ADRY Agent Research Articles

Chemical Inhibitors of Photosystem II

Although more and more precise and efficient methods of scientific research regularly arise, inhibitory analysis still remains far from being the least among the extensively used experimental approaches. Many current investigations aplly chemical inhibitors of photosystem II as instruments of scientific cognition. Novel chemical inhibitors are being screened, educed, and/or designed. New effects of the already known inhibitors are being discovered and/or significantly corrected. The review presents the generalized information on both the known inhibitors, such as diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea), dinoseb (6‑sec-butyl-2,4-dinitrophenol), and ADRY agents (agents causing Acceleration of the Deactivation Reactions of the water-splitting enzyme system Y) and recently revealed chemical inhibitors of PSII (derivatives of perfluoroisopropyldinitrobenzene and metal-organic complexes). The action of carbonic anhydrase inhibitors on a photochemical activity of PSII is also discussed.

The oxidation/reduction kinetics of the plastoquinone pool controls the appearance of the I-peak in the O–J–I–P chlorophyll fluorescence rise: Effects of various electron acceptors

Quantitative analysis of the fluorescence induction (FI) rise was used in this study to elucidate the complex effects of N, N, N′, N′-tetramethyl- p-phenylenediamine (TMPD) on thylakoids. Reduced TMPD molecules, responsible for the ADRY agent effect, caused an increase in the amplitude of the O–J rise. Also, only oxidized TMPD molecules were shown to have the ability to bind the Q B pocket of photosystem II (PSII). On the other hand, the I–P rise was slowed in proportion with the oxidized TMPD concentration, inducing the clear appearance of the I-peak. While this property was previously thought to be unique to TMPD, this study shows that some artificial electron acceptors of PSII, silicomolybdate, 2,5-dichloro- p-benzoquinone, and phenyl- p-benzoquinone, have a similar effect. These results demonstrated a major role of the oxido-reduction kinetics of the PQ-pool in the resolution of J–I and I–P phases in the FI of isolated thylakoids.

Interaction of N,N,N′,N′-tetramethyl- p-phenylenediamine with photosystem II as revealed by thermoluminescence: Reduction of the higher oxidation states of the Mn cluster and displacement of plastoquinone from the Q B niche

The flash-induced thermoluminescence (TL) technique was used to investigate the action of N,N,N′,N′-tetramethyl- p-phenylenediamine (TMPD) on charge recombination in photosystem II (PSII). Addition of low concentrations (μM range) of TMPD to thylakoid samples strongly decreased the yield of TL emanating from S 2Q B − and S 3Q B − (B-band), S 2Q A − (Q-band), and Y D +Q A − (C-band) charge pairs. Further, the temperature-dependent decline in the amplitude of chlorophyll fluorescence after a flash of white light was strongly retarded by TMPD when measured in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Though the period-four oscillation of the B-band emission was conserved in samples treated with TMPD, the flash-dependent yields (Y n) were strongly declined. This coincided with an upshift in the maximum yield of the B-band in the period-four oscillation to the next flash. The above characteristics were similar to the action of the ADRY agent, carbonylcyanide m-chlorophenylhydrazone (CCCP). Simulation of the B-band oscillation pattern using the integrated Joliot–Kok model of the S-state transitions and binary oscillations of Q B confirmed that TMPD decreased the initial population of PSII centers with an oxidized plastoquinone molecule in the Q B niche. It was deduced that the action of TMPD was similar to CCCP, TMPD being able to compete with plastoquinone for binding at the Q B-site and to reduce the higher S-states of the Mn cluster.

Role of Carbonyl Cyanide m-Chlorophenylhydrazone in Enhancing Photobiological Hydrogen Production by Marine Green Alga Platymonas subcordiformis

