Plastoquinone Analogs Research Articles

Contrasted Effects of Inhibitors of Cytochromeb6f Complex on State Transitions inChlamydomonas reinhardtii: THE ROLE OF Qo SITE OCCUPANCY IN LHCII KINASE ACTIVATION

We have investigated the relationship between the occupancy of the Q(o) site in the cytochrome b(6)f complex and the activation of the LHCII protein kinase that controls state transitions. To this aim, fluorescence emission and LHCII phosphorylation patterns were studied in whole cells of Chlamydomonas reinhardtii treated with different plastoquinone analogues. The analysis of fluorescence induction at room temperature indicates that stigmatellin consistently prevented transition to State 2, whereas 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone behaved as an inhibitor of state transitions only after the cells were preilluminated. The same effects were observed on the phosphorylation patterns of the LHCII proteins, while subunit V of the cytochrome b(6)f complex showed a different behavior. These findings are discussed on the basis of a dynamic structural model of cytochrome b(6)f that relates the activation of the LHCII kinase to the occupancy of the Q(o) site and the movement of the Rieske protein.

Chloroplast thylakoid protein phosphorylation is influenced by mutations in the cytochrome bf complex

The effects were studied of the plastoquinone analogs 2,5-dibromo-3-methyl-6-isopropyl- p-benzoquinone (DBMIB) and the 1,3-dinitrophenylether of iodonitrothymol (DNP-INT) on thylakoid protein phosphorylation activated either by light or by reduction in the presence of duroquinol or reduced ferredoxin. Light-activated phosphorylation in the presence of methyl viologen was increased by 0.5–1.0 μM DBMIB or DNP-INT as the reoxidation of plastoquinone and net linear electron flow were inhibited. The phosphorylations of the light-harvesting complex of Photosystem II, of a 32 kDa polypeptide and of the kinase itself (autophosphorylation) were progressively and selectively inhibited at tenfold higher concentrations of either inhibitory analog. Ascorbate, but not duroquinol or reduced ferredoxin, potentiated these selective inhibitions. If the membranes were reductively activated in the dark, however, DBMIB or DNP-INT progressively inhibited the phosphorylation of all thylakoid proteins ( I 50 = 10–15 μM). These observations appear to preclude a simple and direct regulation of protein kinase activity by the cytochrome bf complex at its plastoquinol oxidation (Q Z ) site. Mutants of Zea and Lemna lacking the cytochrome bf complex were tested for the ability to phosphorylate thylakoid proteins. In both cases, the redox-sensitive phosphorylation of light-harvesting chlorophyll a b protein complex of Photosystem II (LHC-II) was abolished, whereas other PS II peptides were phosphorylated as in the wild types. Immune blot analysis showed that this lesion was not due to the absence of the 64-kDa protein kinase. Nor did the mutants possess defective LHC-II, as shown by its utilization as a phosphorylation substrate in a heterogeneous reconstitution assay.

Direct interaction between yeast NADH-ubiquinone oxidoreductase, succinate-ubiquinone oxidoreductase, and ubiquinol-cytochrome c oxidoreductase in the reduction of exogenous quinones.

