The role of reagents accelerating the deactivation reactions of water-splitting enzyme system Y (ADRY reagents) in destabilizing high-potential oxidizing equivalents generated in chloroplast Photosystem II

A class of compounds, usually referred to as ADRY reagents, destabilize intermediates in the photosynthetic water-oxidizing process. The effects of these species on the reduction kinetics of Z^?, the oxidized donor to P-680, have been monitored in Tris-washed chloroplasts by following the decay of EPR Signal IIf. In the presence of ADRY reagents (e.g., sodium picrate, carbonyl cyanide m-chlorophenylhydrazone) this process follows an exponential time course, the decay half-time of which decreases as the ADRY reagent concentration increases. From this pseudo-first-order behavior, the second-order rate constants for four commonly used ADRY reagents have been extracted. The ADRY-induced acceleration in Z^? reduction proceeds independently of conditions imposed on the acceptor side of Photosystem II and shows no synergism with exogenous electron donors. These observations are most easily rationalized in terms of a model which proposes direct reduction of Z^? by the ADRY reagent followed by regeneration of the reduced ADRY reagent in a nonspecific reaction with membrane components such as carotenoids, chlorophyll or protein. A comparison of the second-order rate constants we obtain for ADRY reagents in their reaction with Z^? in Tris-washed chloroplasts with those obtained from the literature for the ADRY- reagent induced deactivation of states S₂ and S₃ in oxygen-evolving chloroplasts reveals a close similarity between the two processes. From this observation, a general model for the action of ADRY reagents in destabilizing the high-potential oxidizing equivalents generated in Photosystem II is proposed.     

Studies on the functional organization of Photosystem II. The effect of protein-modifying procedures and of ADRY agents on the reaction pattern of photosystem II in Tris-washed chloroplasts

The role of the protein matrix embedding the functionally active redox components of Photosystem II reaction centers has been studied by investigating the effects of procedures which modify the structure of proteins. In order to reduce the influence of the electron transport involving secondary donor and acceptor components, Triswashed chloroplasts were used which are completely deprived of their oxygen-evolving capacity. The functional activity was detected via absorption changes, reflecting at 334 and 690 or 834 nm the turnover of the primary plastoquinone acceptor, X320, and of the photochemically active chlorophyll a complex, Chl a_II, respectively, and at 520 nm the transient formation of a transmembrane electric potential gradient. Under repetitive flash excitation of Tris-washed chloroplasts it was found that: (a) The relaxation kinetics at 690 nm become significantly accelerated in the presence of external electron donors. (b) Trypsin treatment blocks to a high degree the turnover of Chl a_II and X320 unless exogenous acceptors are present, which directly oxidize X320^?, such as K₃Fe(CN)₆. (c) In the presence of K₃Fe(CN)₆ the recovery kinetics of Chl a_II and X320 are retarded markedly by trypsin, followed by a progressive decline in the extent thereof. (d) 2-(3-Chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT 2p), known to reduce the lifetime of S₂ and S₃ in normal chloroplasts, significantly accelerates the recovery of Chl a_II. 10 μs kinetics are observed which correspond with the electron-transfer rate from D₁ to Chl a⁺_II. ANT 2p simultaneously retards the decay kinetics of X320^? and of the electrochromic absorption changes. (e) The kinetic pattern of the electrochromic absorption changes is also affected by the salt content of the suspension. Under dark-adapted conditions, the 10 μs relaxation kinetics of the 834 nm absorption change due to the first flash are hardly affected by mild trypsinization of 5–10 min duration, whereas the amplitude decreases by approx. 30%. The data obtained in Tris-washed chloroplasts could consistently be interpreted as a modification of the back reaction between X320^? and Chl a⁺_II which is caused solely by a change in the reactivity of X320 due to trypsin-induced degradation of the native X320-B apoprotein. Furthermore, ADRY agents are inferred to stimulate cyclic electron flow, which leads to reduction of D⁺₁ between the flashes. A simplified scheme is discussed which describes the functional organization of the reaction center complex.     

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.     

