Light-induced changes of absorbance and electron spin resonance in small Photosystem II particles

Photosystem II reaction center components have been studied in small system II particles prepared with digitonin. Upon illumination the reduction of the primary acceptor was indicated by absorbance changes due to the reduction of a plastoquinone to the semiquinone anion and by a small blue shift of absorption bands near 545 nm (C550) and 685 nm. The semiquinone to chlorophyll ratio was between 1/20 and 1/70 in various preparations. The terminal electron donor in this reaction did not cause large absorbance changes but its oxidized form was revealed by a hitherto unknown electron spin resonance (ESR) signal, which had some properties of the well-known signal II but a linewidth and g-value much nearer to those of signal I. Upon darkening absorbance and ESR changes decayed together in a cyclic or back reaction which was stimulated by 3-(3,4 dichlorophenyl)-1,1-dimethylurea. The donor could be oxidized by ferricyanide in the dark.

Illumination in the presence of ferricyanide induced absorbance and ESR changes, rapidly reversed upon darkening, which may be ascribed to the oxidation of a chlorophyll a dimer, possibly the primary electron donor of photosystem II. In addition an ESR signal with 15 to 20 gauss linewidth and a slower dark decay was observed, which may have been caused by a secondary donor.     

Relationships between the photon distribution between the two photosystems, the concentration of system II reaction centers and the intersystem equilibrium constant in Chlorella pyrenoidosa

From Emerson enhancement measurements of O₂ evolution in Chlorella pyrenoidosa, it was possible to establish a relationship between the concentration of photosystem II open reaction centers (E) and the distribution of photons between photosystems I and II [(1 − )/] during steady state. The superposition of lights of two different wavelengths (1 and 2) gives concentrations of E and intermediate between those obtained with light 1 and 2 separately. This relationship extends a previous one based on quantum yield measurements. It has been expressed here by a curve corresponding to a fixed value of the intersystem apparent equilibrium constant (K). Up to 700 nm, K remains equal to 6. Above this wavelength, although the margin of error is rather great, K apparently increases to 12 or more.

The possibility of “spill-over” of light absorbed by System II to System I was studied. There is no probability that this spill-over, if any, exceeds 25% in Chlorella.

The apparent equilibrium constant is decreased by 3(3,4-dichlorophenyl)-1,1-dimethylurea. This is not in favor of the hypothesis of fully independent electron-transfer chains in photosynthesis; it is therefore likely that some communication between those chains exists.     

Fluorescence yield kinetics in the microsecond-range in Chlorella pyrenoidosa and spinach chloroplasts in the presence of hydroxylamine

The kinetics of fluorescence yield inChlorella pyrenoidosa and spinach chloroplasts were studied in the time range of 0.5 μs to several hundreds of microseconds in the presence of hydroxylamine. Fluorescence was excited with a just-saturating xenon flash with a halfwidth of 13 μs (λ = 420 nm). The fast rise of the fluorescence yield which was limited by the rate of light influx, was, in the presence of 10⁻³–10⁻² M hydroxylamine, replaced by a slow component which had a half risetime of 25 μs in essence independent of light intensity. This slow fluorescence yield increase reflects a dark reaction on the watersplitting side of Photosystem II. Simultaneous oxygen evolution measurements suggested that a fast fluorescence component is only present in organisms with intact O₂-evolving system, whereas a slow rise predominantly occurs in organisms with the watersplitting system irreversibly inhibited by hydroxylamine.

The results can be explained by the following hypotheses: (a) The primary donor of Photosystem II in its oxidized state, P⁺, is a fluorescence quencher. (b) Hydroxylamine prevents the secondary electron donor Z from reducing the oxidized reaction center pigment P⁺ rapidly. This inhibition is dependent on hydroxylamine concentration and is complete at a concentration of 10⁻² M. (c) A second donor (not transporting electrons from water) transfers electrons to P⁺ with a half time of roughly 25 μs.     

Interaction of oxidized and reduced N-methylphenazonium methosulfate (PMS) with Photosystem II

In 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) poisoned chloroplasts, the restoration of the fluorescence induction is presumed to be due to a back reaction of the reduced primary acceptor (Q⁻) and the oxidized primary donor (Z⁺) of Photosystem II. Carbonylcyanide m-chlorophenylhydrazone (CCCP) is known to inhibit this back reaction. The influence of reduced N-methylphenazonium methosulfate (PMS) in the absence of CCCP and of oxidized PMS in the presence of CCCP on the back reaction was investigated and the following results were obtained:

1. (1) Reduced PMS at the concentration of 1 μM inhibits the back reaction as effectively as hydroxylamine, suggesting an electron donating function of reduced PMS for System II.

