The extrinsic proteins of Photosystem II

Years of genetic, biochemical, and structural work have provided a number of insights into the oxygen evolving complex (OEC) of Photosystem II (PSII) for a variety of photosynthetic organisms. However, questions still remain about the functions and interactions among the various subunits that make up the OEC. After a brief introduction to the individual subunits Psb27, PsbP, PsbQ, PsbR, PsbU, and PsbV, a current picture of the OEC as a whole in cyanobacteria, red algae, green algae, and higher plants will be presented. Additionally, the role that these proteins play in the dynamic life cycle of PSII will be discussed.     

The extrinsic proteins of Photosystem II

In this review we examine the structure and function of the extrinsic proteins of Photosystem II. These proteins include PsbO, present in all oxygenic organisms, the PsbP and PsbQ proteins, which are found in higher plants and eukaryotic algae, and the PsbU, PsbV, CyanoQ, and CyanoP proteins, which are found in the cyanobacteria. These proteins serve to optimize oxygen evolution at physiological calcium and chloride concentrations. They also shield the Mn(4)CaO(5) cluster from exogenous reductants. Numerous biochemical, genetic and structural studies have been used to probe the structure and function of these proteins within the photosystem. We will discuss the most recent proposed functional roles for these components, their structures (as deduced from biochemical and X-ray crystallographic studies) and the locations of their proposed binding domains within the Photosystem II complex. This article is part of a Special Issue entitled: Photosystem II.     

The functional sites of chlorophylls in D1 and D2 subunits of Photosystem II identified by pulsed EPR

The functional site of Chl_Z, an auxiliary electron donor to P680⁺, was determined by pulsed ELDOR applied to a radical pair of Y_D^• and Chlz⁺ in oriented PS II membranes from spinach. The radical-radical distance was determined to be 29.5 Å and its direction was 50° from the membrane normal, indicating that a chlorophyll on the D2 protein is responsible for the EPR Chlz⁺ signal. Spin polarized ESEEM (Electronin Spin Echo Envelop Modulation) of a ³Chl and Q_A⁻ radical pair induced by a laser flash was observed in reaction center D1D2Cytb₅₅₉ complex, in which Q_A was functionally reconstituted with DBMIB and reduced chemically. Q_A⁻ESEEM showed a characteristic oscillating time profile due to dipolar coupling with ³Chl. By fitting with the dipolar interaction parameters, the distance between ³Chl and Q_A⁻ was determined to be 25.9 Å, indicating that the accessory chlorophyll on the D1 protein is responsible for the ³Chl signal.     

Alterations in Photosystem II electron transport as revealed by thermoluminescence of Cu-poisoned chloroplasts

The Rubisco activase amino acid sequences of spinach and tobacco are 79% identical, yet the tobacco protein does not facilitate the activation of the uncarbamylated, ribulose bisphosphate bound form of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) and vice versa. In contrast, combinations of the spinach Rubisco activase with Rubisco from non-Solanaceae species and combinations of tobacco Rubisco activase with Rubisco from other Solanaceae species are almost as effective as the analogous combination. To examine the basis of the preference of an activase protein for either Solanaceae or non-Solanaceae Rubisco, several recombinant chimeric proteins were obtained by combining regions from the cDNAs of spinach and tobacco activase and expression in Escherichia coli. The chimeric proteins were analyzed for ATP hydrolysis and ability to activate spinach and tobacco Rubisco. Comparisons of Rubisco preference with composition of the various activase chimeras indicate that the major determinants of Rubisco preference seem to be localized in the carboxyl-terminal region.     

EPR studies of the oxygen-evolving enzyme of Photosystem II

The light-induced EPR multiline signal is studied in O₂-evolving PS II membranes. The following results are reported: (1) Its amplitude is shown to oscillate with a period of 4, with respect to the number of flashes given at room temperature (maxima on the first and fifth flashes). (2) Glycerol enhances the signal intensity. This effect is shown to come from changes in relaxation properties rather than an increase in spin concentration. (3) Deactivation experiments clearly indicate an association with the S₂ state of the water-oxidizing enzyme. A signal at g = 4.1 with a linewidth of 360 G is also reported and it is suggested that this arises from an intermediate donor between the S states and the reaction centre. This suggestion is based on the following observations: (1) The g = 4.1 signal is formed by illumination at 200 K and not by flash excitation at room temperature, suggesting that it arises from an intermediate unstable under physiological conditions. (2) The formation of the g = 4.1 signal at 200 K does not occur in the presence of DCMU, indicating that more than one turnover is required for its maximum formation. (3) The g = 4.1 signal decreases in the dark at 220 K probably by recombination with Q^?_AFe. This recombination occurs before the multiline signal decreases, indicating that the g = 4.1 species is less stable than S₂. (4) At short times, the decay of the g = 4.1 signal corresponds with a slight increase in the multiline S₂ signal, suggesting that the loss of the g = 4.1 signal results in the disappearance of a magnetic interaction which diminishes the multiline signal intensity. (5) Tris-washed PS II membranes illuminated at 200 K do not exhibit the signal.     

