Depletion of Photosystem II-extrinsic proteins. I. Effects on O2- and N2-flash yields and steady-state O2 evolution

Wheat O₂-evolving Photosystem II (PS II) membranes having a PS II unit of approx. 200 chlorophylls (Chl), approx. 4 Mn/200 Chl, less than 1 P-700/3000 Chl and an electron-acceptor pool of approx. 2.5 equiv./PS II were analyzed and compared with wheat PS II membranes depleted (at least 90%) of the 17 and 23 kDa proteins by NaCl extraction during Triton X-100 isolation of membranes. Extraction of these proteins caused approx. 50% decrease in O₂ evolution in any light regime and an increase of approx. 2 equiv./PS II of the electron-acceptor pool, but affected neither Mn abundance, photoreduction of DCIP by tetraphenylboron, or N₂ yield (from NH₂OH) from a single flash. Mass spectrometric analyses of O₂ flash yields in the presence of potassium ferricyanide showed that both chloroplasts and the unextracted PS II membranes yielded oscillations compatible with S₀/S₁/S₂/S₃ of 25:75:0:0 and α (0.1) and β (0.05). Depletion of 17 and 23 kDa proteins resulted in a two-fold increase in α, approx. 25–40% disconnection of the S state complex from the PS II trap complex but with no change in β. Preincubation of control or extracted PS II membranes with potassium ferricyanide permitted a significant double-hit on the first flash. In the absence of an added electron acceptor, N₂ flash yields were more sustained with 17 and 23 kDa depleted than with 17 and 23 kDa sufficient PS II membranes. In contrast, no significant O₂ flash yields were observed with extracted PS II preparations under these conditions (control PS II membranes showed a predictable O₂ pattern before damping after only 5–6 flashes). These results suggest that extraction of the 17 and 23 kDa proteins results in an increase of pool size on the PS II acceptor side (seen as unmasking ‘Component C’). ‘Component C’ can mediate electron transfer from Q⁻ to Z⁺ (S₂).     

Slow oxygen release on the first two flashes in chemically stressed Photosystem II membrane fragments results from hydrogen peroxide oxidation

Flash-induced amperometric signals were measured with a Joliot-type O₂ rate electrode in spinach Photosystem II (PS II) membrane fragments exposed to very low concentrations of added hydroxylamine or hydrogen peroxide. In both cases anomalous O₂ signals were observed on the first two flashes, and oscillating four-flash patterns were observed on subsequent flashes. The anomalous signals were eliminated in the presence of catalase but not EDTA. The rise times of the O₂-release kinetics associated with the anomalous signals were slow (ca. 20 ms with NH₂OH and ca. 120 ms with H₂O₂) compared to the kinetics of O₂ release on subsequent flashes and in control membranes (3–6 ms). It is proposed that when the intact PS II O₂-evolving complex is perturbed with small concentrations of added reductant, H₂O₂ can gain access and bind to the complex. Bound H₂O₂ can then reduce lower S states in some centers leading to anomalous O₂ signals on the first two flashes. A model is presented to explain both types of anomalous O₂ production. Oxygen observed on the third and subsequent flashes is due to the normal photosynthetic O₂-evolution process arising from the S₃-state. Anomalous O₂ production could be a protective mechanism in PS II centers subjected to stress conditions.Abbreviations DCIP 2,6-dichlorophenolindophenol - EDTA ethylenediaminetetraacetic acid - MES 4-morpholine-ethanesulfonic acid - OEC oxygen-evolving complex - PS II Photosystem II - Y_i O₂ flash yield on the i^th flash - Y_ss steady-state O₂ flash yield level in algae, chloroplasts, or thylakoids after flash-driven S-state oscillations have been damped Formerly, the Solar Energy Research Institute and operated by the Midwest Research Institute for the US Department of Energy under Contract DE-AC-02-83CH10093. Government and MRI retain non-exclusive, royalty-free license to publish or reproduce published articles, or allow others to do so for Government purposes.     

