Disorganization of the Mn4Ca complex of photosystem II by ruthenium red: a thermoluminescence study

Ruthenium red (RR) is known to be an inhibitor that binds to Ca²⁺ sites. It releases Ca²⁺ and Cl^? together with the extrinsic polypeptide of 17 kDa associated with the oxygen evolving complex of photosystem II. In this work we used thermoluminescence to study S_2/3Q_B^? and S₂Q_A^? charge recombination. It is shown that RR produced a deeper inhibition of oxygen evolution compared with the effect of extrinsic polypeptide or Ca²⁺/Cl^? depletion. Even though Mn is not released, the Mn cluster is disorganized by RR and the S₁ → S₂ transition is inhibited. Copyright © 2008 John Wiley & Sons, Ltd.     

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.     

Stability and oscillation properties of thermoluminescent charge pairs in the O2-evolving system depleted of Cl− or the 33 kDa extrinsic protein

The effects of Cl⁻ depletion and removal of the 33 kDa extrinsic protein on the charge stabilization in O₂-evolving Photosystem II (PS II) particles were studied by curve fitting and deconvolution of thermoluminescence bands. The following results were obtained. (1) Cl⁻ depletion reversibly decreases the redox potential of the S₂ state by 60–80 mV, and thereby elevates the recombination temperature of both S₂Q⁻_B and S₂Q⁻_A charge pairs. (2) Removal of the 33 kDa extrinsic protein specifically elevates the recombination temperature of the S₂Q⁻_A charge pair, with practically no effect on the S₂Q⁻_B pair. This was tentatively interpreted as showing that the protein removal decreases the redox potential of both S₂ and Q⁻_B, but not of Q⁻_A, and, thus, the effects are mutually cancelled for the S₂Q⁻_B pair, but are manifested for the S₂Q⁻_A pair. (3) Deconvolution of glow curves demonstrated that S₃ is not formed in Cl⁻-depleted PS II, but is formed in 33 kDa protein-depleted PS II even at a low (20 mM) Cl⁻ concentration. Analysis of thermoluminescence oscillations confirmed that Cl⁻ depletion interrupts S₂-S₃ transition, whereas the protein removal interrupts S₃-(S₄)-S₀ transition at mM Cl⁻. (4) Cl⁻ depletion by SO²⁻₄ replacement in the absence of 33 kDa protein affected thermoluminescence in a different way from that in the presence of the protein. Based on these findings, the properties of charge pairs in the Cl⁻-depleted PS II particles were discussed in relation to the role of the 33 kDa extrinsic protein.     

Thermoluminescence properties of the S2-state in chloride-depleted water oxidizing complexes after reconstituting treatments with various monovalent anions

Under conditions that assured rebinding of the extrinsic 17 and 23 kDa polypeptides, Cl^--depleted Photosystem II membranes isolated from spinach chloroplasts were subjected to reconstituting treatments in media containing NaF, NaCl, NaBr, NaI or NaNO₃, or they were kept in a medium without any added salt other than the buffer. After removing most of the unbound reconstituting anions by washing, the O₂-evolution activities and thermoluminescence properties of the membranes were compared. While the temperature of maximal thermoluminescence emission was lowest for membranes treated with Cl^-, no uniform correlation was evident between the temperature profile of the thermoluminescence emission and the apparent activating effectiveness of the anions in the membranes' water oxidizing machinery. However, the differences between the thermoluminescence features did conform to a trend according to which the emission temperatures were upshifted as the size of the activating anion increased, and its hydration energy decreased, i.e. Cl^-<Br^-<NO₃ ^-<I^-. The inactive F^- anions were not well retained by the membranes. To explain the experimental data it is suggested that the structural environment of the charge accumulating Mn-center is influenced by the ionic conditions encountered by the Photosystem II membranes after Cl^- removal, further enforced by the binding of compatible anions, and then stabilized by the 17 and 23 kDa extrinsic polypeptides. If, as some concepts imply, the anion binding sites are located at or near the functional Mn, only very exceptional characteristics of the water-oxidizing mechanism may account for the observation that the potentially electron-donating I^- anion can serve as activator and that it stabilizes rather than destabilizes the S₂-state.Abbreviations Chl chlorophyll - Hepes 4-(2-hydroxyethyl)-1-piperazine-ethane sulfonic acid - Mes 2-(N-morpholino)ethane sulfonic acid - Pheo the pheophytin a of the Photosystem II reaction center - PS photosystem     

