Inhibition at Photosystem II

The action of photosynthetic inhibitors has traditionally been monitored in vitro by measuring the so-called 'Hill Reaction'. This is the ability of a thylakoid preparation to evolve oxygen in the presence of a suitable electron acceptor. The natural acceptor, NADP+ , is washed free from isolated thylakoids, so artificial acceptors (A) are used experimentally.

2 thylakoids 2 2

Figure 5.2 Structural (A) and functional (B) models of Photosystem II. 32 refers to the molecular mass of the D1 polypeptide in kDa. Tr, a tyrosine residue acting as an electron donor to p680+, the bold arrow shows the direction of electron transfer; OEC, oxygen-evolving complex; p680, PS II reaction centre; phaeo, phaeophytin a; QA and QB, quinones; PQ, mobile plastoquinone pool (after Rutherford, 1989).

Figure 5.2 Structural (A) and functional (B) models of Photosystem II. 32 refers to the molecular mass of the D1 polypeptide in kDa. Tr, a tyrosine residue acting as an electron donor to p680+, the bold arrow shows the direction of electron transfer; OEC, oxygen-evolving complex; p680, PS II reaction centre; phaeo, phaeophytin a; QA and QB, quinones; PQ, mobile plastoquinone pool (after Rutherford, 1989).

NADPH

NADP+

NADPH

NADP+

Figure 5.3 Structural (A) and functional (B) models of Photosystem I. Numbers 82 and 83 refer to molecular masses of polypeptides in kDa. PC, plastocyanin (the electron donor to p700+); A0 and A1f electron acceptors/donors; bold arrow shows direction of electron transfer. p700, PS I reaction centre; FeS, iron-sulphur centres; Fd, ferredoxin. The final electron acceptor is NADP+ (after Knaff, 1988).

Figure 5.4 Schematic representation of the light-harvesting and photosystem complexes and electron transport chain in the thylakoid membrane (from Lawlor, 2001, by permission of Oxford University Press). The number of protein complexes and their relation is shown. The molecular mass of components is indicated. The components are listed as follows. Key:

Figure 5.4 Schematic representation of the light-harvesting and photosystem complexes and electron transport chain in the thylakoid membrane (from Lawlor, 2001, by permission of Oxford University Press). The number of protein complexes and their relation is shown. The molecular mass of components is indicated. The components are listed as follows. Key:

1 Antenna protein-pigment complex

2 32 kDa, D1 herbicide binding protein of the reaction centre

3 32 kDa, D2 reaction centre protein

4 Cytochrome b559, 9 kDa b559 type 1 and 4 kDa b559 type 2 proteins

5 Light-harvesting antenna

6 10 kDa docking protein

7 22 kDa stabilising protein (intrinsic membrane protein)

8 20 kDa Rieske Fe-S centre

9 Cytochrome b6-f complex with polypeptides

10 Light-harvesting protein-pigment (chlorophyll a and b) complex of PSI and polypeptides

11 PSI reaction centre, with two 70 (?) kDa polypeptides

12 Plastocyanin, 10.5 kDa

13 Plastocyanin binding protein (10 kDa)

14 Fe-S protein A, 18 kDa

15 Fe-S proteins B, 16 kDa

16 Fe S protein 8 kDa

17 Ferredoxin binding protein

18 Ferredoxin

19 Ferredoxin, NADP oxidoreductase

20 Coupling factor, CF0, membrane subunits

A commonly used artificial electron acceptor is potassium ferricyanide which is reduced to ferrocyanide with the evolution of oxygen. Oxygen evolution can be conveniently monitored using an oxygen electrode. Similarly, the blue dye 2,6-dichlorophenolindophe-nol (DCPIP) will accept electrons from the functioning thylakoid and the reduced product is colourless. Thus, photosynthetic electron flow can be measured by following DCPIP decolourisation with a spectrophotometer (Figure 5.5).

Such reactions, however, measure the activity of both photosystems rather than specific sites, and are therefore of limited value in identifying precise sites of photosynthetic inhibition. Nowadays the partial reactions of electron flow can be characterised in great detail using highly specific artificial electron donors and acceptors (e.g. Table 5.1; Trebst, 1980). Indeed, herbicides have proved invaluable in unravelling the flow of electrons in the thylakoid.

It was using DCPIP as an artificial electron acceptor that Wessels and van der Veen (1956) first demonstrated that the urea herbicide diuron could reversibly inhibit photosynthetic electron flow at micromolar concentrations. Subsequent studies in the following decades established that diuron was acting at the reducing side of PS II in the vicinity of Qa and Qb. This was deduced from the observations that (1) PS I activity was insensitive to diuron, (2) electron flow at the cytochrome b6-f complex was similarly unaffected, and (3) diuron had no influence on the photolysis of water, nor on charge separation at p680, but electron flow to plastoquinone was potently inhibited.

