In situ SUSCEPTIBILITIES OF PHOTOSYSTEMS I AND II TO PHOTOSENSITIZED DEACTIVATION via SINGLET OXYGEN MECHANISM

Abstract— A comparative study was carried out on the in situ susceptibilities to photoinactivation of the photosystem I (PS I) and II (PS II) complexes of spinach thylakoids treated with efficient type II sensitizers. While the presence of the exogenous sensitizers caused a substantial increase in the extent of photoinactivation of whole chain electron transport, it did not affect PS I activity of thylakoids in light but exerted an enhanced photoinactivating effect only on PS II. The measurements of the action spectrum for the inhibition of PS II activity of the sensitizer-incorporated thylakoids and that for the generation of singlet oxygen (¹O₂) from them revealed that photosensitized inactivation of PS II is directly related to the photoproduction of ¹O₂ in thylakoid membranes. The results obtained in the present work clearly demonstrate an exceptional sensitivity of PS II to ¹O₂, providing circumstantial evidence that high light-induced damage to PS II may result from photosensitization reactions mediated by ¹O₂, which is not necessarily produced within the PS II complex.     

INACTIVATION OF THE ACCEPTOR SIDE AND DEGRADATION OF THE D1 PROTEIN OF PHOTOSYSTEM II BY SINGLET OXYGEN PHOTOGENERATED FROM THE OUTSIDE

Abstract— The possible association of photodynamic sensitization with photoinhibition damage to the photosystem II complex (PS II) has been investigated using isolated intact thylakoids from pea leaves. For this study singlet oxygen (¹O₂), photoproduced by endogenous chromophores that are independent of the function of PS II, was assumed to be the major reactive intermediate involved in the photoinhibition process. When thylakoid samples preincubated with rose bengal were subjected to exposure to relatively weak green light (500–600 nm) under aerobic conditions, PS II was severely damaged. The pattern of the rose bengal-sensitized inhibition of PS II was similar to that of high light-induced damage to PS II: (1) the secondary quinone (Q_B)-dependent electron transfer through PS II is inactivated much faster than the Q_B-independent electron flow, (2) PS II activity is lost prior to degradation of the D1 protein, (3) diuron, an herbicide that binds to the Q_B domain on the D1 protein, prevents D1 degradation, and (4) PS II is damaged to a greater extent by the deuteration of thylakoid suspensions but to a lesser extent by the presence of histidine. Furthermore, it was observed that destroying thylakoid Fe-S centers resulted in a marked reduction of high light-induced PS II damage. These results may suggest that the primary processes of photoinhibition are mediated by ¹O₂ and that Fe-S centers, which are located in some membrane components, but not in PS II, play an important role in photogenerating the activated oxygen immediately responsible for the initiation of photodamage to PS II.     

THE LIGHT DEPENDENT QUENCHING OF CHLOROPLAST FLUORESCENCE BY COFACTORS OF CYCLIC ELECTRON FLOW IN PHOTOSYSTEM I

Abstract—When 3–(3',4'-dichlorophenyl)-1,1-dimethylurea poisoned, intact thylakoids of isolated chloroplasts are illuminated in salt free suspension media, N -methylphenazinium cations (MP⁺) are reversibly taken up. Simultaneously, the chlorophyll fluorescence is reversibly lowered. When inorganic salts in the reaction medium provide membrane permeant charge balancing ions, the extent of the MP⁺ association with the thylakoids is strongly increased, but the fluorescence lowering is hardly affected. lonophoretically active agents inhibit specifically the salt dependent increment of the MP⁺ interaction with the thylakoids, but have only insignificant effects on the fluorescence lowering provided the experimental conditions do not allow the formation of a proton gradient across the thylakoid membrane. On the basis of these results, and of data obtained from comparative studies with other cofactors of cyclic electron transport in PS I, it is suggested that the 'energy dependent' fluorescence lowering is linked to a binding of cationic cofactors to nucleophilic sites in or at the thylakoid membrane. Such sites appear to become exposed in the wake of a light dependent transport of the cofactor, or of protons, into the thylakoid.     