Temperature and preillumination dependence of delayed fluorescence of spinach chloroplasts

Delayed fluorescence (luminescence) from spinach chloroplasts, induced by short saturating flashes, was studied in the temperature region between 0 and ?40 °C. At these temperatures, in contrast to what is observed at room temperature, luminescence at 40 ms after a flash was strongly dependent, with period four, on the number of preilluminating flashes (given at room temperature, before cooling). At ?35 °C luminescence of chloroplasts preilluminated with two flashes (the optimal preillumination) was about 15 times larger than that of dark-adapted chloroplasts. The intensity of luminescence obtained with preilluminated chloroplasts increased steeply below ?10 °C, presumably partly due to accumulation of reduced acceptor (Q^?), and reached a maximum at ?35 °C.In the presence of 50 mM NH₄Cl the temperature optimum was at ?15 °C; at this temperature luminescence was increased by NH₄Cl; at temperatures below ?20 °C luminescence at 40 ms was decreased by NH₄Cl. At room temperature a strongly enhanced 40-ms luminescence was observed after the third and following flashes. The results indicate that both the S₂ to S₃ and the S₃ to S₄ conversion are affected by NlH₄Cl.Inhibitors of Q^? reoxidation, like 3-(3, 4-dichlorophenyl)-1, 1- dimethylurea, did only slightly affect the preillumination dependence of luminescence at sub-zero temperatures if they were added after the preillumination. This indicates that these substances by themselves do not accelerate the deactivation of S₂ and S₃.     

Evidence from thermoluminescence for bicarbonate action on the recombination reactions involving the secondary quinone electron acceptor of Photosystem II

In this paper, we present the first measurements on thermoluminescence from isolated thylakoids to probe the recombination reactions of S₂ (or possibly S₃) with Q^?_B or Q^?_A, after bicarbonate depletion and its readdition. The effects of bicarbonate depletion on the S₂Q^?_B (or S₃O^?_B) thermoluminescence band was (1) a 6–10°C shift to a higher temperature; (2) a reduction in its intensity upon prolonged depletion; and (3) elimination after the first few flashes of the characteristic period four oscillations in its intensity as a function of the flash number. On the other hand, addition of diuron (3-(3′,4′-dichlorophenyl)-1,1-dimethylurea), which blocks electron flow from Q^?_A to Q_B, produced the same thermoluminescence band, at about + 20°C, assigned to S₂Q^?_A recombination, in both depleted and reconstituted samples. These results suggest (1) the initial effect of bicarbonate depletion is to increase the activation energy for S₂(S₃)Q^?_B recombination; (2) with further depletion, the incidence of this recombination decreases and the cycling of the S₂Q^?_B and S₃Q^?_B recombination is inhibited through effects at the Q_B apoprotein; and (3) the depletion effects are fully reversible. It is suggested that a conformational change of the PS II complex in the region of the Q_B apoprotein is responsible for these effects.     

The effect of herbicides on components of the PS II reaction centre measured by EPR

Incubation of PS II membranes with herbicides results in changes in EPR signals arising from reaction centre components. Dinoseb, a phenolic herbicide which binds to the reaction centre polypeptide, changes the width and form of the EPR signal arising from photoreduced Q^?_AFe. o-Phenanthroline slightly broadens the Q^?_AFe signal. These effects are attributed to changes in the interaction between the semi-quinone and the iron. DCMU, which binds to the 32 kDa protein, has virtually no effect on the width of the Q^?_AFe signal but does give rise to an increase in its amplitude. This could result from a change in redox state of an interacting component. Herbicide effects can also be seen when Q^?_AFe is chemically reduced and these seen to be reflected by changes in splitting and amplitude of the split pheophytin^? signal. Dinoseb also results in the loss of ‘Signal II dark’, the conversion of reduced high-potential cytochrome b₅₅₉ to its oxidized low-potential form and the presence of transiently photooxidized carotenoid after a flash at 25°C; these effects indicate that dinoseb may also act as an ADRY reagent.     

Factors influencing hydroxylamine inactivation of photosynthetic water oxidation

The kinetics of Mn release during NH₂OH inactivation of the water oxidizing reaction is largely insensitive to the S-state present during addition of NH₂OH. This appears to reflect reduction by NH₂OH of higher S-states to a common more reduced state (S₀ or S_?1) which alone is susceptible to NH₂OH inactivation. Sequences of saturating flashes with dark intervals in the range 0.2–5 s^?1 effectively prevent NH₂OH inactivation and the associated liberation of manganese. This light-induced protection disappears rapidly when the dark interval is longer than about 5 s. Under continuous illumination, protection against NH₂OH inactivation is maximally effective at intensities in the range 10³–10⁴ erg · cm^?2 · s^?1. This behavior differs from that of NH₂OH-induced Mn release, which is strongly inhibited at all intensities greater than 10³ erg · cm^?2 · s^?1. This indicates that two distinct processes are responsible for inactivation of water oxidation at high and low intensities. Higher S-states appear to be immune to the reaction by which NH₂OH liberates manganese, although the overall process of water oxidation is inactivated by NH₂OH in the presence of intense light. The light-induced protection phenomenon is abolished by 50 μM DCMU, but not by high concentrations of carbonyl cyanide m-chlorophenylhydrazone, which accelerates inactivation reactions of the water-splitting enzyme, Y (an ADRY reagent). The latter compound accelerates both inactivation of water oxidation and manganese extraction in the dark.     

