Intactness of the oxygen-evolving system in thylakoids and Photosystem II particles

Photosystem II activity of oxygen-evolving membranes can be quantified by their capacity to do charge separation or their capacity to transport electrons. In this study using flash excitation of saturating intensity, charge separation is measured by absorption changes in the ultraviolet region of the spectra associated with primary-quinone reduction, and electron transport is measured by oxygen flash yield. These methods are applied to thylakoids and three different types of Photosystem II particles. In thylakoids electron-transport activity is 75–85% of charge separation activity. In Photosystem II particles this percentage is 60–70%, except for the BBY type (Berthold, D.A., Babcock, G.T. and Yocum, C.F. (1981) FEBS Lett. 135, 231–234), in which it is only 29%. These estimates of non-functional oxygen-evolving centers agree within experimental error, except for the BBY particle, with the quantum requirement for oxygen evolution measured under light-limited conditions. These reaction centers that are non-functional in oxygen evolution occur during sample preparation and are not a result of inhibition by ferricyanide or quinone acceptor systems. In thylakoids on the first flash, absorption changes at 325 nm do not show significant contributions from oxygen evolution S-state transitions. In the presence of ferricyanide the absorption change at 325 nm does have a significant contribution from Q₄₀₀ in thylakoids, but considerably less in Photosystem II particles.     

A backflow of electrons around Photosystem II in Chlorella cells

A study was made with a modulated oxygen electrode of the effect of variations of oxygen concentration on photosynthetic oxygen evolution from algal cells. When Chlorella vulgaris is examined with a modulated 650 nm light at 22°C, both the oxygen yield and the phase lag between the modulated oxygen signal and the light modulations have virtually constant values between 800 and 120 ergs · cm^?1 · s^?1 if the bathing medium is in equilibrium with the air. Similar results are obtained at 32°C between 1600 and 120 ergs · cm^?2 · s^?1. Under anerobic conditions both the oxygen yield and the phase lag decrease if the light intensity is lowered below about 500 ergs · cm^?2 · s^?1 at 22°C or about 1000 ergs · cm^?2 · s^?1 at 32°C. A modulated 706 nm beam also gives rise to these phenomena but only at significantly lower rates of oxygen evolution. The cells of Anacystis nidulans and Porphyridium cruentum appear to react in the same way to anaerobic conditions as C. vulgaris. An examination of possible mechanisms to explain these results was performed using a computer simulation of photosynthetic electron transport. The simulation suggests that a backflow of electrons from a redox pool between the Photosystems to the rate-limiting reaction between Photosystem II and the water-splitting act can cause a decrease in oxygen yield and phase lag. If the pool between the Photosystems is in a very reduced state a significant cyclic flow is expected, whereas if the pool is largely oxidized little or no cyclic flow should occur. It is shown that the effects of 706 nm illumination and removal of oxygen can be interpreted in accordance with these proposals. Since a partial inhibition of oxygen evolution by 3-(3.4-dichlorophenyl)-1,1-dimethylurea (10^?8 M) magnifies the decreases in oxygen yield and phase lag, it is proposed that the pool which cycles back electrons is in front of the site of 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition and is probably the initial electron acceptor pool after Photosystem II.     

Detection of oxygen-evolving Photosystem II centers inactive in plastoquinone reduction

Quite different estimates of the number of Photosystem II centers present in thylakoid membranes are obtained depending on the technique used in making the determination. By using brief saturating light flashes and measuring the electron transport per flash, we have obtained two values for the number of functional centers. When the electrons produced reduce the intersystem plastoquinone pool, there are about 1.7 mmol of active Photosystem II centers per mol chlorophyll, whereas there are at least 3 mmol of active centers per mol chlorophyll when certain halogenated benzoquinones are being reduced. There are also at least 3 mmol of terbutryn binding sites per mol of chlorophyll when this tightly binding herbicide is employed as a specific inhibitor of Photosystem II. Thus only about 60% of the membrane's total complement of Photosystem II centers are able to transfer electrons to Photosystem I at appreciable rates. Many functional assays requiring significant rates of turnover sample only this more active pool, whereas herbicide-binding studies and measurements of changes in the Photosystem II electron donor Z and electron acceptor Q_A performed by other investigators reveal, in addition, a large population of Photosystem II reaction centers that normally have negligible turnover numbers. However, these normally inactive centers readily transfer electrons to the halogenated benzoquinones and are then counted among the active centers. Therefore, it can be concluded that all of herbicide-binding sites represent centers with operative water-oxidizing reactions. It can also be concluded that there are few, if any, centers capable of binding more than a single herbicide molecule.     

