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Chapter 32

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Inhibition of Photosynthesis by Substituted 4-Nitrophenols in Wildtype and Five Mutants of Chlamydomonas reinkardtii Thylakoids Structure-Activity and Molecular Modeling Studies 1

2

1

2

W. Draber , U. Hilp , M. Schindler , and A. Trebst 1

Agrochemical Research, Bayer AG, 5090 Leverkusen, Germany Department of Biology, Ruhr-Universität Bochum, 4630 Bochum, Germany

2

Photosynthetic inhibition data were determined with thylakoids of the green alga Chlamydomonas reinhardtii on fourty-four 2-halo-4-nitro-6alkyl- and 2,4-dinitro-6-alkyl-phenols. For the measurements wildtype and five different mutants of the algae were employed. The mutants had known amino acid changes in the herbicide binding protein D1. Their response to the various inhibitors was quite different from that of the wildtype. Tolerance as well as enhanced inhibitory activity was found in the mutants. This implies that the phenols are bound specifically to the D1 protein. The wealth of data encouraged structure-activity and molecular modelling studies, which were based on a model of the binding niche. It was attempted to explain the enhanced or decreased inhibitory activity of selected compounds by energy calculations. Photosynthesis inhibitors act by displacing the secondary electron acceptor Q , plastoquinone, from its binding proteinD1in photosystem II as suggested first by Velthuys in 1981 (1). Already in 1979 we had emphasized (2) that there is distinction to be made between classical herbicides and phenolic inhibitors. In 1987 it was suggested (3) to divide the inhibitors into two chemical groups that behaved different in many biochemical respects: the classical inhibitors represented by compounds like diuron (DCMU) and atrazine, and a second group which consists mainly of phenol derivatives like the two herbicides dinoseb and bromoxynil. The first group has been called the serine family because these compounds loose their activity in plants and algae in which serine264 in theD1protein is replaced by alanine or glycin. The phenolic inhibitors do not show decrease of inhibitory activity in these mutants. It was assumed that they are oriented towards histidine in theD1binding niche. For that reason they were called the histidine family. In the meantime, however, experimental evidence began to show that this distinction was rather inefficient and needed to be replaced by a more detailed one. This became possible by screening a great number of inhibitors and the availibility of algae mutants, in which the amino acid sequence of theD1protein was changed in a known manner. B

215

0097-6156/94/0551-0449$07.00/0 © 1994 American Chemical Society Hedin et al.; Natural and Engineered Pest Management Agents ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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NATURAL AND ENGINEERED PEST MANAGEMENT AGENTS

Structure-activity studies have proved instrumental in describing the dependence of inhibitory activity on physicochemical and calculated descriptors. A nearly historical example of a correlation study with phenolic photosynthesis inhibitors is shown in Figure 1 (2). In this equation the only parameters employed were the STERIMOL parameters by Verloop et al. (4). In a more recent analysis of the same set of compounds (5) it was found that the hydrophobicity parameter π also contributed to inhibitory activity. Computer power is now no longer a serious limitation for many applications in the area of small molecules. We have carried out studies of molecular modeling and energy calculations using a simplified model of the Dl binding niche, based on the X-ray analysis of the bacterial photosystem by Deisenhofer et al. (6) and using the inhibition data obtained with the phenols. The results, however, have to be regarded as preliminary. Materials and Methods Compounds Studied. Fourty-four 4-nitrophenols were prepared according to methods known in the literature. The structures are listed in Tables I and II. Table I contains twenty-seven 2-halo-4-nitro-6-alkylphenols, some with a methyl group in 3-position. Table II consists of seventeen 2,4-dinitro-6-alkylphenols, one of them with a methyl group in 3-position. Chlamydomonas reinhardtii Mutants. Five different mutants with mutations in the psbA gene that encodes the Dl protein were obtained as described by Wildner et al. (7). The val219ile mutant was first described by Galloway and Mets (8) and the corresponding psbk gene sequenced by Erickson et al. (9). The ala251val, ser264ala and leu275phe mutants were obtained by Johanningmeier et al. (70) from wildtype cells after mutagenesis and metribuzin pressure. They also identified the amino acid substitutions by sequencing the psbk gene. The compounds were assayed for inhibitory activity in isolated thylakoid membranes by measuring photosynthetic DCPIP reduction. Structure Activity Analyses. The PI50 values of wild type and mutant thylakoids were subjected to regression analyses according to the Hansch approach. Experimentally based and calculated descriptors were employed. Partition coefficients logP were obtained by using HPLC retention data that were converted by a calibration program. CLOGP and CMR were calculated with the Pomona software (77). The STERIMOL parameters were taken from Verloop et al. (4,72;. Two indicator variables had to be introduced: n-R which takes 0 for R = H and 1 for R2= Me, and Ph-R which takes 0 for R = all alkyl and cycloalkyl groups and 1 if R = phenyl or benzyl. From the many calculations that were performed, only those are presented with the best statistical criteria F, r , and s. From these, the standard deviation s was considered as the most important one, hence it was related to the respective range (see the last columns of Tables V and VI). 2