Effects of removal and reconstitution of the extrinsic 33, 24 and 16 kDa proteins on flash oxygen yield in Photosystem II particles

The effects of removal and reconstitution of the three extrinsic proteins on the flash O₂ yield were investigated and the following results were obtained. (1) Removal in darkness of the 24 and 16 kDa proteins affected neither the oscillation pattern nor the signal amplitude of the flash O₂ yield. However, the signal amplitude was reduced with a factor of 2 in the presence of EDTA and was restored by excess Ca²⁺. The EDTA treatment did not change the oscillation pattern of the flash O₂ yield, but considerably damped the oscillation pattern of thermoluminescence B band. These results suggest a heterogeneity among the centers in binding affinity for Ca²⁺, and that Ca²⁺ removal induces an all-or-none type inactivation of O₂ evolution but not in the thermoluminescence processes, indicative of an inhibition of the S-state turnover at a specific S-state. (2) Removal in darkness of the 33, 24 and 16 kDa proteins abolished the flash O₂ yield, but the inhibited yield was appreciably restored either by reconstitution with the 33 kDa protein or by inclusion of 200 mM Cl⁻ in the reaction mixture. The flash O₂ yield reconstituted by the 33 kDa protein exhibited a rather normal oscillation pattern accompanied by a slightly increased damping, which could be simulated by assuming a high miss factor (30%) for S₃ → S₀ transition. The Cl⁻-restored flash O₂ yield exhibited a strongly damped oscillation pattern with obscured maxima at the 4th and 8th flashes, which was simulated by assuming a much higher miss factor (70%) for S₃ → S₀ transition. It was indicated that the Cl⁻-restored O₂ evolution considerably differs from the 33 kDa protein-reconstituted O₂ evolution with respect to the mechanism of S-state turnover.     

Inhibition of a respiratory activity by short saturating flashes in Chlamydomonas: Evidence for a chlororespiration

Excitation with short actinic flashes (2 μs) of oxygenated dark-adapted Chlamydomonas cells deposited on a bare O₂ platinum electrode induces an increase of the amperometric signal after the first two flashes. Mass spectrometer experiments performed in the presence of ¹⁸O₂ showed that this signal was not due to the photolysis of water (H₂¹⁶O). The insensitivity of this signal to 10 μM DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea), its stimulation by acetate or high O₂ concentration as well as its inhibition by cyanide indicate that these flash-induced changes in O₂ concentration were related to the inhibition of a respiratory process. Because this rather fast inhibition of respiration is insensitive to antimycin A and to salicyl hydroxamic acid, inhibitors of mitochondrial respiration, and because it occurs on a single flash illumination, we conclude that the related respiratory activity takes place inside the chloroplast (chlororespiration) and not in the mitochondria. This interpretation is confirmed by the quite high K_m(O2) of this process (about 23 μM) compared to those measured for the mitochondrial reactions (0.2 μM for the cytochrome oxidase pathway and 5.5 μM for the alternative pathway). In a mutant lacking Photosystem I activity, no photoinhibition of respiration was observed. We conclude from the above results that the light-induced inhibition of chlororespiration is due to the oxidation by Photosystem I activity of electron carriers common to both photosynthetic and chlororespiratory chains.     

Formation and flash-dependent oscillation of the S2-state multiline EPR signal in an oxygen-evolving Photosystem-II preparation lacking the three extrinsic proteins in the oxygen-evolving system

In PS-II-enriched membranes lacking the three extrinsic water-soluble proteins in the oxygen-evolving system (18, 24 and 33 kDa), but still evolving oxygen to some extent, the formation of the multiline EPR signal originating from the S₂-state is dependent on the concentration of Cl⁻. In 200 mM Cl⁻ the multiline signal was observed after the first flash and oscillated with the flash number with a period of four. At 20 mM Cl⁻ no signal could be observed in this material. These results suggest that the extrinsic proteins are not necessary for multiline signal formation and that complete advancement through the S-states can occur in their absence when sufficient Cl⁻ is present.     

