Wilfried Draber

The Inhibition of Photosynthetic Electron Flow in Chloroplasts by the Dinitrophenylether of Bromo-or Iodo-nitrothymol

Abstract The O -dinitrophenyl derivatives of 2 -bromo-and 2-iodo-4-nitrothymol are inhibitors of photo synthetic electron flow from water to NADP or methylviologen, yielding 50% inhibition at 0.5 μм. Photoreductions by either photosystem I or photosystem II alone are not inhibited. The inhibition site is bypassed by TM PD. The inhibition pattern identical to the one of dibromothymoquinone. It is reduction of plastoquinone.

Lipophilicity and catalysis of photophosphorylation. II. Quinoid compounds as artificial carriers in cyclic photophosphorylation and photoreductions by Photosystem I

Abstract The topography of the chloroplast membrane has been studied using the following pairs of quinoid compounds with similar structure and chemical properties, but with different lipid solubility: phenazine/sulfophenazine, naphthoquinone/naphthoquinone sulfonate, indophenol/sulfoindophenol and lumiflavin/FMN. All these compounds in the oxidized form are able to accept electrons from the photosynthetic electron transport chain in Hill reactions. However, only the lipophilic compounds in the reduced form can donate electrons to Photosystem I, when electron flow from Photosystem II is blocked by inhibitors. This is in agreement with the notation that the oxidizing site of Photosystem I (P 700 + ) and the electron donors for Photosystem I (cytochrome f and plastocyanin) are located inside the lipid barrier of the inner chloroplast membrane. The reducing sites in the Hill reactions must be located on the outer surface, accessible from the suspending medium. It has been known for a long time that N , N ′-tetramethyl- p -phenylenediamine can donate electrons to Photosystem I, but contrary to diaminodurene (2,3,5,6-tetramethyl phenylenediamine) it does not induce ATP formation. Both compounds are lipophilic and have similar redox potentials, but only the latter carries hydrogens which are involved in the redox reaction. For energy conservation, coupled to electon flow in Photosystem I, it therefore seems necessary that the lipophilic redox compound in the reduced form can carry hydrogens through the chloroplast membrane.

QUINOLONES AND THEIR N-OXIDES AS INHIBITORS OF MITOCHONDRIAL COMPLEXES I AND III

4(1H)-quinolones (2-alkyl- (1), 2-alkyl-3-methyl- (2), 2-methyl-3-alkyl- (3), 1-hydroxy-2-methyl-3-alkyl- (4) and 1-hydroxy-2-alkyl- (5)) with n-alkyl side chains varying from C5 to C17 have been synthesized and tested for biological activity in mitochondrial complexes. Whereas all quinolones were efficient inhibitors of electron transport in the cytochrome b/c1-complex from either beef heart or Rhodospirillum rubrum, in complex I from beef heart quinolones 1 and 2 only were highly active. In a Quantitative Structure-Activity Relationship (QSAR) inhibitory activity in the cytochrome b/c1-complexes could be correlated to the physicochemical parameters lipophilicity pi and/or to STERIMOL L. Maximal inhibitory potency was achieved at a carbon chain length of 12-14 A. Oxidant-induced reduction of cytochrome b established that some quinolones are inhibitors of the Qp rather than the Qn site.

Quinolones and their N-oxides as inhibitors of photosystem II and the cytochrome b6/f-complex

4(1H)-quinolones (2-alkyl- (1), 2-alkyl-3-methyl- (2), 2-methyl-3-alkyl- (3), 1-hydroxy-2-methyl-3-alkyl- (4) and 1-hydroxy-2-alkyl- (5)) with n-alkyl side chains varying from C(5) to C(17) have been synthesized and tested for biological activity in photosystem II and the cytochrome b(6)/f-complex. In photosystem II, quinolones 1 and 2 showed only moderate activity, whereas 3<5<4 (increasing activity) were potent inhibitors. Displacement experiments with [(14)C]atrazine indicated that the quinolones share an identical binding site with other photosystem II commercial herbicides. In the cytochrome b(6)/f-complex, only 3<4 showed enhanced activity. Maximal inhibitory potency was achieved at a carbon chain length of 12-14 A. Further increase of the chain length decreased activity. In a quantitative structure-activity relationship inhibitory activity in photosystem II and the cytochrome b(6)/f-complex could be correlated to the physicochemical parameters lipophilicity pi and/or to STERIMOL L.

