8, 2.7, and 3.3-3.4 A) were independent of bound 33 kDa protein.

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.

Structural and Functional Differentiation of Three Groups of Tyrosine Residues by Acetylation of N-Acetylimidazole in Manganese Stabilizing Protein

To study its contribution to the assembly of the green plant manganese stabilizing protein (MSP) into photosystem II (PSII), tyrosine residues were specifically acetylated using N-acetylimidazole (NAI). In soluble MSP, three groups of Tyr residues could be differentiated by NAI acetylation: approximately 5 (actually approximately 5.2) Tyr residues could be easily acetylated (superficial), 1-2 Tyr residues could be acetylated when the NAI concentration was sufficiently high (superficially buried), and 1-2 Tyr residues could only be acetylated in the presence of the denaturant, urea (deeply buried). Acetylation of the 5.2 Tyr residues did not affect the reconstitution or oxygen-evolving activities of the MSP, and far-UV circular dichroism (CD) analysis showed that the altered MSP retained most of its native secondary structure. These results suggested that the 5.2 Tyr residues are not absolutely essential to the function of MSP. However, further modification of the 1-2 superficially buried Tyr residues (for a total acetylation of approximately 6.4 Tyr residues) completely abrogated the MSP rebinding and oxygen evolution activities. Finally, at least one tyrosine residue was inaccessible to NAI until MSP was completely unfolded by 8 M urea. Deacetylation of MSP with 6.4 or 8 acetylated Tyr residues with hydroxylamine restored most of the rebinding and oxygen-evolving activities. A prominent red shift in fluorescence spectra of MSP (excited at 280 or 295 nm) was observed after modification of 6.4 Tyr residues, and a further shift could be found after all 8 Tyr residues were modified, indicating a great loss of native secondary structure. Far-UV CD revealed that MSP was mostly unfolded when 6.4 Tyr residues were modified and completely unfolded when all 8 Tyr residues were modified. Fluorescence and far-UV CD studies revealed that loss of MSP rebinding to PSII membranes following NAI modification correlated well with conformational changes in MSP. Together, these results indicate that different tyrosine residues have different contributions to the binding and assembly of MSP into PSII.

Structural Studies of the Manganese Stabilizing Subunit in Photosystem II

Photosystem II (PSII) is the plant photosynthetic reaction center that carries out the light driven oxidation of water. The water splitting reactions are catalyzed at a tetranuclear manganese cluster. The manganese stabilizing protein (MSP) of PSII stabilizes the manganese cluster and accelerates the rate of oxygen evolution. MSP can be removed from PSII, with an accompanying decrease in activity. Either an Escherichia coli expressed version of MSP or native, plant MSP can be rebound to the PSII reaction center; MSP reconstitution reverses the deleterious effects associated with MSP removal. We have employed Fourier transform infrared (FTIR) spectroscopy and solution small angle x-ray scattering (SAXS) techniques to investigate the structure of MSP in solution and to define the structural changes that occur before and after reconstitution to PSII. FTIR and SAXS are complementary, because FTIR spectroscopy detects changes in MSP secondary structure and SAXS detects changes in MSP size/shape. From the SAXS data, we conclude that the size/shape and domain structure of MSP do not change when MSP binds to PSII. From FTIR data acquired before and after reconstitution, we conclude that the reconstitution-induced increase in β-sheet content, which was previously reported, persists after MSP is removed from the PSII reaction center. However, the secondary structural change in MSP is metastable after removal from PSII, which indicates that this form of MSP is not the lowest energy conformation in solution.

Specific Isotopic Labeling and Photooxidation-linked Structural Changes in the Manganese-stabilizing Subunit of Photosystem II

Photosystem II (PSII) oxidizes water to molecular oxygen; the catalytic site is a cluster of four manganese ions. The catalytic site undergoes four sequential light-driven oxidation steps to form oxygen; these sequentially oxidized states are referred to as the Sn states, where n refers to the number of oxidizing equivalents stored. The extrinsic manganese stabilizing protein (MSP) of PSII influences the efficiency and stability of the manganese cluster, as well as the rates of the S state transitions. To understand how MSP influences photosynthetic water oxidation, we have employed isotope editing and difference Fourier transform infrared spectroscopy. MSP was expressed in Escherichia coli under conditions in which MSP aspartic and glutamic acid residues label at yields of 65 and 41%, respectively. Asparagine and glutamine were also labeled by this approach. GC/MS analysis was consistent with minimal scrambling of label into other amino acid residues and with no significant scrambling into the peptide bond. Selectively labeled MSP was then reconstituted to PSII, which had been stripped of native MSP. Difference Fourier transform infrared spectroscopy was used to probe the S1QA to S2QA- transition at 200 K, as well as the S1QB to S2QB- transition at 277 K. These experiments show that aspargine, glutamine, and glutamate residues in MSP are perturbed by photooxidation of manganese during the S1 to S2 transition.