The roles of the extrinsic subunits in Photosystem II as revealed by EPR

The effects of selective removal of extrinsic proteins on donor side electron transport in oxygen-evolving PS II particles were examined by monitoring the decay time of the EPR signal from the oxidized secondary donor, Z⁺, and the amplitude of the multiline manganese EPR signal. Removal of the 16 and 24 kDa proteins by washing with 1 M NaCl inhibits oxygen evolution, but rapid electron transfer to Z⁺ still occurs as evidenced by the near absence of Signal II_f. The absence of a multiline EPR signal shows that NaCl washing induces a modification of the oxygen-evolving complex which prevents the formation of the S₂ state. This modification is different from the one induced by chloride depletion of PS II particles, since in these a large multiline EPR signal is found. After removal of the 33 kDa protein with 1 M MgCl₂, Signal II_f is generated after a light flash. Readdition of the 33 kDa component to the depleted membranes accelerates the reduction of Z⁺. Added calcium ions show a similar effect. These findings suggest that partial advancement through the oxygen-evolving cycle can occur in the absence of the 16 and 24 kDa proteins. The 33 kDa protein, on the other hand, may be necessary for such reactions to take place.     

The two forms of the S2 state multiline signal in Photosystem II: effect of methanol and ethanol

The characteristic Mn hyperfine ‘multiline’ signal exhibited in the S₂ state of the oxygen-evolving complex (OEC) complex of Photosystem II (PSII) has been shown to be heterogeneous in character. In this study, we have explored the effects that influence the proportions of the two forms of the S₂ state multiline signal present in any sample. The narrow form of the signal is lost upon storage (weeks) at 77 K, whereas the broad form remains. In particular, we explore the roles of ethanol and methanol as well as effects of the second turnover of the enzyme on storage of the sample at 77 K. We find that in samples containing methanol, the narrow form may predominate upon the first flash, but the broad form predominates on the fifth flash and also in samples containing ethanol.     

A comparison of the absorption changes near 325 nm and chlorophyll a fluorescence characteristics of the Photosystem II acceptors Qa and Q400

The stoichiometry of chlorophyll/Photosystem II was determined in pea thylakoids. The concentration of Photosystem II was determined by the absorption change at 325 nm. When the 325 nm measurement was made on the first flash in the presence of ferricyanide, the Photosystem II absorption change was found to increase by up to 100% of the same measurement made in the absence of ferricyanide. The increase in absorption change in the presence of various amounts of ferricyanide was found to correlate well with the increase in area above the Chl a fluorescence induction curve. Also, the dark recovery of both the 325 nm absorption change and the area above the Chl a fluorescence curve are similar and in the order of several minutes. Absorption changes made under repetitive flash excitation showed no increase in signal with the addition of ferricyanide. We conclude that there are two acceptors, Q_a and Q₄₀₀, for each active oxygen-evolving complex and only Q_a is involved in active electron transport to Photosystem I.     

Studies on the polypeptide composition of the cyanobacterial oxygen-evolving complex

Various approaches have been used to investigate the polypeptides required for oxygen evolution in cyanobacteria, in particular the thermophile Phormidium laminosum. Antibodies against the extrinsic 33 kDa protein from spinach Photosystem II cross-reacted clearly in immunoblotting experiments with a corresponding polypeptide in isolated thylakoids and Photosystem II particles from P. laminosum and with whole-cell homogenates of three species of cyanobacteria (Phormidium laminosum, Synechococcus leopoliensis and Anabaena variabilis). In contrast, no cyanobacterial proteins reacted with antibodies against the 23 and 16 kDa proteins of spinach Photosystem II. The lack of cross-reactivity and the absence of these polypeptides from highly active Photosystem II particles of Phormidium laminosum strongly suggest that cyanobacteria do not contain polypeptides corresponding to these two chloroplast proteins. Treatment of P. laminosum Photosystem II particles with 0.8 M alkaline Tris, 1 M NaCl, CaCl₂ or MgCl₂ inhibited O₂ evolution, and quantitatively removed a 9 kDa polypeptide from the particles. None of these treatments removed comparable amounts of the 33 kDa polypeptide, and only Tris treatment removed manganese. The release of the 9 kDa polypeptide upon NaCl treatment correlated well with the deactivation at the donor side of Photosystem II. A direct connection between the 33 kDa polypeptide and O₂ evolution was established by the finding that trypsin treatment digested this polypeptide and inhibited O₂ evolution in parallel.     