PARTICIPATION OF THE TWO PHOTOSYSTEMS IN LIGHT DEPENDENT HYDROGEN EVOLUTION IN Scenedesmus obliquus

Abstract— The H₂-photoproduction in the presence of dithionite measured in wild type and mutant cells of Scenedesmus obliquus demonstrates two sequential phases. In mutants showing only PS I activity phase 1 of H₂-photoproduction is visible with its core activity. When PS II is developed during greening, considerable activity is added to the core of phase I and phase II activity appears. Addition of DCMU reduces H₂-photoproduction by about 90%. The residual activity is completely attributed to the core of phase I. It was concluded that the core of phase I is dependent upon PS I only and can use sources different from water as electron donors. Phase II is dependent upon the capacity of PS II, a functioning photosynthetic apparatus and water as electron donor. The results are supported by studies of wavelength dependent activity of the two separate phases of H₂-photoproduction.     

BASF 13.338 INDUCED CHANGES IN STRUCTURE and FUNCTION OF THE PHOTOSYNTHETIC APPARATUS OF WHEAT SEEDLINGS

Abstract— Growing wheat seedlings in the presence of BASF 13.338 [4-chloro-5-dimethylamino-2-phenyl-3(2H)pyridazinone], a PS II inhibitor of the pyridazinone group, brought about notable changes in the structure and functioning of photosynthetic apparatus. In BASF 13.338 treated plants, there was a decrease in the ratio of Chi a/Chl b, an increase in xanthophyll/carotene ratio and an increase in the content of Cyt b 559 (HP + LP). Chl/p700 ratio increased when measured with the isolated chloroplasts but not with the isolated PS I particles of the treated plants. The SDS-PAGE pattern of chloroplast preparations showed an increase in the CPII/CP I ratio. The F685/F740 ratio in the emission spectrum of chloroplasts at -196°C increased. The difference absorption spectrum of chloroplasts between the control and the treated plants showed a relative increase of a chlorophyll component with a peak absorption at 676 nm and a relative decrease of a chlorophyll component with a peak absorption at 692 nm for the treated plants. The excitation spectra of these chloroplast preparations were similar. Chloroplasts from the treated plants exhibited a greater degree of grana stacking as measured by the chlorophyll content in the 10 K pellet. The rate of electron transfer through photosystem II at saturating light intensity in chloroplast thylakoids isolated from the treated plants increased (by 50%) optimally at treatment of 125 μM BASF 13.338 as compared to the control. This increase was accompanied by an increase in (a) I₅₀ value of DCMU inhibition of photosystem II electron transfer; (b) the relative quantum yield of photosystem II electron transfer; (c) the magnitude of C550 absorbance change; and (d) the rate of carotenoid photobleaching. These observations were interpreted in terms of preferential synthesis of photosystem II in the treated plants. The rate of electron transfer through photosystems I and through the whole chain (H₂O → methyl viologen) also increased, due to an additional effect of BASF 13.338, namely, an increase in the rate of electron transfer through the rate limiting step (between plastoquinol and cytochrome f). This was linked to an enhanced level of functional cytochrome f. The increase in the overall rate of electron transfer occurred in spite of a decrease in the content of photosystem I relative to photosystem II. Treatment with higher concentrations (> 125 μM) of BASF 13.338 caused a further increase in the level of cytochrome f, but the rate of electron transfer was no greater than in the control. This was due to an inhibition of electron transfer at several sites in the chain.     