EPR studies of the water oxidizing complex in the S1 and the higher S states: the manganese cluster and YZ radical

The parallel polarization electron paramagnetic resonance (EPR) method has been applied to investigate manganese EPR signals of native S₁ and S₃ states of the water oxidizing complex (WOC) in photosystem (PS) II. The EPR signals in both states were assigned to thermally excited states with S=1, from which zero-field interaction parameters D and E were derived. Three kinds of signals, the doublet signal, the singlet-like signal and g=11–15 signal, were detected in Ca²⁺-depleted PS II. The g=11–15 signal was observed by parallel and perpendicular modes and assigned to a higher oxidation state beyond S₂ in Ca²⁺-depleted PS II. The singlet-like signal was associated with the g=11–15 signal but not with the Y_Z (the tyrosine residue 161 of the D1 polypeptide in PS II) radical. The doublet signal was associated with the Y_Z radical as proved by pulsed electron nuclear double resonance (ENDOR) and ENDOR-induced EPR. The electron transfer mechanism relevant to the role of Y_Z radical was discussed.     

Inhibition of Photosystem II activity by saturating single turnover flashes in calcium-depleted and active Photosystem II

Inhibition of Photosystem II (PS II) activity induced by continuous light or by saturating single turnover flashes was investigated in Ca²⁺-depleted, Mn-depleted and active PS II enriched membrane fragments. While Ca²⁺- and Mn-depleted PS II were more damaged under continuous illumination, active PS II was more susceptible to flash-induced photoinhibition. The extent of photoinactivation as a function of the duration of the dark interval between the saturating single turnover flashes was investigated. The active centres showed the most photodamage when the time interval between the flashes was long enough (32 s) to allow for charge recombination between the S₂ or S₃ and Q_B ⁻ to occur. Illumination with groups of consecutive flashes (spacing between the flashes 0.1 s followed by 32 s dark interval) resulted in a binary oscillation of the loss of PS II-activity in active samples as has been shown previously (Keren N, Gong H, Ohad I (1995), J Biol Chem 270: 806–814). Ca²⁺- and Mn-depleted PS II did not show this effect. The data are explained by assuming that charge recombination in active PS II results in a back reaction that generates P₆₈₀ triplet and thence singlet oxygen, while in Ca²⁺- and Mn-depleted PS II charge recombination occurs through a different pathway, that does not involve triplet generation. This correlates with an up-shift of the midpoint potential of Q_A in samples lacking Ca²⁺ or Mn that, in term, is predicted to result in the triplet generating pathway becoming thermodynamically less favourable (G.N. Johnson, A.W. Rutherford, A. Krieger, 1995, Biochim. Biophys. Acta 1229, 201–207). The diminished susceptibility to flash-induced photoinhibition in Ca²⁺- and Mn-depleted PS II is attributed at least in part to this mechanism. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of preillumination on the P-680+ reduction kinetics in chloride-free photosystem II membranes

The re-reduction course of P-680⁺, the photooxidized PS II primary donor, was measured as a function of excitation number in Cl⁻-depleted PS II membranes. After the 1st and 2nd excitations the signal amplitude of P-680⁺ is small, indicating a submicrosecond reduction of P-680⁺ by Z, the secondary donor of PS II. After the 3rd excitation, however, a larger P-680⁺ signal with a 40–50 μs half-life is observed. The slow decay of this signal is attributed to a back-reaction with a reduced acceptor in the presence of the Z⁺S₂ state on the donor side. The state Z⁺S₂ has a lifetime longer than 300 ms and its formation was found to depend on the presence of the abnormal S₂ state created by the 1st excitation. The P-680 data and thermoluminescence measurements show that the S-state advancement beyond S₂ is blocked in the absence of Cl⁻ and that the Cl⁻-free abnormal S₂ state has a lifetime about 10-times longer than the normal S₂ state.     