Mode of action of Photosystem II herbicides studied by thermoluminescence

The mode of action of chemically different herbicides (ureas, pyridazinones, phenylcarbamates, triazines, hydroxyquinolines, hydroxybenzonitriles and dinitrophenols) on photosynthetic electron transport was investigated by measurements of oxygen evolution and thermoluminescence. Depending on the particular herbicide used the thermoluminescence band related to Q (the primary acceptor of Photosystem II) appears at +5, 0 or −14°C. It was shown that these three different peak positions can be ascribed to various redox states of Q, the shifts being due to the binding of herbicides to the chloroplast membrane. Both displacement experiments and additive inhibition of herbicide pairs measured by thermoluminescence and oxygen evolution suggested that the sites of action of these herbicides are on the same protein. However, herbicide treatment of trypsinized chloroplasts showed that there were three different binding sites on the same protein, in agreement with the classification of herbicides into three groups based on thermoluminescence measurements. Our results suggest that the primary and secondary acceptors of Photosystem II (Q and B, respectively) are in close proximity and form a common complex with the herbicide-binding protein within the chloroplast membrane.     

Thermoluminescence as a probe of photosystem II. The redox and protonation states of the secondary acceptor quinone and the O2-evolving enzyme

The thermoluminescence band observed in chloroplasts after flash excitation at ambient temperatures has recently been identified as being due to recombination of the electron on the semiquinone form of the secondary plastoquinone acceptor, Q_B, with positive charges on the oxygen-evolving enzyme, S₂ and S₃ (Rutherford, A.W., Crofts, A.R. and Inoue, Y. (1982) Biochim. Biophys. Acta 682, 457–465). Further investigation of this thermoluminescence confirms this assignment and provides information on the function of PS II. The following data are reported: (1) Washing of chloroplasts with ferricyanide lowers the concentration of Q_B⁻ in the dark and predictable changes in the extent of the thermoluminescence band are observed. (2) The thermoluminescence intensity arising from S₂Q_B⁻ is approximately one half of that arising from S₃Q_B⁻. (3) Preflash treatment followed by dark adaptation results in changes in the intensity of the thermoluminescence band recorded after a series of flashes. These changes can be explained according to the above assignments for the origin of the thermoluminescence and if Q_B⁻ provides an important source of deactivating electrons for the S states. Computer simulations of the preflash data are reported using the above assumptions. Previously unexplained data already in the literature (Läufer, A. and Inoue, Y. (1980) Photobiochem. Photobiophys. 1, 339–346) can be satisfactorily explained and are simulated using the above assumptions. (4) Lowering the pH to pH 5.5 results in a shift of the S₂Q_B⁻ thermoluminescence band to higher temperatures while that arising from S₃Q_B⁻ does not shift. This effect is interpreted as indicating that Q_B⁻ is protonated and the S₂ to S₃ reaction involves deprotonation while the S₁ to S₂ reaction does not.     

Refined purification and further characterization of oxygen-evolving and Tris-treated Photosystem II particles from the thermophilic Cyanobacterium synechococcus sp.

