The existence of a high photochemical turnover rate at the reaction centers of System II in Tris-washed chloroplasts

In Tris-washed chloroplasts the kinetics of the primary electron acceptor X 320 of reaction center II has been investigated by fast repetitive flash spectroscopy with a time resolution of $\text{≈ 1 μs}$. It has been found that X 320 is reduced by a flash in $\text{? 1 μs}$. The subsequent reoxidation in the dark occurs mainly by a reaction with a 100–200 μs kinetics. The light-induced difference spectrum confirms X 320 to be the reactive species. From these results it is concluded that in Tris-washed chloroplasts the reaction centers of System II are characterized by a high photochemical turnover rate mediated either via rapid direct charge recombination or via fast cyclic electron flow.     

Interaction of exogenous benzoquinone with Photosystem II in chloroplasts. The semiquinone form acts as a dichlorophenyldimethylurea-insensitive secondary acceptor

Chloroplasts were submitted to a sequence of saturating short flashes and then rapidly mixed with dichlorophenyldimethylurea (DCMU). The amount of singly reduced secondary acceptor (B^?) present was estimated from the DCMU-induced increase in fluorescence in the dark caused by the reaction: QB^?

Q^?B. By varying the time interval between the preillumination and the mixing, the time course of B^? reoxidation by externally added benzoquinone was investigated. It was found that benzoquinone oxidizes B^? in a bimolecular reaction, and does not interact directly with Q^?. When a sufficient delay after the preillumination was allowed in order to let benzoquinone reoxidize B^? before the injection of DCMU, the fluorescence increase caused by one subsequent flash fired in the presence of DCMU was followed by a fast decay phase ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 100 μ}\text{s}$). The amplitude of this phase was proportional to the amount of B^? produced by the preillumination. This fast decay was observed only after the first flash in the presence of DCMU. These results are interpreted by assuming a binding of the singly reduced benzoquinone to Photosystem II where it acts as an efficient, DCMU-insensitive, secondary (exogenous) acceptor.     

The reduction of the oxygen-evolving system in chloroplasts by thylakoid components

Using thoroughly dark-adapted thylakoids and an unmodulated Joliot-type oxygen electrode, the following results were obtained. (i) At high flash frequency (4 Hz), the oxygen yield at the fourth flash (Y₄) is lower compared to Y₃ than at lower flash frequency. At 4 Hz, the calculated S₀ concentration after thorough dark adaptation is found to approach zero, whereas at 0.5 Hz the apparent $\text{S}{}_0\text{(}\text{S}{}_0\text{+}\text{S}{}_1\text{)}$ ratio increases to about 0.2. This is explained by a relatively fast donation ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.0–1.5}\text{s}$) of one electron by an electron donor to S₂ and S₃ in 15–25% of the Photosystem II reaction chains. The one-electron donor to S₂ and S₃ appears to be rereduced very slowly, and may be identical to the component that, after oxidation, gives rise to ESR signal II_s. (ii) The probability for the fast one-electron donation to S₂ and S₃ has nearly been the same in triazine-resistant and triazine-susceptible thylakoids. However, most of the slow phase of the S₂ decay becomes 10-fold faster ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 5–6}\text{s}$) in the triazine-resistant ones. In a small part of the Photosystem II reaction chains, the S₂ decay was extremely slow. The S₃ decay in the triazine-resistant thylakoids was not significantly different from that in triazine-susceptible thylakoids. This supports the hypothesis that S₂ is reduced mainly by Q^?_A, whereas S₃ is not. (iii) In the absence of CO₂/HCO^?_A and in the presence of formate, the fast one-electron donation to S₂ and S₃ does not occur. Addition of HCO^?₃ restores the fast decay of part of S₂ and S₃ to almost the same extent as in control thylakoids. The slow phase of S₂ and S₃ decay is not influenced significantly by CO₂/HCO^?₃. The chlorophyll a fluorescence decay kinetics in the presence of DCMU, however, monitoring the Q^?_A oxidation without interference of Q_B, were 2.3-fold slower in the absence of CO₂/HCO^?₃ than in its presence. (iv) An almost 3-fold decrease in decay rate of S₂ is observed upon lowering the pH from 7.6 to 6.0. The kinetics of chlorophyll a fluorescence decay in the presence of DCMU are slightly accelerated by a pH change from 7.6 to 6.0. This indicates that the equilibrium Q^?_A concentration after one flash is decreased (by about a factor of 4) upon changing the pH from 7.6 to 6.0. When direct or indirect protonation of Q^?_B is responsible for this shift of equilibrium Q^?_A concentration, these data would suggest that the pK_a value for Q^?_B protonation is somewhat higher than 7.6, assuming that the protonated form of Q^?_B cannot reduce Q_A.     