The reduction of the following exogenous quinones by succinate and NADH was studied in mitochondria isolated from both wild type and ubiquinone (Q)-deficient strains of yeast: ubiquinone-0 (Q0), ubiquinone-1 (Q1), ubiquinone-2 (Q2), and its decyl analogue 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (DB), duroquinone (DQ), menadione (MQ), vitamin K1 (2-methyl-3-phytyl-1,4-naphthoquinone), the plastoquinone analogue 2,3,6-trimethyl-1,4-benzoquinone (PQOc1), plastoquinone-2 (PQ2), and its decyl analogue (2,3-dimethyl-6-decyl-1,4-benzoquinone). Reduction of the small quinones DQ, Q0, Q1, and PQOc1 by NADH occurred in both wild type and Q-deficient mitochondria in a reaction inhibited more than 50% by myxothiazol and less than 20% by antimycin. The reduction of these small quinones by succinate also occurred in wild type mitochondria in a reaction inhibited more than 50% by antimycin but did not occur in Q-deficient mitochondria suggesting that endogenous Q6 is involved in their reduction. In addition, the inhibitory effects of antimycin and myxothiazol, specific inhibitors of the cytochrome b-c1 complex, on the reduction of these small quinones suggest the involvement of this complex in the electron transfer reaction. By contrast, the reduction of Q2 and DB by succinate was insensitive to inhibitors and by NADH was 20-30% inhibited by myxothiazol suggesting that these analogues are directly reduced by the primary dehydrogenases. The dependence of the sensitivity to the inhibitors on the substrate used suggests that succinate-ubiquinone oxidoreductase interacts specifically with center i (the antimycin-sensitive site) and NADH ubiquinone oxidoreductase preferentially with center o (the myxothiazol-sensitive site) of the cytochrome b-c1 complex. The NADH dehydrogenase involved in the myxothiazol-sensitive quinone reduction faces the matrix side of the inner membrane suggesting that center o may be localized within the membrane at a similar depth as center i.

The effect of ring substituents on the mechanism of interaction of exogenous quinones with the mitochondrial respiratory chain

In uncoupled pig-heart mitochondria the rate of the reduction of duroquinone by succinate in the presence of cyanide is inhibited by about 50% by antimycin. This inhibition approaches completion when myxothiazol is also added or British anti-Lewisite-treated (BAL-treated) mitochondria are used. If mitochondria are replaced by isolated succinate:cytochrome c oxidoreductase, the inhibition by antimycin alone is complete. The reduction of a plastoquinone homologue with an isoprenoid side chain (plastoquinone-2) is strongly inhibited by antimycin with either mitochondria or succinate:cytochrome c reductase. The reduction by succinate of plastoquinone analogues with an n-alkyl side chain in the presence of mitochondria is inhibited neither by antimycin nor by myxothiazol, but is sensitive to the combined use of these two inhibitors. On the other hand, the reduction of the ubiquinone homologues Q 2, Q 4, Q 6 and Q 10 and an analogue, 2,3-dimethoxyl-5- n-decyl-6-methyl-1,4-benzoquinone, is not sensitive to any inhibitor of QH 2:cytochrome c reductase tested or their combined use, either in normal or BAL-treated mitochondria or in isolated succinate:cytochrome c reductase. It is concluded that quinones with a ubiquinone ring can be reduced directly by succinate:Q reductase, whereas those with a plastoquinone ring can not. Reduction of the latter compounds requires participation of either center i or center o (Mitchell, P. (1975) FEBS Lett. 56, 1–6) or both, of QH 2:cytochrome c oxidoreductase. It is proposed that a saturated side chain promotes, while an isoprenoid side chain prevents reduction of these compounds at center o.

Inhibition by plastoquinone analogues of ferricyanide reduction by a Photosystem II chloroplast preparation

There is wide agreement that inhibition of plastoquinone function by plastoquinone analogues, of which the best known is dibromothymoquinone (DBMIB), inhibits electron transport from Photosystem II (PS II) to Photosystem I (PS I), but does not inhibit electron transport that is dependent on PS II alone. In contrast, we have recently presented evidence in support of a hypothesis for a hitherto unrecognized additional role of plastoquinone in the conductance of protons derived from the photooxidation of water by PS II (Arnon, D.I. and Tang, G.M.-S. (1985) Biochim. Biophys. Acta 809, 167–172). Since the transport of water-derived electrons and protons is coupled, this hypothesis predicts that photosynthetic electron transport by PS II alone would still be sensitive to DBMIB inhibition. We now report that this prediction has been verified by obtaining inhibition by low concentrations of DBMIB of electron transport from water to ferricyanide in a PS II preparation depleted of plastocyanin and PS I. The preparation consisted of inside-out vesicles isolated by the aqueous polymer two-phase partition method. The pattern of DBMIB inhibition in the PS II preparation was the same as that in unfractionated chloroplasts: inhibition of ferricyanide reduction was relieved by the addition of catalytic amounts of one of several lipophilic acceptors that appear to bypass the site of DBMIB inhibition at the Rieske FeS center of the cytochrome f b 6 complex. The bearing of these findings on the role of plastoquinones in the conductance of protons released by the photooxidation of water is discussed.