Several classes of herbicide including the ureas, triazines and phenols are now known to inhibit PS II activity by displacing plastoquinone from the QB site and so preventing electron flow from QA-. Although QA is tightly bound to the D2 protein, QB is not firmly bound to D1, and so herbicides successfully compete with QB for this site. Inhibition by the ureas and triazines is characteristically reversible and competitive. Furthermore, it was found that there was only one binding site for each PS II reaction centre, and that binding/ dissociation constants were very similar to inhibition constants, hence occupancy of this site is required for photosynthetic inhibition.

Table 5.1 Examples of the measurement of photosystem activity in vitro by oxygen exchange.

Donor

Acceptor

Inhibitor

Reactions measured

Oxygen

H2O

Ferricyanide

Absent

PS II and PS I (H2O ^ FeS centres)

Evolution

h2o

Dimethyl-benzoquinone

Absent

PS II only (H2O ^ plastoquinone)

Evolution

h2o

Silicomolybdate

Absent

PS II only (H2O ^ phaeophytin)

Evolution

Ascorbate + DCPIP

Methyl viologen

(plastocyanin ^ ferredoxin)

Uptake

Studies in the 1970s established that thylakoids treated with trypsin became insensitive to diuron suggesting a stromal - facing proteinaceous binding site (Renger, 1976) . This protein was further characterised using radiolabelled herbicides as a rapid turnover polypeptide (molecular mass 32 kDa) that was encoded in the chloroplast psbA genome (Mattoo et al., 1981). Conclusive evidence that this protein had a dominant role in herbicide binding came from its analysis in plants showing resistance to PS II herbicides. Hirschberg and colleagues (1984) cloned the psbA gene from atrazine-resistant and susceptible biotypes of Solanum nigrum and Amaranthus retroflexus- and detected a single base substitution from serine to glycine at amino acid 264 to be the basis of resistance to the herbicide. Resistance is therefore achieved by the substitution of one amino acid in a protein containing 353 amino acid residues!

Figure 5.6 presents the interaction between the quinones and atrazine at protein D1.

A separate binding site specific to the phenols (such as dinoseb) and the hydroxyben-zonitriles (e.g. ioxynil) has been proposed with a molecular mass of 41-47 kDa that is less susceptible to trypsin digestion, and therefore more deeply located in the thylakoid. It is argued that these compounds bind to a different site on the D1 protein.

Our understanding of this protein was vastly enhanced when the reaction centre of the photosynthetic bacterium Rhodopseudomonas viridis was first crystallised and then its structure resolved by Michel and Deisenhofer in 1984 (see Michel and Deisenhofer, 1988) . This seminar work, for which the Nobel Prize for Chemistry was awarded in 1988, pointed to clear similarities and sequence homologies between the bacterial reaction centre and PS II, and provided three-dimensional detail of the amino acids involved in this quinone-binding herbicide niche on the D1 protein. Trebst -1987) utilised this X-ray data and, with information gained from photoaffinity labelling of herbicides and site-directed mutagenesis, proposed a detailed model of the herbicide binding niche. This model shows that the D1 polypeptide contains five transmembrane helical spans (1-5) and two parallel helices (A and B), and that helices 4, 5 and B specifically participate in herbicide binding (Figure 5.7A). The model predicts the orientation of residues at the quinone niche (Figure 5.7B) and envisages QB binding via two hydrogen bridges at His 215 and close to Ser 264.

Following closer examination of the QB binding niche, Trebst (1987) suggested that inhibitors with a carbonyl or equivalent group (e.g. ureas, triazines, triazinones) were orientated towards the peptide bond close to Ser 264 and could form a hydrogen bridge to this peptide bond. However, the phenol group of inhibitors cannot form this bridge and are therefore thought to bind towards His 215, where they are bound more strongly to the membrane. In this way, Trebst envisages two families of PS II herbicides, namely the serine and the histidine families.