2. (2) The inhibition of the back reaction by CCCP is regenerated to a high degree by oxidized PMS which led to assume a cyclic System II electron flow catalysed by PMS.

3. (3) At concentrations of reduced PMS higher than 1 μM it is shown that both the fast initial emission and more significantly the variable emission are quenched.

Light-induced changes of absorbance and electron spin resonance in small photosystem II particles.

Photosystem II reaction center components have been studied in small system II particles prepared with digitonin. Upon illumination the reduction of the primary acceptor was indicated by absorbance changes due to the reduction of a plastoquinone to the semiquinone anion and by a small blue shifts of absorption bands near 545 nm (C550) and 685 nm. The semiquinone to chlorophyll ratio was between 1/20 and 1/70 in various preparations. The terminal electron donor in this reaction did not cause large absorbance changes but its oxidized form was revealed by a hitherto unknown electron spin resonance (ESR) signal, which had some properties of the well-known signal II but a linewidth and g-value much nearer to those of signal I. Upon darkening absorbance and ESR changes decayed together in a cyclic or back reaction which was stimulated by 3-(3,4 dichlorophenyl)-1,1-dimethylurea. The donor could be oxidized by ferricyanide in the dark. Illumination in the presence of ferricyanide induced absorbance and ESR changes, rapidly reversed upon darkening, which may be ascribed to the oxidation of a chlorophyll a dimer, possibly the primary electron donor of photosystem II. In addition an ESR signal with 15 to 20 gauss linewidth and a slower dark decay was observed, which may have been caused by a secondary donor.     

Probing fluorescence induction in chloroplasts on a nanosecond time scale utilizing picosecond laser pulse pairs

The fluorescence induction and other fluorescence properties of spinach chloroplasts at room temperature were probed utilizing two 30-ps wide laser pulses (530 nm) spaced Δt (s) apart in time (Δt = 5–110 ns). The energy of the first pulse (P₁) was varied (10¹²–10¹⁶ photons · cm⁻²), while the energy of the second (probe) pulse (P₂) was held constant (5 · 10¹³ photons · cm⁻²). A gated (10 ns) optical multichannel analyzer-spectrograph system allowed for the detection of the fluorescence generated either by P₁ alone, or by P₂ alone (preceded by P₁). The dominant effect observed for the fluorescence yield generated by P₁ alone is the usual singlet-singlet exciton annihilation which gives rise to a decrease in the yield at high energies. However, when the fluorescence yield of dark-adapted chloroplasts is measured utilizing P₂ (preceded by pulse P₁) an increase in this yield is observed. The magnitude of this increase depends on Δt, and is characterized by a time constant of 28 ± 4 ns. This rise in the fluorescence yield is attributed to a reduction of the oxidized (by P₁) reaction center P-680⁺ by a primary donor. At high pulse energies (P₁ = 4 · 10¹⁴ photons · cm⁻²) the magnitude of this fluorescence induction is diminished by another quenching effect which is attributed to triplet excited states generated by intense P₁ pulses. Assuming that the P₁ pulse energy dependence of the fluorescence yield rise reflects the closing of the reaction centers, it is estimated that about 3–4 photon hits per reaction center are required to close completely the reaction centers, and that there are 185–210 chlorophyll molecules per Photosystem II reaction center.     

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

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

Reactions of b-cytochromes in the red alga Porphyridium aerugineum

1. 1. The kinetics of light-induced absorbance changes due to oxidation and reduction of cytochromes were measured in a suspension of intact cells of the unicellular red alga Porphyridium aerugineum. Absorbance changes in the region 540–570 nm upon alternating far-red light and darkness indicated the oxidation of cytochrome ƒ and reduction of cytochrome b₅₆₃ upon illumination. The relative efficiencies of far-red and orange light indicated that both reactions were driven by Photosystem I.

2. 2. Experiments with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), with anaerobic cells and in alternating far-red and orange light indicated that cytochrome b₅₆₃ reacts in a cyclic chain around Photosystem I, and that the reduced cytochrome does not react with oxygen or with another oxidized product of Photosystem II. The quantum requirement for the photoreduction was about 6 quanta/equiv at 700 nm. A low concentration of N-methylphenazonium methosulphate (PMS) enhanced the rate of reoxidation of cytochrome b₅₆₃ in the dark. In the presence of higher concentrations of PMS a photooxidation, driven by Photosystem I, instead of reduction was observed. These observations suggest that PMS enhances the rate of reactions between reduced cytochrome b₅₆₃ and oxidized products of Photosystem I.