Uncouplers enhance photosynthetic electron transport from water to NADP + in the presence of plastoquinone inhibitors

Uncouplers have been previously observed to relieve appreciably the inhibition of photosynthetic electron transport from water to NADP + by the plastoquinone analogues, dibromothymoquinone (DBMIB) and dinitrophenyl ether of iodonitrothymol (DNP-INT). These results were now extended by demonstrating that the reversal by uncouplers of DBMIB and DNP-INT inhibition occurred under conditions when the uncouplers did not stimulate or inhibit NADP + reduction in control treatments without the plastoquinone analogues. Since effects of uncouplers on photosynthetic electron transport depend on external pH, we determined for each of the four uncouplers, gramicidin, nigericin, FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and SF 6847 (a ditertiary phenol derivative) its effect on oxygenic electron transport (H 2O to NADP +) over a range of external pH from 6.7 to 8.7. The effect of each uncoupler on counteracting the inhibition of DBMIB and DNP-INT was then measured at its crossover external pH at which the uncoupler had little or no effect on electron transport in the uninhibited controls. Under these controlled conditions, uncouplers increased the rate of plastoquinone-inhibited electron transport, in some cases by almost 300%. To explain these results, a role for plastoquinone in processing protons released by the oxidation of water is postulated.

Differential inhibition by plastoquinone analogues of photoreduction of cytochrome b-559 in chloroplasts

The high-potential form of cytochrome b-559 ( b-559 HP) is closely linked to the oxygenic photosystem (photosystem II) but its relation to other redox components of the photosynthetic apparatus, including plastoquinone, is still obscure. We investigated the photoreduction of cytochrome b-559 HP by isolated chloroplasts in the presence of 3 antagonists of plastoquinone, of which, DBMIB (dibromothymoquinone) and DNP-INT (dinitrophenyl ether of iodonitrothymol) are known to inhibit the oxidation of the plastoquinone pool (PQ) by the FeS-cytochrome ƒ/ b 6 complex and one, UHDBT (5- n-undecyl-6-hydroxy-4,7-dioxobenzothiazole) is known to inhibit the reduction of PQ by Q B.Q B is a protein-bound plastoquinone that serves as a two-electron gate for the reduction of PQ. We found that DBMIB and DNP-INT did not inhibit but low concentrations of UHDBT severely inhibited the photoreduction of cytochrome b-559 HP. These results suggest that the electron donor for the reduction of cytochrome b-559 HP was either Q B or a portion of the PQ pool that was oxidized by a new pathway free of binding sites for DBMIB and DNP-INT.

Structure Activity Correlation of Herbicides Affecting Plastoquinone Reduction by Photosystem II: Electron Density Distribution in Inhibitors and Plastoquinone Species

Molecular orbital calculations of the net charge and the π charge distribution in several inhibitors and herbicides of the functionally related group of the diuron and dinoseb type are reported. They confirm the model that urea, aminotriazinone and triazine herbicides all have in common a positive π-charge at a particular atom considered to be essential for binding. Phenol type inhibitors have different charge distribution and a model for their essential features is presented. The calculations support the finding that two different subunits with different binding characteristics are involved in inhibitor and plastoquinone function on the acceptor side of photosystem II. Force-field model building and MO calculations of the charge distribution of a plastoquinone analogue with a butenyl side chain, of two of its semiquinone forms and of the hydroquinone, are reported, as well as their conformation with the lowest energy content and their likely anionic forms.

