N-terminus of the photosystem II manganese stabilizing protein: effects of sequence elongation and truncation

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 DeltaG3M, 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 DeltaE10M, 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.     

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.     

Probing the N-terminal sequence of spinach PsbO: evidence that essential threonine residues bind to different functional sites in eukaryotic photosystem II

The N-terminal 1E-?L domain of the manganese-stabilizing protein (PsbO) from spinach prevents non-specific binding of the subunit to photosystem II (PSII) and deletions of the 1E-?T or 1E-1?T sequences from the PsbO N-terminus reduce or impair, respectively, functional binding of PsbO to PSII (Popelkova et al., Biochemistry 42:6193-6200, 2003). The work presented here provides deeper insights into the interaction of PsbO with PSII. The data show that a single mutation, 1?T → A in mature PsbO from spinach reduces the stoichiometry of its functional binding from two to one subunit per PSII and decreases reconstitution of activity to about 45 % of the wild-type control. Replacement of the 1E-?L domain with ?M in the T15A PsbO mutant has no additional negative effect on recovery of O? evolution activity, but it significantly weakens both functional and nonspecific binding of the truncated mutant to PSII. These results suggest that the 1?T side-chain by itself is essential for binding of one of two PsbO subunits to eukaryotic PSII and that specific PSII-binding sites for PsbO are distinguishable; one PSII-binding site does not require PsbO-1?T and probably interacts with the other N-terminal domain of PsbO. Identity of the latter domain is revealed by a requirement for the presence of the 1E-?L sequence that is shown here to be necessary for high-affinity binding of PsbO to PSII. When combined with previous results, the data presented here lead to a more detailed model for PsbO binding in eukaryotic PSII.     

Identification of functional domains of the extrinsic 12 kDa protein in red algal PSII by limited rroteolysis and directed mutagenesis.

The extrinsic 12 kDa protein in red algal photosystem II (PSII) functions to minimize the chloride and calcium requirement of oxygen-evolving activity [Enami et al. (1998) Biochemistry 37: 2787]. In order to identify functional domains of the 12 kDa protein, we prepared the 12 kDa protein lacking N-terminal peptides or C-terminal peptides or both by limited proteolysis and directed mutagenesis. The resulting 12 kDa protein fragments were examined for their binding and functional properties by reconstitution experiments. (1) A peptide fragment from Gly-6 to C-terminus of the 12 kDa protein was prepared by V8 protease. This fragment rebound to PSII completely, and it reactivated oxygen evolution partially in the absence of Cl(-) and Ca(2+) ions but significantly in the presence of Cl(-) ion. (2) A peptide from Leu-10 to Phe-83 was obtained by chymotrypsin treatment. This peptide rebound to PSII effectively, but the rebinding did not restore oxygen evolution in both the absence and presence of Cl(-) and Ca(2+) ions. (3) Two mutant proteins, one lacking five residues and the other lacking nine residues of the N-terminus, were able to bind to PSII effectively. Recovery of oxygen evolution by their binding was almost the same as that reconstituted with the V8 protease-treated peptide. (4) Three mutant proteins lacking ten, seven or three residues of the C-terminus effectively rebound to PSII, but their binding did not result in recovery of the oxygen evolution. In contrast, reconstitution with a mutant protein lacking one residue of the C-terminus showed the same high restoration of oxygen evolution as reconstitution with the full-length 12 kDa protein. (5) These results indicate that two residues from lysine of the C-terminus of the 12 kDa protein constitute an important domain for minimizing the chloride and calcium requirement of oxygen evolution. In addition, the N-terminus of the protein, at least five residues, has a secondary function for the chloride requirement.     

A domain of the manganese-stabilizing protein from Synechococcus elongatus involved in functional binding to photosystem II.

Site-directed mutagenesis was performed to investigate whether the two protease-sensitive sequences Phe(156)-Gly(163) and Arg(184)-Ser(191), of the manganese-stabilizing protein (MSP) from a thermophilic cyanobacterium, Synechococcus elongatus (Motoki, A., Shimazu, T., Hirano, M., and Katoh, S. (1998) Biochim. Biophys. Acta 1365, 492-502), are involved in functional interaction with photosystem II (PSII). The ability of MSP to bind to its functional site on the PSII complex and to reactivate oxygen evolution was dramatically reduced by the substitution of Arg(152), Asp(158), Lys(160), or Arg(162) with uncharged residues, by insertion of a single residue between Phe(156) and Leu(157), or by deletion of Leu(157). Substitution of each of the four charged residues with an identically charged residue showed that the charges at Asp(158), and possibly Lys(160), are important for the electrostatic interaction with PSII. The reactivating ability was also strongly affected by the alteration of Phe(156) to Leu. Replacement of Lys(188), the only strictly conserved charged residue in the Arg(184)-Ser(191) sequence, by Gln had only a marginal effect on the function of MSP. High affinity binding of MSP to PSII was also affected significantly by mutation at Arg(152), which is located in a region (Val(148)-Arg(152)) strictly conserved among the 14 sequences so far reported. These results imply that the Val(148)-Gly(163) sequence, which is well conserved among MSPs from cyanobacteria to higher plants, is a domain of MSP for functional interaction with 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 beta-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.     