Two possible 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive sites in Photosystem II of spinach chloroplasts

Two possible 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive sites were found in PS II of spinach chloroplasts, depending on the pH of the assay medium used. The low site (pH 6) can be inhibited by certain quinolines, such as 8-hydroxyquinoline at concentrations less than 50 μM. The high pH site (pH 8) can be inhibited by disodium cyanamide, folic acid, or 5,6-benzoquinoline at concentrations from 50 μM to 5 mM. With the exception of orthophenanthroline, which stimulates the high pH site but does not show much inhibition at low pH, all other inhibitors gave opposite effects at the pH values used, i.e., they stimulated at low pH or inhibited at high pH, or vice versa. Several mechanisms for the observed effects are discussed.     

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⁻.     

Effects of chloride depletion on electron donation from the water-oxidizing complex to the photosystem II reaction center as measured by the microsecond rise of chlorophyll fluorescence in isolated pea chloroplasts

The role of Cl^? in the electron transfer reactions of the oxidizing side of Photosystem II (PS II) has been studied by measuring the fluorescence yield changes corresponding to the reduction of P⁺-680, the PS II reaction center chlorophyll, by the secondary PS II donor, Z. In Cl^?-depleted chloroplasts, a rapid rise in fluorescence yield was observed following the first and second flashes, but not during the third or subsequent flashes. These results indicate that there exists an additional endogenous electron donor beyond P-680 and Z in Cl^?-depleted systems. In contrast, the terminal endogenous donor on the oxidizing side of PS II in Tris-washed preparations has previously been shown to be Z, the component giving rise to EPR signals II_f and II_vf. The rate of reduction of P⁺-680 in the Cl^?-depleted chloroplasts was as rapid as that measured in uninhibited systems, within the time resolution of our instrument. Again, this is in contrast to Tris-washed preparations in which a dramatic decrease in the rate if this reaction has been previously reported. We have also carried out a preliminary study on the rate of rereduction of Z⁺ in the Cl^?-depleted system. Under steady-state conditions, the reduction half-time of Z⁺ in uninhibited systems was about 450 μs, while in the Cl^?-depleted chloroplasts, the reduction of Z⁺ was biphasic, one phase with a half-time of about 120 ms, and a slower phase with a half-time of several seconds. The appearance of the quenching state due to P⁺-680 observed following the third flash on excitation of Cl^?-depleted chloroplasts was delayed by two flashed when low concentrations of NH₂OH (20–50 μM) were included in the medium. Hydrazine at somewhat higher concentrations showed the same effect. This is taken to indicate that the reactions leading to PS II oxidation of NH₂OH or NH₂NH₂ are uninhibited by Cl^? depletion. Addition of NH₂OH at low concentrations to Tris-washed chloroplasts did not alter the pattern of the fluorescence yield, indicating that the reactions leading to the NH₂OH oxidation present in Cl^?-depleted systems are absent following Tris inhibition. The results are discussed in terms of an inhibition by Cl^? depletion of the reactions of the oxygen-evolving complex. It is suggested that no intermediary redox couple exists between the oxygen-evolving complex and Z, and that Z⁺ is reduced directly by Mn of the complex. In terms of the S-state model, Cl^? depletion appears to inhibit the advancement of the mechanism beyond S₂, but not to inhibit the transitions from S₀ to S₁, or from S₁ to S₂.     

Fluorescence induction in intact spinach chloroplasts

Under conditions in which the Photosystem II quencher is rapidly reduced upon illumination, either after a preillumination or following treatment with dithionite, the fluorescence-induction curve of intact spinach chloroplasts (class I type) displays a pronounced dip. This dip is probably identical with that observed after prolonged anaerobic incubation of whole algal cells (“I-D dip”). It is inhibited by 3(3,4-dichlorophenyl)-1,1-dimethylurea and occurs in the presence of dithionite, sufficient to reduce the plastoquinone pool. It is influenced by far red light, methylviologen, anaerobiosis and uncouplers in a manner consistent with the interpretation that it represents a photochemical quenching of fluorescence by an electron transport component situated between the Photosystem II quencher and plastoquinone. Glutaraldehyde inhibition may indicate that protein structural changes are involved.     