Herbicide binding at photosystem II. A new azido-triazinone photoaffinity label

Azido-triazinone (3-dimethylamino-4-methyl-6-(3′-azidophenyl)-1,2,4-triazin-5-one) was found to be an efficient inhibitor of Photosystem II electron transport. This compound has an I50 value of 69 nM (extrapolated to zero chlorophyll concentration), a high-affinity binding constant of 12.6 nM, and a number of binding sites of 1.9 nmol/mg chlorophyll. This corresponds to 550–580 molecules of chlorophyll per bound inhibitor; i.e., one molecule inhibitor per electron transport chain. In isolated spinach thylakoids, [14C]azido-triazinone upon ultraviolet illumination covalently binds almost exclusively to a 34 kDa protein. Covalent binding is prevented in the presence of other Photosystem II inhibitors. The protein labeled by azido-triazinone is identical to the 34 kDa herbicide-binding protein which is tagged by another photoaffinity label azido-atrazine (2-azido-4-(ethylamino)-6-(isopropylamino)-s-triazine).

STRUCTURE-ACTIVITY CORRELATION IN THE AZOLES

Numerous highly potent 1-substituted imidazoles and 1.2.4.-triazoles have been developed to date for the control of fungi pathogenic to plants and humans, and have been successfully introduced into practice. Some of these compounds stem from the tritylazole and phenoxy-triazolylmethane groups. This overview presents structure-activity relationships among the trityl-imidazoles, structure-activity correlations among the phenoxy-triazolyl-methanes and also the influence of stereochemistry of the latter upon biological activity. Not only QSAR studies but also statistical methods such as Spearmans rank correlation are discussed as methods for describing the influences of physicochemical parameters upon biological activity. Possibilities, problems and limitations of structure-activity correlations among the azoles are illustrated by the above examples.

The Effect of Analogues of Dibromothymoquinone and of Bromonitrothymol on Photosynthetic Electron Flow

Abstract Alkyl substituted derivatives of halogenated p-benzoquinones, of halogenated p-nitrophenols, and of 2,4-dinitrophenols were tested in the inhibition of photosynthetic electron flow in chloroplasts. The effect of the compounds on photoreductions by photosystem I or II, on a TMPD bypass in NADPH formation and the reversibility of the inhibition by dithiothreitol is used to distinguish between an inhibition site before or after plastoquinone function, i. e. between a DBMIB versus a DCMU inhibition pattern. It is shown, that different isopropyl and t-butyl substituted halogenated p-benzoquinones are as effective and specific as DBMIB in the inhibition of plastoquinone function. Alkyl substituted p-nitrophenols, with an additional halogen-or nitro-group at C-2, are shown to be effective electron flow inhibitors. The new potent nitrophenol derivatives inhibit at the site of DCMU action, nevertheless they do not contain the basic chemical element essential for inhibition common to DCMU and its many herbicidal analogues. Small changes in the ring-substitution can alter the inhibition pattern from a DCMU typ to a DBMIB typ inhibition.