4

2

4

4

2

Molecular Modeling and Energy Calculations. An ESV 30/33 RISC workstation was used for molecular modeling and energy calculations with the software package SYBYL (Tripos Ass., St. Louis, USA). Figures 2 - 7 were drawn after photographs from the screen by using the software Aldus FreeHand 3.1 (Aldus Corporation).

Hedin et al.; Natural and Engineered Pest Management Agents ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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DRABER E T A L .

Inhibition of Photosynthesis by 4-Nitrophenols 451

Figure 1. The First Regression Analysis of 4-Nitrophenols. (Reproduced with permission from reference 2. Copyright 1979.)



452

Table I. The Inhibition of Photosynthetic Electron Flow by Substituted Phenols in Wildtype and Five Mutants of Chlamydomonas

rh.

N02

4-Nitrophenols pi 50 values in Chlamydomonas chloroplasts R2

No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

CI Br Br Br Br Br Br Br J Η Cl Cl Br Br Br Br Br Br Br Br Br Br Br CI Br J Br

H H H H H Me H H H Me H Me H Me H H H H H H H H H H H H H

R3

NO2 NO2 NU2 Νθ2 NOz Νθ2 Νθ2 Νθ2 Νθ2 Νθ2 Νθ2 NOz Νθ2 Νθ2 Νθ2 Νθ2 Νθ2 Νθ2 Νθ2 Νθ2 Νθ2 ΝΟζ Νθ2 ΝΟζ ΝΟζ NÛ2 ΝΟζ

R

4

wt

Cl Br Me Et n-C H CHMe2 n-C H CHMeEt CH CHMe2 CMe CMe CMe CMe CMe 11-C5H11 n-C H n-C7Hi5 n-C H n-CçH^ç n-Ci H i n-C H 5 3

7

4

9

2

3

3 3 3 3

6

13

8

17

0

12

2

2

C-C5H9

c-C Hn Ph Ph Ph CH Ph 6

2

4.4 5.0 4.4 4.6 6.0 7.3 6.5 6.3 6.7 7.1 6.8 8.2 7.2 8.4 7.0 7.3 7.5 8.0 8.1 8.1 7.6 7.6 8.0 7.1 7.7 7.5 7.2

rh.

mutants val ala 219 251 île val

phe 255 tyr

ser 264 ala

leu 275 phe

4.4 5.4 4.6 4.2 4.7 7.0 5.8 5.4 5.5 6.7 6.6 8.2 7.1 8.0 5.8 6.4 7.0 6.8 7.6 7.4 7.1 6.5 6.4 5.8 6.4 6.0 5.5

4.3 4.7 5.2 4.6 5.5 7.5 6.6 6.1 6.7 6.5 6.0 7.0 7.2 7.8 7.0 7.2 7.8 7.7 7.8 7.8 7.2 7.5 7.2 7.2 7.3 7.0 7.2

5.0 6.0 6.3 5.7 6.6 7.7 7.3 7.2 7.2 6.8 6.8 8.0 7.5 8.4 7.4 7.6 8.0 8.4 8.0 8.3 7.8 7.8 8.1 7.3 8.0 7.5 7.6

6.1 6.5 5.3 5.6 6.8 7.1 7.5 6.1 7.1 7.0 5.4 7.0 5.8 7.2 7.7 8.0 8.5 8.3 8.7 8.6 8.0 7.1 7.0 7.0 7.5 7.5 7.1

4.8 5.7 4.7 5.6 6.4 6.4 7.6 6.8 7.8 7.0 7.2 7.0 7.1 7.8 7.6 8.0 8.1 8.4 8.6 8.7 8.0 7.7 8.1 7.7 8.4 8.2 7.4


DRABER E T AL.

Inhibition of Photosynthesis by 4-Nitrophenols

453

Table II. The Inhibition of Photosynthetic Electron Flow by Substituted Phenols in Wildtype and Five Mutants of

„

Chlamydomonas

rh.

OH

2,4-Dinitrophenols pi50 values in Chlamydomonas chloroplasts R2

No.