Binding of manganese stabilizing protein to photosystem II: identification of essential N-terminal threonine residues and domains that prevent nonspecific binding.

The N-terminus of spinach photosystem II manganese stabilizing protein (MSP) contains two amino acid sequences, (4)KRLTYD(10)E and (15)TYL(18)E, that are necessary for binding of two copies of this subunit to the enzyme [Popelkova et al. (2002) Biochemistry 41, 10038-10045]. To better understand the basis of MSP-photosystem II interactions, the role of threonine residues in the highly conserved motifs T(Y/F)DE and TY has been characterized. Deletion mutants lacking the first 5, 6, 7, and 15 amino acid residues at the N-terminus of the protein were examined for their ability to reconstitute activity in MSP-depleted photosystem II. The results reported here show that truncations of five and six amino acid residues (mutants DeltaR5M and DeltaL6M mutants) have no negative effect on recovery of oxygen evolution activity or on binding of MSP to photosystem II. Deletion of seven residues (mutant DeltaT7M) decreases reconstitution activity to 40% of the control value and reduces functional binding of the mutant protein to photosystem II from two to one copy. Deletion of 15 amino acid residues (mutant DeltaT15M) severely impairs functional binding of MSP, and lowers O(2) evolution activity to less than 20% of the control. DeltaT7M is the only mutant that exhibited neither nonspecific binding to photosystem II nor changes in tertiary structure. These, and previous results, show that the highly conserved Thr7 and Thr15 residues of MSP are required for functional binding of two copies of the eukaryotic protein to photosystem II. Although the N-terminal domains, (1)EGGKR(6)L, (8)YDEIQS(14)K, and (16)YL(18)E of spinach MSP are unnecessary for specific, functional binding interactions, these sequences are necessary to prevent nonspecific binding of the protein to photosystem II.

Comparative studies on the properties of the extrinsic manganese-stabilizing protein from higher plants and of a synthetic peptide of its C-terminus

The present study describes a comparative analysis on the fluorescence properties of the manganese-stabilizing protein (MSP), a synthetic peptide corresponding to its C terminus and a 7:1 (molar ratio) mixture of N-acetyl-tyrosine and N-acetyl-tryptophan, respectively, together with reconstitution experiments of oxygen evolution in MSP-depleted photosystem II (PS II) membrane fragments. It is found: (i) at neutral pH, the fluorescence from Trp 241 is strongly diminished in MSP solutions, whereas it highly dominates the overall emission from the C-terminus peptide; (ii) at alkaline pH, the emission of Tyr and Trp is quenched in both, MSP and C-terminus peptide, with increasing pH but the decline curve is shifted by about two pH units towards the alkaline region in MSP; (iii) a drastically different pattern emerges in the 7:1 mixture where the Trp emission even slightly increases at high pH; (iv) the anisotropy of the fluorescence emission is wavelength-independent (310–395 nm) and indicative of one emitter type (Trp) in the C-terminus peptide and of two emitter types (Tyr, Trp) in MSP; and (v) in MSP-depleted PS II membrane fragments the oxygen evolution is restored (up to 85% of untreated control) by rebinding of MSP but not by the C-terminus peptide, however, the presence of the latter diminishes the restoration effect of MSP. A quenching mechanism of Trp fluorescence by a next neighbored tyrosinate in the peptide chain is proposed and the relevance of the C terminus of MSP briefly discussed.

Inhibition of photosynthetic electron transport by palmitoleic acid is partially correlated to loss of thylakoid membrane proteins

Palmitoleic acid (16:1 Δ9), a monounsaturated fatty acid, is found to inhibit electron transport. Inhibition occurs rapidly (within 30 s). The oxidizing side of photosystem (PS) II is ~ 90% inactivated, whereas no inhibition occurs on the reducing side of the PSII complex. PSI activity is ∼ 65% inhibited. Inhibition of electron transport is not correlated to lipid peroxidation. 16:1 causes the loss of proteins from the thylakoid membrane which is exacerbated by the light. The loss of five proteins, viz. plastocyanin (PC), manganese stabilizing protein (MSP), cytochrome f (Cyt f), D1 and D2 is examined. The proteins are found to be lost in the following order: PC (< 1 min), MSP (~5 min), Cyt f (~10 min), D1 (~60 min) and D2 (~60 min). The timing of the loss of a PSI associated protein, PC, overlaps with that of the inhibition of PSI. Also, preventing the loss of PC with spermine (spm) treatment can partially protect PSI from 16:1 inhibition. Thus, the results suggest that the loss of PC is the cause of PSI inhibition. On the other hand, the loss of MSP from the oxygen-evolving complex is not likely to be the cause of the 16:1 inhibition on the oxidizing side of PSII, as the MSP loss occurs later than the inhibition. Also, increased retention of MSP with spm treatment does not relieve the 16:1 inhibition of PSII. Instead, inhibition of PSII by 16:1 is prevented by Mn 2+ treatment, suggesting that loss of Mn 2+ ions is the cause of PSII inhibition.

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.