Selective extraction of 22 kDa and 10 kDa polypeptides from Photosystem II without removal of 23 kDa and 17 kDa extrinsic proteins

Selective solubilization of Photosystem II membranes with the non-ionic detergent octyl thioglucopyranoside has allowed the isolation of a PS II system which has been depleted of the 22 and 10 kDa polypeptides but retains all three extrinsic proteins (33, 23 and 17 kDa). The PS II membranes which have been depleted of the 22 and 10 kDa species show high rates of oxygen evolution activity, external calcium is not required for activity and the manganese complex is not destroyed by exogenous reductants. When we compared this system to control PS II membranes, we observed a minor modification of the reducing side, and a conversion of the high-potential to the low-potential form of cytochrome b ₅₅₉.Abbreviations Chl- chlorophyll - DCBQ- 2,5-dichloro-p-benzoquinone - DCMU- 3-(3,4-dichlorophenyl)-1,1-dimethylurea - ESR- electron spin resonance - MES- 2-(N-morpholino)ethanesulfonic acid - OTG- octyl--d-thioglucopyranoside - PS II- Photosystem II - PEG- polyethylene glycol, M_r=6000 - Tris- 2-amino-2-hydroxyethylpropane-1,3-diol     

Effect of the manganese complex on the binding of the extrinsic proteins (17, 23 and 33 kDa) of Photosystem II

Selective extraction-reconstitution experiments with the extrinsic Photosystem II polypeptides (33 kDa, 23 kDa and 17 kDa) have demonstrated that the manganese complex and the 33 kDa polypeptide are both necessary structural elements for the tight binding of the water soluble 17 and 23 kDa species. When the manganese complex is intact the 33 kDa protein interacts strongly with the rest of the photosynthetic complex. Destruction of the Mn-complex has two dramatic effects: i) The binding of the 33 kDa polypeptide is weaker, since it can be removed by exposure of the PS II system to 2 M NaCl, and ii) the 17 and 23 kDa species do not rebind to Mn-depleted Photosystem II membranes that retain the 33 kDa protein.Abbreviations Chl chlorophyll - HQ hydroquinone - MES 2(N-morpholino)ethanesulfonic acid - PS II Photosystem II - Tris 2-amino-2-hydroxymethylpropane-1,3-diol     

Manganese binding to the 23 kDa extrinsic protein of Photosystem II

The recombinant form of the extrinsic 23 kDa protein (psbP) of Photosystem II (PSII) was studied with respect to its capability to bind Mn. The stoichiometry was determined to be one manganese bound per protein. A very high binding constant, K_A = 10^− 17 M^− 1, was determined by dialysis of the Mn containing protein against increasing EDTA concentration. High Field EPR spectroscopy was used to distinguish between specific symmetrically ligated Mn(II) from those non-specifically Mn(II) attached to the protein surface. Upon Mn binding PsbP exhibited fluorescence emission with maxima at 415 and 435 nm when tryptophan residues were excited. The yield of this blue fluorescence was variable from sample to sample. It was likely that different conformational states of the protein were responsible for this variability. The importance of Mn binding to PsbP in the context of photoactivation of PSII is discussed.     

The structure and function of the 33 kDa extrinsic protein of Photosystem II: A critical assessment

In this review the structure and function of the 33 kDa protein of Photosystem II is examined. Significant controversies exist concerning the solution secondary structure of the protein, the location of its binding site(s) within Photosystem II, the amino acid residues of the 33 kDa protein required for binding and its stoichiometry within the photosystem. The studies which examine these topics are considered from a critical perspective. A hypothetical model of the folding of the 33 kDa extrinsic protein which is supported by site-specific labeling studies and site-directed mutagenesis experiments is presented. Additionally, the function of the protein within the photosystem is unclear. We present a hypothesis that the 33 kDa protein is involved in maintaining the chloride associated with photosynthetic oxygen evolution in close proximity to the oxygen-evolving site.     

COOH-terminal residues of D1 and the 44 kDa CPa-2 at spinach photosystem II core complex

The COOH-termini of the 32 kDa D1 and 44 kDa CPa-2 were determined by protein sequencing of peptides from trypsinized photosystem II core complexes. COOH-terminal fragments were isolated by affinity chromatography using anhydrotrypsin-agarose. One peptide had a sequence corresponding to the segment from Asn at position 335 to Ala at position 344 of the sequence deduced from the psbA gene coding for D1. Nine amino acids may be cleaved from the COOH-terminus of pre-D1 during maturation. In contrast, CPa-2 was not modified at its COOH-terminus.     