We demonstrated that a significant volume of H(2) gas could be photobiologically produced by a marine green alga Platymonas subcordiformis when an uncoupler of photophosphorylation, carbonyl cyanide m-chlorophenylhydrazone (CCCP), was added after 32 h of anaerobic dark incubation, whereas a negligible volume of H(2) gas was produced without CCCP. The role of CCCP in enhancing photobiological H(2) production was delineated. CCCP as an ADRY agent (agent accelerating the deactivation reactions of water-splitting enzyme system Y) rapidly inhibited the photosystem II (PSII) activity of P. subcordiformis cells, resulting in a markedly decline in the coupled oxygen evolution. The mitochondrial oxidative respiration was only slightly inactivated by CCCP, which depleted O(2) in the light. As a result, anaerobiosis during the stage of photobiological H(2) evolution was established, preventing severe O(2) inactivation of the reversible hydrogenase in P. subcordiformis. The uncoupling effect of CCCP accelerates electron transfer from water due to a disruption of the proton motive force and release of DeltapH across the thylakoid membrane and thus enhances the accessibility of electron and H(+) to hydrogenase. The electrons for hydrogen photoevolution are mainly from the photolysis of water (90%). Upon the addition of CCCP, Chl a/b ratio increased, which implies a decrease in the light-harvesting PSII antennae or an increase in PSII/PSI ratio, possibly resulting in higher efficiency of utilization of light energy. The enhancement of H(2) evolution by the addition of CCCP is mostly due to the combination of the above three mechanisms. However, the disruption of the proton gradient across the thylakoid membrane may prevent a sustained photobiological H(2) evolution due to a shortfall of ATP generation essential for the maintenance and repair functions of the cells.

Photoreduction of silicomolybdate in chloroplasts by agents accelerating the deactivation reactions of the water-oxidizing system

Uncouplers of photosynthetic phosphorylation, CCCP, TTFB and PCP, inhibited light-induced O 2 evolution in the Hill reaction with SiMo ( I 50 ∼20, 3 and 45 μM, respectively), but only insignificantly diminished SiMo photoreduction by pea chloroplasts. The same properties were exhibited by the ADRY agent ANT2p. CCCP, TTFB and PCP are oxidizable compounds with redox potentials of +1.17, +1.18 and +1.09 V (pH 6.0), as determined by cyclic voltammetry. Similarly to NH 2OH, the tested uncouplers can apparently serve as electron donors for photosystem II.

Interaction of carbonyl cyanide m-chlorophenylhydrazone with the photosystem II acceptor side

We show that CCCP, known as an uncoupler of photophosphorylation and an ADRY agent, inhibits FeCy photoreduction and coupled O 2 evolution by isolated chloroplasts equally (1 50 ∼ 2μM), but is practically without effect on the O 2 evolution coupled with SiMo reduction within the 0.2–10μM concentration range. CCCP has no effect on the nanosecond chlorophyll fluorescence in chloroplasts incubated at low light intensity, but decreases it at high light intensity. The electron transfer from reduced TMPD or duroquinol to methylviologen is resistant to CCCP. The efficiency of the CCCP inhibitory action on the FeCy photoreduction depends on the rate of electron flow, which is controlled by the light intensity. The data obtained show that CCCP is oxidized by the photosystem II donor side and is reduced by Q p, competing for electrons with FeCy and the cytochrome blf complex.

Studies on the electron transfer from Tyr-161 of polypeptide D-1 to P680+ in PS II membrane fragments from spinach

The functional connection between redox component Y z identified as Tyr-161 of polypeptide D-1 (Debus et al. 1988) and P680(+) was analyzed by measurements of laser flash induced absorption changes at 830 nm in PS II membrane fragments from spinach. It was found that neither DCMU nor the ADRY agent 2-(3-chloro-4-trifluoromethyl) anilino-3,5-dinitrothiophene (ANT 2p) affects the rate of P680(+) reduction by Y z under conditions where the catalytic site of water oxidation stays in the redox state S1. In contrast to that, a drastic retardation is observed after mild trypsin treatment at pH=6.0. This effect which is stimualted by flash illumination can be largely reversed by Ca(2+). The above mentioned data lead to the following conclusions: (a) the segment of polypeptide D-1 containing Tyr-161 and coordination sites of P680 is not allosterically affected by structural changes due to DCMU binding at the QB-site which is also located in D-1. (b) ANT 2p as a strong protonophoric uncoupler and ADRY agent does not modify the reaction coordinate of P680(+) reduction by Y z , and (c) Ca(2+) could play a functional role for the electronic and vibrational coupling between the redox groups Y z and P680. The electron transport from Y z to P680(+) is discussed within the framework of a nonadiabatic process. Based on thermodynamic considerations the reorganization energy is estimated to be in the order of 0.5 V.

