Kinetics of chlorophyll fluorescence at 77 K in Chlorella and chloroplasts. Effects of CCCP,ferricyanide and DCMU

The kinetics of chlorophyll fluorescence at 77 K were studied in Chlorella cells and spinach chloroplasts.During a first illumination, the rise is polyphasic with at least three phases. The slowest one is irreversible and corresponds to the cytochrome oxidation.The dark regeneration of half the variable fluorescence is biphasic, the fast phase being inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) both in Chlorella and chloroplasts.The fluorescence rise during a second illumination is still biphasic.Carbonyl cyanide m-chlorophenylhydrazone (CCCP) slows down the fluorescence rise in Chlorella but has no effect on the dark regeneration. It does not affect the fluorescence of chloroplasts.Ferricyanide which oxidizes cytochrome b-559 at room temperature produces a quenching of the variable fluorescence and an acceleration of the fluorescence rise during the first illumination.Our results fit the idea of the heterogeneity of the Photosystem II centers at low temperature.     

Effects of carbonyl cyanide m-chlorophenylhydrazone and hydroxylamine on the Photosystem II electron exchange mechanism in 3-(3,4-dichlorophenyl)-1,1-dimethylurea treated algae and chloroplasts

The effects of NH₂OH and carbonyl cyanide m-chlorophenylhydrazone (CCCP) on 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated algae and chloroplasts were studied. In the presence of DCMU, the photochemically separated charges can only disappear through a recombination back reaction; both substances induce an irreversible reduction of the donor side and after sufficient illumination their action in the presence of DCMU leads to the formation of a permanent fluorescent state.

In the DCMU + CCCP system, a fast fluorescence induction curve is observed. The fluorescence yield is brought to its maximum by two flashes. The luminescence emission is strongly inhibited and most centers reach their permanent fluorescent state after one flash.

In the DCMU + NH₂OH system, a slow fluorescence rise is observed and several saturating flashes are needed for the fluorescence yield to reach its maximum. The exhaustion of the NH₂OH oxidizing capacity and the complete transformation to a permanent fluorescent state also require a large number of flashes.

The reduction pathway catalyzed by CCCP appears to be a good competitor to the back reaction, while NH₂OH seems to be a relatively inefficient donor.

In addition the action of NH₂OH and CCCP on fluorescence suggests that the donor side influences the quenching properties of Photosystem II centers. A possible mechanism is proposed.     

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.     

Restoration by silicotungstic acid of DCMU-inhibited photoreactions in spinach chloroplasts

The restoration by silicotungstic acid of the reversible light-induced pH rise mediated by pyocyanine in EDTA-treated chloroplasts corresponds to an irreversible fixation of the acid. The proton uptake is linearly related to the amount of fixed acid (4 protons per molecule of acid) as long as the amount of silicotungstic acid does not exceed 200 nmoles/mg of chlorophyll.In the same conditions silicotungstic acid partly restores ferricyanide reduction and O₂ evolution in chloroplasts suspensions supplemented with DCMU. These photoreactions are observed only with chloroplasts and these chloroplasts must have an unimpaired water-splitting mechanism.Silicotungstic acid does not impair DCMU fixation on the specific sites. More likely in its presence the properties of the membrane change and ferricyanide can accept electrons from a part of the electron transport chain, between the Photosystem II reaction center and the block of the electron flow by DCMU.     

Studies on the slow fluorescence decline in isolated chloroplasts

Data presented here indicate that the slow fluorescence decline in osmotically disrupted chloroplasts is not associated with the well known divalent cation effect on fluorescence yield. Thus the two phenomena have markedly different magnesium concentration requirements, magnesium addition after the fluorescence decline did not stimulate the dark reversal, and the characteristics of the fluorescence induction kinetics of the two processes are not similar.At pH 7.6 the slow fluorescence decline was stimulated by several uncouplers demonstrated to greatly reduce proton pumping, and at pH 9.2 it was stimulated by all uncouplers tested. Acid-base transition was strongly inhibitory, and this inhibition was relieved by uncoupler. Thus the pH gradient seems to inhibit the process. The involvement of coupling factor is suggested by experiments in which phosphorylation substrates were inhibitory, and this inhibition was prevented by uncoupler. These data are explained in terms of coupling factor structural changes which in an unknown manner influence Photosystem II fluorescence emission.Fluorescence induction curves indicate that the slow quenching decreased only the variable fluorescence. The half rise time was decreased along with the sig-moidicity of the rise curve. These data can be accomodated in terms of a model recently proposed by Butler and Kitajima (Biochim. Biophys Acta (1975) 376, 116–125), involving the transfer of energy from the excited, but closed, reaction centres II to the light harvesting chlorophyll system. The slow fluorescence decline is suggested to represent a decrease of this process.     

Isolation of a fast decay in submillisecond Chlorella luminescence

Using a simple He-Ne (632.8-nm) laser phosphoroscope steady-state luminescence from Chlorella pyrenoidosa was studied from 50 μs to 1.1 ms between 1 ms long exciting flashes. The following results were obtained: (1) prior freezing or ultraviolet irradiation changed the time course of the luminescence to a rapid decay with a half-time of about 110 μs; (2) 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) suppressed the 110-μs luminescence; (3) spectrally, all observed luminescence was, within possible error, identical to fluorescence; (4) no effect on the luminescence intensity from pulsed magnetic fields up to 30 kgauss was observed; (5) the relative fluorescence yield, measured simultaneously with luminescence, was found to be constant.Our principal conclusions, supported mainly by experiments with DCMU, are: (1) the 110-μs decay is a distinct component of the total steady-state luminescence; (2) prior freezing or ultraviolet irradiation isolates this component of the luminescence by suppressing all other components; (3) the half-time and intensity of this component are temperature independent in the interval 0–22 °C.     