Properties of the proteinaceous component acting as apoenzyme for the functional plastoquinone redox groups on the acceptor side of system II

The electron-transfer reactions between the plastoquinone molecules of the acceptor side of photosystem II have been inferred to be regulated by a proteinaceous component (apoenzyme), which additionally contains the receptor site for DCMU-type inhibitors (Renger, G., (1976) Biochim. Biophys. Acta 440, 287–300). In order to reveal the functional properties of this apoenzyme, the effect of procedures which modify the structure of proteins on the photosystem II electron transport have been investigated in isolated spinach chloroplasts by comparative measurements of O₂ evolution and absorption changes at 334 nm induced by repetitive flash excitation and of fluorescence induction curves caused by continuous actinic light. It was found that: (1) The release of blockage of O₂ evolution by the DCMU-type inhibitor SN 58132 due to mild tryptic digestion correlates kinetically with the deterioration of the binding properties. (2) Glutaraldehyde fixation of chloroplasts does not markedly modify the reoxidation kinetics of the reduced primary plastoquinone acceptor component, X320^?, of photosystem II, but it greatly reduces the fluorescence yield of the antenna chlorophylls and slightly retards the ADRY effect. Furthermore, it prevents the attack of trypsin on the apoenzyme. (3) Incubation of chloroplasts in ‘low’ salt medium markedly diminishes the ability of trypsin to release the blockage of O₂ evolution by SN 58132 and completely presents the effect on inhibition by DCMU. Based on these results and taking into account recent findings of other groups, the functional mechanism of the electron transport on the acceptor side of photosystem II is discussed. Assuming a tunnel mechanism, the apoprotein is inferred to act as a dynamic regulator rather than changing only the relative levels of the redox potentials of the plastoquinone molecules involved in the transfer steps. It is further concluded that salt depletion does not only cause grana unstacking and a change of the excitation energy transfer probabilities, but it additionally modifies the orientation of functional membrane proteins of photosystem II and their structural interaction within the thylakoid membrane.     

Effect of pH on the thermoluminescence of spinach chloroplasts in the presence and absence of Photosystem II inhibitors

The glow curve of chloroplasts excited by continuous light of high intensity (500 W · m⁻²) at pH 7.5 during cooling from +2 to −80°C consisted of seven bands appearing at about −30°C (TL₋₃₀), −15°C (TL₋₁₀), +10°C (TL₊₁₀), +30°C (TL₊₃₀), +50°C (TL₊₅₀), +65°C (TL₊₆₅) and +85°C (TL₊₈₀), in which TL stands for thermoluminescence. In the pH range from 5.5 to 9.0 the peak positions of the TL₋₃₀, TL₋₁₀, TL₊₅₀, TL₊₆₅ and TL₊₈₀ bands were independent of pH. On the other hand the peak positions of the TL₊₁₀ and TL₊₃₀ bands were gradually shifted from +25 to −5°C and from +20 to +40°C, respectively, as the pH was decreased from 9.0 to 5.5. The same pH-induced shift (from +25 to −5°C) was observed for the TL₊₁₀ band when electron transport was inhibited by DCMU. In dinoseb-treated chloroplasts the peak position of the main thermoluminescence band also exhibited pH dependency, and shifted from +20 to −20°C upon lowering the pH from 9.0 to 5.5. After the water-splitting system had been inactivated by Tris or NH₂OH treatment no pH-induced shifts were observed in the peak positions of the thermoluminescence bands of DCMU and dinoseb-treated chloroplasts. The results suggest that the effect of pH on the thermoluminescence of untreated and inhibitor-treated chloroplasts is associated with protonation/deprotonation reactions occurring at the donor and acceptor sides of Photosystem II during the S₁ → S₂ transition of the water-splitting system.     