Highly active, monomeric and dimeric Photosystem II complexes were purified from the thermophilic cyanobacterium Synechococcus sp. by two sucrose density gradients, and the size, shape and mass of these complexes have been estimated (Rögner, M., Dekker, J.P., Boekema, E.J. and Witt, H.T. (1987) FEBS Lett. 219, 207–311). (1) Further purification could be obtained by ion-exchange chromatography, by which the 300 kDa monomer could be separated into a highly active, O₂-evolving fraction, and a fraction without O₂-evolving capacity, which has lost its extrinsic 34 kDa protein. Both showed very high reaction center activities as measured by the photoreduction of the primary quinone acceptor, Q_A, at 320 nm, being up to one reaction center per 31 Chl a molecules. (2) Tris-treatment yielded homogeneous 300 kDa particles which had lost their extrinsic 34 kDa polypeptide. Electron microscopy of this complex revealed very similar dimensions compared to the oxygen-evolving 300 kDa particle, except that the smallest dimension was decreased from about 6.5 nm to about 5.8 nm. This difference is attributed to the missing extrinsic 33 kDa protein, and the smallest dimension is attributed to the distance across the membrane. (3) Experiments are presented, allowing an estimation for the contribution of detergent to the other dimensions being about 2 × 1.5 nm for dodecyl β- -maltoside. This leads to dimensions, corrected for detergent size, of 12.3 × 7.5 nm for the monomeric form of PS II and 12 × 15.5 nm for the dimeric form. (4) From some extracts a 35 kDa, chlorophyll-binding complex could be isolated which lacks the characteristic absorbance changes of Q_A and of Chl a_II (P-680) and is therefore supposed to be a light-harvesting complex of cyanobacteria. (5) A model for the in vivo organization of PS II in cyanobacteria is discussed.     

Directed inactivation of the psbl gene does not affect Photosystem II in the cyanobacterium Synechocystis sp. PCC 6803

PsbI is a small, integral membrane protein component of photosystem II (PSII), a pigment-protein complex in cyanobacteria, algae and higher plants. To understand the function of this protein, we have isolated the psbI gene from the unicellular cyanobacterium Synechocystis sp. PCC 6803 and determined its nucleotide sequence. Using an antibiotic-resistance cartridge to disrupt and replace the psbI gene, we have created mutants of Synechocystis 6803 that lack the PsbI protein. Analysis of these mutants revealed that absence of the PsbI protein results in a 25–30% loss of PSII activity. However, other PSII polypeptides are present in near wild-type amounts, indicating that no significant destabilization of the PSII complex has occurred. These results contrast with recently reported data indicating that PsbI-deficient mutants of the eukaryotic alga Chlamydomonas reinhardtii are highly light-sensitive and have a significantly lower (80–90%) titer of the PSII complex. In Synechocystis 6803, PsbI-deficient cells appear to be slightly more photosensitive than wild-type cells, suggesting that this protein, while not essential for PSII biogenesis or function, plays a role in the optimization of PSII activity.     

Kinetics of the oxygen-evolving complex in salt-washed photosystem II preparations

The kinetics of flash-induced electron transport were investigated in oxygen-evolving Photosystem II preparations, depleted of the 23 and 17 kDa polypeptides by washing with 2 M NaCl. After dark-adaptation and addition of the electron acceptor 2,5-dichloro-p-benzoquinone, in such preparations approx. 75% of the reaction centers still exhibited a period 4 oscillation in the absorbance changes of the oxygen-evolving complex at 350 nm. In comparison to the control preparations, three main effects of NaCl-washing could be observed: the half-time of the oxygen-evolving reaction was slowed down to about 5 ms, the misses and double hits parameters of the period 4 oscillation had changed, and the two-electron gating mechanism of the acceptor side could not be detected anymore. EPR-measurements on the oxidized secondary donor Z⁺ confirmed the slower kinetics of the oxygen-releasing reaction. These phenomena could not be restored by readdition of the released polypeptides nor by the addition of CaCl₂, and are ascribed to deleterious action of the highly concentrated NaCl. Otherwise, the functional coupling of Photosystem II and the oxygen-evolving complex was intact in the majority of the reaction centers. Repetitive flash measurements, however, revealed P⁺Q⁻ recombination and a slow Z⁺ decay in a considerable fraction of the centers. The flash-number dependency of the recombination indicated that this reaction only appeared after prolonged illumination, and disappeared again after the addition of 20 mM CaCl₂. These results are interpreted as a light-induced release of strongly bound Ca²⁺ in the salt-washed preparations, resulting in uncoupling of the oxygen-evolving system and the Photosystem II reaction center, which can be reversed by the addition of a relatively high concentration of Ca²⁺.     

