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.

New results on the properties of photosystem II centers blocked by 3-(3,4-dichlorophenyl)-1,1-dimethylurea in their different photoactive states

We have studied the 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) action on the different S states by oxygen, fluorescence and luminescence measurements.We show that no oxygen is evolved during a flash following the addition of DCMU to centers in their S₃ state. This suggests that oxygen inhibition cannot be attributed solely to a blocking between Q and A. For all the photoinactive states, the only remaining pathway for the quencher reoxidation, in the presence of DCMU, appears to proceed through a back reaction. Therefore, the complete quencher regeneration still occurring when the fourth positive charge is formed in the presence of DCMU is also an indication of an action by DCMU at the donor side.The data well fit the model in which the oscillations of the fluorescence yield and their damping are attributed to a fast equilibrium between two forms of the centers: a photoactive and a photoinactive form, both of which are quenchers. The equilibrium constant depends on the number of positive charges stored and DCMU changes the characteristics of this equilibrium.     

Inhibition of the reoxidation of the secondary electron acceptor of Photosystem II by bicarbonate depletion

In bicarbonate-depleted chloroplasts, the chlorophyll a fluorescence decayed with a halftime of about 150 ms after the third flash, and appreciably faster after the first and second flash of a series of flashes given after a dark period. After the fourth to twentieth flashes, the decay was also slow. After addition of bicarbonate, the decay was fast after all the flashes of the sequence. This indicates that the bicarbonate depletion inhibits the reoxidation of the secondary acceptor R²⁻ by the plastoquinone pool; R is the secondary electron acceptor of pigment system II, as it accepts electrons from the reduced form of the primary electron acceptor (Q⁻). This conclusion is consistent with the measurements of the DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea)-induced chlorophyll a fluorescence after a series of flashes in the presence and the absence of bicarbonate, if it is assumed that DCMU not only causes reduction of Q if added in the state QR⁻, but also if added in the state QR²⁻.     

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

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

Electron paramagnetic resonance Signal II in spinach chloroplasts. II. Alternative spectral forms and inhibitor effects on kinetics of Signal II in flashing light

Linewidth and hyperfine structure measurements of the EPR spectrum of Signal II in spinach chloroplasts show that the signal reflects two alternative states. One state is characterized by a 16-G linewidth and four partially resolved hyperfine components. The other state has 19 G linewidth and five partially resolved hyperfine components. It is possible to interconvert these two states by changing the ionic strength of the chloroplast suspension. Both states of Signal II show similar light-induced increases in dark-adapted chloroplasts and respond to 10-μs white light flashes with identical kinetics.

In chloroplasts at room temperature, Signal II dark decays to 50% of its total light-induced level in about 1 h. Single flashes increase the spin concentration in these aged chloroplasts but with decreased effectiveness compared with fresh, dark-adapted chloroplasts. Carbonyl cyanide-m-chlorophenylhydrazone (CCCP) decreases the decay time of Signal II from hours to seconds without appreciably altering the level of Signal II formed in saturating continuous light. However, both the formation time constant and the extent of Signal II increase stimulated by a single saturating flash are decreased in CCCP-treated chloroplasts.

These results are interpreted in terms of the model, proposed in the preceding paper, in which Signal II is generated by oxidation-reduction reactions on the water side of Photosystem II.     

Effect of low temperature (−30 to −60°C) on the reoxidation of the Photosystem II primary electron acceptor in the presence and absence of 3(3,4-dichlorophenyl)-1,1-dimethylurea

The fluorescence yield has been measured on spinach chloroplasts at low temperature (−30 to −60°C) for various dark times following a short saturating flash. A decrease in the fluorescence yield linked to the reoxidation of the Photosystem II electron acceptor Q is still observed at −60°C. Two reactions participate in this reoxidation: a back reaction or charge recombination and the transfer of an electron from Q⁻ to Pool A. The relative competition between these two reactions at low temperature depends upon the oxidation state of the donor side of the Photosystem II center:

1. (1) In dark-adapted chloroplasts (i.e. in States S₀+S₁ according to Kok, B., Forbush, B. and McGloin, M. (1970) Photochem. Photobiol. 11, 457–475), Q, reduced by a flash at low temperature, is reoxidized by a secondary acceptor and the positive charge is stabilized on the Photosystem II donor Z. Although this reaction is strongly temperature dependent, it still occurs very slowly at −60°C.