Characterization of inhibitory effects of NH2OH and its N-methyl derivatives on the O2-evolving complex of Photosystem II

Inorganic cofactors (Mn, Ca²⁺ and Cl^-) are essential for oxidation of H₂O to O₂ by Photosystem II. The Mn reductants NH₂OH and its N-methyl derivatives have been employed as probes to further examine the interactions between these species and Mn at the active site of H₂O oxidation. Results of these studies show that the size of a hydroxylamine derivative regulates its ability to inactivate O₂ evolution activity, and that this size-dependent inhibition behavior arises from the protein structure of Photosystem II. A set of anions (Cl^-, F^- and SO₄ ^2-) is able to slow NH₂OH and CH₃NHOH inactivation of intact Photosystem II membranes by exerting a stabilizing influence on the extrinsic 23 and 17 kDa polypeptides. In contrast to this non-specific anion effect, only Cl^- is capable of attenuating CH₃NHOH and (CH₃)₂NOH inhibition in salt-washed preparations lacking the 23 and 17 kDa polypeptides. However, Cl^- fails to protect against NH₂OH inhibition in salt-washed membranes. These results indicate that the attack by NH₂OH and its N-methyl derivatives on Mn occurs at different sites in the O₂-evolving complex. The small reductant NH₂OH acts at a Cl^--insensitive site whereas the inhibitions by CH₃NHOH and (CH₃)₂NOH involve a site that is Cl^- sensitive. These findings are consistent with earlier studies showing that the size of primary amines controls the Cl^- sensitivity of their binding to Mn in the O₂-evolving complex.Abbreviation MES 4-morpholinoethanesulfonic acid - PS II Photosystem II     

Water binding to the oxygen-evolving system of chloroplasts; effects of isotope substitution on the S2 state EPR spectrum

Replacement of H₂O by ²H₂O in oxygen-evolving Photosystem II preparations caused an increased resolution of the fine structure of the S₂ state EPR spectrum. In both ²H₂O and H₂O samples, comparison of the S₂ spectra generated by illumination at 200 and 283 K (10°C) showed a difference in the fine structure on the hyperfine lines. A reduction in the spacing of the outer hyperfine lines was also observed when samples illuminated at 283 K were compared to those where S₂ was formed by 200 K illumination. The observations are interpreted as due to proton binding, perhaps as water, at or near the manganese complex giving rise to the S₂ signal.     

Perturbations at the chloride site during the photosynthetic oxygen-evolving cycle

Photosystem II (PSII) catalyzes the oxidation of water to O₂ at the manganese-containing, oxygen-evolving complex (OEC). Photoexcitation of PSII results in the oxidation of the OEC; four sequential oxidation reactions are required for the generation and release of molecular oxygen. Therefore, with flash illumination, the OEC cycles among five S _n states. Chloride depletion inhibits O₂ evolution. However, the binding site of chloride in the OEC is not known, and the role of chloride in oxygen evolution has not as yet been elucidated. We have employed reaction-induced FT-IR spectroscopy and selective flash excitation, which cycles PSII samples through the S state transitions. On the time scale employed, these FT-IR difference spectra reflect long-lived structural changes in the OEC. Bromide substitution supports oxygen evolution and was used to identify vibrational bands arising from structural changes at the chloride-binding site. Contributions to the vibrational spectrum from bromide-sensitive bands were observed on each flash. Sulfate treatment led to an elimination of oxygen evolution activity and of the FT-IR spectra assigned to the S₃ to S₀ (third flash) and S₀ to S₁ transitions (fourth flash). However, sulfate treatment changed, but did not eliminate, the FT-IR spectra obtained with the first and second flashes. Solvent isotope exchange in chloride-exchanged samples suggests flash-dependent structural changes, which alter protein dynamics during the S state cycle. Supported by NSF MCB 03-55421.     