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.     

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.     

Deletion mutagenesis in Synechocystis sp. PCC6803 indicates that the Mn-stabilizing protein of photosystem II is not essential for O2 evolution

The photosystem II (PSII) reaction center complex coordinates a cluster of Mn 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 O2 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 MSP 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 that in contrast to previous reports, MSP is not required for the assembly of active PSII complexes nor is it essential for H2O-splitting activity in vivo.     

N-Bromosuccinimide modification of tryptophan 241 at the C-terminus of the manganese stabilizing protein of plant photosystem II influences its structure and function

Life on earth depends upon the ability of oxygenic photosynthesis to oxidize water to molecular oxygen. This process is catalyzed by water–plastoquinone oxido-reductase complex. In addition to the photosystem II (PSII) reaction core, it includes a manganese stabilizing protein (MSP) that plays an important regulatory role in the process in plants and algae. Tryptophan 241, located at the carboxyl-terminus of the MSP, is its sole tryptophan. Modification of MSP by N-bromosuccinimide (NBS) was carried out to explore the role of Trp241 in maintaining its structure and function. Data and arguments are presented to show that it is Trp241, not other tyrosines in MSP, that is involved in the modification and changes observed in this study. Further, the pH-dependence of the modification and the comparison of features of fluorescence spectra of MSP suggested that Trp241 is buried in the hydrophobic interior of the protein. Hydropathy analysis revealed that Trp241 is located in the middle of the hydrophobic region at the C-terminus of MSP. Circular dichroism spectroscopy showed that NBS modification of Trp241 dramatically modified the protein structure. The affinity of MSP to PSII decreased greatly after the modification of Trp241, and no oxygen-evolving activity was recovered after its reconstitution. This study provides a novel demonstration that Trp241 at the C-terminus hydrophobic region of the MSP is critical for maintaining appropriate structure and function of 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.     

Reconstitution of the spinach oxygen-evolving complex with recombinant Arabidopsis manganese-stabilizing protein

The psbO gene of cyanobacteria, green algae and higher plants encodes the precursor of the 33 kDa manganese-stabilizing protein (MSP), a water-soluble subunit of photosystem II (PSII). Using a pET-T7 cloning/expression system, we have expressed in Escherichia coli a full-length cDNA clone of psbO from Arabidopsis thaliana. Upon induction, high levels of the precursor protein accumulated in cells grown with vigorous aeration. In cells grown under weak aeration, the mature protein accumulated upon induction. In cells grown with moderate aeration, the ratio of precursor to mature MSP decreased as the optical density at induction increased. Both forms of the protein accumulated as inclusion bodies from which the mature protein could be released under mildly denaturing conditions that did not release the precursor. Renatured Arabidopsis MSP was 87% as effective as isolated spinach MSP in restoring O₂ evolution activity to MSP-depleted PSII membranes from spinach; however, the heterologous protein binds to spinach PSIIs with about half the affinity of the native protein. We also report a correction to the previously published DNA sequence of Arabidopsis psbO (Ko et al., Plant Mol Biol 14 (1990) 217–227).     

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.     

The PsbQ protein stabilizes the functional binding of the PsbP protein to photosystem II in higher plants

PsbP and PsbQ proteins are extrinsic subunits of photosystem II (PSII) and optimize the oxygen evolution reaction by regulating the binding properties of the essential cofactors Ca(2+) and Cl(-). PsbP induces conformational changes around the catalytic Mn cluster required for Ca(2+) and Cl(-) retention, and the N-terminal region of PsbP is essential for this reaction. It was reported that PsbQ partially restores the functional defect of N-terminal truncated PsbP [Ifuku and Sato (2002) Plant Cell Physiol. 43, 1244-1249]; however, the mechanism of this restoration is yet to be clarified. In this study, we demonstrate that PsbQ is able to restore the functional binding of mutated PsbPs. In the presence of PsbQ, ?15-PsbP, a truncated PsbP lacking 15 N-terminal residues, was able to specifically bind to NaCl-washed spinach PSII membranes and significantly restore the oxygen evolving activity. Furthermore, PsbQ was also able to compensate for the impaired ion-retention of H144A-PsbP, in which a conserved histidine at position 144 in the C-terminal domain was substituted with an alanine. Fourier transform infrared (FTIR) difference spectroscopy showed that PsbQ restored the ability of ?15- and H144A-PsbP to induce proper conformational changes during S(1) to S(2) transition. These data suggest that the major function of PsbQ is to stabilize PsbP binding, thereby contributing to the maintenance of the catalytic Mn cluster of the water oxidation machinery in higher plant PSII. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Binding stoichiometry and affinity of the manganese-stabilizing protein affects redox reactions on the oxidizing side of photosystem II