Thermoluminescence study of charge recombination in Photosystem II at low temperatures. II. Oscillatory properties of the Zv and A thermoluminescence bands in chloroplasts dark-adapted for various time periods

The oscillations of the Z_V and A thermoluminescence bands were investigated in spinach chloroplasts which had been dark-adapted for various time periods and subjected to a series of flashes at +2°C before continuous illumination at various low temperatures. When excited with continuous light below −65°C, the Z_V band exhibited period-4 oscillation, with maxima on preflashes 0, 4 and 8. Above −65°C, the oscillation pattern depended greatly on the dark-adaptation period of the chloroplasts. In preilluminated samples (15 s light followed by 3 min dark), when the Q_B pool is half oxidized, the oscillation of the thermoluminescence intensity measured at −50°C was similar to that observed below −65°C. However, after the thorough dark-adaptation of the chloroplasts (6 h), when the major fraction of the Q_B pool is assumed to be oxidized, a binary oscillation appeared in the oscillation pattern, with maxima at odd flash numbers. Below −65°C, period-2 oscillation of the Z_V band could not be induced by the dark-adaptation of the chloroplasts, suggesting an inhibition of electron exchange between Q_A and Q_B. Upon excitation of the chloroplasts with continuous light at −30°C, the A band oscillated with a periodicity of 4 with maxima at preflash numbers 2 and 6. At pH 7.5, the period-4 oscillation was converted into a period-2 oscillation by thorough dark-adaptation of the chloroplasts (24 h). Model calculations of the oscillatory patterns suggest that the period-4 oscillations of the Z_V and A bands are determined by the concentrations [S₀] + [S₁] and [S₂] + [S₃], respectively, which are present after the preflashes prior to the low-temperature continuous illumination. The period-2 oscillations in the amplitudes of the Z_V and A bands reflect the changes occurring in the redox state of the Q_B pool in a sequence of flashes. The possible relationship between the characteristics of the Z_V and A bands and the temperature-dependence of the S state transitions was investigated. Comparison of the amplitudal changes of the B (S₂Q⁻_B and S₃Q⁻_B recombination) and Q (S₂Q⁻_A recombination) thermoluminescence bands as a function of the excitation temperature suggests that the S₂ → S₃ and S₃ → S₄ transitions are blocked at about −65 and −40°C, respectively. It is also concluded that the thermoluminescence intensity emitted by the reaction center is about twice as high in the S₃ state as in the S₂ state.     

The chloride requirement for photosynthetic oxygen evolution. Analysis of the effects of chloride and other anions on amine inhibition of the oxygen-evolving complex

In the presence of Cl^?, the severity of ammonia-induced inhibition of photosynthetic oxygen evolution is attenuated in spinach thylakoid membranes (Sandusky, P.O. and Yocum, C.F. (1983) FEBS Lett. 162, 339–343). A further examination of this phenomenon using steady-state kinetic analysis suggests that there are two sites of ammonia attack, only one of which is protected by the presence of Cl^?. In the case of Tris-induced inhibition of oxygen evolution only the Cl^? protected site is evident. In both cases the mechanism of Cl^? protection involves the binding of Cl^? in competition with the inhibitory amine. Anions (Br^? and NO^?₃) known to reactive oxygen evolution in Cl^?-depleted membranes also protect against Tris-induced inhibition, and reactivation of Cl^?-depleted membranes by Cl^? is competitively inhibited by ammonia. Inactivation of the oxygen-evolving complex by NH₂OH is impeded by Cl^?, whereas Cl^? does not affect the inhibition induced by so-called ADRY reagents. We propose that Cl^? functions in the oxygen-evolving complex as a ligand bridging manganese atoms to mediate electron transfer. This model accounts both for the well known Cl^? requirement of oxygen evolution, and for the inhibitory effects of amines on this reaction.     