Response in the inhibitor efficiency of substituted phenols on ps II activity in six mutants of the D1 protein subunit in Chlamydomonas reinhardtii

Abstract The D1 protein subunit of the reaction centre of photosystern II is also a herbicide-binding protein. Blockage of the Q B binding site in the D1 protein is the mode of action of many herbicides grouped as classical diuron atrazine type or as phenol group. Tolerance of photosynthetic electron flow in PS II to classical herbicides like diuron or atrazine is observed in several mutants of higher plants and algae with amino acid substitutions in the D1 protein. But in most of these mutants, the few phenol-typeinhibitors tested were not tolerated, some of them even displaying supersensitivity. This seemed to indicate a substantial change of orientation of the phenolic inhibitors in the Q B site when compared to the classical herbicides. However, we report here on the appreciable tolerance of mutants to phenol-type inhibitors of the D1 protein. We investigated the effect of 47 derivatives of 6-alkyl-substituted 2-halogeno-4-nitrophenols and of 2,4-dinitrophenols on photosynthetic electron flow in six mutants of Chlamydomonas reinhardtii . The mutants were obtained either by site-selected screening towards metribuzin or diuron or by site-directed mutagenesis. With the exception of an asn266thr mutant, all the mutants responded toward the phenol inhibitors either by decreased or enhanced sensitivity compared with the wild type. Greatest effects were observed with a val219ile mutant. Tolerance ranged from zero to 1.7 pI 50 units depending on the substituent at position 6 of the phenol ring. Similarly, but somewhat lower, is the tolerance in a phe255tyr mutant. A leu275phe and an ala251val mutant show a mixed behaviour of tolerance to some, and supersensitivity to other derivatives. Many phenols display supersensitivity in the ser264ala mutant, up to 100-fold, and there is never tolerance. It is possible to rationalize the cross resistance of phenol-type inhibitors of PS II with their substitution pattern, particularly in the val2l9ile mutant. The extent of tolerance depends entirely on the size and shape of the substituent in position 6. The maximum is found with 2-bromo-4-nitro-6 benzylphenol with a 50-fold decrease in inhibitor potency in this mutant. The data allow us to orient now also the phenol-type inhibitors in the herbicide binding niche in the D1 protein in a way not too dissimilar from that of the classical inhibitors. The concept that not only the classical herbicides but also phenol-type inhibitors act by binding to the D1 protein is now greatly supported by the observation of tolerance if the sequence of the D1 protein is changed. The principle difference of the phenols to the classical herbicides can now be specified clearly: it is the response to the ser264ala mutant where there is no tolerance of phenol-type inhibitors in contrast to the classical herbicides. This is not invalidated by observations that some phenols may bind to other subunits of PS II as well, nor that many of them are uncouplers. These are to be considered as secondary effects not contributing to the high affinity binding to the D1 protein.

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.

Mode of action and mo calculation of two classes of herbicides interacting with the reducing side of photosystem II

Abstract Two chemically different classes of herbicides - represented by diuron and by dinoseb - are inhibitors of photosynthetic electron flow at the same functional site between the primary acceptor of photosystem II and plastoquinone. Recent functional and binding studies with radioactive labelled compounds with intact membranes from chloroplasts from susceptible and herbicide resistant plants or algae gave important new data on the biochemistry involved in the mode of action of these herbicides. In particular, photoaffinity labels identified the peptide substructure of the herbicide target: the B-protein on the thylakoid membrane. This membrane protein catalyses electron flow from photosystem II to plastoquinone and is composed of two subunits: a 32 kDa peptide carrying the binding site for diuron, triazine, amino-triazinone and related herbicides and a 42 kDa peptide carrying the binding site for phenol herbicides, like dinoseb and ioxynil. Already QSAR studies had let to quite diverse types of correlations for the two classes of herbicides. Molecular orbital calculations presented here provide further evidence that the two classes of photosystem II inhibitors differ significantly in the charge distribution at the atoms essential for binding to the membrane. π charge density maps of the basic structure reveal an asymmetric distribution in an amino-triazinone with a large positive charge at the nitrogen in the 4-position. The phenolic compounds in contrast are rather symmetric. The net charge distribution of compounds binding to the 42 kDa peptide is also quite different from those binding to the 32 kDa peptide.