Low Redox Potential Promotes Sulphide- and Light-dependent Hydrogen Evolution in Oscillatoria limnetica

Summary: Anoxygenic photosynthetic electron transport from sulphide, culminating in either H2 evolution or CO2 photoassimilation, was shown to include the segment from plastoquinone to ferredoxin in the cyanobacterium Oscillatoria limnetica. Both sulphide-dependent H2 evolution and CO2 photoassimilation were inhibited by plastoquinone analogues. In the former reaction, the block was bypassed by reduced N,N,N”,N' -tetramethyl-p-phenylenediamine (TMPD). The link between this segment of electron transport and the hydrogenase enzyme was shown to limit the rate of sulphide-dependent H2 evolution. The rate of flow of electrons through this pathway was lower than would be expected either from the amounts of available enzyme, as measured by the in vitro oxidation of reduced methyl viologen, or from rates of electron transport to CO2 photoassimilation. When the strong reductant sodium dithionite was added to intact cells, the resulting low redox potential significantly improved photosynthetic sulphide-dependent H2 evolution. Hydrogenase activity in cell free extracts was similarly affected by dithionite. It is suggested that ambient redox potential controls electron flow through the hydrogenase, so that surplus reducing power is removed via H2 evolution.

Enhanced sensitivity to plastoquinone inhibitors of ferredoxin photoreduction by photosystem II in inside-out chloroplast vesicles

New evidence is presented in support of the concept that reducing power for photosynthesis is generated solely by photosystem II (the oxygenic photosystem) when it transfers electrons from water to ferredoxin without the collaboration of photosystem I, the anoxygenic photosystem responsible for cyclic photophosphorylation. Membrane vesicles of opposite sidedness were prepared from spinach chloroplasts by the two-phase partition method: inside-out-vesicles greatly enriched in photosystem II and right-side-out vesicles containing both photosystems and having the same sidedness orientation as unfractionated chloroplast membranes. In both types of vesicles, plastoquinone analogues were used to inhibit light-induced electron transport from water to ferredoxin and from water to native photosystem I acceptors, the membrane-bound iron-sulfur centers A and B. In right-side-out vesicles the photoreduction of iron-sulfur centers A and B was more sensitive to plastoquinone inhibitors than the photoreduction of ferredoxin, whereas the converse was found in inside-out vesicles in which a greatly enhanced sensitivity of ferredoxin reduction to plastoquinone inhibitors was detected: the photoreduction of ferredoxin was about 80% inhibited at low concentrations of plastoquinone inhibitors that had practically no effect on the photoreduction of iron-sulfur centers A and B. These findings appear to exclude the possibility that these photosystem I contaminants were involved in the photoreduction of ferredoxin by the PSII-enriched inside-out vesicles.

Competition between plastoquinone and 3-(3,4-dichlorophenyl)-1,1-dimethylurea at the acceptor side of Photosystem II

In plastoquinone-depleted thylakoids, as obtained by n-hexane extraction, a high specifically labeled 3-(3,4-[ 3H]dichlorophenyl)-1,1-dimethylurea ([ 3H]DCMU) is displaced competitively from the membrane by the short-chain plastoquinone analogue plastoquinone-1. A binding constant K b = 51 ± 19 μM could be calculated for plastoquinone-1. Similarly, [ 3H]DCMU is also competitively displaced by the Photosystem II acceptor dichlorophenolindophenol ( K b = 20 ± 3 μM).