Kinetics of EPR signal IIvf in chloroplast Photosystem II

The risetime of EPR signal II_vf (S II_vf) has been measured in oxygen-evolving Photosystem II particles from spinach chloroplasts at pH 6.0. The EPR signal shows an instrument-limited rise upon induction ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 3 μ}\text{s}$). These data are consistent with a model where the species Z responsible for S II_vf is the immediate electron donor to P-680⁺ in spinach chloroplasts. A new, faster decay component of S II_vf has also been detected in these experiments.     

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 origin of split EPR signals in the Ca2+-depleted Photosystem II

A light-driven reaction model for the Ca²⁺-depleted Photosystem (PS) II is proposed to explain the split signal observed in electron paramagnetic resonance (EPR) spectra based on a comparison of EPR assignments with recent x-ray structural data. The split signal has a splitting linewidth of 160 G at around g = 2 and is seen upon illumination of the Ca²⁺-depleted PS II in the S₂ state associated with complete or partial disappearance of the S₂ state multiline signal. Another g=2 broad ESR signal with a 110 G linewidth was produced by 245 K illumination for a short period in the Ca²⁺-depleted PS II in S₁ state. At the same time a normal Y_Z^· radical signal was also efficiently trapped. The g=2 broad signal is attributed to an intermediate S₁X^· state in equilibrium with the trapped Y_Z^· radical. Comparison with x-ray structural data suggests that one of the split signals (doublet signal) is attributable to interaction between His 190 and the Y_Z^· radical, and other signals is attributable to interaction between His 337 and the manganese cluster, providing further clues as to the mechanism of water oxidation in photosynthetic oxygen evolution.     

Rise time of EPR signal IIvf in chloroplast Photosystem II

The rise time of the photoinduced, reversible EPR Signal II_vf in spinach chloroplasts is found using flash excitation to be 20 ± 10 μs. The results are interpreted as evidence that the Signal II_vf radical is an electron carrier on the donor side of Photosystem II, but probably does not result from the first donor to P680⁺.     

Photosystem II single crystals studied by transient EPR: the light-induced triplet state

Transient electron paramagnetic resonance (TR EPR) at 9.8 GHz has been used to study the light-induced triplet state in single crystals of Photosystem II (PS II). The crystals were grown from a solution of PS II core complexes from the thermophilic cyanobacterium Synechococcus elongatus. The core complexes contain at least 17 subunits, including the water-oxidizing complex, and 32 chlorophyll a molecules per PS II complex. The PS II complexes are active in light-induced electron transfer and water oxidation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with four dimers of PS II complexes per unit cell. Laser excitation was used to generate the recombination triplet state in PS II which was then studied by EPR at low temperatures (10 K). The crystal spectra show the same magnitude of the zero-field splitting (ZFS) values D, E as spectra obtained earlier for the triplet state of PS II in frozen solution. The orientation of the ZFS tensor D of the triplet state with respect to the crystallographic axes has been deduced from the analysis of angular-dependent EPR spectra. Knowledge of the orientation of the D tensor component perpendicular to the plane of the chlorophyll (D(Z)) allows an assignment on which chlorophyll of the reaction centre the triplet state is localized at low temperatures. Furthermore, the orientation of the D(X) and D(Y) components of the D tensor yielded the in-plane orientation of the respective chlorophyll in the reaction centre providing first experimental evidence for the orientation of this molecule in the PS II.     

The structure of the secondary donor of Photosystem II investigated by EPR at 9 and 35 GHz

Signal II of plant photosynthesis, which is generally thought to be connected to the secondary donor complex of Photosystem II, has been investigated with EPR spectroscopy at 9 and 35 GHz. From the spectrum at 35 GHz of deuterated Chlorella vulgaris, the principle values of the g-tensor are determined to be g_xx = 2.0074, g_yy = 2.0044 and g_zz = 2.0023. Proton hyperfine coupling tensor elements and orientations were determined from spectral simulation of random and oriented samples, assuming that Signal II is due to a plastose-miquinone cation having its π-electrons in an antisymmetric orbital as proposed by P.J. O'Malley, G.T. Babcock and R.C. Prince (Biochim. Biophys. Acta 765 (1984) 370–379). In contrast to their work, it is found that most hyperfine interaction is due to the methylene group at ring position 5 and to both hydroxyl groups. One of the hydroxyl groups shows bond bending of 35°. We presume that this is due to hydrogen bonding and that this bond stabilizes the antisymmetric orbital of the π-electrons.     