Gene sequence for the 9 kDa component of Photosystem II from the cyanobacterium Phormidium laminosum indicates similarities between cyanobacterial and other leader sequences

Summary A 9 kDa polypeptide which is loosely attached to the inner surface of the thylakoid membrane and is important for the oxygen-evolving activity of Photosystem II in the thermophilic cyanobacterium Phormidium laminosum has been purified, a partial amino acid sequence obtained and its gene cloned and sequenced. The derived amino acid sequence indicates that the 9 kDa polypeptide is initially synthesised with an N-terminal leader sequence of 44 amino acids to direct it across the thylakoid membrane. The leader sequence consists of a positively charged N-terminal region, a long hydrophobic region and a typical cleavage site. These features have analogous counterparts in the thylakoid-transfer domain of lumenal polypeptides from chloroplasts of higher plants. These findings support the view of the proposed function of this domain in the two-stage processing model for import of lumenal, nuclear-encoded polypeptides. In addition, there is striking primary sequence homology between the leader sequences of the 9 kDa polypeptide and those of alkaline phosphatase (from the periplasmic space of Escherichia coli) and, particularly in the region of the cleavage site, the 16 kDa polypeptide of the oxygen-evolving apparatus in the thylakoid lumen of spinach chloroplasts.     

On the role of the N-terminus of the extrinsic 33 kDa protein of Photosystem II

The role of the N-terminus of the extrinsic 33 kDa protein of Photosystem II has been investigated by means of site-directed mutagenesis and cross-linking. Replacement of Asp-9 resulted in a dramatic increase in proteolytic sensitivity leading to the degradation of the protein forming a 31 kDa fragment with an undefined N-terminus. This fragment was unable to restore oxygen evolution. However, the variants of the 33 kDa protein which remained intact could reconstitute oxygen evolution as effectively as the wild-type protein. Cross-linking experiments with a water-soluble carbodiimide revealed that mutagenesis of residue D9 led to the disruption of an intramolecular salt bridge. Therefore we suggest that the N-terminus of the 33 kDa protein is necessary for maintaining the binding ability of the protein to Photosystem II but might not be involved in binding itself.     

Structural predictions on the 33 kDa extrinsic protein associated to the oxygen evolving complex of photosynthetic organisms

Modern computational methods for protein structure prediction have been used to study the structure of the 33 kDa extrinsic membrane protein, associated to the oxygen evolving complex of photosynthetic organisms. A multiple alignment of 14 sequences of this protein from cyanobacteria, algae and plants is presented. The alignment allows the identification of fully conserved residues and the recognition of one deletion and one insertion present in the plant sequences but not in cyanobacteria. A tree of similarity, deduced from pair-wise comparison and cluster analysis of the sequences, is also presented. The alignment and the consensus sequence derived are used for prediction the secondary structure of the protein. This prediction indicates that it is a mainly-beta protein (25–38% of -strands) with no more than 4% of -helix. Fold recognition by threading is applied to obtain a topological 2D model of the protein. In this model the secondary structure elements are located, including several highly conserved loops. Some of these conserved loops are suggested to be important for the binding of the 33 kDa protein to Photosystem II and for the stability of the manganese cluster. These structural predictions are in good agreement with experimental data reported by several authors.     

Variable chlorophyll a fluorescence from P-700 enriched Photosystem I particles dependent on the redox state of the reaction centre

1. Photosystem I particles enriched in P-700 prepared by Triton X-100 treatment of chloroplasts show a light-induced increase in fluorescence yield of more than 100% in the presence of dithionite but not in its absence.2. Steady state light maintains the P-700, of these particles, in the oxidised state when ascorbate is present but in the presence of dithionite only a transient oxidation occurs.3. EPR data show that, in these particles, the primary electron acceptor (X) is maintained in the reduced state by light at room temperature only when the dithionite is also present. In contrast, the secondary electron acceptors are reduced in the dark by dithionite.4. Fluorescence emission and excitation spectra and fluorescence lifetime measurements for the constant and variable fluorescence indicate a heterogeneity of the chlorophyll in these particles.5. It is concluded that the variable fluorescence comes from those chlorophylls which can transfer their energy to the reaction centre and that the states PX and P⁺X are more effective quenchers of chlorophyll fluorescence than PX^?, where P is P-700.     