Studies on the reaction mechanism of tetraphenylboron at the Photosystem II donor side in isolated spinach chloroplasts

In the present study the effect of low tetraphenylboron (TPB −) concentrations on the flash-induced oxygen yield pattern in dark-adapted spinach chloroplasts has been analysed. The following was found. (1) TPB − at a concentration of less than ten molecules per Photosystem II strongly damps the characteristic oscillation pattern. (2) TPB − induces an accelerated decay of S 2 and S 3 that is concentration dependent. In contrast to the acceleration of the deactivation reactions of the water-splitting enzyme system Y agent (ADRY agent) 2-(3-chloro-4-trifluoromethyl)-anilino-3,5-dinitrothiophene (ANT 2p) the acceleration by TPB − disappears after preflashing. (3) TPB − does not induce a bivalent reduction of and S 3 of the type S i+2 + TPB − → S 1 + oxidation products. (4) The effect of TPB − on the decay kinetics of S 2 and S 3 can be quantitatively described by a mobile-type mechanism as discussed recently for ANT 2p (Hanssum B., Dohnt G. and Renger, G. (1985) Biochim. Biophys. Acta 806, 210–220) if one additionally accounts for the irreversible oxidative consumption of TPB −. (5) Cyclic voltammetry in buffer solution reveals that TPB − becomes irreversibly oxidized with redox potential of the order of +0.7 and +1.0 V, respectively. Similar measurements with different 2-anilino-3,5-dinitrothiophene derivatives support the idea that also ANT 2p becomes irreversibly oxidized. Based on these data the mechanism of TPB − and ADRY agents on the donor side of system II is discussed.

On the mechanism of ADRY agent interaction with the Photosystem II donor side

The effect of 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT 2p), known to be the most powerful compound among ADRY agents (Renger, G. (1972) Biochim. Biophys. Acta 256, 42–439), on the decay kinetics of the redox states S 2 and S 3 of the water-oxidizing enzyme system and on the donor component D 1 in Tris-washed chloroplasts has been investigated. The following was found. (a) In isolated spinach chloroplasts without ANT 2p, the decay of S 3 and S 2 can be completely described by a sequence of one-electron-transfer steps: S 3→S 2→S 1. (b) In the dark state, S 0 is slowly auto-oxidizable with a half-life of more than 5 min in isolated spinach chloroplasts without ANT 2p. (c) Only one ANT 2p molecule per 40 reaction centers of system II is sufficient to induce a significant acceleration of the S 2 and S 3 decay. (d) The dependence on ANT 2p concentration of S 2- and S 3-decay rates can be consistently described by a simple kinetic scheme, assuming that ANT 2p acts as a very mobile catalyst within the thylakoid membrane reacting with the water-oxidizing enzyme system independent of its redox state forming a transient complex. The S 2 and S 3 decay induced by ANT 2p occurs via a one-electron-transfer sequence in normal chloroplasts. (e) The numerical evaluation of the scheme reveals a high ANT 2p affinity for the water-oxidizing enzyme system, with a dissociation constant of the order of 1 · 10 −7 M −1. This value closely corresponds with an estimation based on thermoluminescence measurements (Renger, G. and Inoue, Y. (1983) Biochim. Biophys. Acta 725, 146−154). The kinetic parameters also suggest a rapid exchange of ANT 2p between the binding sites. (f) Fluorescence measurements obtained in normal chloroplasts in the presence of DCMU led to rate constants for the S 2 decay which correspond to values derived from oxygen yield data. The ANT-2p-induced reduction of D OX 1, the donor to P-680 +, was inferred to occur with a larger rate constant than the S 2 decay. Based on these data, ANT 2p is confirmed to be the most efficient reactant with the water-oxidizing enzyme system known so far which allows a very specific kinetic labeling of redox states S 2 and S 3.