EPR and ENDOR studies of the water oxidizing complex of Photosystem II

A comparative study of X-band EPR and ENDOR of the S₂ state of photosystem II membrane fragments and core complexes in the frozen state is presented. The S₂ state was generated either by continuous illumination at T=200 K or by a single turn-over light flash at T=273 K yielding entirely the same S₂ state EPR signals at 10 K. In membrane fragments and core complex preparations both the multiline and the g=4.1 signals were detected with comparable relative intensity. The absence of the 17 and 23 kDa proteins in the core complex preparation has no effect on the appearance of the EPR signals. ¹H-ENDOR experiments performed at two different field positions of the S₂ state multiline signal of core complexes permitted the resolution of four hyperfine (hf) splittings. The hf coupling constants obtained are 4.0, 2.3, 1.1 and 0.6 MHz, in good agreement with results that were previously reported (Tang et al. (1993) J Am Chem Soc 115: 2382–2389). The intensities of all four line pairs belonging to these hf couplings are diminished in D₂O. A novel model is presented and on the basis of the two largest hfc's distances between the manganese ions and the exchangeable protons are deduced. The interpretation of the ENDOR data indicates that these hf couplings might arise from water which is directly ligated to the manganese of the water oxidizing complex in redox state S₂.Abbreviations cw continuous wave - ENDOR electron nuclear double resonance - EPR electron paramagnetic resonance - hf hyperfine - hfc hyperfine coupling - MLS multiline signal - PS II Photosystem II - rf radio frequency - WOC water oxidizing complex     

Requirements for different combinations of the extrinsic proteins in specific cyanobacterial Photosystem II mutants

The crystallographic data available for Photosystem II (PS II) in cyanobacteria has now provided complete structures for loop E from CP43 and CP47 as well as the extrinsic subunits PsbO, PsbU and PsbV. Protein interactions between these subunits are essential for stable water splitting and there is evidence that the binding of PsbU facilitates optimal energy transfer from the phycobilisome. Interactions between PsbO and CP47 may also play a role in dimer stabilization while loop E of CP43 contributes directly to the water-splitting reaction. Recent evidence also suggests that homologs of PsbP and PsbQ play key roles in cyanobacterial PS II, and under nutrient-deficient conditions PsbQ appears essential for photoautotrophic growth.     

EPR evidence for a modified S-state transition in chloride-depleted Photosystem II

The role of chloride on the S-state transition in spinach Photosystem II (PS II) particles was investigated by EPR spectroscopy at low temperature and the following results were obtained. (1) After excitation by continuous light at 200 K, chloride-depleted particles did not show the EPR multiline signal associated with the S₂ state, but only showed the broad signal at g = 4.1. The S₂ multiline signal was completely restored upon chloride repletion. (2) In the absence of chloride the S₂ multiline signal was not induced by a single flash excitation at 0°C. However, upon addition of chloride after the flash the signal was developed in darkness. (3) The amplitude of the multiline S₂ signal thus developed upon chloride addition after flash illumination did not show oscillations dependent upon flash number. These results indicate that the O₂-evolving complex in chloride-depleted PS II membranes is able to store at least one oxidizing equivalent, a modified S₂ state, which does not give rise to the multiline signal. Addition of chloride converts this oxidizing equivalent to the normal S₂ state which gives rise to the multiline signal. The modified S₂ state is more stable than the normal S₂ state, showing decay kinetics about 20-times slower than those of the normal S₂ state, and the formation of higher S states is blocked.     

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.     

Conformational changes in photosystem II supercomplexes upon removal of extrinsic subunits

Photosystem II is a multisubunit pigment-protein complex embedded in the thylakoid membranes of chloroplasts. It consists of a large number of intrinsic membrane proteins involved in light-harvesting and electron-transfer processes and of a number of extrinsic proteins required to stabilize photosynthetic oxygen evolution. We studied the structure of dimeric supercomplexes of photosystem II and its associated light-harvesting antenna by electron microscopy and single-particle image analysis. Comparison of averaged projections from native complexes and complexes without extrinsic polypeptides indicates that the removal of 17 and 23 kDa extrinsic subunits induces a shift of about 1.2 nm in the position of the monomeric peripheral antenna protein CP29 toward the central part of the supercomplex. Removal of the 33 kDa extrinsic protein induces an inward shift of the strongly bound trimeric light-harvesting complex II (S-LHCII) of about 0.9 nm, and in addition destabilizes the monomer-monomer interactions in the central core dimer, leading to structural rearrangements of the core monomers. It is concluded that the extrinsic subunits keep the S-LHCII and CP29 subunits in proper positions at some distance from the central part of the photosystem II core dimer to ensure a directed transfer of excitation energy through the monomeric peripheral antenna proteins CP26 and CP29 and/or to maintain sequestered domains of inorganic cofactors required for oxygen evolution.     