Functional characterisation of a purified homogeneous Photosystem II core complex with high oxygen evolution capacity from spinach

The functional properties of a purified homogeneous spinach PS II-core complex with high oxygen evolution capacity (Haag et al. 1990a) were investigated in detail by measuring thermoluminescence and oscillation patterns of flash induced oxygen evolution and fluorescence quantum yield changes. The following results were obtained:

1. Depending on the illumination conditions the PS II-core complexes exhibit several thermoluminescence bands corresponding to the A band, Q band and Z_v band in PS II membrane fragments. The lifetime of the Q band (T_max=10°C) was determined to be 8s at T=10°C. No B band corresponding to S₂Q_B ^? or S₃Q_B ^? recombination could be detected.
2. The flash induced transient fluorescence quantum yield changes exhibit a multiphasi relaxation kinetics shich reflect the reoxidation of Q _A ^? . In control samples without exogenous acceptors this process is markedly slower than in PS II membrane fragments. The reaction becomes significantly retarded by addition of 10 μM DCMU. After dark incubation in the presence of K₃[Fe(CN)₆
3. Excitation of dark-adapted samples with a train of short saturating flashes gives rise to a typical pattern dominated by a high O₂ yield due to the third flash and a highly damped period four oscillation. The decay of redox states S₂ and S₃ are dominated by short life times of 4.3 s and 1.5 s, respectively, at 20°C.

The results of the present study reveal that in purified homogeneous PS II-core complexes with high oxygen evolution isolated from higher plants by β-dodecylmaltoside solubilization the thermodynamic properties and the kinetic parameters of the redox groups leading to electron transfer from water to Q_A are well preserved. The most obvious phenomenon is a severe modification of the Q_B binding site. The implications of this finding are discussed.     

Photo-inactivation of System II centers by carbonyl cyanide m-chlorophenylhydrazone in Chlorella pyrenoidosa

In the presence of a high concentration of carbonyl cyanide m-chlorophenylhydrazone (CCCP) (4 · 10^?6 M), the S₂ and S₃ dark decays are accelerated and become biphasic with a first half-time of 0.6 s. The first fast phase of the decays does not correspond to a simple reduction of S₂, S₃ back to S₀, S₁ (i.e. to an acceleration of the deactivation reaction), but to a decrease in the number of oxygen-evolving System II centers. This photo-inactivation produced by CCCP is rapidly reversible in the dark.     

Thermoluminescence and fluorescence study of changes in Photosystem II photochemistry in desiccating barley leaves

An effect of desiccation (a decrease of relative water content from 97% to 10% within 35 h) on Photosystem II was studied in barley leaf segments (Hordeum vulgare L. cv. Akcent) using chlorophyll a fluorescence and thermoluminescence (TL). The O-J-I-P fluorescence induction curve revealed a decrease of F_P and a slight shift of the J step to a shorter time with no change in its height. The analysis of the fluorescence decline after a saturating light flash revealed an increased portion of slow exponential components with increasing desiccation. The TL bands obtained after excitation by continuous light were situated at about –27°C (Z_v band – recombination of P680⁺Q_A ⁻), –14 °C (A band – S₃Q_A ⁻), +12 °C (B band – S_2/3Q_B ⁻) and +45 °C (C band – TyrD⁺Q_A ⁻). The bands related to the S-states of oxygen evolving complex (A and B) were reduced by desiccation and shifted to higher and lower temperatures, respectively. In accordance with this, the band observed at about +27 °C (S₂Q_B ⁻) after excitation by 1 flash fired at –10 °C and band at about +20 °C (S_2/3Q_B ⁻) after 2 flashes decreased with increasing water deficit and shifted to lower temperatures. A new band around 5 °C appeared in both regimes of TL excitation for a relative water content of under 42% and was attributed to the Q band (S₂Q_A ⁻). It is suggested that under desiccation, an inhibition of the formation of S₂- and S₃-states in OEC occurred simultaneously with a lowering of electron transport on the acceptor side of PS II. The temperature down-shift of the TL bands obtained after the flash excitation was induced at the initial phases of water stress, indicating a decrease of the activation energy for the S_2/3Q_B ⁻recombination. This revised version was published online in June 2006 with corrections to the Cover Date.     

CALB-catalyzed kinetic resolution of (RS)-3-benzoylthio-2-methylpropyl azolides: kinetic and thermodynamic analysis