Quantitative analysis of membrane components in a highly active O2-evolving photosystem II preparation from spinach chloroplasts

Stoichiometry of membrane components associated with Photosystem II was determined in a highly active O₂-evolving Photosystem II preparation isolated from spinach chloroplasts by the treatment with digitonin and Triton X-100. From the analysis with sodium dodecyl sulfate polyacrylamide gel electrophoresis and Triton X-114 phase partitioning, the preparation was shown to contain the reaction center protein (43 kDa), the light-harvesting chlorophyll-protein complex (the main band, 27 kDa), the herbicide-binding protein (32 kDa) and cytochrome b-559 (10 kDa) as hydrophobic proteins, and three proteins (33, 24 and 18 kDa) which probably constitute the O₂-evolution enzyme complex as hydrophilic proteins. These proteins were associated stoichiometrically with the Photosystem II reaction center: one Photosystem II reaction center, approx. 200 chlorophyll, one high-potential form of cytochrome b-559, one low-potential form of cytochrome b-559, one 33 kDa protein, one (to two) 24 kDa protein and one (to two) 18 kDa protein. Measurement of fluorescence induction showed the presence of three electron equivalents in the electron acceptor pool on the reducing side of Photosystem II in our preparation. Three molecules of plastoquinone A were detected per 200 chlorophyll molecules with high-performance liquid chromatography. The Photosystem II preparation contained four managanese atoms per 200 chlorophyll molecules.     

Electron donation in Photosystem II

The electron transfer resulting from illumination and dark storage of PS II has been studied using EPR signals from several electron carriers. The recombination of D⁺ (Signal II) and Q⁻_A formed by illumination occurred during dark storage at 77 K and was used to deplete reaction centres of D⁺. The donor D was then shown to be oxidized in the dark by the S₂ state of the oxygen-evolving complex. A slow change which occurred during dark storage of PS II samples was detected using the power saturation characteristics of D. We interpret this effect on D to be an indirect result of a rearrangement of the manganese complex during long-term dark adaptation. A role for D in the stability, protection and perhaps initial manganese binding of the oxygen-evolving complex is suggested.     

The S-state dependence of Cl binding to plant Photosystem II

A combined single-turnover flash and ³⁵Cl NMR technique has been used to monitor S-state dependence of Cl⁻ binding to PS-II particles derived from mangrove (Avicennia marina). No detectable high-affinity binding was found to particles in the S₀ and S₁ states, but binding with an affinity comparable to that which activates O₂ evolution was found in the S₂ and S₃ states.     

Optical and EPR characterizations of oxygen-evolving Photosystem II subchloroplast fragments isolated from the thermophilic blue-green alga Phormidium laminosum