28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44

N0 N0 N0 N0 N0 N0 N0 N0 N0 N0 N0 N0 N0 N0 N0 N0 N0

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

Η Η Η Η Η Η Η Me Η Η Η Η Η Η Η Η Η

R3

R

4

wt

NQj Me NQ* Et ΝΟ2 n-C H ΝΟ2 n-C H ΝΟ2 CHMeEt ΝΟ2 CH CHMe ΝΟζ CMe ΝΟζ CMe Νθ2 n-C Hn Νθ2 n-C Hi 3

7

4

9

2

3

3

5

6

3

Νθ2 n-C Hi5 7

NC^ ΝΟζ ΝΟζ ΝΟζ

n-C H n-CçHiç n-Ci H i 8

17

0

2

n-Ci H 5 2

2

Νθ2 c-C Hn Νθ2 Ph 6

2

4.2 4.7 5.1 5.6 5.8 5.3 6.0 7.1 5.7 6.7 7.0 7.4 7.7 7.7 7.4 6.5 6.1

rh.

mutants val ala 219 251 iie val

phe 255 tyr

ser 264 ala

leu 275 phe

4.1 4.5 4.7 5.4 5.2 5.0 5.5 7.3 5.2 6.0 6.5 6.7 7.4 7.3 7.5 5.7 4.7

5.3 4.8 5.1 5.8 5.4 5.0 5.7 6.4 6.0 6.3 7.0 7.0 7.2 7.3 7.3 6.3 6.5

4.8 5.3 5.7 6.4 6.3 5.8 6.0 7.1 6.4 6.7 7.4 7.7 8.0 8.0 8.0 7.3 6.5

5.1 5.8 5.5 6.5 5.7 6.2 5.7 6.7 6.1 7.0 7.7 7.8 8.3 8.3 8.1 6.2 6.2

4.8 5.8 6.0 7.0 6.3 6.7 7.0 7.4 6.8 7.3 7.8 8.0 8.5 8.1 8.2 7.3 7.0


454


Ser222

Ala277 Figure 2.

Compound 6 in the Wildtype Binding Niche.

In Figures 2 - 7 the Amino Acids where Mutations can Occur are Figured as Filled Circles. The Amino Acids at the Ends of the Binding Niche and His215 are Represented as Open Circles.


32.

DRABERETAL.



455

456

NATURAL AND ENGINEERED PEST M A N A G E M E N T AGENTS

Ser222

Ala277 Figure 4.



32.

DRABERETAL.


Ser222

Figure 5.

Compound 38 in the Ala251Val Mutant Binding Niche.


457

458

NATURAL AND ENGINEERED PEST M A N A G E M E N T AGENTS

Ala277 Figure 6.



32.

DRABER ET AL.

Figure 7.

Inhibition of Photosynthesis by 4-Nitrophenoh

Compound 27 in the Val219Ile Mutant Binding Niche.


459

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NATURAL A N D ENGINEERED PEST M A N A G E M E N T AGENTS

Results. Biochemistry. From the inhibition assays with the wildtype (wt) and mutant algae it becomes evident that there are large differences in sensitivity not only to the diverse nitrophenols but also among the various mutants. The range of the pi 50 values from Tables I and II is listed in Table III. Table III. Spread of pi 5 0 Values in Wildtype and Mutant Chloroplasts

Mutant

Most Active Compound No. pl50

Least Active Compound No. pl50

wt val219ile ala251val phe255tyr ser264ala leu275phe

14 12 20 17 14 19

28 28 1 1 28 28

8.4 8.2 8.7 8.7 8.4 8.7

4.2 4.1 4.8 4.3 4.8 5.1

Aplso

4.2 4.1 3.9 4.4 3.6 4.6

In Table IV the various mutant data are compared to those of the wildtype. The compounds are listed in same order as in Tables I and II. The Aplsos are the differences of the wildtype values to the corresponding mutant values (= plsrjwildtype - plsOmutant)Table IV presents the complicated pattern of enhanced and decreased sensitivity to the 4-nitrophenols compared with the wildtype. There is no uniform response to the compounds in the sense that in one mutant all plso values are shifted in the same direction. However, a remarkable feature from Table IV is that almost all phenols show tolerance in the val219ile mutant whereas most compounds have increased sensitivity in the ser264ala mutant. There are only few exceptions to this rule (val219ile: No.s 2, 3, 42; ser264ala: 12, 19, no change: 11, 14, 26, 34, 35, 37). In this context one has to note that classical inhibitors show the opposite behaviour in the ser264ala mutant. They get tolerant to a great extent. Atrazine for example has a resistance factor (i. e. the ratio of the I50 values in resistant and susceptible chloroplasts) of 160 and diuron (DCMU) one of 200 (7) which corresponds to a Δρΐ5ο = 2.2 or 2.3 respectively (10). In the other mutants the 4-nitrophenols display a mixed behaviour. In the ala251val mutant enhanced sensitivity prevails, whereas


32.


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Natural and Engineered Pest Management Agents - American

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