Structural organization of the oxidizing side of photosystem II. Exogenous reductants reduce and destroy the Mn-complex in photosystems II membranes depleted of the 17 and 23 kDa polypeptides

Removal of 23 and 17 kDa water-soluble polypeptides from PS II membranes causes a marked decrease in oxygen-evolution activity, exposes the oxidizing side of PS II to exogenous reductants (Ghanotakis, D.F., Babcock, G.T. and Yocum, C.F. (1984) Biochim. Biophys. Acta 765, 388–398) and alters a high-affinity binding site for Ca²⁺ in the oxygen-evolving complex (Ghanotakis, D.F., Topper, J.N., Babcock, G.T. and Yocum, C.F. (1984) FEBS Lett. 170, 169–173). We have examined further the state of the functional Mn complex in PS II membranes from which the 17 and 23 kDa species have been removed by high-salt treatment. These membranes contain a structurally altered Mn complex which is sensitive to destruction by low concentrations of NH₂OH which cannot, in native PS II membranes, cause extraction of functional Mn. In addition to NH₂OH, a wide range of other small (H₂O₂, NH₂NH₂, Fe²⁺) and bulky (benzidine, hydroquinone) electron donors extract Mn (up to 80%) from the polypeptide-depleted PS II preparations. This extraction is due to reduction of the functional Mn complex since light, which would generate higher oxidation states within the Mn complex, prevents Mn release by reductants. Release of Mn by reductants does not extract the 33 kDa water-soluble protein implicated in Mn binding to the oxidizing side of PS II, although the protein can be partially or totally extracted from Mn-depleted preparations by exposure to high ionic strength or to high (0.8 M) concentrations of Tris. We view our results as evidence for a shield around the Mn complex of the oxygen-evolving complex comprised of the 33 kDa polypeptide along with the 23 and 17 kDa proteins and tightly bound Ca²⁺.     

Light saturation curves show competence of the water splitting complex in inactive Photosystem II reaction centers

Photosystem II complexes of higher plants are structurally and functionally heterogeneous. While the only clearly defined structural difference is that Photosystem II reaction centers are served by two distinct antenna sizes, several types of functional heterogeneity have been demonstrated. Among these is the observation that in dark-adapted leaves of spinach and pea, over 30% of the Photosystem II reaction centers are unable to reduce plastoquinone to plastoquinol at physiologically meaningful rates. Several lines of evidence show that the impaired reaction centers are effectively inactive, because the rate of oxidation of the primary quinone acceptor, Q_A, is 1000 times slower than in normally active reaction centers. However, there are conflicting opinions and data over whether inactive Photosystem II complexes are capable of oxidizing water in the presence of certain artificial electron acceptors. In the present study we investigated whether inactive Photosystem II complexes have a functional water oxidizing system in spinach thylakoid membranes by measuring the flash yield of water oxidation products as a function of flash intensity. At low flash energies (less that 10% saturation), selected to minimize double turnovers of reaction centers, we found that in the presence of the artificial quinone acceptor, dichlorobenzoquinone (DCBQ), the yield of proton release was enhanced 20±2% over that observed in the presence of dimethylbenzoquinone (DMBQ). We argue that the extra proton release is from the normally inactive Photosystem II reaction centers that have been activated in the presence of DCBQ, demonstrating their capacity to oxidize water in repetitive flashes, as concluded by Graan and Ort (Biochim Biophys Acta (1986) 852: 320–330). The light saturation curves indicate that the effective antenna size of inactive reaction centers is 55±12% the size of active Photosystem II centers. Comparison of the light saturation dependence of steady state oxygen evolution in the presence of DCBQ or DMBQ support the conclusion that inactive Photosystem II complexes have a functional water oxidation system.Abbreviations DCBQ 2,6-dichloro-p-benzoquinone - DMBQ 2,5-dimethyl-p-benzoquinone - F_o initial fluorescence level using dark-adapted thylakoids - Inactive reaction centers reaction centers inactive in plastoquinone reduction - PS II Photosystem II - Q_A primary quinone acceptor of Photosystem II - Q_B secondary quinone acceptor of Photosystem II Department of Plant Biology, University of IllinoisDepartment of Physiology & Biophysics, University of Illinois     

Enhancement studies on algae and isolated chloroplasts. Part I. Variability of photosynthetic enhancement in Chlorella phyrenoidosa