THE PHOTOSYNTHETIC APPARATUS OF Acetabularia mediterranea GROWN UNDER RED OR BLUE LIGHT. BIOPHYSICAL QUANTIFICATION and CHARACTERIZATION OF PHOTOSYSTEM II and ITS CORE COMPONENTS

Abstract— The photosynthetic activity of white light-grown Acetabularia mediterranea Lamouroux (= A. acetabulum (L.) Silva) decreases under continuous red light to less than 20% within 3 weeks. Subsequent blue light reactivates photosynthesis within a relatively short period of 3 days. In a former publication (Wennicke and Schmid, Plant Physiol. 84 ,1252–1256, 1987) we have shown that the regulated rate limiting step, which is an immediate light driven reaction, is part of photosystem II (PS II). The following biophysical properties of PS II were analyzed in thylakoids isolated from algae grown 3 weeks under either blue or red light with or without subsequent 3 days of blue light illumination: (a) fluorescence induction in the short time domain dominated by Q_A reduction, (b) the slow fluorescence decline reflecting pheophytin photoaccumulation, (c) absorption changes at 320 and 830 nm under repetitive flash excitation as indicator for the turnover of Q_A and P₆₈₀, respectively, (d) oscillation pattern of the oxygen yield by a flash train in dark adapted samples and (e) the binding capacity for atrazine. None of these PS II functions were severely affected, but a minor impairment of20–30% was observed in the thylakoids from algae grown for 3 weeks in red irradiation. The changes do not fully account for the drastic reduction of the electron transport through PS II which was 80% after red light treatment. Therefore, the regulated rate-limiting step appears to not be mainly located in the PS II core complex itself. It seems likely that the regulation process predominantly comprises the antenna system.     

CHLOROPHYLL b: AN INTEGRAL COMPONENT OF PHOTOSYSTEM I OF HIGHER PLANT CHLOROPLASTS

Abstract— An undissociated photosystem I complex may be isolated from spinach thylakoids by mild gel electrophoresis (CP1a) or Triton X-100. CP1a has a Chl a / b ratio of 11 and a Chl/P700 ratio of 120. while the Triton X-100 PS I complex (Chl a / b ratio of 5.9) has a larger antenna unit size (Chl/P700 ratio of 180). None of the Chl a / b -proteins of the main light-harvesting complex (apoproteins of 30–27 kD) are present in CP1a, and they account for less than 10% of the total chlorophyll in the Triton X-100 PS I complex. Instead, these PS I complexes have specific, but as yet little characterized, Chi a / b -proteins (apoproteins in the 26–21 kD range). With both PS I complexes, Chi b transfers light excitation to the 735 nm low temperature fluorescence band characteristic of photosystem I. We suggest that Chi b is an integral but minor component of photosystem I.     

Separation and determination of minor photosynthetic pigments by reversed-phase HPLC with minimal alteration of chlorophylls.

Reversed-phase HPLC conditions for separation of chlorophyll (Chl) a, Chl a' (the C132-epimer of Chl a), pheophytin (Pheo) a (the primary electron acceptor of photosystem (PS) II), and phylloquinone (PhQ) (the secondary electron acceptor of PS 1), have been developed. Pigment extraction conditions were optimized in terms of pigment alteration and extraction efficiency. Pigment composition analysis of light-harvesting complex II, which would not contain Chl a' nor Pheo a, showed the Chl a'/Chl a ratio of 3-4 x 10(-4) and the Pheo a/Chl a ratio of 4-5 x 10(-4), showing that the conditions developed here were sufficiently inert for Chl analysis. Preliminary analysis of thylakoid membranes with this analytical system gave the PhQ/Chl a' ratio of 0.58 +/- 0.03 (n = 4), in line with the stoichiometry of one molecule of Chl a' per PS I.     

DETERMINATION OF THE KINETICS OF THE BACK REACTION OF PHOTOSYSTEM II IN THE PRESENCE OF 3–(3,4-DICHLORPHENYL)-1,1-DIMETHYLUREA FROM LUMINESCENCE MEASUREMENTS