3. 3. In the presence of carbonylcyanide m-chlorophenylhydrazone (CCCP) a light-induced decrease of absorption at 560 nm occurred. Spectral evidence suggested the photooxidation of cytochrome b₅₅₉ under these conditions. Inhibition by DCMU and a relatively efficient action of orange light suggested that this photooxidation is driven by Photosystem II.

Flash-induced absorption changes of the primary donor of photosystem II at 820 nm in chloroplasts inhibited by low pH or tris-treatment.

A comparative study is made, at 15 degrees C, of flash-induced absorption changes around 820 nm (attributed to the primary donors of Photosystems I and II) and 705 nm (Photosystem I only), in normal chloroplasts and in chloroplasts where O2 evolution was inhibited by low pH or by Tris-treatment. At pH 7.5, with untreated chloroplasts, the absorption changes around 820 nm are shown to be due to P-700 alone. Any contribution of the primary donor of Photosystem II should be in times shorter than 60 mus. When chloroplasts are inhibited at the donor side of Photosystem II by low pH, an additional absorption change at 820 nm appears with an amplitude which, at pH 4.0, is slightly higher than the signal due to oxidized P-700. This additional signal is attributed to the primary donor of Photosystem II. It decays (t 1/2 about 180 mus) mainly by back reaction with the primary acceptor and partly by reduction by another electron donor. Acid-washed chloroplasts resuspended at pH 7.5 still present the signal due to Photosystem II (t 1/2 about 120 mus). This shows that the acid inhibition of the first secondary donor of Photosystem II is irreversible. In Tris-treated chloroplasts, absorption changes at 820 nm due to the primary donor of Photosystem II are also observed, but to a lesser extent and only after some charge accumulation at the donor side. They decay with a half-time of 120 mus.     

Primary reactions, plastoquinone and fluorescence yield in subchloroplast fragments prepared with deoxycholate

1. In subchloroplast fragments prepared with the detergent deoxycholate the primary reactions of Photosystem II could be studied at room temperature, because the secondary reactions were largely or completely inhibited.

2. The main quencher of chlorophyll fluorescence in these particles was the photosynthetically active pool of plastoquinone in its oxidized form. Its photoreduction in the presence of artificial electron donors was accompanied by a shift of a chlorophyll a absorption band. Its reoxidation in the dark was very slow, even in the presence of ferricyanide.

3. Of all the artificial electron donors tested MnCl₂ was by far the most efficient.

4. Measurements at room temperature of the C550 absorbance change confirmed its correlation with the primary electron acceptor. Its difference spectrum was broader and its extinction coefficient correspondingly lower than at liquid-N₂ temperature. In chloroplasts the C550 concentration was about 1:360 chlorophylls.

5. In the dark C550 was largely in the reduced state and its oxidation by plastoquinone took place in the presence of an artificial electron donor only, suggesting that the redox potential of C550 was increased by accumulated positive charges at the donor side of the reaction center.

6. The free radical 1,1′-diphenyl-2-picrylhydrazyl oxidized C550 directly in a 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-insensitive reaction. A DCMU-insensitive oxidation of C550 was observed at high ferricyanide concentrations as well, but probably in this case an endogenous electron donor was oxidized, which in turn oxidized C550 via the back reaction of the photochemical reaction.

7. The oxidized form of the primary electron donor, P680⁺, accumulated in the light in the presence of deoxycholate and a low ferricyanide concentration. In chloroplasts the P680 concentration was about 1:360 chlorophylls.

8. The P 680 absorption difference spectrum and electron spin resonance could be explained by the oxidation of a chlorophyll a dimer. Repeated deoxycholate treatments progressively changed the spectra to those of a monomer. The monomer was still photochemically active.

9. A new interpretation of the difference spectrum of P700 is proposed: it may be the same as that of the difference spectrum of P680 if the bleaching at 700 nm is attributed to a band shift.     