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.     

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.     

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.     

A rapid,light-induced transient in electron paramagnetic resonance signal II activated upon inhibition of photosynthetic oxygen evolution

A rapid, light-induced reversible component in Signal II is observed upon inhibition of oxygen evolution in broken spinach chloroplasts. The inhibitory treatments used include Tris washing, heat, treatment with chaotropic agents, and aging. This new Signal II component is in a 1 : 1 ratio with Signal I (P700). Its formation corresponds to a light-induced oxidation which occurs in less than 500 μs. The subsequent decay of the radical results from a reduction which occurs more rapidly as the reduction potential of the chloroplast suspension is decreased. The formation of this free radical component is complete following a single 10-μs flash, and it occurs with a quantum efficiency similar to that observed for Signal I formation. Red light is more effective than far-red light in the generation of this species, and, in preilluminated chloroplasts, 3-(3,4-dichlorophenyl)-1,1-dimethylurea blocks its formation. Inhibition studies show that the decline in oxygen evolution parallels the activation of this Signal II component.These results are interpreted in terms of a model in which two pathways, one involving water, the other involving the rapid Signal II component, compete for oxidizing equivalents generated by Photosystem II. In broken chloroplasts this Signal II pathway is deactivated and water is the principal electron donor. However, upon inhibition of oxygen evolution, the Signal II pathway is activated.     

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.     

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⁻.     

Changes in photosynthetic activity in the cyanobacterium Chlorogloea fritschii following transition from dark to light growth

The cyanobacterium Chlorogloea fritschii loses Photosystem II activity, measured by delayed fluorescence and oxygen evolution, during dark heterotrophic growth, but retains Photosystem I, measured as light induced EPR signals. Following transition to the light, Photosystem II recovers in two stages, the first of which does not require protein synthesis. New Photosystem I reaction centres are not synthesised until after net chlorophyll synthesis has commenced. Carbon dioxide fixation recovery commences immediately, the initial rate being unaffected by chloramphenicol. The recovery of carbon dioxide fixation is not directly related to oxygen evolution rate and is only inhibited slightly by 3-(3,4-dichlorophyenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone.     

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.     

Determination and modification of the redox state of the secondary acceptor of Photosystem II in the dark

The redox state of the secondary electron acceptor B of Photosystem II was studied using fluorescence measurements. Preillumination of algae or chloroplasts with a variable number of short saturating flashes followed rapidly by the addition of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea induces oscillations of the initial level of fluorescence. The phase of these oscillations is characteristic of a given $\text{B}\text{B}{}^{\mathrm{?}}$ ratio in the dark-adapted samples.We conclude from our results that about 50% of the secondary electron acceptors are singly reduced in the dark in Chlorella cells, but that more than 70% are fully oxidized in the dark adapted chloroplasts.Benzoquinone treatment modifies this distribution in Chlorella leading to the same situation as in chloroplasts, i.e. more than 70% of the secondary acceptors are oxidized in the dark.The same ratio is observed if these algae are illuminated and then dark-adapted, unless an artificial donor (hydroxylamine) is added before this illumination. In that case about 50% B^? is generated and stabilized in the dark.     

Electron paramagnetic resonance Signal II in spinach chloroplasts. I. Kinetic analysis for untreated chloroplasts

An analysis of electron paramagnetic resonance Signal II in spinach chloroplasts has been made using both continuous and flashing light techniques. In order to perform the experiments we developed a method which allows us to obtain fresh, untreated chloroplasts with low dark levels of Signal II. Under these conditions a single 10-μs flash is sufficient to generate greater than 80% of the possible light-induced increase in Signal II spin concentration. The risetime for this flash-induced increase in Signal II is approx. 1 s. The close association of Signal II with Photo-system II is confirmed by the observations that red light is more effective than is far red light in generating Signal II, and that 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) does not inhibit the formation of the radical. Single flash saturation curves for the flash-induced increase in Signal I and Signal II indicate that the quantum efficiency for Signal II formation is close to that for Signal I. While one or two flashes (spaced 10 ms apart) are quite efficient in generating Signal II, three or four flashes are much less effective. However, if this spacing is decreased to 100 μs, three or four flashes become as efficient as one or two flashes. From observations of a deficiency of O₂ evolved during the initial flashes of dark-adapted chloroplasts, we conclude that the species which gives rise to Signal II is able to compete with water for oxidizing equivalents generated by Photosystem II. On the basis of these results we postulate a model in which Signal II arises from an oxidized radical which is produced by a slow electron transfer to the specific states S₂ and S₃ on the water side of Photo-system II.     

Photooxidation of cytochrome b559 and the electron donors in chloroplast Photosystem II

The effect of light on the reaction center of Photosystem II was studied by differential absorption spectroscopy in spinach chloroplasts.

At − 196 °C, continuous illumination results in a parallel reduction of C-550 and oxidation of cytochrome b₅₅₉ high potential. With flash excitation, C-550 is reduced, but only a small fraction of cytochrome b₅₅₉ is oxidized. The specific effect of flash illumination is suppressed if the chloroplasts are preilluminated by one flash at 0 °C.

At − 50 °C, continuous illumination results in the reduction of C-550 but little oxidation of cytochrome b₅₅₉. However, complete oxidation is obtained if the chloroplasts have been preilluminated by one flash at 0 °C. The effect of preillumination is not observed in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea.

A model is discussed for the reaction center, with two electron donors, cytochrome b₅₅₉ and Z, acting in competition. Their respective efficiency is dependent on temperature and on their states of oxidation. The specific effect of flash excitation is attributed to a two-photon reaction, possibly based on energy-trapping properties of the oxidized trap chlorophyll.