Abstract— An alternative method to that used by Mar and Roy (1974) for the determination of the kinetics of the back reaction of photosystem II from the luminescence decay curve in the presence of 3–(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) has been suggested. The new theory relies upon two hypotheses: the well-known recombination hypothesis of luminescence and the assumption that the luminescence yield in the seconds region is given by the variable part of the live fluorescence yield. The second hypothesis was introduced since assuming a constant luminescence yield results in kinetic data that are not consistent with measurements of the kinetics of the back reaction by the restoration of the area over the fluorescence rise curve. The dependence of the live fluorescence yield from the concentration of closed PS II traps was assumed to be represented by Delosme's expression originally derived for the rise curve of the fluorescence yield in the presence of DCMU.
The theory is based on the fact that then the partial and total light sums of luminescence are simple functions of the concentration of the primary electron acceptor Q^- of PS II. Thus, after integrating the luminescence decay curve the theory permits a convenient evaluation of the kinetics of the back reaction [Q^-]( t ) in terms of the partial and total light sum.
This method was applied in order to determine the kinetics of the back reaction in Chlorella fusca in the presence of DCMU. It is shown that the kinetics of deactivation of the S, state can be described using the expression for the kinetics of the back reaction derived by Mar and Roy. As an alternative explanation, a biphasic first order decay of S₂ is proposed.     

AFFINITY FOR OXYGEN IN PHOTOREDUCTION OF MOLECULAR OXYGEN AND SCAVENGING OF HYDROGEN PEROXIDE IN SPINACH CHLOROPLASTS

Abstract— The apparent K _m for O₂ in the photoreduction of molecular oxygen by spinach class II chloroplasts and photosystem I subchloroplast fragments was determined. In both cases, a value of 2 ∼ 3 μ M O₂ was obtained. The reaction rate constant between O₂ and P-430, the primary electron acceptor of PS I, is estimated to be ∼ 1.5 × 10⁷ M ^-1 s^-1 and the factors affecting the production of superoxide by the photoreduction of O₂ in chloroplasts are discussed. Preliminary evidence is presented indicating the occurrence of an azide-insensitive scavenging system for H₂O₂ in chloroplast stroma.     

Reversed-phase HPLC determination of chlorophyll a' and naphthoquinones in photosystem I of red algae: existence of two menaquinone-4 molecules in photosystem I of Cyanidium caldarium.

Chlorophyll (Chl) a', the C13(2)-epimer of Chl a, is one of the two Chl molecules constituting the primary electron donor (P700) of photosystem (PS) I of a thermophilic cyanobacterium Synechococcus elongatus. To examine whether PS I of other oxygenic photosynthetic organisms in general contain one Chl a' molecule in P700, the pigment composition of thylakoid membranes and PS I preparations isolated from red algae Porphyridium purpureum and Cyanidium caldarium was examined by reversed-phase HPLC with particular attention to Chl a' and phylloquinone (PhQ), the secondary electron acceptor of PS I. The two red algae contained one Chl a' molecule at the core part of PS I. In PS I of C. caldarium, two menaquinone-4 (MQ-4) molecules were detected in place of PhQ used by higher plants and cyanobacteria. The 1:2:1 stoichiometry among Chl a', PhQ (MQ-4) and P700 in PS I of the red algae indicates that one Chl a' molecule universally exists in PS I of oxygenic photosynthetic organisms, and two MQ-4 molecules are associated with PS I of C. caldarium.     

The action of oxygen on chlorophyll fluorescence quenching and absorption spectra in pea thylakoid membranes under the steady-state conditions