An oxygen-evolving, Photosystem II particle was isolated from the thermophilic, blue-green alga, Phormidium laminosum, according to the procedure of Stewart and Bendall (Stewart, A.C. and Bendall, D. (1979) FEBS Lett. 107, 308–312). Our particle has an oxygen-evolution activity of 1500–1600 μmol O₂/mg chlorophyll per h. The oxygen-evolution activity has a pH optimum at 5–6, and is abolished at pH 9. Maximum oxygen evolution occurs at approx. 47°C in whole cells, but at 29°C in the particles. The activity decreases to 50% when the cells are heated for 30 min at 55°C; with the particles, 50% inactivation occurred at 47°C for the same heating time of 30 min. Flash excitation of the particle at 100 K produced absorbance changes whose difference spectrum in the ultraviolet-to-near infrared region shows photochemical charge separation and recombination of P-680⁺ and Q⁻ in the dark with of 1.75 ms. An EPR spectrum for the P-680⁺ free radical, with g 2.0027 and ΔH_pp = 8 G, was constructed from flash-induced EPR changes under conditions identical to those used for obtaining P-680 absorbance changes. The actinic light-induced variable fluorescence yield is 5-fold that induced by the weak probing beam alone. Addition of dithionite to the particle brings the fluorescence to the same maximum level. Under the reducing condition, strong actinic light caused the fluorescence to decrease. This observation is consistent with the notion that variable fluorescence yield in Photosystem II originates, as in green-plant chloroplasts, from recombination luminescence, the attenuation of which corresponds to photoaccumulation of reduced pheophytin under these conditions. Broad segments (300 nm) of the difference spectrum for pheophytin photoreduction were recorded by an intensified photodiode array in conjunction with a phosphoroscopic photometer. Kinetic spectrophotometric assays together with chemical analysis showed a rather clean and simple stoichiometry in these particles, namely, 1 P-680:1 Ph:1 Q:4 Mn:44 Chl. Initial investigation failed to reveal the doublet EPR spectrum previously observed for Ph⁻·Q⁻ Fe in spinach subchloroplast particles (Klimov, V.V., Dolan, E. and Ke, B. (1980). FEBS Lett. 118, 97–100). A hyperfine EPR spectrum consisting of 16–20 lines and presumably associated with the manganese clusters in the oxygen-evolving protein has been confirmed in these particles. Tris washing but not washing with EDTA eliminates this signal. Active oxygen-evolving particles also yield the II_vf signal with a of approx. 800 μs. Upon Tris washing, the II_f signal appears which decays in 23.5 ms.     

CI-NMR measurement of chloride binding to the oxygen-evolving complex of spinach Photosystem II

The mechanism by which Cl⁻ activates the oxygen-evolving complex (OEC) of Photosystem II (PS II) in spinach was studied by ³⁵Cl-NMR spectroscopy and steady-state measurements of oxygen evolution. Measurements of the excess ³⁵Cl-NMR linewidth in dark-adapted, Cl⁻-depleted thylakoid and Photosystem II membranes show an overall hyperbolic decrease which is interrupted by sharp increases in linewidth (linewidth maxima) at approx. 0.3 mM, 0.75 mM, 3.25 mM (2.0 mM in PS II membranes), and 7.0 mM Cl⁻. The rate of the Hill reaction (H₂O → 2,6-dichlorophenolindophenol) at low light intensities (5% of saturation) as a function of [Cl⁻] in thylakoids shows three intermediary plateaus in the concentration range between 0.1 and 10 mM Cl⁻ indicating kinetic cooperativity with respect to Cl⁻. The presence of linewidth maxima in the ³⁵Cl-NMR binding curve indicates that Cl⁻ addition exposes four types of Cl⁻ binding site that were previously inaccessible to exchange with Cl⁻ in the bulk solution. These results are best explained by proposing that Cl⁻ binds to four sequestered (salt-bridged) domains within the oxygen-evolving complex. Binding of Cl⁻ is facilitated by the presence of H⁺ and vice versa. The pH dependence of the excess ³⁵Cl-NMR linewidth at 0.75 mM Cl⁻ shows that Cl⁻ binding has a maximum at pH 6.0 and two smaller maxima at pH 5.4 and 6.5 which may suggest that as many as three groups (perhaps histidine) with pK_a values in the region may control the binding.     

A study of dark luminescence in Chlorella. Background luminescence, 3-(3,4-dichlorophenyl)-1,1-dimethylurea-triggered luminescence and hydrogen peroxide chemiluminescence

