Studies on the Functional Mechanism of System II Herbicides in Isolated Chloroplasts

The effect of specific proteolytic enzymes on variable fluorescence, p-benzoquinone-mediated oxygen evolution, PS II herbicide (atrazine and bromoxynil) binding, and protein degradation has been analyzed in isolated class II pea chloroplasts. It was found that: 1. Trypsin and a lysine-specific protease effectively reduce the maximum chlorophyll-a fluorescence yield, whereas the initial fluorescence remains almost constant. At the same number of enzymatic activity units both proteases have practically the same effect. 2. Trypsin and a lysine-specific protease inhibit the p-benzoquinone-mediated flash-induced oxygen evolution with trypsin being markedly more effective at the same number of activity units of both enzymes. Unstacked thylakoids exhibit a higher sensitivity to proteolytic degradation by both enzymes. 3. Trypsin and a lysine-specific protease reduce the binding capacity of [14C]atrazine, but enhance that of [14C]bromoxynil (at long incubation times trypsin treatment also impairs bromoxynil binding). At the same specific activity a markedly longer treatment is required for the lysine-specific protease in order to achieve the same degree of modification as with trypsin. 4. Trypsin was found to attack the rapidly-turned-over 32 kDa-protein severely, whereas the lysine-specific protease does not modify this polypeptide. On the other hand, the lysine-specific protease attacks the light harvesting complex II. 5. Under our experimental conditions an arginine-specific protease did not affect chlorophyll-a fluorescence yield, p-benzoquinone-mediated oxygen evolution, herbicide binding and the poly- peptide pattern. Based on these results a mechanism is proposed in which an as yet unidentified polypeptide with exposable lysine residues, as well as the lysine-free “QB-protein” regulate the electron transfer from Q-A to QB and are involved in herbicide binding

Herbicide/Quinone Binding Interactions in Photosystem II

Many inhibitors prevent the oxidation of the primary electron-accepting quinone (QA) by the secondary quinone (Qв) in photosystem II by displacement of Qв from its binding site. On the other hand, plastoquinone-1 and 6-azido-5-decyl-2,3-dim ethoxy-p-benzoquinone displace herbicides. Binding studies show the herbicide/quinone interaction to be (apparently) competitive.The herbicide binding is influenced differentially by various treatments. In this paper it is shown that the affinity of, for example, bromoxynil is decreased by thylakoid unstacking or by light-or reductant-induced reduction of certain thylakoid components, whereas atrazine affinity remains unchanged. Furthermore, absence of HCO-3 in the presence of form ate leads to an affinity decrease of bromoxynil and atrazine, but to an increase in i-dinoseb affinity. Other differential photosystem II herbicide effects are known from the literature.Since different and unrelated groups of Q-A oxidation inhibitors have been found, and because of the above-mentioned dissimilarities in binding characteristics for different inhibitor groups, the hypothesis of non-identical, but “overlapping” binding sites for different herbicide groups and the native quinone must be more extensively defined. In this manuscript we evaluate both the competitive herbicide/quinone binding model, and a model in which binding of one ligand alters the protein conformation resulting in a dramatic decrease in the binding affinity of ligands from other chemical groups; in this model ligands from the same or related chemical groups bind competitively. Thus, the latter model proposes that only one herbicide or quinone molecule can be bound with high affinity to the herbicide/quinone binding environment, but it depends on the chemical structure of the ligands whether the binding interaction between two ligands is truly competitive or more indirect (allosteric), mediated through the protein conformation

Lifetimes of photosystem I and II proteins in the cyanobacterium Synechocystis sp. PCC 6803

AbstractThe half-life times of photosystem I and II proteins were determined using 15N-labeling and mass spectrometry. The half-life times (30–75h for photosystem I components and <1–11h for the large photosystem II proteins) were similar when proteins were isolated from monomeric vs. oligomeric complexes on Blue-Native gels, suggesting that the two forms of both photosystems can interchange on a timescale of <1h or that only one form of each photosystem exists in thylakoids in vivo. The half-life times of proteins associated with either photosystem generally were unaffected by the absence of Small Cab-like proteins

The psbC start codon in Synechocystis sp. PCC 6803

AbstractThe translation start codon for psbC, the gene encoding CP43, a chlorophyll-binding protein of photosystem II, has been identified for the cyanobacterium Synechosystis sp. PCC 6803 using site-directed mutagenesis. An AUG codon, about 50 bases upstream from the end of psbD-I had previously been assumed to be the translation start site of psbC. However, the fact that the AUG codon is not present in psbC from several other organisms, whereas a GUG codon 14 bases upstream from the end of psbD-I is strictly conserved suggests that CP43 translation starts at the latter codon. Mutation of GUG, but not of AUG, led to a loss of CP43 and photoautotrophic growth, indicating that the GUG codon is the sole initiation site for translation of the CP43 protein in Synechocystis sp. PCC 6803

A Dedicated Type II NADPH Dehydrogenase Performs the Penultimate Step in the Biosynthesis of Vitamin K\u3csub\u3e1\u3c/sub\u3e in \u3ci\u3eSynechocystis\u3c/i\u3e and Arabidopsis

Mutation of Arabidopsis thaliana NAD(P)H DEHYDROGENASE C1 (NDC1; At5g08740) results in the accumulation of demethylphylloquinone, a late biosynthetic intermediate of vitamin K1. Gene coexpression and phylogenomics analyses showed that conserved functional associations occur between vitamin K biosynthesis and NDC1 homologs throughout the prokaryotic and eukaryotic lineages. Deletion of Synechocystis ndbB, which encodes for one such homolog, resulted in the same defects as those observed in the cyanobacterial demethylnaphthoquinone methyltransferase knockout. Chemical modeling and assay of purified demethylnaphthoquinone methyltransferase demonstrated that, by virtue of the strong electrophilic nature of S-adenosyl-Lmethionine, the transmethylation of the demethylated precursor of vitamin K is strictly dependent on the reduced form of its naphthoquinone ring. NDC1 was shown to catalyze such a prerequisite reduction by using NADPH and demethylphylloquinone as substrates and flavine adenine dinucleotide as a cofactor. NDC1 displayed Michaelis-Menten kinetics and was markedly inhibited by dicumarol, a competitive inhibitor of naphthoquinone oxidoreductases. These data demonstrate that the reduction of the demethylnaphthoquinone ring represents an authentic step in the biosynthetic pathway of vitamin K, that this reaction is enzymatically driven, and that a selection pressure is operating to retain type II NAD(P)H dehydrogenases in this process