The effect of oxygen concentration on both absorption and chlorophyll fluorescence spectra was investigated in isolated pea thylakoids at weak actinic light under the steady-state conditions. Upon the rise of oxygen concentration from anaerobiosis up to 412 microM a gradual absorbance increase around both 437 and 670 nm was observed, suggesting the disaggregation of LHCII and destacking of thylakoids. Simultaneously, an increase in oxygen concentration resulted in a decline in the Chl fluorescence at 680 nm to about 60% of the initial value. The plot of normalized Chl fluorescence quenching, F(-O(2))/F(+O(2)), showed discontinuity above 275 microM O(2), revealing two phases of quenching, at both lower and higher oxygen concentrations. The inhibition of photosystem II by DCMU or atrazine as well as that of cyt b(6)f by myxothiazol attenuated the oxygen-induced quenching events observed above 275 microM O(2), but did not modify the first phase of oxygen action. These data imply that the oxygen mediated Chl fluorescence quenching is partially independent on non-cyclic electron flow. The second phase of oxygen-induced decline in Chl fluorescence is diminished in thylakoids with poisoned PSII and cyt b(6)f activities and treated with rotenone or N-ethylmaleimide to inhibit NAD(P)H-plastoquinone dehydrogenase. The data suggest that under weak light and high oxygen concentration the Chl fluorescence quenching results from interactions between oxygen and PSI, cyt b(6)f and Ndh. On the contrary, inhibition of non-cyclic electron flow by antimycin A or uncoupling of thylakoids by carbonyl cyanide m-chlorophenyl hydrazone did not modify the steady-state oxygen effect on Chl fluorescence quenching. The addition of NADH protected thylakoids against oxygen-induced Chl fluorescence quenching, whereas in the presence of exogenic duroquinone the decrease in Chl fluorescence to one half of the initial level did not result from the oxygen effect, probably due to oxygen action as a weak electron acceptor from PQ pool and an insufficient non-photochemical quencher. The data indicate that mechanism of oxygen-induced Chl fluorescence quenching depends significantly on oxygen concentration and is related to both structural rearrangement of thylakoids and the direct oxygen reduction by photosynthetic complexes.     

QUANTITATION OF PHOTOSYSTEM II ACTIVITY IN SPINACH CHLOROPLASTS. EFFECT OF ARTIFICIAL QUINONE ACCEPTORS

Quantitation of photosystem II (PSII) activity in spinach chloroplasts is presented. Rates of PSII electron-transport were estimated from the concentration of PSII reaction-centers (Chl/PSII = 380:1 when measured spectrophotometrically in the ultraviolet [ΔA₃₂₀] and green [ΔA_540–550] regions of the spectrum) and from the rate of light utilization by PSII under limiting excitation conditions. Rates of PSII electron-transport were measured under the same light-limiting conditions using 2,5-dimethylbenzoquinone or 2,5-dichlorobenzoquinone as the PSII artificial electron acceptors. Evaluation is presented on the limitations imposed in the measurement of PSII electron flow to artificial quinones in chloroplasts. Limitations include the static quenching of excitation energy in the pigment bed by added quinones, the fraction of PSII centers (PSII_β) with low affinity to native and added quinones, and the loss of reducing equivalents to molecular oxygen. Such artifacts lowered the yield of steady-state electron transport in isolated chloroplasts and caused underestimation of PSII electron-transport capacity. The limitations described could explain the low PSII concentration estimates in higher plant chloroplasts (Chl/PSII = 600 ± 50) resulting from proton flash yield and/or oxygen flash-yield measurements. It is implied that quantitation of PSII by repetitive flash-yield methods requires assessment of the slow turnover of electrons by PSII_β and, in the presence of added quinones, assessment of the PSII quantum yield.     

LIGHT-INDUCED QUENCHING OF PHOTOSYSTEM II FLUORESCENCE AT 77 K

Abstract— Light-induced quenching of the low temperature fluorescence emission from photosystem II (PS II) at 695 nm ( F ₆₉₅) has been observed in chloroplasts and whole leaves of spinach. Photosystem I (PS I) fluorescence emission at 735 nm ( F ₇₃₅) is quenched to a lesser degree but this quenching is thought to originate from PS II and is manifest in a reduced amount of excitation energy available for spillover to PS I. Differential quenching of these two fluorescence emissions leads to an increase in the F ₇₃₅/ F ₆₈₅ ratio on exposure to light at 77 K. Rewarming the sample from -196°C discharges the thermoluminescence Z-band and much of the original unquenched fluorescence is recovered. The relationship between the thermoluminescence Z-band and the quenching of the low temperature fluorescence emission ( F ₆₉₅) is discussed with respect to the formation of reduced pheophytin in the PS II reaction center at 77 K.     