Photosystem II in a mutant of Chlamydomonas reinhardtii lacking the 23 kDa psbP protein shows increased sensitivity to photoinhibition in the absence of chloride

The psbP gene product, the so called 23 kDa extrinsic protein, is involved in water oxidation carried out by Photosystem II. However, the protein is not absolutely required for water oxidation. Here we have studied Photosystem II mediated electron transfer in a mutant of Chlamydomonas reinhardtii, the FUD 39 mutant, that lacks the psbP protein. When grown in dim light the Photosystem II content in thylakoid membranes of FUD 39 is approximately similar to that in the wild-type. The oxygen evolution is dependent on the presence of chloride as a cofactor, which activates the water oxidation with a dissociation constant of about 4 mM. In the mutant, the oxygen evolution is very sensitive to photoinhibition when assayed at low chloride concentrations while chloride protects against photoinhibition with a dissociation constant of about 5 mM. The photoinhibition is irreversible as oxygen evolution cannot be restored by the addition of chloride to inhibited samples. In addition the inhibition seems to be targeted primarily to the Mn-cluster in Photosystem II as the electron transfer through the remaining part of Photosystem II is photoinhibited with slower kinetics. Thus, this mutant provides an experimental system in which effects of photoinhibition induced by lesions at the donor side of Photosystem II can be studied in vivo.Abbreviations Chl chlorophyll - DCIP 2,6-dichlorophenolindophenol - DPC 2,2-diphenylcarbonic dihydrazide - HEPES 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid - P₆₈₀ the primary electron donor to PS II - PpBQ phenyl-p-benzoquinone - PS II Photosystem II - Q_A the first quinone acceptor of PS II - Q_B the second quinone acceptor of PS II - SDS sodium dodecyl sulfate - Tris tris(hydroxymethyl)aminomethane - Tyr_D accessory electron donor on the D₂-protein - Tyr_Z tyrosine residue, acting as electron carrier between P₆₈₀ and the water oxidizing system     

Effect of photosystem II reaction center closure on nanosecond fluorescence relaxation kinetics

The fluorescence decay of chlorophyll in spinach thylakoids was measured as a function of the degree of closure of Photosystem II reaction centers, which was set for the flowed sample by varying either the preillumination by actinic light or the exposure of the sample to the exciting pulsed laser light. Three exponential kinetic components originating in Photosystem II were fitted to the decays; a fourth component arising from Photosystem I was determined to be negligible at the emission wavelength of 685 nm at which the fluorescence decays were measured. Both the lifetimes and the amplitudes of the components vary with reaction center closure. A fast (170–330 ps) component reflects the trapping kinetics of open Photosystem II reaction centers capable of reducing the plastoquinone pool; its amplitude decreases gradually with trap closure, which is incompatible with the concept of photosynthetic unit connectivity where excitation energy which encounters a closed trap can find a different, possibly open one. For a connected system, the amplitude of the fast fluorescence component is expected to remain constant. The slow component (1.7–3.0 ns) is virtually absent when the reaction centers are open, and its growth is attributable to the appearance of closed centers. The middle component (0.4–1.7 ns) with approximately constant amplitude may originate from centers that are not functionally linked to the plastoquinone pool. To explain the continuous increase in the lifetimes of all three components upon reaction center closure, we propose that the transmembrane electric field generated by photosynthetic turnover modulates the trapping kinetics in Photosystem II and thereby affects the excited state lifetime in the antenna in the trap-limited case.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - HEPES 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid - PQ plastoquinone - PSI and PSII Photosystem I and II - Q_A and Q_B primary and secondary quinone acceptor of PSII     

The sensitivity of Photosystem II to damage by UV-B radiation depends on the oxidation state of the water-splitting complex

The water-oxidizing complex of Photosystem II is an important target of ultraviolet-B (280-320 nm) radiation, but the mechanistic background of the UV-B induced damage is not well understood. Here we studied the UV-B sensitivity of Photosystem II in different oxidation states, called S-states of the water-oxidizing complex. Photosystem II centers of isolated spinach thylakoids were synchronized to different distributions of the S(0), S(1), S(2) and S(3) states by using packages of visible light flashes and were exposed to UV-B flashes from an excimer laser (lambda=308 nm). The loss of oxygen evolving activity showed that the extent of UV-B damage is S-state-dependent. Analysis of the data obtained from different synchronizing flash protocols indicated that the UV-sensitivity of Photosystem II is significantly higher in the S(3) and S(2) states than in the S(1) and S(0) states. The data are discussed in terms of a model where UV-B-induced inhibition of water oxidation is caused either by direct absorption within the catalytic manganese cluster or by damaging intermediates of the water oxidation process.     