Kinetics of reduction of the oxidized primary electron donor of Photosystem II in spinach chloroplasts and in Chlorella cells in the microsecond and nanosecond time ranges following flash excitation

Absorption changes (ΔA) at 820 nm, following laser flash excitation of spinach chloroplasts and Chlorella cells, were studied in order to obtain information on the reduction time of the photooxidized primary donor of Photosystem II at physiological temperatures.In the microsecond time range the difference spectrum of ΔA between 750 and 900 nm represents a peak at 820 nm, attributable to a radical-cation of chlorophyll a. In untreated dark-adapted material the signal can be attributed solely to P⁺?700; it decays in a polyphasic manner with half-times of 17 μs, 210 μs and over 1 ms. The oxidized primary donor of Photosystem II (P⁺_II) is not detected with a time resolution of 3 μs. After treatment with 3–10 mM hydroxylamine, which inhibits the donor side of Photosystem II, P⁺_II is observed and decays biphasically (a major phase with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 20–40 μ}\text{s}$, and a minor phase with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{? 200 μ}\text{s}$), probably by reduction by an accessory electron donor.In the nanosecond range, which was made accessible by a new fast-response flash photometer operating at 820 nm, it was found the P⁺_II is reduced with a half-time of 25–45 ns in untreated dark-adapted chloroplasts. It is assumed that the normal secondary electron donor is responsible for this fast reduction.     

Potentiometric titration of Photosystem II fluorescence decay kinetics in spinach chloroplasts

The fluorescence yield of chloroplasts reflects the redox state of the electron acceptor of the Photosystem II reaction center, with increasing yield as the acceptor is reduced. Chemical reductive titrations of fluorescence yield in chloroplasts at room temperature indicate two distinct midpoint potentials, suggesting the possibility of Photosystem II electron acceptor heterogeneity. We have carried out a potentiometric titration of the fluorescence decay kinetics in spinach chloroplasts using a continuous mode-locked dye laser with low-intensity excitation pulses and a picosecond-resolution single-photon timing system. At all potentials the fluorescence decay is best described by three exponential components. As the potential is lowered, the slow phase changes 30-fold in yield with two distinct midpoint potentials, accompanied by a modest (3-fold) increase in the lifetime. The titration curve for the slow component of the fluorescence decay of spinach chloroplasts is best characterized by two single-electron redox reactions with midpoint potentials at pH 8.0 of +119 and ?350 mV, with corresponding relative contributions to the fluorescence yield of 49 and 51%, respectively. There is little change in the fast and middle components of the fluorescence decay. We found that the oxidized form of the redox mediator 2-hydroxy-1,4-naphthoquinone preferentially quenches the fluorescence, causing an anomalous decrease in the apparent midpoint of the high-potential transition. This effect accounts for a significant difference between the midpoint potentials that we observe and some of those previously reported. The selective effect of reduction potentials on particular fluorescence decay components provides useful information about the organization and distribution of the Photosystem II electron acceptor.     

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.     

Changes in the redox state of the secondary acceptor of Photosystem II associated with light-induced thylakoid protein phosphorylation

Thylakoid membrane protein phosphorylation affects photochemical reactions of Photosystem II. Incubation of thylakoids in the light with ATP leads to: (1) an increase in the amplitude of three components (4–6, 25–45 and 280–300 μs) of delayed light emission after a single flash without any change in their kinetics; (2) a reduction of the flash-dependent binary oscillations of chlorophyll a fluorescence yield associated with electron transfer from the primary quinone acceptor, Q, to the secondary quinone acceptor, B; (3) an increase in the $\text{B}{}^{\mathrm{?}}\text{B}$ ratio resulting from an increase in stability of the semiquinone anion during dark adaptation; and (4) no change in the redox state of the plastoquinone pool as determined by flash-induced photooxidation of the Photosystem I reaction center, P-700. All the above observations are reversible upon dephosphorylation of the thylakoid membranes. These data are explained by a protein phosphorylation-induced stabilization of the bound semiquinone anion, B^?. It is proposed that this increased stability may be due to an alteration in the accessibility of an endogenous reductant to B, or to an increase in dissipative cycling of charge around Photosystem II.     