Studies on the mechanism of ADRY agents (agents accelerating the deactivation reactions of water-splitting enzyme system Y) on thermoluminescence emission

The effect of 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT-2p), known to be the most powerful ADRY agent (Renger, G. (1972) Biochim. Biophys. Acta 256, 428–439), on thermoluminescence has been investigated. Two thermoluminescence bands were analyzed: (a) the emission peaking at about 20–30°C caused by warming up of untreated chloroplasts, illuminated with a single 5 μs flash at room temperature and frozen rapidly to 77 K; and (b) the band emitted in the range of −10 up 10°C after warming of chloroplast suspensions containing 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) which were illuminated with a single 5 μs flash at −15°C and frozen rapidly at 77 K. These bands were attributed to the recombination of the B −S 2(S 3) and X-320 −S 2 states, respectively (Rutherford, A.W., Crofts, A.R. and Inoue, Y. (1982) Biochim. Biophys. Acta 682, 457–465). It was found that: (1) The B −S 2(S 3) band is markedly diminished at very low ANT-2p concentrations of less than one molecule per 2000 chlorophylls. (2) The inhibition of the X-320 −S 2 band requires significantly higher concentrations of ANT-2p (50% peak reduction at one ANT-2p molecule per 100 chlorophylls). (3) Preflashing at room temperature before cooling to −15°C diminishes the X-320 −S 2 band significantly in the presence of ANT-2p, while almost no effect is observed in its absence. (4) The state X-320 −S 2 decays monoexponentially with a half-lifetime of 2 min at −15°C in the absence of ANT-2p. In the presence of one ANT-2p molecule per 800 chlorophylls the decay becomes biphasic with half-lifetimes of 0.5 and 2 min and an amplitude ratio of 2:3, respectively. The results obtained can be explained consistently by the function of ANT-2p as an ADRY agent acting as a mobile species within the thylakoid membrane at room temperature. At subzero temperatures, a ‘fixed-place’ mechanism appears to be operative. The implications for the ADRY effect and thermoluminescence are discussed.

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 K 3Fe(CN) 6. (c) In the presence of K 3Fe(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 S 2 and S 3 in normal chloroplasts, significantly accelerates the recovery of Chl a II. 10 μs kinetics are observed which correspond with the electron-transfer rate from D 1 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.

Gel electrophoresis of chloroplast membranes of mutants of Chlamydomonas reinhardii which have impaired Photosystem II function and lack photosynthetic cytochromes

Photosystem (PS) II-enriched particles or chloroplast fragments of the wild type and of three nonphotosynthetic mutants of Chlamydomonas reinhardii, which lack chloroplast cytochromes, were analyzed by lithium dodecyl sulfate polyacrylamide gel electrophoresis at 4°C to locate which chlorophyll complexes and which proteins are associated with cytochrome b-559. Two mutants, Fl 39 and Fl 50, have previously been shown to contain, respectively, 3.6- and 2.7-times less hydroquinone-reducible high-potential cytochrome b-559 than the wild type. They have impaired PS II functions. In the presence of ADRY agents: carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT 2p) or 2-(3,4,5-trichloro)-anilino-3,5-dinitrothiophene (ANT 2s), Fl 50 carried out photo-oxidation of cytochrome b-559 with half the amplitude of that of the wild type. No photo-oxidation was observed with Fl 39. We show here that in both these mutants chlorophyll-protein complexes CP III, CP IV and CP V were missing. There were traces of the corresponding apoproteins (45 000, 42 000 and 33 000 daltons, respectively) in Fl 50 but none in Fl 39. In addition, a 19 000 dalton protein was missing in Fl 39 and present in a very small amount in Fl 50. In another mutant, Fl 9, previously characterized as lacking both cytochromes b-563 and c-553 with a normal cytochrome b-559 content, CP III-CP V and the 19 000 dalton protein were detected. CP I (110 000 daltons) and CP II (24 000 daltons) were present in all strains. These observations confirmed the close relationship between deficiencies in cytochrome b-559, lack of CP III and CP IV and anomalies in the photochemistry of PS II. They provided additional evidence that CP V and a 19 000 dalton protein are also involved in this PS II photochemistry. Staining of the gels with 3,3′,5,5′-tetramethylbenzidine and H 2O 2 allowed us to distinguish clearly four heme protein bands having peroxidase activity. Three of these bands (45 000, 42 000 and 19 000 daltons), which were shown in wild-type, Fl 39 and Fl 50 preparations but not in Fl 9, appeared related to cytochromes b-563 and c-553. The fourth heme protein (14 000 daltons) occurred in wild type and Fl 9 but was missing in Fl 39 and Fl 50; it appeared related to cytochrome b-559.