2. (2) When chloroplasts are placed in the S₂+S₃ states by a two-flash preillumination at room temperature, the reoxidation of Q⁻ after a flash at low temperature is mainly due to a temperature-independent back reaction which occurs with non-exponential kinetics.

3. (3) Long continuous illumination of a frozen sample at −30°C causes 6–7 reducing equivalents to be transferred to the pool. Thus, a sufficient number of oxidizing equivalents should have been generated to produce at least one O₂ molecule.

4. (4) A study of the back reaction in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) shows the superposition of two distinct non-exponential reactions one temperature dependent, the other temperature independent.

Binding of the inhibitor NH3 to the oxygen-evolving apparatus of spinach chloroplasts

Experiments are described on flash-induced luminescence of isolated spinach chloroplasts after addition of NH₄Cl. The results indicate a binding of NH₃, presumably in competition with water, in the oxidation states S₂ and S₃, i.e. the states reached upon illumination of dark-adapted material with one and two flashes, respectively. In the initial state S₁, no binding of NH₃ occurs. In state S₂ the binding of ammonia is rapid (half-time about 0.5 s) and rapidly reversible; in state S₃ the binding is slower (half-time about 10 s) and slowly reversible. NH₃ bound to S₄ prevents the oxidation of water. NH₃ bound to S₂ decreases the rate of the back reaction of reduced primary acceptor (Q⁻), indicating a charge stabilization, i.e. a decrease in the redox potential of S₂ due to interaction with ammonia. In Tris-washed chloroplasts, the stability of the positive charge generated in a flash is much smaller than in normal chloroplasts and not increased by NH₃. On the basis of these observations it is postulated that, in the absence of NH₃, states S₂ and S₃ are stabilized by manganese-coordinated, bound water.     

Chlorophyll a fluorescence as a monitor of nanosecond reduction of the photooxidized primary donor P-680 of Photosystem II

1. Changes in the fluorescence yield of aerobic Chlorella vulgaris have been measured in laser flashes of 15 ns, 30 ns and 350 ns half time. The kinetics after the first flash given after a 3 min dark period could be simulated on a computer using the hypothesis that the oxidized acceptor Q and primary donor P⁺ are fluorescence quenchers, and Q⁻ is a weak quencher, and that the reduction time for P⁺ is 20–35 ns.

2. The P⁺ reduction time for at least an appreciable part of the reaction centers was found to be longer after the second and subsequent flashes. In the first 5 flashes an oscillation was observed. Under steady state conditions, with a pulse separation of 3 s, a reduction time for P⁺ of about 400 ns for all reaction centers gave the best correspondence between computed and experimental fluorescence kinetics.     

Charge accumulation at the reducing side of system 2 of photosynthesis

A study was made of the reactions between the primary and secondary electron acceptors of Photosystem 2 by measurements of the increase of chlorophyll fluorescence induced in darkness by dithionite or by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). The experiments were done either with chloroplasts to which hydroxylamine or carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) was added, or with chloroplasts treated with tris(hydroxymethyl)aminomethane (Tris) to which phenylenediamine and ascorbate were added as donor system. Under these conditions the fluorescence increase induced by dithionite or DCMU added after illumination with short light flashes was dependent on the flash number with a periodicity of two; it was large after an uneven number of flashes, and small after a long darktime or after an even number of flashes. The results are interpreted in terms of a model which involves a hypothetical electron carrier situated between Q and plastoquinone; this electron carrier is thought to equilibrate with plastoquinone in a two-electron transfer reaction; the results obtained with DCMU are explained by assuming that its midpoint potential is lowered by this inhibitor.     