Primary donor recovery kinetics in reaction centers from Rhodopseudomonas viridis. The influence of ferricyanide as a rapid oxidant of the acceptor quinones

In reaction centers from Rhodopseudomonas viridis that contain a single quinone, the decay of the photo-oxidized primary donor, P+, was found to be biphasic when the bound, donor cytochromes were chemically oxidized by ferricyanide. The ratio of the two phases was dependent on pH with an apparent pK of 7.6. A fast phase, which dominated at high pH (t1/2 = 1 ms at pH 9.5), corresponded to the expected charge recombination of P+ and the primary acceptor QA-. A much slower phase dominated at low pH and was shown to arise from a slow reduction of P+ by ferrocyanide in reaction centers where QA- has been rapidly oxidized by ferricyanide. The rate of QA- oxidation was linear with respect to ferricyanide activity and was strongly pH-dependent. The second-order rate constant, corrected for the activity coefficient of ferricyanide, approached a maximum of 2 X 10(8) M-1 X s-1 at low pH, but decreased steadily as the pH was raised above a pK of 5.8, indicating that a protonated state of the reaction center was involved. The slow reduction of P+ by ferrocyanide was also second-order, with a maximum rate constant at low pH of 8 X 10(5) M-1 X s-1 corrected for the activity coefficient of ferrocyanide. This rate also decreased at higher pH, with a pK of 7.4, indicating that ferrocyanide also was most reactive with a protonated form of the reaction center. The oxidation of QA- by ferricyanide was unaffected by the presence of o-phenanthroline, implying that access to QA- was not via the QB-binding site. In reaction centers supplemented with ubiquinone, oxidation of reduced secondary quinone, QB-, by ferricyanide was observed but was substantially slower than that for QA-. It is suggested that Q-B may be oxidized via QA so that the rate is modulated by the equilibrium constant for QA-QB in equilibrium with QAQB-.     

Lipophilicity and catalysis of photophosphorylation. II. Quinoid compounds as artificial carriers in cyclic photophosphorylation and photoreductions by Photosystem I

The topography of the chloroplast membrane has been studied using the following pairs of quinoid compounds with similar structure and chemical properties, but with different lipid solubility: phenazine/sulfophenazine, naphthoquinone/naphthoquinone sulfonate, indophenol/sulfoindophenol and lumiflavin/FMN.

All these compounds in the oxidized form are able to accept electrons from the photosynthetic electron transport chain in Hill reactions. However, only the lipophilic compounds in the reduced form can donate electrons to Photosystem I, when electron flow from Photosystem II is blocked by inhibitors. This is in agreement with the notation that the oxidizing site of Photosystem I (P₇₀₀⁺) and the electron donors for Photosystem I (cytochrome f and plastocyanin) are located inside the lipid barrier of the inner chloroplast membrane. The reducing sites in the Hill reactions must be located on the outer surface, accessible from the suspending medium.

It has been known for a long time that N,N′-tetramethyl-p-phenylenediamine can donate electrons to Photosystem I, but contrary to diaminodurene (2,3,5,6-tetramethyl phenylenediamine) it does not induce ATP formation. Both compounds are lipophilic and have similar redox potentials, but only the latter carries hydrogens which are involved in the redox reaction. For energy conservation, coupled to electon flow in Photosystem I, it therefore seems necessary that the lipophilic redox compound in the reduced form can carry hydrogens through the chloroplast membrane.     

Study of fumarase activity in non-conventional media. Part I

Fumarase catalysed hydration of fumarate was investigated in water/organic solvent one-phase systems. The organic solvents used were ethylene glycol, glycerol and dimethylformamide. The effects of the amount of organic solvent on the maximum velocity (V_max), the Michaelis-Menten constant (K_M) and the equilibrium constant (K_eq) were studied in all the reaction media. Together with a denaturing power of the solvent evidenced by a systematic decrease of V_max also a surprising decrease of the K_M was registered as the percentage of organic solvent in the reaction media was increased. While the equilibrium constant of the reaction (K_eq = [l-malate]/[fumarate]) decreased when the percentage of organic solvent was raised. An interpretation of these facts was given. Time-dependent denaturation was also investigated and glycerol resulted the less denaturing of the solvents used, while the aprotic DMF exhibited the highest deactivation.     

The role of chloride ion in photosystem II. I. Effects of chloride ion on photosystem II electron transport and on hydroxylamine inhibition.