Mn2+ reduces Yz + in manganese-depleted Photosystem II preparations

Manganese in the oxygen-evolving complex is a physiological electron donor to Photosystem II. PS II depleted of manganese may oxidize exogenous reductants including benzidine and Mn²⁺. Using flash photolysis with electron spin resonance detection, we examined the room-temperature reaction kinetics of these reductants with Y_z ⁺, the tyrosine radical formed in PS II membranes under illumination. Kinetics were measured with membranes that did or did not contain the 33 kDa extrinsic polypeptide of PS II, whose presence had no effect on the reaction kinetics with either reductant. The rate of Y_z ⁺ reduction by benzidine was a linear function of benzidine concentration. The rate of Y_z ⁺ reduction by Mn²⁺ at pH 6 increased linearly at low Mn²⁺ concentrations and reached a maximum at the Mn²⁺ concentrations equal to several times the reaction center concentration. The rate was inhibited by K⁺, Ca²⁺ and Mg²⁺. These data are described by a model in which negative charge on the membrane causes a local increase in the cation concentration. The rate of Y_z ⁺ reduction at pH 7.5 was biphasic with a fast 400 s phase that suggests binding of Mn²⁺ near Y_z ⁺ at a site that may be one of the native manganese binding sites.Abbreviations PS II Photosystem II - Y_D tyrosine residue in Photosystem II that gives rise to the stable Signal II EPR spectrum - Y_z tyrosine residue in Photosystem II that mediates electron transfer between the reaction center chlorophyll and the site of water oxidation - ESR electron spin resonance - DPC diphenylcarbazide - DCIP dichlorophenolindophenol     

Chemical oxidation and reduction of the O2-evolution center in Photosystem II

Treatment of Photosystem II (PS II) with low concentrations of hydroxylamine is known to cause a two-flash delay in the O₂-evolution pattern, and in the formation of the S₂-state multiline EPR signal, due to the two-electron reduction of the S₁-state by hydroxylamine to form the S_-1-state. Past work has shown that these delays are not reversed by washing out the hydroxylamine nor by adding DCBQ or ferricyanide to oxidize the residual hydroxylamine, but are reversed by illumination with two saturating flashes followed by a 30-min dark incubation. We have examined the effects of treatments aimed at restoring the normal flash-induced O₂-evolution pattern and S₂-state multiline EPR signal after treatment of PS II with 40 M hydroxylamine. In agreement with past work, we find that the two-flash delay in O₂ evolution is not reversed when the hydroxylamine is removed by three cycles of centrifugation and resuspension in hydroxylamine-free buffer nor by adding ferricyanide or DCBQ to oxidize the unreacted hydroxylamine. However, the normal flash-induced O₂-evolution pattern is restored by illumination with two saturating flashes followed by a 30-min dark incubation (after the sample was first treated with 40 M hydroxylamine and the unreacted hydroxylamine was removed); illumination with one saturating flash followed by a 30-min dark incubation is only partially effective. These results show that ferricyanide and DCBQ are not effective at oxidizing the S_-1-state to the S₁-state. In contrast, adding hypochlorite (OCl^-) after treatment with hydroxylamine restored the normal flash-induced O₂-evolution pattern and also restored the formation of the S₂-state multiline EPR signal by illumination at 200 K. We conclude that hypochlorite is capable of oxidizing the S_-1-state to the S₁-state. This is the first example of a chemical treatment that advances the delayed flash-induced O₂ evolution pattern.Abbreviations DCBQ 2,5-dichloro-p-benzoquinone - OEC O₂-evolving center     

EPR studies on the photosystem II donor side in salt-washed and reconstituted inside-out thylakoids

EPR measurements on inside-out thylakoids revealed that salt-washing, known to inhibit oxygen evolution and release a 23 and a 16 kDa protein, induced a Signal II_f and decreased the EPR signal from state S₂. Readdition of the released 23 kDa protein restored the oxygen evolution and decreased the Signal II_f, but did not relieve the decrease in the state S₂ signal. It is suggested that salt-washing inhibits the electron transfer from the oxygen-evolving site to Z, the physiological donor to P680. In inhibited photosystem II units lacking Signal II_f, Z⁺ is rapidly reduced, possibly by a modified S-cycle unable to evolve oxygen.     