EVIDENCE FOR A HETEROGENOUS ORGANIZATION OF VIOLAXANTHIN IN THYLAKOID MEMBRANES

Abstract Two functionally different species of violaxanthin have been observed in thylakoid membranes, one that can be de-epoxidised to zeaxanthin under light and one not available for light-induced zeaxanthin formation (Siefermann, D. and H. Y. Yamamoto, 1974, Biochim. Biophys. Acta 357 , 144–150). Here the distribution of available and unavailable violaxanthin is examined between membrane subfractions obtained from Triton X-100 solubilized spinach thylakoids by isoelectric focusing: (1) Only 40% of the available violaxanthin is detected in isolated Chl-proteins, while the residual 60% occur in a fraction of'free'pigments; (2) Almost 80% of the unavailable violaxanthin is recovered from the light-harvesting Chl a/b -protein complex (36%) and from photochemically active complexes containing photosystem I (20%) or photosystem II (20%). The results suggest a heterogenous organization of available and unavailable violaxanthin in thylakoid membranes.     

INFLUENCE OF RUTHENIUM, RHODIUM AND VANADIUM IONS ON PHOTOPRODUCTION OF HYDROGEN BY Chlorella fusca

Abstract Ru³⁺ and Rh³⁺ supplied at a concentration of 1 x 10⁻⁵ mol/l in the nutrient medium are incorporated preferably by the chloroplasts of Chlorella fusca cells as shown by electron micrographs without using any contrasting agent. Both elements cause a significant increase of production rate as well as of total hydrogen production. In experiments with synchronized C. fusca the maturing cells yielded maximal hydrogen production. Inhibition of hydrogen photoproduction by DCMU indicates that both photosystems are involved. Ru(bipy)3²⁺, an essential component of photocatalysts, also is incorporated to a great extent and preferably bound to the thylakoid membranes of the chloroplasts but shows a negative influence on hydrogen production. V³⁺ is incorporated only to a small extent without any enrichment to the chloroplasts and shows no effect on hydrogen production.     

ON THE FORMATION OF PHOTOSYNTHETIC MEMBRANES IN BEAN PLANTS*

Abstract— The formation of lamellar chlorophyll-protein complexes I and II, solubilized by sodium dodecyl sulfate, was studied by hydroxylapatite column chromatography during greening of etiolated Phaseohis vulgaris leaves.
The protein moiety of both complexes preexists in the prolamellar body of etiolated tissue. The complex II to complex I protein ratio is of the order of 0.5. During greening in intermittent illumination the 'proto'-chloroplast is agranal, and contains 'primary' thylakoids and chlorophyll a (Chl a ). At this stage the complex II to complex I protein ratio increases only slightly. Further greening of the plant tissue in continuous illumination results in grana, Chi b (chlorophyll b ) and more Chl a formation. The complex II to complex I protein ratio in unfractionated thylakoids is now of the order of 2.5, while in grana it is of the order of 4.0.
The binding of chlorophyll formed during greening to the protein moiety of the two complexes is found to be selective. The Chi a selectively formed under intermittent illumination is more strongly bound to the complex I protein. The Chi b and Chl a formed in continuous illunination are found bound to both complex I and complex II proteins.
Analysis by hydroxylapatite column chromatography of subchloroplast fractions obtained by different fractionation procedures have shown that these two chlorophyll-protein complexes are most probably derived from the PSI (photosystem I) and PSII (photosystem II) particles of the photosynthetic membrane. These findings suggest that PSI units are assembled ahead of PSII units. Moreover, they indicate that the complex I protein is the main protein component in the prolamellar body membranes, the 'primary' thylakoids. and the stroma lamellae, while in the grana membranes the major protein is the complex II protein. Finally our results show that formation of the photosynthetic membranes is a multi-step process.     