Light-induced changes of fluorescence and absorbance in spinach chloroplasts at −40 °C

1. 1. Spinach chloroplasts were stored in the dark for at least 1 h, rapidly cooled to −40 °C, and illuminated with continuous light or short saturating flashes. In agreement with the measurements of Joliot and Joliot, chloroplasts that had been preilluminated with one or two flashes just before cooling showed a less efficient increase in the yield of chlorophyll a fluorescence upon illumination at −40 °C than dark-adapted chloroplasts. The effect disappeared below −150 °C, but reappeared again upon warming to −40 °C. Little effect was seen at room temperature in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), added after the preillumination.

2. 2. Light-induced absorbance difference spectra at −40 °C in the region 500–560 nm indicated the participation of two components, the socalled 518-nm change (P518) and C-550. After preillumination with two flashes the absorbance change at 518 nm was smaller, and almost no C-550 was observed. After four flashes, the bands of C-550 were clearly visible again.

3. 3. The fluorescence increase and the absorbance change at 518 nm showed the same type of flash pattern with a minimum after the second and a maximum at the fourth flash. In the presence of 100 μM hydroxylamine, the fluorescence response was low after the fourth and high again after the sixth flash, which confirmed the hypothesis that the flash effect was related to the so-called S-state of the electron transport pathway from water to Photosystem 2.

4. 4. The kinetics of the light-induced absorbance changes were the same at each wavelength, and, apart from the size of the deflection, they were independent of preillumination. Flash experiments indicated that the absorbance changes were a one-quantum reaction. This was also true for the fluorescence increase in dark-adapted chloroplasts, but with preilluminated chloroplasts several flashes were needed to approximately saturate the fluorescence yield.

5. 5. The results are discussed in terms of a mechanism involving two electron donors and two electron acceptors for System 2 of photosynthesis.

Carotenoid triplet states in reaction centers from Rhodopseudomonas sphaeroides and Rhodospirillum rubrum

Purified photochemical reaction centers from three strains of Rhodopseudomonas sphaeroides and two of Rhodospirillum rubrum were reduced with Na₂S₂O₄ so as to block their photochemical electron transfer reactions. They then were excited with flashes lasting 5–30 ns. In all cases, absorbance measurements showed that the flash caused the immediate formation of a transient state (P^F) which had been detected previously in reaction centers from Rps. sphaeroides strain R26. Previous work has shown that state P^F is an intermediate in the photochemical electron transfer reaction in the reaction centers of that particular strain, and the present work generalizes that conclusion.

In the reaction centers from two strains that lack carotenoids (Rps. sphaeroides R26 and R. rubrum G9), the decay of P^F yields a longer-lived state (P^R) which is probably a triplet state of the bacteriochlorophyll of the reaction center. In the R26 preparation, the decay of P^F was found to have a half-time of 10±2 ns. The decay kinetics rule out the identification of P^F as the fluorescent excited singlet state of the reaction center.

In the reaction centers from three strains that contain carotenoids (Rps sphaeroides 2.4.1 and Ga, and R. rubrum S1), state P^R was not detected, and the decay of P^F generated triplet states of carotenoids. The efficiency of the coupling between the decay of P^F and the formation of the carotenoid triplet appeared to be close to 100% at room temperature, but somewhat lower at 77 °K. Taken with previous results, this suggests that the coupling is direct and does not require the intermediate formation of state P^R. This conclusion would be consistent with the view that P^F is a biradical which can be triplet in character.     

The back reaction in the primary electron transfer couple of photosystem II of photosynthesis

Changes of C-550, cytochrome b₅₅₉ and fluorescence yield induced in chloroplasts by single saturating flashes were studied at low temperature. A single saturating flash at −196°C was quite ineffective in reducing C-550, oxidizing cytochrome b₅₅₉ or increasing the fluorescence yield, presumably because most of the charge separation induced by the flash was dissipated by a direct back reaction in the primary electron transfer couple. The back reaction, which competes with the dark reduction of the oxidized primary electron donor by a secondary electron donor, becomes increasingly important as the temperature is lowered because of the temperature coefficient of the reaction with the secondary donor. The effect of the back reaction is to lower the quantum yield for the production of stable photochemical products by steady irradiation. Assuming a quantum yield of unity for the photoreduction of C-550 at room temperature, the quantum yield for the reaction is about 0.40 at −100°C and 0.27 at −196°C.     