More recently, additional mutants have been identified with altered amino acids in the stroma-exposed P-helix, particularly in algae, and other substitutions noted that produce herbicide resistance (Table 5.2) . Substitutions at position 264 from serine to glycine yields atrazine resistance, as demonstrated in Table 12.2- while serine to alanine or threonine produces additional resistance to diuron. This observation has led to the proposal that a serine hydroxyl group may be essential for atrazine binding. Changes at nearby residues 255 and 256 also cause triazine resistance, although changes at positions 219 and 275 give resistance to diuron and also to bromoxynil in the latter example. Resistance may then be due to conformational changes in the binding niche, an absence

Oh Ser 264

CH3 CH3

CH3 CH3

Phe 255

Plastoquinone (PQ)

His 215 R

CH3 CH3 OH

Plastoquinol (PQH2)

Ser264

H(CH2-C=CH- CH2)9yVCH3

C2H5

PQ cannot bind

Cl atrazine

Phe 255

His 215 R

Figure 5.6 Proposed interaction between plastoquinone (A) and atrazine (B) with the QB site of protein D1. Dashes represent hydrogen bonds and dots represent hydrophobic interactions (after Fuerst and Norman, 1991, from the journal Weed Science, courtesy of the Weed Science Society of America). (A) PQ binds to the D1 protein, accepts two electrons and two protons, and is released as PQH2. (B) Atrazine binding to the D1 protein prevents the binding of PQ.

Figure 5.7 The D1 polypeptide, (a) Schematic arrangement of the protein in the thylakoid. (b) The quinone/herbicide binding niche viewed from above (after Trebst, 1987).
Table 5.2 Characteristics of mutants with amino acid substitutions in D1 ( modified from Gressel, 2002).

Mutation

Organism

Primary resistances

Negative cross-resistance

Phe211^Ser

Cyanobacteria

Atrazine x9

Val219^Ile

Alga + cyano Poa annua

Metribuzin x200 Metabenzthiazuron x62 Metribuzin and/or diuron

Ketonitrile x 0.6

Tyr237^Phe

Cyanobacterium

Diuron/ioxynil x5

BNT x0.2

Lys238^Val

Cyanobacterium

loxynil x2.3

BNT x0.4

Ile248^Thr

Cyanobacterium

Metribuzin x28

Ala250^Arg

Alga

Phenmedipham x6.3

Bromoxynil x0.3

Ala250^Asn

Alga

Metamitron x5

Bromoxynil x0.2/ atrazine x0.3

Ala25o^Asp

Alga

Phenmedipham x5

-oxynils x0.2/atrazine x0.25

Ala250^His

Alga

Phenmedipham x10

Ala25o^Ile

Alga

Phenmedipham x2.5

Metribuzin x0.25

Ala25o^Tyr

Alga

Phenmedipham x20

Bromoxynil x0.4

Ala251^Cys

Alga

Metamitron/bromoxynil x6.3

Ala251^Gly

Alga

Metamitron x10

Ala251^Ile

Alga

Diuron x0.6

Ala251^Leu

Alga

Metribuzin x108 Bromacil x26

Ala251^Val

Alga / cyano

Metribuzin x1000

Ketonitrile x0.5

Phe255^Tyr

Alga / cyano

Cyanoacrylate x39

Metamitron x0.3

Gly256^Asp

Alga

Bromacil x10

Arg257^Val

Cyanobacterium

Atrazine / diuron x34

BNT x0.3

Ala263^Pro

Cyanobacterium

Atrazine x2000/metribuzin x1600

Ser264^Ala

Alga / cyano / Euglena

Metribuzin x>3000 Chloroxuron x480 / atrazine variable

Ser264^Gly

Weeds / cyano

Most s- and as-triazines x>500

-oxynils/pyridate x<0.5

Ser264^Asn

Tobacco cells

Terbutryn

Ser264^Pro

Cyanobacterium

Atrazine x10,000

Ser264^Thr

Plant cells / Euglena Portulaca oleracea

Atrazine x>50 Linuron

Dinoseb/-oxynils/BNT x<0.3

Asn266^Asp

Cyanobacterium

loxynil x2.5

Asn266^Thr

Cyanobacterium

Bromoxynil x15

Ser268^Pro

Soybean cells

Atrazine x50

Arg269^Gly

Alga

Terbutryn x8

Ioxynil x0.2

Leu275^Phe

Alga

Metamitron x63

atrazine

^ BW314

Figure 5.8 The structures of atrazine and BW314.

of a specific residue so that a herbicide is unable to bind or the introduction of steric hindrance to prevent herbicide access to the binding niche.

A new group of triazine herbicides, the 2-(4-halogenobenzyl amino)-4-methyl-6-trif-luoromethyl-1,3,5-triazines, were shown in 1998 to inhibit photosynthetic electron flow at the D1 protein. Interestingly, they have shown activity against atrazine-resistant species, such as Chenopodium album L. and Solanum nigrum, presumably by binding to different amino acids in the D1 niche (Kuboyama et al., 1999; Kohno et al., 2000). Figure 5.8 shows the structure of one example, BW 314, with the structure of atrazine also presented for comparison.

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