Abstract

Zofenopril as an ACE inhibitor expired recently was found to have a favourable safety profile in comparison with other ACE inhibitors in treating high blood pressure, congestive heart failure, and acute myocardial infarction. It can be synthesised from the key building blocks of (S)-3-benzoylthio-2-methylpropanoic acid and (4S)-phenylthio-L-proline. In this report, an efficient hydrolytic resolution via Candida antarctic lipase B (CALB) for preparing the former block in isopropyl ether (IPE) containing (RS)-3-benzoylthio-2-methylpropyl pyrazolide (1) and water was developed. Quantitative improvements of the enzyme activity and enantioselectivity in terms of k_2SK_mS^?1?=?5.726?L h^?1 g^?1 and E?=?217 at 45?°C were found from the kinetic analysis. Insights into the CALB performance via thermodynamic analysis were then addressed and compared with those by using (RS)-3-benzoylthio-2-methylpropyl 1,2,4-triazolide (2) as the substrate. A putative thermodynamic model was moreover hypothesised for elucidating the more enthalpy reduction of 68.92-70.86?kJ mol^?1 from the acyl part of (S)-1 and (S)-2 as well as that of 23.69-25.63?kJ mol^?1 from the triad imidazolium to Ser105 and leaving 1,2,4-triazole moiety of (R)-2 and (S)-2 on stabilising the corresponding transition states.     

A Tris-induced change in the midpoint potential of Z,the donor to photosystem II,as determined by the kinetics of the back reaction

A new method of measuring the rate of the back reaction from the state Z⁺ P680 Q_A^? in Tris-washed chloroplasts is described. By using ratios of back reaction rates we demonstrate a Tris-induced change in the equilibrium between Z and P680 and attribute this change to an alteration of the midpoint potential of Z by Tris treatment. We also demonstrate that the previously observed inhibition of the back reaction by ADRY reagents can be localized at Z and understood in terms of electron donation to Z⁺ by ADRY reagents.     

Comparative EPR and thermoluminescence study of anoxic photoinhibition in Photosystem II particles

Photosystem II particles were exposed to 800 W m^–2 white light at 20 °C under anoxic conditions. The F_o level of fluorescence was considerably enhanced indicating formation of stable-reduced forms of the primary quinone electron acceptor, Q_A. The F_m level of fluorescence declined only a little. The g=1.9 and g=1.82 EPR forms characteristic of the bicarbonate-bound and bicarbonate-depleted semiquinone-iron complex, Q_A ^–Fe²⁺, respectively, exhibited differential sensitivity against photoinhibition. The large g=1.9 signal was rapidly diminished but the small g=1.82 signal decreased more slowly. The S₂-state multiline signal, the oxygen evolution and photooxidation of the high potential form of cytochrome b-559 were inhibited approximately with the same kinetics as the g=1.9 signal. The low potential form of oxidized cytochrome b-559 and Signal II_slow arising from Tyr_D ⁺ decreased considerably slower than the g=1.9 semiquinone-iron signal. The high potential form of oxidized cytochrome b-559 was diminished faster than the low potential form. Photoinhibition of the g=1.9 and g=1.82 forms of Q_A was accompanied with the appearance and gradual saturation of the spin-polarized triplet signal of P 680. The amplitude of the radical signal from photoreducible pheophytin remained constant during the 3 hour illumination period. In the thermoluminescence glow curves of particles the Q band (S₂Q_A ^– charge recombination) was almost completely abolished. To the contrary, the C band (Tyr_D ⁺Q_A ^– charge recombination) increased a little upon illumination. The EPR and thermoluminescence observations suggest that the Photosystem II reaction centers can be classified into two groups with different susceptibility against photoinhibition.Abbreviations C band thermoluminescence band associated with Tyr-D⁺Q _a ^– charge recombination - Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - EPR electron paramagnetic resonance - F_o initial fluorescence - F_m maximum fluorescence - Q band thermoluminescence band originating from S₂Q _a -charge recombination - Q _a the primary quinone electron acceptor of PS II - P 680 the primary electron donor chlorophyll of PS II - S₂ oxidation state of the water-splitting system - Phe pheophytin - TL thermoluminescence - Tyr _d redox active tyrosine-160 of the D₂ protein     

Effect of phenolic herbicides on the oxygen-evolving side of Photosystem II. Formation of the carotenoid cation

Phenolic herbicides were added to suspensions of spinach chloroplasts or to oxygen-evolving Photosystem II membranes. Flash absorption spectroscopy at 21°C around 1000 nm reveals that these chemicals lead to a flash-induced absorption increase attributed to the radical-cation of a carotenoid. The herbicides studied can be arranged in the following order of decreasing efficiency for the reported effect: i-dinoseb, bromonitrothymol, trinitrophenol, ioxynil, dinitroorthocresol, 2,4-dinitrophenol. A similar effect was not observed with atrazine, DCMU or o-phenanthroline. For a given herbicide concentration, the amount of flash-induced carotenoid cation increases sharply when the pH is lowered below 5.5. A similar effect does not take place with other molecules which induce the formation of a carotenoid cation: tetraphenylboron, FCCP, 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT-2p). The previous effects are observed in both oxygen-evolving Photosystem II and in preparations in which oxygen evolution is inhibited with alkaline Tris. In untreated material, the carotenoid cation is formed with a half-time of 10–35 μs. After Tris treatment, this half-time is a little longer at low than at high pH. These results indicate the existence of a specific site where phenolic inhibitors interact in the oxygen-evolving site of Photosystem II     