The 16 and 23 kDa extrinsic polypeptides and the associated Ca2+ and Cl- modify atrazine interaction with the photosystem II core complex

The oxygen evolving complex of photosystem II (PS II) contains three extrinsic polypeptides of approximate molecular weights 16, 23 and 33 kDa. These polypeptides are associated with the roles of Cl^-, Ca²⁺ and Mn²⁺ in oxygen evolution. We have shown that selective removal of 16 and 23 kDa polypeptides from the above complex by NaCl washing of PS II enriched membrane fragments renders the PS II core complex more susceptible to the herbicide atrazine. On the other hand, when both native and depleted preparations were resupplied with exogenous Ca²⁺ and Cl^-, we obtained a reduction of atrazine inhibition which was much stronger in the depleted preparations than in the native ones. It is concluded that removal of 16 and 23 kDa polypeptides in general, and disorganization of associated Ca²⁺ and Cl^- in particular, enhances atrazine penetration to its sites of action in the vicinity of the PS II complex. The above could be interpreted if we assume a reduced plastoquinone affinity at the Q_B (secondary plastoquinone electron acceptor) pocket of D1 polypeptide following transmembranous modifications caused by the depletion of these polypeptides.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - Chl chlorophyll - DCIP 2,6-dichlorophenolindophenol - MES 2-(N-morpholino)ethanesulfonic acid - PMSF phenylmethylsul-phonyfluoride - PS II photosystem II - PAGE polyacrilamide gel electrophoresis     

Structural and functional modulation of the manganese cluster in Ca(2+)-depleted photosystem II induced by binding of the 24-kilodalton extrinsic protein.

Depletion of functional Ca2+ from photosystem (PS) II membranes impairs O2 evolution. Redox properties of the Mn cluster as probed by thermoluminescence were modified differently in Ca(2+)-depleted PSII depending on the procedure for Ca2+ extraction. Ca2+ depletion by low-pH treatment gave rise to an abnormally modified S2 state exhibiting a thermoluminescence band with elevated peak temperature accompanied by a marked upshift in threshold temperature for its formation, whereas Ca2+ depletion by NaCl washing in the light followed by the addition of EDTA could generate a similarly modified S2 state only when the Ca(2+)-depleted PSII was reconstituted with the 24-kDa extrinsic proteins. These results indicated that manifestation of the abnormal properties of the Ca(2+)-depleted S2 state is significantly contributed by the association of the 24-kDa extrinsic protein to PSII. It was inferred that the 24-kDa extrinsic protein regulates the structure and function of the Mn cluster in the absence of functional Ca2+ through a conformational modulation of the intrinsic protein(s) that bind(s) both Mn and Ca. Features of the extrinsic protein-dependent modulation of the Mn cluster were discussed in relation to the function of Ca2+ in O2 evolution.     

Drastic changes in the ligand structure of the oxygen-evolving Mn cluster upon Ca depletion as revealed by FTIR difference spectroscopy

A Fourier transform infrared (FTIR) difference spectrum of the oxygen-evolving Mn cluster upon the S₁-to-S₂ transition was obtained with Ca²⁺-depleted photosystem II (PSII) membranes to investigate the structural relevance of Ca²⁺ to the Mn cluster. Previously, Noguchi et al. [Biochim. Biophys. Acta 1228 (1995) 189] observed drastic changes in the carboxylate stretching region of the S₂/S₁ FTIR spectrum upon Ca²⁺ depletion, whereas Kimura and co-workers [Biochemistry 40 (2001) 14061; ibid. 41 (2002) 5844] later claimed that these changes were not ascribed to Ca²⁺ depletion itself but caused by the interaction of EDTA to the Mn cluster and/or binding of K⁺ at the Ca²⁺ site. In the present study, the preparation of the Ca²⁺-depleted PSII sample and its FTIR measurement were performed in the absence of EDTA and K⁺. The obtained S₂/S₁ spectrum exhibited the loss of carboxylate bands at 1587/1562 and 1364/1403 cm^− 1 and diminished amide I intensities, which were identical to the previous observations in the presence of EDTA and K⁺. This result indicates that the drastic FTIR changes are a pure effect of Ca²⁺ depletion, and provides solid evidence for the general view that Ca²⁺ is strongly coupled with the Mn cluster.     