Dark luminescence, defined as the ability of completely relaxed (darkadapted) photosynthetic systems to emit light, has been studied in Chlorella. Three main effects have been demonstrated. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea elicits a weak emission L_D of very long lifetime (several minutes); it is believed to result from a negative shift of redox potential of the secondary System II electron acceptor B producing in some centers a state Q⁻ (reduced primary acceptor), as postulated by Velthuys and Amesz ((1974) Biochim. Biophys. Acta 333, 85–94), which can recombine with an oxidizing equivalent in a state S₂ present in very small amount. As in photoinduced luminescence, this recombination excites chlorophyll which then emits light. A much stronger emission L_H is observed after injection of H₂O₂. Both signals are modified or suppressed by treatments specific of the oxygen emission system, such as: thermal denaturation at 50°C, NH₂OH, etc. In addition, a weak, permanent background luminescence L₀ has been observed; like L_D and L_H, it is a System II property and requires the integrity of the oxygen-evolving system. It is believed to reflect a very slow back flow of electrons from an endogeneous reductant pool to oxygen through part of the photosynthetic chain. Using flash preillumination, it is demonstrated that H₂O₂ is able to oxidize S₀ into S₂, the latter giving rise to L_H; H₂O₂ does not act on S₁ (or much less). The reactive site of H₂O₂ seems to be the same as the binding site of NH₂OH. Evidence is given that the strong L_H signal in particular reveals a stable, low pH of the intrathylakoid phase in Chlorella.     

Isolation of an oxygen-evolving Photosystem II preparation containing only one tightly bound calcium atom from a chlorophyll b-deficient mutant of rice

Oxygen-evolving Photosystem II (PS II) particles were prepared from the thylakoid membranes of a chlorophyll b-less rice mutant, which totally lacks light-harvesting chlorophyll a/b proteins, after solubilization with β-octylglucoside. The preparation was essentially free of Photosystem I as judged from its low-temperature fluorescence spectrum and polypeptide composition. The PS II particles contained all the major subunit polypeptides of the PS II reaction center core complexes and the three extrinsic proteins related to oxygen evolution. The relative abundances of the 33, 21 and 15 kDa proteins were 100, 64 and 20%, respectively, of the corresponding proteins in the mutant thylakoids. The chlorophyll-to-Q_A ratio was 53 and there was only one bound Ca²⁺ per Q_A. Thus, one of the two bound Ca²⁺ present in the oxygen-evolving PS II membrane preparations from wild-type rice (Shen J.-R., Satoh, K. and Katoh, S. (1988) Biochim. Biophys. Acta 933, 358–364) is missing. The mutant PS II particles were highly active in oxygen evolution in the absence of exogenously added Ca²⁺, although addition of 5 mM Ca²⁺ enhanced the activity by 30%. When the 21 and 15 kDa proteins were supplemented to the particles, the Ca²⁺-effect disappeared and the rate of oxygen evolution increased to a level exceeding 1000 μmol O₂ per mg chlorophyll per h. The results indicate that the number of Ca²⁺ needed to promote a high rate of oxygen evolution is one per PS II in higher plants.     

The influence of anions on oxygen evolution by isolated spinach chloroplasts

A study has been made of the onset of chloride deprivation on the oxygen-evolving characteristics of isolated spinach chloroplasts. Using a modulated oxygen electrode it is found that the type of inhibition depends on the anion replacing chloride in the bathing medium. With nitrate a large increase in phase lag accompanies a relatively small inhibition which can be shown to be consistent with a decrease in the rate constant of the reaction which limits the rate of electron transport between water and Photosystem II. With sulphate there is a very small phase change but a larger inhibition which suggests that replacing chloride with sulphate in an electron-transport chain shuts off that chain. With acetate there is a moderate increase in phase lag and the largest inhibitory effect. The phase-lag increase suggests that acetate is affecting the same chloride-sensitive site as nitrate. However, the inhibition cannot be explained by this effect alone and points to the existence of a second chloride-sensitive site. Of the four forward reactions associated with the Kok model of oxygen evolution (Kok, B., Forbush, B. and McGloin, M. (1970) Photochem. Photobiol. 11, 457–475) only S1₃ → S₀ is slowed down when chloride is replaced by nitrate. This reaction is not slowed down by replacing chloride with sulphate.     