Comparative EPR and thermoluminescence study of anoxic photoinhibition in Photosystem II particles

Photosystem II particles were exposed to 800 W m^–2 white light at 20 °C under anoxic conditions. The F_o level of fluorescence was considerably enhanced indicating formation of stable-reduced forms of the primary quinone electron acceptor, Q_A. The F_m level of fluorescence declined only a little. The g=1.9 and g=1.82 EPR forms characteristic of the bicarbonate-bound and bicarbonate-depleted semiquinone-iron complex, Q_A ^–Fe²⁺, respectively, exhibited differential sensitivity against photoinhibition. The large g=1.9 signal was rapidly diminished but the small g=1.82 signal decreased more slowly. The S₂-state multiline signal, the oxygen evolution and photooxidation of the high potential form of cytochrome b-559 were inhibited approximately with the same kinetics as the g=1.9 signal. The low potential form of oxidized cytochrome b-559 and Signal II_slow arising from Tyr_D ⁺ decreased considerably slower than the g=1.9 semiquinone-iron signal. The high potential form of oxidized cytochrome b-559 was diminished faster than the low potential form. Photoinhibition of the g=1.9 and g=1.82 forms of Q_A was accompanied with the appearance and gradual saturation of the spin-polarized triplet signal of P 680. The amplitude of the radical signal from photoreducible pheophytin remained constant during the 3 hour illumination period. In the thermoluminescence glow curves of particles the Q band (S₂Q_A ^– charge recombination) was almost completely abolished. To the contrary, the C band (Tyr_D ⁺Q_A ^– charge recombination) increased a little upon illumination. The EPR and thermoluminescence observations suggest that the Photosystem II reaction centers can be classified into two groups with different susceptibility against photoinhibition.Abbreviations C band thermoluminescence band associated with Tyr-D⁺Q _a ^– charge recombination - Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - EPR electron paramagnetic resonance - F_o initial fluorescence - F_m maximum fluorescence - Q band thermoluminescence band originating from S₂Q _a -charge recombination - Q _a the primary quinone electron acceptor of PS II - P 680 the primary electron donor chlorophyll of PS II - S₂ oxidation state of the water-splitting system - Phe pheophytin - TL thermoluminescence - Tyr _d redox active tyrosine-160 of the D₂ protein     

A new EPR signal attributed to the primary plastosemiquinone acceptor in Photosystem II

A study of signals, light-induced at 77 K in O₂-evolving Photosystem II (PS II) membranes showed that the EPR signal that has been attributed to the semiquinone-iron form of the primary quinone acceptor, Q^?_AFe, at g = 1.82 was usually accompanied by a broad signal at g = 1.90. In some preparations, the usual g = 1.82 signal was almost completely absent, while the intensity of the g = 1.90 signal was significantly increased. The g = 1.90 signal is attributed to a second EPR form of the primary semiquinone-iron acceptor of PS II on the basis of the following evidence. (1) The signal is chemically and photochemically induced under the same conditions as the usual g = 1.82 signal. (2) The extent of the signal induced by the addition of chemical reducing agents is the same as that photochemically induced by illumination at 77 K. (3) When the g = 1.82 signal is absent and instead the g = 1.90 signal is present, illumination at 200 K of a sample containing a reducing agent results in formation of the characteristic split pheophytin^? signal, which is thought to arise from an interaction between the photoreduced pheophytin acceptor and the semiquinone-iron complex. (4) Both the g = 1.82 and g = 1.90 signals disappear when illumination is given at room temperature in the presence of a reducing agent. This is thought to be due to a reduction of the semiquinone to the nonparamagnetic quinol form. (5) Both the g = 1.90 and g = 1.82 signals are affected by herbicides which block electron transfer between the primary and secondary quinone acceptors. It was found that increasing the pH results in an increase of the g = 1.90 form, while lowering the pH favours the g = 1.82 form. The change from the g = 1.82 form to the g = 1.90 form is accompanied by a splitting change in the split pheophytin^? signal from approx. 42 to approx. 50 G. Results using chloroplasts suggest that the g = 1.90 signal could represent the form present in vivo.     

Positions of QAand ChlZRelative to Tyrosine YZand YDin Photosystem II Studied by Pulsed EPR

PELDOR (Pulsed Electron eLectron DOuble Resonance) was applied to determinethe distance of between Y_Zand Q_A ^-inY_D-less mutant of Chlamydomonas reinhardtiiin Tris-treatedand Zn-substituted preparation of photosystem II. The value of distance wasfound to be 34.5 ± 1 Â. A 2+1 electron spin echo method has beenapplied to measure the orientation of the radius-vector RfomY_Dto Chl_Zin a membrane-oriented photosystem II. The anglebetween Rand the membrane normal nwas determined to be 50 ±5^°, using the distance 29.4 ± 0.5 Â determined in non-orientedPS II.