Charge accumulation and recombination in Photosystem II studied by thermoluminescence. I. Participation of the primary acceptor Q and secondary acceptor B in the generation of thermoluminescence of chloroplasts

In the glow curves of chloroplasts excited by a series of flashes at +1°C the intensity of the main thermoluminescence band appearing at +30°C (B band; B, secondary acceptor of Photosystem II) exhibits a period-4 oscillation with maxima on the 2nd and 6th flashes indicating the participation of the S₃ state of the water-splitting system in the radiative charge recombination reaction. After long-term dark adaptation of chloroplasts (6 h), when the major part of the secondary acceptor pool (B pool) is oxidized, a period-2 contribution with maxima occurring at uneven flash numbers appears in the oscillation pattern. The B band can even be excited at ?160°C as well as by a single flash in which case the water-splitting system undergoes only one transition (S₁ → S₂). The experimental observations and computer simulation of the oscillatory patterns suggest that the B band originates from charge recombination of the S₂B^? and S₃B^? redox states. The half-time of charge recombination responsible for the B band is 48 s. When a major part of the plastoquinone pool is reduced due to prolonged excitation of the chloroplasts by continuous light, a second band (Q band; Q, primary acceptor of Photosystem II) appears in the glow curve at +10°C which overlaps with the B band. In chloroplasts excited by flashes prior to DCMU addition only the Q band can be observed showing maxima in the oscillation pattern at flash numbers 2, 6 and 10. The Q band can also be induced by flashes after DCMU addition which allows only one transition of the water-splitting system (S₁ → S₂). In the presence of DCMU, electrons accumulate on the primary acceptor Q, thus the Q band can be ascribed to the charge recombination of either the S₂Q^? or S₃Q^? states depending on whether the water-splitting system is in the S₂ or the S₃ state. The half-time of the back reaction of Q^? with the donor side of PS II (S₂ or S₃ states) is 3 s. It was also observed that in a sequence of flashes the peak positions of the Q and B bands do not depend on the advancement of the water-splitting system from the S₂ state to the S₃ state. This result implies that the midpoint potential of the water-splitting system remains unmodified during the S₂ → S₃ transition.     

Multiple effects of linolenic acid addition to pea thylakoids

The addition of linolenic acid to thylakoids produces various pH-dependent effects. We have demonstrated a binding site near the Photosystem (PS) II center with a pK_a of 6.5: when linolenic acid is unprotonated it induces in the dark a rise of the initial fluorescence level, the latter being similar to the maximum fluorescence obtained during illumination of untreated thylakoids. The comparison of the fluorescence lifetimes in the presence and absence of linolenic acid leads us to conclude that the charge stabilisation on the primary acceptor, Q, is prevented by linolenic acid. A second binding site on the protein carrying B, the secondary acceptor of PS II, has also been demonstrated for linolenic acid. It has a 3-(3,4-dichlorophenyl)-1,1-dimethylurea-type effect both in the protonated and unprotonated forms. Finally, measurements of electrophoretic mobility of the thylakoids indicate several other sites of linolenic acid inclusion with an average pK_a of 5.7. At alkaline pH the presence of unprotonated linolenic acid increases the charge density on the membrane. As a result a higher concentration of divalent cations is needed to obtain fluorescence and stacking changes than for untreated thylakoids. The presence, at acidic pH values, of the unprotonated form of linolenic acid leads to the inhibition of cation-induced fluorescence changes, probably by preventing the movement of chlorophyll-protein complexes in the membrane.     

Photosystem II energy coupling in chloroplasts with H2O2 as electron donor

NH₂OH-treated, non-water-splitting chloroplasts can oxidize H₂O₂ to O₂ through Photosystem II at substantial rates (100–250 μequiv · h^?1 · mg^?1 chlorophyll with 5 mM H₂O₂) using 2,5-dimethyl-p-benzoquinone as an electron acceptor in the presence of the plastoquinone antagonist dibromothymoquinone. This H₂O₂ → Photosystem II → dimethylquinone reaction supports phosphorylation with a $\text{P}\text{e}{}_2$ ratio of 0.25–0.35 and proton uptake with $\text{H}{}^{\mathrm{+}}\text{e}$ values of 0.67 (pH 8)–0.85 (pH 6). These are close to the $\text{P}\text{e}{}_2$ value of 0.3–0.38 and the $\text{H}{}^{\mathrm{+}}\text{e}$ values of 0.7–0.93 found in parallel experiments for the H₂O → Photosystem II → dimethylquinone reaction in untreated chloroplasts. Semi-quantitative data are also presented which show that the donor → Photosystem II → dibromothymoquinone (→O₂) reaction can support phosphorylation when the donor used is a proton-releasing reductant (benzidine, catechol) but not when it is a non-proton carrier (I^?, ferrocyanide).     