Studies on the mechanism of destabilization of the positive charges trapped in the photosynthetic water-splitting enzyme system Y by a deactivation-accelerating agent.

Abstract The mechanism of the 2-(3,4,5-trichloro)anilino-3,5 dinitrothiophene (ANT 2S)-induced cyclic electron flow leading to the discharge of the higher-trapped-hole accumulation states S2 and S3 in the photosynthetic water-splitting enzyme system Y of chloroplasts has been investigated. It was found: 1. 1. Under normal conditions the ANT 2s-catalyzed cycle includes both light reactions. 2. 2. By selective kinetical inhibition of the electron flow through P700—either by histone treatment or by 2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone blockage—the ANT 2s-induced deactivation of S2 and S3 is not significantly changed. Hence, System I activity is not a functional prerequisite for the ANT 2s-catalyzed discharge of S2 and S3. 3. 3. The reciprocal half time of the ANT 2s-induced decay of the relative average oxygen yield per flash, as a function of the time td between the flashes representing the degree of the Acceleration of the Deactivation Reactions of the water-splitting enzyme system (ADRY) effect, is nearly linearly related to the ANT 2s concentration within the range of 10−7–10−6 M. 4. 4. In respect to the mode of action of ANT 2s two different types of mechanism have been discussed: fixed-place mechanism and mobile-catalyst mechanism. 5. 5. Based on the experimental data the conclusion has been drawn that the ADRY agent ANT 2s probably acts as a mobile catalyst.

The modification of the trapping properties within the photosynthetic watersplitting enzyme system Y.

The modification of the trapping properties for oxidizing equivalents (holes) within the watersplitting enzyme system Y by chemicals (ADRY agents) has been investigated. It is shown: (1) The ADRY agents decrease significantly the storage life time of the higher trapped hole accumulation states. (2) The acidic NH-or OH-group is a functional indispensable element for substances to be able to act as ADRY agents. (3) The trapping efficiency of the watersplitting enzyme system Y for holes is not correlated with the transmembrane electrochemical gradient. (4) As electron donor for the discharge of the trapped holes acts an andogeneous carrier of the electron transport chain located either between the photosystems I and II respectively or on the reducing side of system I. A special electron donor is not required for the ADRY effect.

Requirement of an acidic proton in substances which act as accelerators of the deactivation reactions in the water-splitting enzyme system of photosynthesis

Recently it was found [1,2], that the positive charges generated by photosystem II, which are responsible for water oxidation (these charges are designated as trapped holes, cf. [3] ), can be destabilized by a class of substances, designated ADRYagents* [4]. The molecular mechanism of the ADRY effect remains to be elucidated, but in the last year results were obtained leading to the conclusion that the acidic NH-group, present in all ADRY-agents so far known, is an important functional element [4]. In this paper it will be shown that substances which contain an acidic OH-group instead of an acidic NH. group, can act as ADRY agents. Furthermore, it was found that the N-methylderivatives of the ADRY agents ANT 2a and ANT 2p do not act as ADRY substances. From these results it is concluded that the presence of an acidic group in the

The action of 2-anilinothiophenes as accelerators of the deactivation reactions in the watersplitting enzyme system of photosynthesis

The effect of a number of 2-anilinothiophene derivates on the O 2 yield per flash obtained by excitation with repetitive flashes and repetitive double flash groups has been investigated. It was found that: 1. 1. The decrease of the average O 2 yield per flash at long times ( t d) between the flashes caused by the natural deactivation reactions of the watersplitting enzyme system is strongly accelerated by various derivatives of 2-anilinothiophene. 2. 2. The action of these agents as Acceleration of the Deactivation Reactions of the watersplitting Enzyme System Y (ADRY) substances is unequivocally proved. 3. 3. The ADRY effect is dependent on the acidity of the imino group, present in all ADRY agents so far known. These observations are discussed in connection with the mechanism of the activation—deactivation reactions of the watersplitting Enzyme System Y. A new class of photosynthesis effectors is established: the class of the ADRY agents. These substances provide a tool both to label and to modify the oxidizing equivalents which are responsible for the oxidation of water, in a similar way as the uncouplers can be used for the characterization of the high energy state in the phosphorylation process.