Thermoluminescence studies on the function of Photosystem II in the desiccation tolerant lichen Cladonia convoluta

The effect of desiccation and rehydration on the function of Photosystem II has been studied in the desiccation tolerant lichen Cladonia convoluta by thermoluminescence. We have shown that in functional fully hydrated thalli thermoluminescence signals can be observed from the recombination of the S₂₍₃₎Q_B ^– (B band), S₂Q_A ^– (Q band), Tyr-D⁺Q_A ^– (C band) and Tyr-Z⁺(His⁺)Q_A ^– (A band) charge stabilization states. These thermoluminescence signals are completely absent in desiccated thalli, but rapidly reappear on rehydration. Flash-induced oscillation in the amplitude of the thermoluminescence band from the S₂₍₃₎Q_B ^– recombination shows the usual pattern with maxima after 2 and 6 flashes when rehydration takes place in light. However, after rehydration in complete darkness, there is no thermoluminescence emission after the 1 st flash, and the maxima of the subsequent oscillation are shifted to the 3rd and 7th flashes. It is concluded that desiccation of Cladonia convoluta converts PS II into a nonfunctional state. This state is characterized by the lack of stable charge separation and recombination, as well as by a one-electron reduction of the water-oxidizing complex. Restoration of PS II function during rehydration can proceed both in the light and in darkness. After rehydration in the dark, the first charge separation act is utilized in restoring the usual oxidation state of the water-oxidizing comples.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DT desiccation tolerant - PS II Photosystem II - TL thermoluminescence - P₆₈₀ reaction center Chl of PS II - Q_A and Q_B puinone electron acceptors of PS II - S₀,...,S₄ the redox states of the water-oxidizing complex - Tyr-Z and Tyr-D redox-active tyrosine electron donors of PS II     

Thermoluminescence study of the in vivo effects of bicarbonate depletion and acetate/formate presence in the two algae Chlamydobotrys stellata and Chlamydomonas reinhardtii

We investigated the influence of CO₂/HCO₃ ^–-depletion and of the presence of acetate and formate on the in vivo photosynthetic electron transport in the two green algae Chlamydobotrys stellata and Chlamydomonas reinhardtii by means of thermoluminescence technique and mathematical glow curve analysis. The main effects of the removal of CO₂ from the algal cultures was: (1) A shift of the glow curve peak position to lower temperatures resulting from a decrease of the B band and an increase of the Q band. (2) Treatment of CO₂-deficient Chl. stellata with DCMU yielded two thermoluminescence bands in the Q band region peaking at around +12°C and +5°C; in case of Chl. reinhardtii DCMU treatment induced only one band with an emission maximum at +5°C. The presence of acetate or formate in CO₂-depleted algal cultures lowered the intensities of all of the individual TL bands but that of a HT band (TL+37). The effects of CO₂-depletion and of the presence of anions were fully reversible.Abbreviations DCMU 3-(3,4)-dichlorophenyl-1,1-dimethylurea - HT band high temperature TL band - P₆₈₀ reaction center chlorophyll of PS II - Q_A and Q_B primary and secondary quinone acceptors of PS II, respectively - PS II Photosystem II - S_2/3 redox states of the oxygen evolving complex of PS II - TL thermoluminescence     

Calcium depletion alters energy transfer and prevents state changes in intact Anacystis

A time-dependent loss of Photosystem II (PS II) activity seen in Anacystis nidulans grown without Ca²⁺ was paralleled by a loss in chlorophyll (Chl) a fluorescence of variable yield which reflects inhibition of Q reduction and of state changes. Both inhibitions were fully reversed by the addition of Ca²⁺ to the growth medium. The lack of state changes in Ca²⁺-depleted cells was confirmed in 77 K fluorescence difference spectra of light versus dark-adapted cells.Absorption spectra of control and of Ca²⁺-depleted cells were identical whether measured at room temperature or at 77 K. Fluorescence emission spectra measured at 39°C (cell growth temperature) demonstrated higher yields in Ca²⁺-depleted cells compared to controls. Fluorescence emission spectra at 77 K also produced higher yields in Ca²⁺-depleted cells but the increased fluorescence at this temperature occurred principally at 683 nm. The increased relative fluorescence yield in Ca²⁺-depleted samples results from light absorbed by phycocyanin (PC), but not from light absorbed almost exclusively by Chl. The 683 run fluorescence peak probably represents increased allophycocyanin (APC) emission as intact phycobilisomes become energetically disassociated from the photosynthetic apparatus. This inferred disassociation occurred only after PSII activity was mostly inhibited in Ca²⁺-depleted cells, and was not fully reversible.Abbreviations APC Allophycocyanin - Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - EDTA ethylenediaminotetraacetic acid - PC phycocyanin - PS photosystem - Q primary quinone electron acceptor of Photosystem II also a quencher of Chl a fluorescence DPB-CIW Publ. No. 817     

Correlation of the cytochrome c 2 content of cyanobacterial Photosystem II with the EPR properties of the oxygen-evolving complex