Studies of the variability of enhancement in Chlorella pyrenoidosa confirm the existence of two types of variability: a very slow diurnal variation linked to the growth cycle and a much more rapid adaptive response to the immediate incident light conditions (State I–State II transitions). Measurements of the wavelength dependencies and relative contributions of these two types of variability suggest that they may be linked.A close examination of the enhancement signals associated with the State I–State II transition reveals that the transitions can take place in any one of three ways: by a change in Photosystem II efficiency alone, by a change in Photosystem I efficiency alone or by a simultaneous change in the efficiencies of both photosystems.Measurements of the rates of transition between State I, State II and the dark adapted state, Dark, suggest that the behaviour of State II and Dark are normally, but not always, identical. The transitions between the three states were found to be first order. For those samples exhibiting the same behaviour in Dark and State II, the rate of the State I–State II transition was found to be independent of the wavelength of Light II, suggesting that the return from State I to State II is essentially a dark process and that the driving force for the adaptive transition is the over-stimulation of Photosystem I.Finally, a model is proposed, involving an antagonistic control of the quantum yields of photochemistry of the two photosystems, that is capable of explaining the links between the two types of variability, their wavelength dependencies and the shapes of the individual enhancement signals.     

Fluorescence and Fourier-transform infrared spectroscopic studies on the role of disulfide bond in the calcium binding in the 33 kDa protein of Photosystem II

The 33 kDa protein of Photosystem II has one intrachain disulfide bond. Fluorescence spectroscopy shows that the major groups in the protein that bind to Ca²⁺ should be the carboxylic side groups of glutamic acid and/or aspartic acid. Fluorescence and Fourier-transform infrared (FTIR) spectroscopic studies indicate that the conformation of the 33 kDa protein is altered upon reduction, while the reduced protein still retains the secondary structure. FTIR spectroscopy also shows that the metal ions induce a relative decrease of unordered structure and -sheet, and a substantial increase of -helix in both the intact and the reduced 33 kDa protein. This indicates that the addition of cations results in a much more compact structure and that both the intact and the reduced 33 kDa proteins have the ability to bind calcium. The above results may suggest that the disulfide bridge is not essential for calcium binding.Abbreviations CD circular dichroism - FTIR Fourier transform infrared - La lanthanum - PS photosystem - Tb terbium     

Subunit proteins of photosystem II

Recipient of the Society Award for Young Scientists 1991.     

Light-induced quenching of chlorophyll fluorescence at 77 K in leaves,chloroplasts and Photosystem II particles

The light-induced chlorophyll (Chl) fluorescence decline at 77 K was investigated in segments of leaves, isolated thylakoids or Photosystem (PS) II particles. The intensity of chlorophyll fluorescence declines by about 40% upon 16 min of irradiation with 1000 μmol m⁻² s⁻¹ of white light. The decline follows biphasic kinetics, which can be fitted by two exponentials with amplitudes of approximately 20 and 22% and decay times of 0.42 and 4.6 min, respectively. The decline is stable at 77 K, however, it is reversed by warming of samples up to 270 K. This proves that the decline is caused by quenching of fluorescence and not by pigment photodegradation. The quantum yield for the induction of the fluorescence decline is by four to five orders lower than the quantum yield of Q_A reduction. Fluorescence quenching is only slightly affected by addition of ferricyanide or dithionite which are known to prevent or stimulate the light-induced accumulation of reduced pheophytin (Pheo). The normalised spectrum of the fluorescence quenching has two maxima at 685 and 695 nm for PS II emission and a plateau for PS I emission showing that the major quenching occurs within PS II. ‘Light-minus-dark’ difference absorbance spectra in the blue spectral region show an electrochromic shift for all samples. No absorbance change indicating Chl oxidation or Pheo reduction is observed in the blue (410–600 nm) and near infrared (730–900 nm) spectral regions. Absorbance change in the red spectral region shows a broad-band decrease at approximately 680 nm for thylakoids or two narrow bands at 677 and 670–672 nm for PS II particles, likely resulting also from electrochromism. These absorbance changes follow the slow component of the fluorescence decline. No absorbance changes corresponding to the fast component are found between 410 and 900 nm. This proves that the two components of the fluorescence decline reflect the formation of two different quenchers. The slow component of the light-induced fluorescence decline at 77 K is related to charge accumulation on a non-pigment molecule of the PS II complex. This revised version was published online in June 2006 with corrections to the Cover Date.     

Two new components of 9 and 14 kDa from spinach photosystem I complex

Two formerly-uncharacterized subunits of 9 kDa and 14 kDa were found in spinach PSI complex. The 9 kDa subunit was released upon removal of antenna chlorophyll complex, whereas the 14 kDa subunit was tightly bound to the core complex. We determined the N-terminal amino acid sequence of the 9 kDa, and an internal sequence of the 14 kDa subunit after protease treatment, since the N-terminus of the latter protein was blocked. These partial sequences suggested that both subunits are new PSI components.     