Partial degradation of the extrinsic 23-kDa protein of the Photosystem II complex of spinach

Chymotrypsin eliminated nine amino acid residues at the amino-terminal side of the extrinsic 23-kDa protein of the oxygen-evolving Photosystem II complex of spinach. The resultant 22-kDa fragment was able to bind to the Photosystem II complex but with lowered binding affinity. However, once the 22-kDa fragment bound to the complex, it retained most functions of the 23-kDa protein; the fragment provided a binding site for the extrinsic 18-kDa protein, preserved a tight trap for Ca²⁺ in the complex, and shifted the optimum Cl⁻ concentration for oxygen evolution from 30 to 10 mM, although it was less effective in sustaining oxygen evolution at Cl⁻ concentrations below 10 mM. These observations suggest that the elimination of nine amino acid residues at the amino-terminal region of the 23-kDa protein does not significantly alter the conformation of the protein, except for partial modification of its binding site and its interaction with Cl⁻.     

Spin-label studies of the lipid regions of spinach thylakoids and a detergent-derived oxygen-evolving Photosystem II preparation

We have compared the fluidity of thylakoid membranes with the membrane present in a Triton X-100-derived, oxygen-evolving Photosystem II (PS II) preparation using two different spin labels. Data obtained with 2,2,6,6-tetramethylpipiridine-N-oxyl (TEMPO) shows that the PS II preparation contains less fluid membrane than the thylakoid. The TEMPO partition parameter (f) is about 2.5-times greater for the thylakoids at 6 mg chlorophyll/ml than for the PS II preparation at the same chlorophyll concentration. Similarly, the rotational correlation time, τ, of TEMPO residing in the membrane of the PS II preparation is about 2-times longer than the τ for TEMPO in the thylakoid membrane. A spin label which partitions more completely into the bilayer, 2-heptyl-2-hexyl-5,5-dimethyloxazolidine-N-oxyl (7N14), indicates a much greater fluidity in the thylakoid membrane than the membrane of the PS II preparation. The PS II preparation appears to have a hydrocarbon phase which approaches the rigid limit of EPR detectable motion. These results are discussed in terms of possible lipid depletion in the PS II preparation and in terms of lateral heterogeneity of hydrocarbon fluidity in the thylakoid membrane caused by the lateral heterogeneity in protein components.     

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.     

Interaction of 1,4-benzoquinones with Photosystem II in thylakoids and Photosystem II membrane fragments from spinach

The interaction of exogenous quinones with the Photosystem II (PS II) acceptor side has been analyzed by measurements of flash-induced 320 nm absorption changes, transient flash-induced variable fluorescence changes, thermoluminescence emission and oxygen yield in dark-adapted thylakoids and PS II membrane fragments. Two classes of 1,4-benzoquinones were shown to give rise to remarkably different reaction patterns. (A) Phenyl-p-benzoquinone (Ph-p-BQ) -type compounds give rise to a marked binary oscillation of the initial amplitudes of 320 nm absorption changes induced by a flash train in dark-adapted PS II membrane fragments and a retardation of the decay kinetics of the flash-induced variable fluorescence. The electron transfer reactions to these type of quinones are severely inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). (B) In the presence of tribromotoluquinone (TBTQ) a different oscillation pattern of the 320 nm absorption changes is observed characterized by a marked relaxation after the first flash in the 5 ms domain. This relaxation is insensitive to 10 μM DCMU. Likewise the decay of the flash-induced variable fluorescence in TBTQ-treated samples is much less sensitive to DCMU than in control. The thermoluminescence emission exhibits an oscillation in samples incubated for 5 min with TBTQ before addition of 30 μM DCMU. Under the same conditions a significant flash-induced oxygen evolution is observed only after the third and fourth flash, respectively, whereas in the presence of TBTQ alone a normal oscillation pattern is observed. The different functional patterns of PS II caused by the two types of classes of exogenous quinones are interpreted by their binding properties: a noncovalent association with the Q_B-site of Ph-p-BQ-type quinones versus a tight (covalent?) binding in the vicinity of Q_A (possibly also at the Q_B-site) in the case of halogenated 1,4-benzoquinones. The mechanistic implications of these findings are discussed.