Light-dependent inhibitory action of p-nitrothiophenol on Photosystem II in relation to the redox state of electron carriers

The photosystem-II activity of chloroplasts was inhibited by the treatment with p-nitrothiophenol (NphSH) in the light, and the inhibition was accompanied by a change of the fluorescence spectrum. Aromatic mercaptans examined were active in causing this inhibition and fluorescence change. These effects of p-nitrothiophenol were highly accelerated by blocking the electron transport on the oxidation side of photosystem II by carbonyl cyanide-m-chlorophenylhydrazone (CCCP) or Tris · HCl or heat pre-treatment, whereas these were suppressed by blocking the transport on the reduction side by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). It was deduced that the site of NphSH action in the electron transport chain is closer to the reaction center of photosystem II that the blocking site of CCCP or Tris · HCl or heat, and that such a site in photosystem II is exposed to be modified with NphSH when electron carriers on the oxidation side of photosystem II are oxidized by illumination.     

Primary Events, Energy Transfer, and Reactions in Photosynthetic Units: 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU) Inhibition of System II and Light-Induced Regulatory Changes in Energy Transfer Efficiency

Oxygen pulses produced in Chlorella by a xenon flash of 15 μsec half-width were measured by means of a rapid oxygen polarograph. Under appropriate conditions the height of the pulse caused by a saturating flash was a measure of the number of active reaction centers in system II. In pigment state II, caused by illumination during several minutes with light II, the number of active centers II was the same as in pigment state I. Oxygen pulses produced by about half-saturating flashes were diminished by about 7-10% in state II, showing that the fluorescence decrease in light II was at least partly caused by a decrease in energy transfer to reaction center II. After addition of 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), only the first flash produced oxygen which gives additional support for the hypothesis that DCMU inhibits between Q and system I.     

Identification of S2 as the sensitive state to alkaline photoinactivation of Photosystem II in chloroplasts

1. Chloroplasts have been preilluminated by a sequence of n short saturating flashes immediately before alkalinization to pH 9.3, and brought back 2 min later to pH 7.8. The assay of Photosystem II activity through dichlorophenolindophenol photoreduction, oxygen evolution, fluorescence induction, shows that part of the centers is inactivated and that this part depends on the number of preilluminating flashes (maximum inhibition after one flash) in a way which suggests identification of state S₂ as the target for alkaline inactivation.2. As shown by Reimer and Trebst ((1975) Biochem. Physiol. Pflanz. 168, 225–232) the inactivation necessitates the presence of gramicidin, which shows that the sensitive site is on the internal side of the thylakoid membrane.3. The electron flow through inactivated Photosystem II is restored by artificial donor addition (diphenylcarbazide or hydroxylamine); this suggests that the water-splitting enzyme itself is blocked. The inactivation is accompanied by a solubilization of bound Mn²⁺ and by the occurrence of EPR Signal II “fast”.4. Glutaraldehyde fixation before the treatment does not prevent the inactivation which thus does not seem to involve a protein structural change.     