Alternative Photosystem I-Driven Electron Transport Routes: Mechanisms and Functions

In addition to the linear electron transport, several alternative Photosystem I-driven (PS I) electron pathways recycle the electrons to the intersystem electron carriers mediated by either ferredoxin:NADPH reductase, NAD(P)H dehydrogenase, or putative ferredoxin:plastoquinone reductase. The following functions have been proposed for these pathways: adjustment of ATP/NADPH ratio required for CO(2) fixation, generation of the proton gradient for the down-regulation of Photosystem II (PS II), and ATP supply the active transport of inorganic carbon in algal cells. Unlike ferredoxin-dependent cyclic electron transport, the pathways supported by NAD(P)H can function in the dark and are likely involved in chlororespiratory-dependent energization of the thylakoid membrane. This energization may support carotenoid biosynthesis and/or maintain thylakoid ATPase in active state. Active operation of ferredoxin-dependent cyclic electron transport requires moderate reduction of both the intersystem electron carriers and the acceptor side of PS I, whereas the rate of NAD(P)H-dependent pathways under light depends largely on NAD(P)H accumulation in the stroma. Environmental stresses such as photoinhibition, high temperatures, drought, or high salinity stimulated the activity of alternative PS I-driven electron transport pathways. Thus, the energetic and regulatory functions of PS I-driven pathways must be an integral part of photosynthetic organisms and provides additional flexibility to environmental stress.     

Two-dimensional separation of chloroplast membrane proteins by isoelectri focusing and electrophoresis in sodium dodecyl sulphate

Proteins of chloroplast subfragments enriched in Photosystem I and Photosystem II electron flow activity have been analyzed by two-dimensional polyacrylamide gel electrophoresis. In the first dimension, polyacrylamide gel isoelectric focusing (pH 5–7) was used in the presence of Triton X-100, followed at right angle by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Characteristic fingerprints were obtained for the Photosystem I and II fractions and a correlation between the major proteins separated by isoelectric focusing and the major polypeptides separated by undimensional SDS electrophoresis was established. Two dominant spots of 68 000 and 60 000 daltons appeared in the two-dimensional patterns of Photosystem I fractions $\text{p}\text{I}$ values about 5.6; two spots with molecular weights of 33 000 and 23 000 were characteristics for Photosystem II fractions $\text{p}\text{I}$ values about 5.3 and 6.3). Photosystem I fractions were furthermore characteristics by a series of spots in the 44 000–33 000 range $\text{p}\text{I}$ values from about 5.9 to 6.8). The two-dimensional system revealed that (a) several SDS-polypeptides have multiple forms differing in charge only, (b) some proteins separated by isoelectric focusing are resolved in the second dimensional into polypeptides of different size. The two-dimensional method combining Triton X-100 isoelectric focusing' and SDS electrophoresis provides a higher degree of resolution than either of the unidimensional methods thus allowing a detailed analysis of chloroplast membrane proteins.     

Quantification of photosystem I and II in different parts of the thylakoid membrane from spinach

Electron paramagnetic resonance (EPR) was used to quantify Photosystem I (PSI) and PSII in vesicles originating from a series of well-defined but different domains of the thylakoid membrane in spinach prepared by non-detergent techniques. Thylakoids from spinach were fragmented by sonication and separated by aqueous polymer two-phase partitioning into vesicles originating from grana and stroma lamellae. The grana vesicles were further sonicated and separated into two vesicle preparations originating from the grana margins and the appressed domains of grana (the grana core), respectively. PSI and PSII were determined in the same samples from the maximal size of the EPR signal from P700⁺ and Y_D, respectively. The following PSI/PSII ratios were found: thylakoids, 1.13; grana vesicles, 0.43; grana core, 0.25; grana margins, 1.28; stroma lamellae 3.10. In a sub-fraction of the stroma lamellae, denoted Y-100, PSI was highly enriched and the PSI/PSII ratio was 13. The antenna size of the respective photosystems was calculated from the experimental data and the assumption that a PSII center in the stroma lamellae (PSIIβ) has an antenna size of 100 Chl. This gave the following results: PSI in grana margins (PSIα) 300, PSI (PSIβ) in stroma lamellae 214, PSII in grana core (PSIIα) 280. The results suggest that PSI in grana margins have two additional light-harvesting complex II (LHCII) trimers per reaction center compared to PSI in stroma lamellae, and that PSII in grana has four LHCII trimers per monomer compared to PSII in stroma lamellae. Calculation of the total chlorophyll associated with PSI and PSII, respectively, suggests that more chlorophyll (about 10%) is associated with PSI than with PSII.