Thermoluminescence as a probe of Photosystem II photochemistry. The origin of the flash-induced glow peaks

A single flash given at − 15°C to chloroplasts results in charge separation in Photosystem II to form a stable state which, upon warming, recombines giving rise to luminescence. This recombination occurs at 25°C in untreated chloroplasts but is shifted to 0°C in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea or weak concentrations of a reducing agent. The luminescence at 0°C is attributed to recombination of the S₂Q⁻_A state while that at 25°C is attributed to recombination of S₂Q_AQ⁻_B (and S₃Q_AQ⁻_B upon further flash illumination). The identification of the thermoluminescence at 25°C is based upon the following experimental evidence: (1) illumination of chloroplasts in the presence of methyl viologen with 710 nm light before and after flash illumination has no effect on the extent or temperature of the thermoluminescence. This is taken as evidence that the plastoquinone pool is not involved in the recombination reaction. (2) Calculations of the extent of thermoluminescence expected after a number of flashes, assuming that S₂Q_AQ⁻_B and S₃Q_AQ⁻_B are the thermoluminescent reactants, give a good fit to the experimental results. (3) The effect of continuous illumination at 77 K (i.e., donation from cytochrome b-559 to Q_A and thence to Q_B or Q⁻_B) results in predictable changes in the extent of flash-induced thermoluminescence.     

Seasonal CO2 exchange patterns of developing peach (Prunus persica) fruits in response to temperature, light and CO2 concentration

CO₂ exchange rates per unit dry weight, measured in the field on attached fruits of the late-maturing Cal Red peach cultivar, at 1200 μmol photons m^?2S^?1 and in dark, and photosynthetic rates, calculated by the difference between the rates of CO₂ evolution in light and dark, declined over the growing season. Calculated photosynthetic rates per fruit increased over the season with increasing fruit dry matter, but declined in maturing fruits apparently coinciding with the loss of chlorophyll. Slight net fruit photosynthetic rates ranging from 0. 087 ± 0. 06 to 0. 003 ± 0. 05 nmol CO₂ (g dry weight)^?1 S^?1 were measured in midseason under optimal temperature (15 and 20°C) and light (1200 μmol photons m^?2 S^?1) conditions. Calculated fruit photosynthetic rates per unit dry weight increased with increasing temperatures and photon flux densities during fruit development. Dark respiration rates per unit dry weight doubled within a temperature interval of 10°C; the mean seasonal O₁₀ value was 2. 03 between 20 and 30°C. The highest photosynthetic rates were measured at 35°C throughout the growing season. Since dark respiration rates increased at high temperatures to a greater extent than CO₂ exchange rates in light, fruit photosynthesis was apparently stimulated by high internal CO₂ concentrations via CO₂ refixation. At 15°C, fruit photosynthetic rates tended to be saturated at about 600 μmol photons m^?2 S^?1. Young peach fruits responded to increasing ambient CO₂ concentrations with decreasing net CO₂ exchange rates in light, but more mature fruits did not respond to increases in ambient CO₂. Fruit CO₂ exchange rates in the dark remained fairly constant, apparently uninfluenced by ambient CO₂ concentrations during the entire growing season. Calculated fruit photosynthetic rates clearly revealed the difference in CO₂ response of young and mature peach fruits. Photosynthetic rates of younger peach fruits apparently approached saturation at 370 μl CO₂1^?2. In CO₂ free air, fruit photosynthesis was dependent on CO₂ refixation since CO₂ uptake by the fruits from the external atmosphere was not possible. The difference in photosynthetic rates between fruits in CO₂-free air and 370 μl CO₂ 1^?1 indicated that young peach fruits were apparently able to take up CO₂ from the external atmosphere. CO₂ uptake by peach fruits contributed between 28 and 16% to the fruit photosynthetic rate early in the season, whereas photosynthesis in maturing fruits was supplied entirely by CO₂ refixation.