The site of manganese function in photosynthetic electron transport system

The effects of Mn²⁺ on aerobic photobleaching of carotenoids, on photoreduction of 2,6-dichlorophenolindophenol (DCIP) and on fluorescence above 600 mμ of spinach chloroplasts washed with 0.8 M Tris-HC1 buffer were investigated. Carotenoids (mostly carotenes, lutein and violaxanthin) in the Tris-washed chloroplasts were irreversibly bleached by illumination with red light, while carotenoids in normal chloroplasts prepared with a low concentration of Tris-HC1 underwent no bleaching upon illumination. The photobleaching of carotenoids observed with Tris-washed chloroplasts was inhibited by Mn²⁺ (MnCl₂ or MnSO₄) as well as by some inhibitors of the Hill reaction such as dichlorophenyl-1,1-dimethylurea (DCMU), methylthio-4,6-bis-isopropylamino-s-triazine and o-phenanthroline or by reducing agents such as ascorbate plus tetramethyl-p-phenylene diamine (TMPD). DCIP photoreduction, which was deactivated by Tris, was reactivated to 50–80% of the rate for normal chloroplasts upon addition of Mn²⁺. The restored photoreduction of DCIP was inhibited by DCMU and carbonylcyanide m-chlorophenylhydrazone (CCCP). The steady-state fluorescence yield of normal chloroplasts measured at room temperature was lowered by Tris treatment, and the decreased yield was restored by adding Mn²⁺ as well as ascorbate plus TMPD. CCCP also lowered the yield; the yield was recovered by adding ascorbate plus TMPD. Determination of manganese in normal and Tris-washed chloroplasts showed that 30% of the manganese in chloroplast was removed with Tris. It was postulated that Mn²⁺ functions in the electron transport on the oxidizing side of Photosystem II at a site between water and an electron carrier (Y). CCCP as well as Tris inhibits the reduction of Y⁺ by Mn²⁺, and carotenoids are oxidized by Y⁺ which is reduced by ascorbate plus TMPD.     

New results on the mode of action of 3,-(3,4-dichlorophenyl)-1,1-dimethylurea in spinach chloroplasts

Simultaneous measurements of hydroxylamine photo-oxidation and fluorescence induction were performed in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). The results provide a justification for the common use of fluorescence data to estimate the concentration of active System II centers in the presence of inhibitors.The addition of DCMU to dark-adapted chloroplasts under special conditions induces a large increase of the initial yield of fluorescence. A reversible inactivation of part of the System II centers is responsible for this effect. Similar data were obtained with other classical inhibitors of oxygen evolution.     

The rapid component of electron paramagnetic resonance signal II: A candidate for the physiological donor to Photosystem II in spinach chloroplasts

Rapid light-induced transients in EPR Signal IIf (F^?+) are observed in 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated, Tris-washed chloroplasts until the state F P680 Q^? is reached. In the absence of exogenous redox mediators several flashes are required to saturate this photoinactive state. However, the Signal IIf transient is observed on only the first flash following DCMU addition if an efficient donor to Signal IIf, phenylenediamine or hydroquinone, is present. Complementary polarographic measurements show that under these conditions oxidized phenylenediamine is produced only on the first flash of a series. The DCMU inhibition of Signal IIf can be completely relieved by oxidative titration of a one-electron reductant with E′_08.0 = +480 mV. At high reduction potentials the decay time of Signal IIf is constant at about 300 ms, whereas in the absence of DCMU the decay time is longer and increases with increasing reduction potential.A model is proposed in which Q^?, the reduced Photosystem II primary acceptor, and D, a one-electron 480 mV donor endogenous to the chloroplast suspension, compete in the reduction of Signal IIf (F^?+). At high potentials D is oxidized in the dark, and the (Q^? + F^?+) back reaction regenerates the photoactive F P680 Q state. The electrochemical and kinetic evidence is consistent with the hypothesis that the Signal IIf species, F, is identical with Z, the physiological donor to P680.     

Delayed fluorescence induction in chloroplasts. Irradiance dependence

We have studied the delayed fluorescence in spinach chloroplasts produced 0.5 ms after each of a pair of (sub)-microsecond flashes. We observe an increase in the delayed fluorescence from the second flash relative to that produced by the first. This increase is proportional to the product of the first and second flash irradiances, appearing as an I² dependence if both flashes are increased together. The enhancement is observable at very weak flash levels (roughly 1 photon absorbed/100 PS II centers). If the irradiance of the first flash is increased, but the irradiance of the second held constant, the delayed fluorescence from the second flash is observed to increase, but then to saturate well below the first flash irradiance at which the delayed fluorescence from the first flash itself saturates. For most experiments, the dark time between flashes was 30 ms. If the dark time is varied, the enhancement changes, reaching a half-maximal value for a dark time of approx. 300 μs. The enhancement is stopped by hydroxylamine, but not by gramicidin, valinomycin, DCMU, or mild heating. These experiments are consistent with the notion that there are two different types of Photosystem II centers if we assume that only one type is responsible for the induction we see and has an optical cross-section about 4-times the size of the other type of center.