Interaction of the Quinone Analogue, DBMIB, with the Photosynthetic Rieske Iron‐Sulfur Center

AbstractAddition of the plastoquinone analogue, DBMIB, to chloroplasts results in the appearance of an EPR signal at g = 1.94 which is observed at cryogenic temperatures. Studies with subchloroplast fragments which either contain or lack the Rieske iron‐sulfur center indicate the presence of this electron carrier is required for the DBMIB‐induced EPR signal. A correlation between the disappearance of the Rieske center signal at g = 1.89 and the appearance of the DBMIB‐induced signal at g = 1.94 suggests DBMIB alters the Rieske center to produce the new signal. Similar effects were observed in photosynthetic membrane fragments from a blue‐green alga and a photosynthetic bacterium. It has been possible to use the DBMIB‐induced signal to test for interaction of other inhibitors in the region of the Rieske center. These results are considered in terms of models in which quinone and the Rieske center function as a donor‐acceptor pair during physiological electron transport.

A dual requirement for plastoquinone in chloroplast electron transport

When plastoquinone is extracted from lyophilized chloroplasts, electron transport from Photosystem II is inhibited. This inhibition can occur at two levels. One involves the accepted site on the intermediate electron transport chain between the photosystems. The other is associated with the oxidizing side of photosystem II, presumably involving the water oxidation mechanism. Evidence for a dual-site role for plastoquinone is demonstrated by the following findings: 1. 1. Extraction at least 75% of total plastoquinone inhibits all photosynthetic electron transport employing water as the electron source. 2. 2. The photosystem II reaction using iodide as the electron donor and dimethyl-methylene dioxybenzoquinone as a class III acceptor takes place in plastoquinone-extracted chloroplasts. This indicates that class III acceptors intercept electron flow before the plastoquinone pool, and the hypothesized site on the water-splitting side of photosystem II is between the site of water oxidation and iodide donation. 3. 3. Artificial donors, including I −, do not photoreduce class I acceptors such as methyl viologen in extracted chloroplasts, supporting the previous conclusions that plastoquinone functions between the photosystems. 4. 4. Reconstitution with either plastoquinone A or C in combination with β-carotene restores both the plastoquinone site between the photosystems and water oxidation. β-Tocopherol quinone is also effective in this role. 5. 5. Reconstitution with trimethyl benzoquinone is ineffective in restoring electron transport either from water or artificial donors, indicating a requirement for an isoprenoid or phytol side chain. Consistent with the work of Okayama (Plant Cell. Physiol. (1974) 15, 95–101), these findings are strong evidence for a dual role for plastoquinone in the photosynthetic electron transport scheme. The nature of the requirement implicated in the water oxidation mechanism is still unclear. However, some tentative conclusions based on the effectiveness of plastoquinone analogs in reconstitutions can be made. These lend support to the notion that this function is relatively specific with regard to the structure of the plastoquinone aromatic ring but independent of the length of the isoprene chain beyond one phytol unit.

Synthesis of plastoquinone analogs and inhibition of photosynthetic and mammalian enzyme systems.

New 5-hydroxy- and 5-chloro-6-alkyl-1,4-benzoquinones with one or two methyl groups on the nucleus were synthesized as potential antimetabolites of plastoquinones for biological research on photosynthetic and mammalian enzyme systems; the primary emphasis was on photosynthesis.2,3-Dimethyl-5-hydroxy-6-phytyl-1,4-benzoquinone completely inhibited in chloroplasts the water-dependent electron transport, but photosystem I was insensitive to this analog. The data are consistent with the interpretation that this analog inhibits electron transport in the chain prior to the site of electron donation from the ascorbate-dichlorophenolindophenol couple. Concentrations of 70 muM and 120 muM of this analog caused about 50 and 100% inhibition, respectively, of cyclic photophosphorylation.2,3-Dimethyl-5-hydroxy-6-phytyl-1,4-benzoquinone is a new type of inhibitor of photosynthetic electron transport that specifically inhibits the rate-limiting step between photosystems I and II. Structurally related analogs caused inhibitions in the range of 50-100% in chloroplasts. These analogs showed marginal inhibition in mitochondrial coenzyme Q(10)-oxidase systems from beef heart.