It has been reported previously that the two subunits of PsbO, the photosystem II (PSII) manganese stabilizing protein, have unique functions in relation to the Mn, Ca(2+), and Cl(-) cofactors in eukaryotic PSII [Popelkova; (2008) Biochemistry 47, 12593]. The experiments reported here utilize a set of N-terminal truncation mutants of PsbO, which exhibit altered subunit binding to PSII, to further characterize its role in establishing efficient O(2) evolution activity. The effects of PsbO binding stoichiometry, affinity, and specificity on Q(A)(-) reoxidation kinetics after a single turnover flash, S-state transitions, and O(2) release time have been examined. The data presented here show that weak rebinding of a single PsbO subunit to PsbO-depleted PSII repairs many of the defects in PSII resulting from the removal of the protein, but many of these are not sustainable, as indicated by low steady-state activities of the reconstituted samples [Popelkova; (2003) Biochemistry 42 , 6193]. High affinity binding of PsbO to PSII is required to produce more stable and efficient cycling of the water oxidation reaction. Reconstitution of the second PsbO subunit is needed to further optimize redox reactions on the PSII oxidizing side. Native PsbO and recombinant wild-type PsbO from spinach facilitate PSII redox reactions in a very similar manner, and nonspecific binding of PsbO to PSII has no significance in these reactions.     

Oligonucleotide-directed mutagenesis of psbB,the gene encoding CP47, employing a deletion mutant strain of the cyanobacterium Synechocystis sp. PCC 6803

A mutant strain of the cyanobacterium Synechocystis sp. PCC (Pasteur Culture Collection) 6803 has been developed in which psbB, the gene coding for the chlorophyl a-binding protein CP47 in Photosystem II (PSII), has been deleted. This deletion mutant can be used for the reintroduction of modified psbB into the cyanobacterium. To study the role of a large hydrophilic region in CP47, presumably located on the lumenal side of the thylakoid membrane between the fifth and sixth membrane-spanning regions, specific deletions have been introduced in psbB coding for regions within this domain. One psbB mutation leads to deletion of Gly-351 to Thr-365 in CP47, another psbB mutation was targeted towards deletion of Arg-384 to Val-392 in this protein. The deletion from Gly-351 to Thr-365 results in a loss of PSII activity and of photoautotrophic growth of the mutant, but the deletion between Arg-384 and Val-392 retains PSII activity and the ability to grow photoautotrophically. The mutant strain with the deletion from Gly-351 to Thr-365 does not assemble a stable PSII reaction center complex in its thylakoid membranes, and exhibits diminished levels of CP47 and of the reaction center proteins D1 and D2. In contrast to the Arg-384 to Val-392 portion of this domain, the region between Gly-351 and Thr-365 appears essential for the normal structure and function of photosystem II.     

Copper effect on the protein composition of photosystem II

We provide data from in vitro experiments on the polypeptide composition, photosynthetic electron transport and oxygen evolution activity of intact photosystem II (PSII) preparations under Cu(II) toxicity conditions. Low Cu(II) concentrations (Cu(II) per PSII reaction centre unit≤230) that caused around 50% inhibition of variable chlorophyll a fluorescence and oxygen evolution activity did not affect the polypeptide composition of PSII. However, the extrinsic proteins of 33, 24 and 17 kDa of the oxygen-evolving complex of PSII were removed when samples were treated with 300 μ M CuCl₂ (Cu(II) per PSII reaction centre unit=1 400). The LHCII antenna complex and D1 protein of the reaction centre of PSII were not affected even at these Cu(II) concentrations. The results indicated that the initial inhibition of the PSII electron transport and oxygen-evolving activity induced by the presence of toxic Cu(II) concentrations occurred before the damage of the oxygen-evolving complex. Indeed, more than 50% inhibition could be achieved in conditions where its protein composition and integrity was apparently preserved.     