The Mn₄ cluster of PS II advances through a series of oxidation states (S states) that catalyze the breakdown of water to dioxygen in the oxygen-evolving complex. The present study describes the engineering and purification of highly active PS II complexes from mesophilic His-tagged Synechocystis PCC 6803 and purification of PS II core complexes from thermophilic wild-type Synechococcus lividus with high levels of the extrinsic polypeptide, cytochrome c ₅₅₀. The g = 4.1 S₂ state EPR signal, previously not characterized in untreated cyanobacterial PS II, is detected in high yields in these PS II preparations. We present a complete characterization of the g = 4.1 state in cyanobacterial His-tagged Synechocystis PCC 6803 PS II and S. lividus PS II. Also presented are a determination of the stoichiometry of cytochrome c ₅₅₀ bound to His-tagged Synechocystis PCC 6803 PS II and analytical ultracentrifugation results which indicate that cytochrome c ₅₅₀ is a monomer in solution. The temperature-dependent multiline to g = 4.1 EPR signal conversion observed for the S₂ state in cyanobacterial PS II with high cytochrome c ₅₅₀ content is very similar to that previously found for spinach PS II. In spinach PS II, the formation of the S₂ state g = 4.1 EPR signal has been found to correlate with the binding of the extrinsic 17 and 23 kDa polypeptides. The finding of a similar correlation in cyanobacterial PS II with the binding of cytochrome c ₅₅₀ suggests a functional homology between cytochrome c ₅₅₀ and the 17 and 23 kDa extrinsic proteins of spinach PS II. This revised version was published online in June 2006 with corrections to the Cover Date.     

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).     

Requirements for the photoligation of Mn2+ in PS II membranes and the expression of water-oxidizing activity of the polynuclear Mn-catalyst

Ligation of Mn²⁺ into the polynuclear Mn-catalyst of water oxidation was shown using PS II membranes depleted of their Mn and the 17, 23 and 33 kDa extrinsic proteins. This process specifically required light and Ca²⁺ concentrations of 5&#x0303;0 mM. Evidence was obtained indicating Mn²⁺/Ca²⁺ competition for Ca²⁺ and Mn²⁺ binding sites essential for the photoligation of Mn. Photoligation of Mn did not result in an increase of water oxidation capacity; however, water oxidation capacity was expressed following dark reconstitution minimally with the 33 kDa protein. The results represent the first observation of photoactivation of water oxidation in a system that excludes simple light-driven Mn²⁺ transport across membrane(s).     

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     

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.     

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.     

Studies on the electron transfer from Tyr-161 of polypeptide D-1 to P680+ in PS II membrane fragments from spinach

The functional connection between redox component Y _z identified as Tyr-161 of polypeptide D-1 (Debus et al. 1988) and P680⁺ was analyzed by measurements of laser flash induced absorption changes at 830 nm in PS II membrane fragments from spinach. It was found that neither DCMU nor the ADRY agent 2-(3-chloro-4-trifluoromethyl) anilino-3,5-dinitrothiophene (ANT 2p) affects the rate of P680⁺ reduction by Y _z under conditions where the catalytic site of water oxidation stays in the redox state S₁. In contrast to that, a drastic retardation is observed after mild trypsin treatment at pH=6.0. This effect which is stimualted by flash illumination can be largely reversed by Ca²⁺. The above mentioned data lead to the following conclusions: (a) the segment of polypeptide D-1 containing Tyr-161 and coordination sites of P680 is not allosterically affected by structural changes due to DCMU binding at the Q_B-site which is also located in D-1. (b) ANT 2p as a strong protonophoric uncoupler and ADRY agent does not modify the reaction coordinate of P680⁺ reduction by Y _z , and (c) Ca²⁺ could play a functional role for the electronic and vibrational coupling between the redox groups Y _z and P680. The electron transport from Y _z to P680⁺ is discussed within the framework of a nonadiabatic process. Based on thermodynamic considerations the reorganization energy is estimated to be in the order of 0.5 V.Abbreviations ADRY acceleration of the deactivation reactions of the water splitting enzyme system Y - ANT 2p 2-(3-chloro-4-trifluoromethyl)anilino-3,5 dinitrothiophene - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - MES 2[N-Morpholino]ethanesulfonic acid - PS II photosystem II - Q_A, Q_B primary and secondary plastoquinone acceptor of photosystem II - S _i redox states of the catalytic site of water oxidation - Y _z redox active Tyr-161 of polypeptide D-1