Desiccation tolerant lichens facilitate in vivo H/D isotope effect measurements in oxygenic photosynthesis

We have used the desiccation-tolerant lichen Flavoparmelia caperata, containing the green algal photobiont Trebouxia gelatinosa, to examine H/D isotope effects in Photosystem II in vivo. Artifact-free H/D isotope effects on both PSII primary charge separation and water oxidation yields were determined as a function of flash rate from chlorophyll-a variable fluorescence yields. Intact lichens could be reversibly dehydrated/re-hydrated with H₂O/D₂O repeatedly without loss of O₂ evolution, unlike all isolated PSII preparations. Above a threshold flash rate, PSII charge separation decreases sharply in both D₂O and H₂O, reflecting loss of excitation migration and capture by PSII. Changes in H/D coordinates further slow charge separation in D₂O (?23% at 120?Hz), attributed to reoxidation of the primary acceptor Q_A^?. At intermediate flash rates (5–50?Hz) D₂O decreases water oxidation efficiency (O₂ evolution) by ?2–5%. No significant isotopic difference is observed at slow flash rates (<5?Hz) where charge recombination dominates. Slower D₂O diffusion, changes in hydrogen bonding networks, and shifts in the pK_a's of ionizable residues may all contribute to these systematic variations of H/D isotope effects. Lichens' reversible desiccation tolerance allows highly reproducible H/D exchange kinetics in PSII reactions to be studied in vivo for the first time.     

Lipoperoxide radical production during oxidation of cardiolipin in the complex with cytochrome c

The key stage of apoptosis is lipid peroxidation which causes cytochrome c efflux from mitochondria. Cardiolipin-bound cytochrome c on the surface of the inner mitochondrial membrane is supposed to be a main lipoperoxidation catalyst. In this work, lipoperoxide radical (LOO·) production in the complex of cytochrome c (Cyt C) with bovine heart cardiolipin (BCL) was investigated with the method of chemiluminescence (CL) in the presence of a physical activator, coumarin dye C-525. It was shown that a CL flash with a half quenching time of 1.12 min was observed after the addition of Cyt C to a BCL+C-525 solution in the absence of hydrogen peroxide. At H₂O₂ concentrations of 0.1–0.5 mM, quenching time reduced at constant CL flash amplitude and at H₂O₂ concentrations of 1–5 mM, the amplitude of CL increased with the growth of peroxide concentration. It testifies to different mechanisms of BCL oxidation: the lipoxygenase mechanism in the absence of H₂O₂ and at low H₂O₂ concentrations, and the peroxidase mechanism at higher H₂O₂ concentrations. When small H₂O₂ amounts were added, another CL flash was observed in the course of a lipoxygenase reaction whose light sum increased with time in parallel with the extent of the following inhibition of CL. Iron chelators EDTA and o-phenanthroline made no significant effect on the CL associated with cytochrome c lipoxygenase action, while desferal, a well-known peroxidase and lipoxygenase inhibitor, inhibited CL by half in a concentration of 18 μM. A scheme of reactions resulting in LOO· radical production on BCL oxidation by the Cyt C-cardiolipin complex in the absence and in the presence of H₂O₂ was suggested.     

Photoproduction of hydrogen peroxide in Photosystem II membrane fragments: A comparison of four signals

The present study describes the formation of different forms of peroxide in Photosystem II (PS II) by using a chemiluminescence detection technique. Four chemiluminescence signals (A, B, C and D) of the luminolperoxidase (Lu-Per) system, which detects peroxide, are found in illuminated O₂-evolving Photosystem II (PS II) membrane fragments isolated from spinach. Signal A (free peroxide) peaking around 0.2–0.3 s after mixing PS II membrane fragments with Lu-Per is eliminated by catalase or removal of oxygen from the suspension and ascribed to O₂ interaction with reduced PS II electron acceptors. In contrast, signal B peaking around 1.5 min remains largely unaffected under anaerobic conditions, as well as in the presence of catalase (20 g/ml). Under flash illumination the extent of this signal exhibits a weak period four oscillation (maximum at third and 7th flash). Its yield increases up to the third flash, but is close to zero in the fourth flash. An analogous behaviour is observed in flashes 5 to 8. Signal B is ascribed to Lu-Per interaction with the water-oxidizing system being in S₂ and/or S₃-state. Signal C (bound peroxide) detected as free peroxide after acid decomposition of illuminated PS II particles is observed on the 1 st flash and oscillates with period 2 with superposition of period 4. It is evidently related to peroxide either released from S₂ or formed at S₂ upon acid shock treatment. Signal D (slowly released peroxide) peaking around 2–3 s after mixing is observed in samples after various treatments (LCC-incubation, washing with 1 M NaCl at pH 8 or with 1 M CaCl₂, Cl^--depletion) that lead to at least partial removal of the extrinsic proteins of 18, 24 and 33 kDa without Mn extraction. The average amplitude of this signal corresponds with a yield of about 0.2 H₂O₂ molecules per RC and flash. In a flash train, the extent of signal D exhibits an oscillation pattern with a minimum at the 3rd flash. We assume that these treatments increase the release of bound peroxide (upon injection into the Lu-Per assay) either formed in the normal oxidative pathway of the water oxidase in the S₂ or the S₃-state or give rise to peroxide formation due to higher accessibility of the Mn-cluster to water molecules.Abbreviations DCPIP 2,6-dichlorophenolindophenol - DPC diphenylcarbazide - LCC lauroylcholine chloride - Lu-Per luminol peroxidase - PS II Photosystem II - RC reaction center - S₂, S₃ redox states of the water oxidizing system - TEMED-N,N,N,N tetramethylethylenediamine     