External electric field effects on prompt and delayed fluorescence in chloroplasts

An electric field pulse was applied to a suspension of osmotically swollen spinach chloroplasts after illumination with a saturating flash in the presence of DCMU. In addition to the stimulation of delayed fluorescence by the electric field, discovered by Arnold and Azzi (Arnold, W.A. and Azzi, R. (1971) Photochem. Photobiol. 14, 233–240) a sudden drop in fluorescence yield was observed. The kinetics of this fluorescence change were identical to those of the integrated delayed fluorescence emission induced by the pulse. The S-state dependence of the stimulated emission was very similar to that of the normal luminescence. We assume that the membrane potential generated by the pulse changes the activation energy for the back reaction in Photosystem II. On this basis, and making use of data we obtained earlier from electrochromic absorbance changes induced by the pulse, the kinetics of the field-induced prompt and delayed fluorescence changes, and also the amplitude of the fluorescence decrease, which was about 12% for a nearly saturating pulse, are explained. Our results indicate that in those reaction centers where a decrease of the activation energy occurs the effect of a pulse can be quite spectacular: the back reaction, which normally takes seconds, is completed in a few hundred microseconds when a sufficiently strong pulse is applied. Measurements of the polarization of the stimulated luminescence supported the interpretation given above.Only 2.8% of the back reaction was found to proceed via transition of reexcited chlorophyll to the ground state, both during the field pulse and in the absence of the field.     

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.     

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.     

Stimulation of microsecond-delayed fluorescence from spinach chloroplasts by uncouplers and by phosphorylation

Delayed fluorescence, as measured with a laser phosphoroscope, is stimulated not inhibited by uncouplers during the first 100 μs after the light is turned off. This is true only wen uncouplers cause an increase in the rate of electron transport. When ADP and P_i cause an increase in the electron transport rate, microsecond-delayed fluorescence is also increased. Indeed, there is a complex quantitative relationship between the rate of electron transport and the initial intensity of delayed fluorescence under a wide range of conditions.

Uncouplers or ADP and P_i also increase the rate of decay of delayed fluorescence so that after about 150 μs they become inhibitory, as already reported by many authors.

Microsecond-delayed fluorescence continues to rise with rising light intensities long after the rate of reduction of exogenous acceptor is light-saturated.

These observations suggest a correlation of the rate of electron transport both with the intensity of the 5–100 μs-delayed fluorescence and with the rate of decay in the intensity of delayed fluorescence. The data imply that the decrease in intensity of millisecond-delayed fluorescence which has often been noted with uncouplers is probably not due to the elimination of a membrane potential. It seems more likely that the decrease in millisecond-delayed fluorescence is a reflection of the rate of disappearance of some other electron transport-generated condition, a condition which is uncoupler-insensitive. Certainly stimulations of microsecond-delayed fluorescence by electron transport which has been uncoupled by gramicidin suggest that ion gradients are not an essential component of the conditions responsible for delayed fluorescence.     

Oxygen-evolution patterns from spinach Photosystem II preparations

Patterns of O₂ evolution resulting from sequences of short flashes are reported for Photosystem (PS) II preparations isolated from spinach and containing an active, O₂-evolving system. The results can be interpreted in terms of the S-state model developed to explain the process of photosynthetic water splitting in chloroplasts and algae. The PS II samples display damped, oscillating patterns of O₂ evolution with a period of four flashes. Unlike chloroplasts, the flash yields of the preparations decay with increasing flash number due to the limited plastoquinone acceptor pool on the reducing side of PS II. The optimal pH for O₂ evolution in this system (pH 5.5–6.5) is more acidic than in chloroplasts (pH 6.5–8.0). The O₂-evolution, inactivation half-time of dark-adapted preparations was 91 min (on the rate electrode) at room temperature. Dark-inactivation half-times of 14 h were observed if the samples were aged off the electrode at room temperature. Under our conditions (experimental conditions can influence flash-sequence results), deactivation of S₃ was first order with a half-time of 105 s while that of S₂ was biphasic. The half-times for the first-order rapid phase were 17 s (one preflash) and 23 s (two preflashes). The longer S₂ phase deactivated very slowly (the minimum half-time observed was 265 s). These results indicate that deactivation from S₃ → S₂ → S₁, thought to be the dominant pathway in chloroplasts, is not the case for PS II preparations. Finally, it was demonstrated that the ratio of S₁ to S₀ can be set by previously developed techniques, that S₀ is formed mostly from activated S₃ (S₄), and that both S₀ and S₁ are stable in the dark.