Biophysical characterization of the DNA binding domain of gpNu1, a viral DNA packaging protein

Terminase enzymes are common to double-stranded DNA viruses. These enzymes "package" the viral genome into a pre-formed capsid. Terminase from bacteriophage lambda is composed of gpA (72.4 kDa) and gpNu1 (20.4 kDa) subunits. We have described the expression and biochemical characterization of gpNu1DeltaK100, a construct comprising the N-terminal 100 amino acids of gpNu1 (Yang, Q., de Beer, T., Woods, L., Meyer, J., Manning, M., Overduin, M., and Catalano, C. E. (1999) Biochemistry 38, 465-477). Here we present a biophysical characterization of this construct. Thermally induced loss of secondary and tertiary structures is fully reversible. Surprisingly, although loss of tertiary structure is cooperative, loss of secondary structure is non-cooperative. NMR and limited proteolysis data suggest that approximately 30 amino acids of gpNu1DeltaK100 are solvent-exposed and highly flexible. We therefore constructed gpNu1DeltaE68, a protein consisting of the N-terminal 68 residues of gpNu1. gpNu1DeltaE68 is a dimer with no evidence of dissociation or further aggregation. Thermally induced unfolding of gpNu1DeltaE68 is reversible, with concomitant loss of both secondary and tertiary structure. The melting temperature increases with increasing protein concentration, suggesting that dimerization and folding are, at least in part, coupled. The data suggest that gpNu1DeltaE68 represents the minimal DNA binding domain of gpNu1. We further suggest that the C-terminal approximately 30 residues in gpNu1DeltaK100 adopt a pseudo-stable alpha-helix that extends from the folded core of the protein. A model describing the role of this helix in the assembly of the packaging apparatus is discussed.     

The importance of protein–protein interactions for optimising oxygen activity in photosystem II: reconstitution with a recombinant thioredoxin—manganese stabilising protein

In this paper we describe how photosystem II (PSII) from higher plants, which have been depleted, of the extrinsic proteins can be reconstituted with a chimeric fusion protein comprising thioredoxin from Escherichia coli and the manganese stabilising protein from Thermosynechococcus elongatus. Surprisingly, even though E. coli thioredoxin is completely unrelated to PSII, the fusion protein restores higher rates of activity upon rebinding to PSII than either the native spinach MSP, or T. elongatus MSP. PSII reconstituted with the fusion protein also has a lower requirement for calcium than PSII with the small extrinsic proteins removed, or PSII reconstituted with spinach or T. elongatus MSP. The MSP portion of the fusion protein is less thermally stable compared to isolated MSP from T. elongatus, which could be the key to its superior activation capability through greater flexibility. This work reveals the importance of protein–protein interactions in the water splitting activity of PSII and suggests that conformational configurations, which increase flexibility in MSP, are essential to its function, even when these are induced by an unrelated protein.     

Characterization of high temperature induced stress impairments in thylakoids of rice seedlings.

Exposure of isolated thylakoids or intact plants to elevated temperature is known to inhibit photosynthesis at multiple sites. We have investigated the effect of elevated temperature (40 degrees C) for 24 hr in dark on rice seedlings to characterize the extent of damage by in vivo heat stress on photofunctions of photosystem II (PSII). Chl a fluorescence transient analysis in the intact rice leaves indicated a loss in PSII photochemistry (Fv) and an associated loss in the number of functional PSII units. Thylakoids isolated from rice seedlings exposed to mild heat stress exhibited >50% reduction in PSII catalyzed oxygen evolution activity compared to the corresponding control thylakoids. The ability of thylakoid membranes from heat exposed seedlings to photooxidize artificial PSII electron donor, DPC, subsequent to washing the thylakoids with alkaline Tris or NH2OH was also reduced by approximately 40% compared to control Tris or NH2OH washed thylakoids. This clearly indicated that besides the disruption of oxygen evolving complex (OEC) by 40 degrees C heat exposure for 24 hr, the PSII reaction centers were impaired by in vivo heat stress. The analysis of Mn and manganese stabilizing protein (MSP) contents showed no breakdown of 33 kDa extrinsic MSP and only a marginal loss in Mn. Thus, we suggest that the extent of heat induced loss of OEC must be due to disorganization of the OEC complex by in vivo heat stress. Studies with inhibitors like DCMU and atrazine clearly indicated that in vivo heat stress altered the acceptor side significantly. [14C] Atrazine binding studies clearly demonstrated that there is a significant alteration in the QB binding site on D1 as well as altered QA to QB equilibrium. Thus, our results show that the loss in PSII photochemistry by in vivo heat exposure not only alters the donor side but significantly alters the acceptor side of PSII.