Structural,biochemical and biophysical characterization of four oxygen-evolving Photosystem II preparations from spinach

Four procedures utilizing different detergent and salt conditions were used to isolate oxygen-evolving Photosystem II (PS II) preparations from spinach thylakoid membranes. These PS II preparations have been characterized by freeze-fracture electron microscopy, SDS-polyacrylamide gel electrophoresis, steady-state and pulsed oxygen evolution, 77 K fluorescence, and room-temperature electron paramagnetic resonance. All of the O₂-evolving PS II samples were found to be highly purified grana membrane fractions composed of paired, appressed membrane fragments. The lumenal surfaces of the membranes and thus the O₂-evolving enzyme complex, are directly exposed to the external environment. Biochemical and biophysical analyses indicated that all four preparations are enriched in the chlorophyll $\text{a}\text{b-}\text{light-harvesting}$ complex and Photosystem II, and depleted to varying degrees in the stroma-associated components, Photosystem I and the CF₁-ATPase. The four PS II samples also varied in their cytochrome f content. All preparations showed enhanced stability of oxygen production and oxygen-rate electrode activity compared to control thylakoids, apparently promoted by low concentrations of residual detergent in the PS II preparations. A model is presented which summarizes the effects of the salt and detergent treatments on thylakoid structure and, consequently, on the configuration and composition of the oxygen-evolving PS II samples.     

Further investigations on structural and catalytic properties of O2 evolving preparations from tobacco and two chlorophyll deficient tobacco mutants

Previous investigations (Specht, S., Pistorius, E.K. and Schmid, G.H.: Photosynthesis Res. 13, 47–56, 1987) of Photosystem II membranes from tobacco (Nicotiana tabacum L. cv. John William's Broadleaf) which contain normally stacked thylakoid membranes and from two chlorophyll deficient tobacco mutants (Su/su and Su/su var. Aurea) which have low stacked or essentially unstacked thylakoids with occasional membrane doublings, have been extended by using monospecific antisera raised against the three extrinsic polypeptides of 33,21 and 16 kDa. The results show that all three peptides are synthesized as well in wild type tobacco as in the two mutants to about the same level and that they are present in thylakoid membranes of all three plants. However, in the mutants the 16 and 21 kDa peptides (but not the 33 kDa peptide) are easily lost during solubilization of Photosystem II membranes. In the absence of the 16 and 21 kDa peptide Photosystem II membranes from the mutants have a higher O₂ evolving activity without addition of CaCl₂ than the wild type Photosystem II membranes. On the other hand, after removal of the 33 kDa peptide no significant differences in the binding of Mn could be detected among the three plants. The results also show that reaction center complexes from wild type tobacco and the mutant Su/su are almost identical to the Triton-solubilized Photosystem II membranes from the mutant Su/su var. Aurea.Abbreviations PS photosystem - chl chlorophyll - LHCP light harvesting chlorophyll a/b protein complex - WT wild type - OEE1, OEE2 and OEE3 oxygen evolution enhancing complex of 29–36 kDa, 21–24 kDa and 16–18 kDa, respectively