1. Chloroplasts washed with Cl--free, low-salt media (pH 8) containing EDTA, show virtually no DCMU-insensitive silicomolybdate reduction. The activity is readily restored when 10 mM Cl- is added to the reaction mixture. Very similar results were obtained with the other Photosystem II electron acceptor 2,5-dimethylquinone (with dibromothymoquinone), with the Photosystem I electron acceptor FMN, and also with ferricyanide which accepts electrons from both photosystems. 2. Strong Cl--dependence of Hill activity was observed invariably at all pH values tested (5.5--8.3) and in chloroplasts from three different plants: spinach, tobacco and corn (mesophyll). 3. In the absence of added Cl- the functionally Cl--depleted chloroplasts are able to oxidize, through Photosystem II, artificial reductants such as catechol, diphenylcarbazide, ascorbate and H2O2 at rates which are 4--12 times faster than the rate of the residual Hill reaction. 4. The Cl--concentration dependence of Hill activity with dimethylquinone as an electron acceptor is kinetically consistent with the typical enzyme activation mechanism: E(inactive) + Cl- in equilibrium E . Cl- (active), and the apparent activation constant (0.9 mM at pH 7.2) is unchanged by chloroplast fragmentation. 5. The initial phase of the development of inhibition of water oxidation in Cl--depleted chloroplasts during the dark incubation with NH2OH (1/2 H2SO4) is 5 times slower when the incubation medium contains Cl- than when the medium contains NH2OH alone or NH2OH plus acetate ion. (Acetate is shown to be ineffective in stimulating O2 evolution).     

Primary reactions of Photosystem II at low pH. 2. Light-induced changes of absorbance and electron spin resonance in spinach chloroplasts

The effects of lowering the pH on Photosystem II have been studied by measuring changes in absorbance and electron spin resonance in spinach chloroplasts.At pH values around 4 a light-induced dark-reversible chlorophyll oxidation by Photosystem II was observed. This chlorophyll is presumably the primary electron donor of system II. At pH values between 5 and 4 steady state illumination induced an ESR signal, similar in shape and amplitude to signal II, which was rapidly reversed in the dark. This may reflect the accumulation of the oxidized secondary donor upon inhibition of oxygen evolution. Near pH 4 the rapidly reversible signal and the stable and slowly decaying components of signal II disappeared irreversibly concomitant with the release of bound manganese.The results are discussed in relation to the effects of low pH on prompt and delayed fluorescence reported earlier (van Gorkom, H. J., Pulles, M. P. J., Haveman, J. and den Haan, G. A. (1976) Biochim. Biophys. Acta 423, 217–226).     

Turnover of the D1 protein and of Photosystem II in a Synechocystis 6803 mutant lacking Tyrz

The Photosystem II reaction center is rapidly inactivated by light, particularly at higher light intensity. One of the possible factors causing this phenomenon is the oxidized primary donor, P680⁺, which may be harmful to Photosystem II because of its highly oxidizing nature. However, no direct evidence specificially implicating P680⁺ in photoinhibition has been obtained yet. To investigate whether P680⁺ is harmful to Photosystem II, turnover of the D1 protein and of the Photosystem II reaction center complex were measured in vivo in a mutant of the cyanobacterium Synechocystis sp. PCC 6803, in which the physiological donor to P680⁺, Tyr_z, was genetically deleted. In this mutant, D1 degradation in the light is an order of magnitude faster than in wild type. The most straightforward explanation of this phenomenon is that accumulation of P680⁺ leads to an increased rate of turnover of the Photosystem II reaction center complex, which is compatible with the hypothesis of destructive oxidation by P680⁺ that is damaging to the Photosystem II complex.     

Photoreduction of pheophytin in reaction centers of the photosystem II in intact cells of green algae and cyanobacteria under anaerobic conditions

Reversible photoreduction of pheophytin (Pheo) accompanied by a decrease in the chlorophyll fluorescence yield is observed in Photosystem 2 of the intact cells of green algae and cyanobacteria under anaerobic conditions. The photoreaction is inhibited by DCMU and reactivated upon subsequent addition of either ascorbate of dithionite. It is suggested that as a result of electron donation from the water splitting system being in the state S(3), to the reaction centre of Photosystem 2 in the state [P(+)(680)Pheo(-)] Q(-) after the primary photoreaction there occurs formation of the long-living state [P(680)Pheo(-)] Q(-). It was found that oxidized NADP, benzyl viologen and methyl viologen accelerate oxidation of Pheo reduced int he Photosystem 2 in the light indicating that these electron acceptors (typical for Photosystem 1) can accept an election from Pheo in Photosystem 2.