STOICHIOMETRY and KINETICS OF PROTON RELEASE UPON PHOTOSYNTHETIC WATER OXIDATION

Abstract— We studied the magnitude and the rise kinetics of proton release into the interior of thylakoids by flash spectrophotometty with neutral red as pH indicator. Excitation of dark-adapted thylakoids by a series of between 4 and 11 flashes produced a complex pattern of proton release into the thylakoid lumen. Proton release upon each flash was time resolved.
A slow component of proton release oscillated weakly in magnitude with period of two as function of flash number. It exhibited a half-rise time of approximately 20 ms from the very first flash on, and it was abolished by inhibitors of plastohydroquinone oxidation. This component was attributed to the oxidation of plastohydroquinone by PS I via the Cytb₆/f complex.
Additionally, rapid and multiphasic proton release was observed with half-rise times of less than 2 ms which exhibited a pronounced and damped oscillation with period of four as function of flash number. This rapid proton release was attributed to water oxidation. A detailed kinetic analysis suggested that proton release occurred with the following stoichiometry and with the following half-rise times during the transitions S₁ S_i+1 of water oxidation: 1 H⁺(250 μs, S₀→₁): 0 H⁺(S₁→ S₂):1 H⁺(200 μs, S₂→S₃):2 H⁺(1.2 ms, S₃→ S₄→ S₀) . Proton release and proton rebinding upon oxidation and reduction of the intermediate electron carrier Z, respectively, may have influenced the kinetics of the respective proton yields but not the stoichiometric pattern.     

KINETIC STUDIES ON THE STABILIZATION OF THE PRIMARY RADICAL PAIR P680+ Pheo- IN DIFFERENT PHOTOSYSTEM II PREPARATIONS FROM HIGHER PLANTS*

Abstract— The stabilization of the primary radical pair P680+ pheophytin (Pheo)^- through rapid electron transfer from Pheo to the special plastoquinone of photosystem II (PS II), Q_A, was analyzed on the basis of time-resolved (40 ps) UV-absorption changes detected in different PS II preparations from higher plants. Lifetime measurements of¹Chi* fluorescence by single photon counting and a numerical analysis of the redox reactions revealed (1) at exciton densities required for light saturation of the stable charge separation, annihilation processes dominate the excited state decay leading to very similar lifetimes of ¹Chi* in systems with open and closed reaction centers and (2) the difference of absorption changes induced by actinic flashes of comparatively high photon density in samples with open and photochemically closed reaction centers, respectively, provides a suitable measure of the rate constant of QA formation. Conclusion 2 was confirmed in PS II membrane fragments by measurements at three wavelengths (280 nm, 292 nm and 325 nm) where the difference spectrum of Q^-_A formation exhibits characteristic features. The numerical evaluation of the experimental data led to the following results: (1) the rate constant of Q^-_A formation was found to be (300 ± 100 ps)^-1 in PS II membrane fragments and PS II core complexes deprived of the distal and proximal antenna and (2) an iron depletion treatment of membrane fragments does not affect these kinetics. The implications of these results are briefly discussed in terms of the PS II reaction pattern.     

KINETIC ANALYSIS OF THE FLUORESCENCE INDUCTION IN 3-(3,4-DICHLOROPHENYL)-1,1-DIMETHYLUREA POISONED CHLOROPLASTS

Abstract— The size of the area over the fluorescence rise curve of chloroplasts is a measure of the total number of quanta utilized in photosystem II during the fluorescence induction, while the growth of the area reflects the progress of photochemical events. In the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), the growth kinetics of the area are affected by the reoxidation of the primary acceptor Q ^- with stored oxidizing charges on the donor side of system II.
At low light intensities, a slow component of this back reaction may limit the steady state fluorescence emission. At higher intensities, however, the fluorescence rise is limited solely by photochemical events, although fast thermochemical reactions like the immediate recombination of photochemically separated charges may affect the efficiency of the photochemistry.
A kinetic analysis of the area growth at moderate light intensities revealed that it occurred in two first order phases which were described by the rate constants k _α and k _β. The biphasic nature suggested a sequential two-electron reduction of the primary acceptor Q , or the presence of two different types of photochemical centers in system II. The rate constants were light intensity dependent. They also were affected by changes in pH, by an addition of NH₂OH, or by a preillumination with short flashes prior to addition of DCMU. It is suggested that the pH of the medium, the presence of NH₂OH, and the flash induced state S_n of the water splitting enzyme, control the values of k _α and k _β by changing the rate constants of electron carrier interactions in the reaction center complex, with a resulting modification of the frequency of back reactions between the primary donor and the primary acceptor.