Binding of plastoquinone B (QB) within D1 protein of Photosystem II

This Key Event describes the competitive displacement of the endogenous electron carrier plastoquinone B (Q_B) from its binding site in the D1 protein (encoded by psbA) by an exogenous stressor, typically a Photosystem II (PSII) inhibitor. Under normal conditions, a plastoquinone molecule enters the Q_B site, accepts two electrons from the primary quinone Q_A and two protons from the stroma, becomes plastoquinol PQH₂, and then leaves the site to continue the electron transport chain. Stressor molecules share structural similarities with the quinone ring of plastoquinone. They enter the Q_B pocket and form stable hydrogen bonds with specific amino acid residues, most critically Serine 264 and Histidine 215. Because the stressor binds with higher affinity or slower dissociation than the natural quinone. This physically blocks the transfer of electrons from Q_A to Q_B, and thus reduced photosynthetic efficiency..

The binding of PSII inhibitors to the Q_B site in the D1 protein can be detected and quantified using a combination of computational, biochemical, biophysical, and functional approaches, each providing complementary lines of evidence.

In silico approaches, including quantitative structure–activity relationship (QSAR) modelling and molecular docking, are widely used to predict the affinity and binding orientation of PSII inhibitors within the Q_B niche of the D1 protein. These methods support chemical screening and mechanistic interpretation of structure–binding relationships (Arnaud et al., 1994; Battaglino, Grinzato and Pagliano, 2021).

Radioligand binding assays represent a classical and highly specific experimental approach, in which radiolabelled herbicides are used to directly quantify competitive displacement of plastoquinone or reference inhibitors from isolated thylakoid membranes or PSII preparations (Tischer and Strotmann, 1977; Vermaas, Renger and Arntzen, 1984). These assays provide quantitative binding constants and competitive inhibition profiles.

Fluorescence-based techniques, including chlorophyll a fluorescence measurements, are frequently applied as indirect but sensitive indicators of QB-site occupation. Inhibitor binding disrupts electron transfer from Q_A to Q_B, leading to characteristic changes in fluorescence yield and PSII photochemical efficiency (Sundby, Chow and Anderson, 1993; Maxwell and Johnson, 2000). These methods are particularly useful for whole-cell or intact-tissue assessments.

Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) provide label-free, real-time measurements of binding kinetics and thermodynamics between PSII inhibitors and PSII protein complexes or isolated D1 fragments. These approaches allow direct determination of binding affinity, stoichiometry, and energetics (Piletska, Piletsky and Rouillon, 2006; Zimmermann et al., 2006).

Resistance mutant analyses, based on naturally occurring or experimentally induced mutations in the psbA gene encoding the D1 protein, offer strong mechanistic evidence for Q_B-site binding. Altered sensitivity to PSII inhibitors in mutants affecting key QB-site residues (e.g. Ser-264/268) directly links chemical binding to functional inhibition and photosynthetic impairment (Sundby, Chow and Anderson, 1993; Alfonso et al., 1996; Oettmeier, 1999).

Finally, structural biology approaches, including X-ray crystallography of PSII complexes, have provided direct visualization of herbicide occupancy within the QB site, confirming binding modes and key amino-acid interactions at atomic resolution (Broser et al., 2011; Zimmermann et al., 2006).

This KE is applicable to oxygenic photosynthetic organisms that possess a functional PSII complex containing the D1 protein encoded by psbA and a conserved Q_B binding site. The scientific basis for this domain of applicability is the high structural and functional conservation of the Q_B niche within the D1 protein across cyanobacteria, algae, and higher plants, which underpins both endogenous plastoquinone binding and competitive binding by PSII-inhibiting chemicals.

The Weight of Evidence supporting this KE is high, based on:

• Direct evidence from radioligand binding, structural studies, and biophysical measurements demonstrating inhibitor occupancy of the QB site;

• Indirect functional evidence from chlorophyll fluorescence and electron transport inhibition assays; and

• Biological plausibility and consistency demonstrated by resistance mutations and cross-species conservation of the binding site.

The strong mechanistic understanding and consistency across experimental systems support high confidence in both the domain of applicability and the causal role of this KE within PSII inhibition-related adverse outcome pathways.

Alfonso, M., Pueyo, J.J., Gaddour, K., Etienne, A.-L., Kirilovsky, D. and Picorel, R. (1996). Induced new mutation of D1 serine-268 in soybean photosynthetic cell cultures produced atrazine resistance, increased stability of S2QB- and S3QB-states, and increased sensitivity to light stress. Plant Physiology, 112(4), 1499–1508.

Arnaud, L., Taillandier, G., Kaouadji, M., Ravanel, P. and Tissut, M. (1994). Photosynthesis inhibition by phenylureas: A QSAR approach. Ecotoxicology and Environmental Safety, 28(2), 121–133.

Battaglino, B., Grinzato, A. and Pagliano, C. (2021). Binding properties of photosynthetic herbicides with the QB site of the D1 protein in plant photosystem II: A combined functional and molecular docking study. Plants, 10(8).

Broser, M., Glöckner, C., Gabdulkhakov, A., Guskov, A., Buchta, J., Kern, J., Müh, F., Dau, H., Saenger, W. and Zouni, A. (2011). Structural basis of cyanobacterial photosystem II inhibition by the herbicide terbutryn. Journal of Biological Chemistry, 286(18), 15964–15972.

Giardi, M.T. and Pace, E. (2006). Photosynthetic proteins for technological applications. In: Giardi, M.T. and Piletska, E.V. (eds), Biotechnological Applications of Photosynthetic Proteins: Biochips, Biosensors and Biodevices. Springer, Boston, MA, pp. 147–154.

Oettmeier, W. (1999). Herbicide resistance and supersensitivity in photosystem II. Cellular and Molecular Life Sciences, 55(10), 1255–1277.

Piletska, E.V., Piletsky, S.A. and Rouillon, R. (2006). Sensor systems for photosystem II inhibitors. In: Giardi, M.T. and Piletska, E.V. (eds), Biotechnological Applications of Photosynthetic Proteins: Biochips, Biosensors and Biodevices. Springer, Boston, MA, pp. 130–146.

Sundby, C., Chow, W.S. and Anderson, J.M. (1993). Effects on photosystem II function, photoinhibition, and plant performance of the spontaneous mutation of serine-264 in the photosystem II reaction center D1 protein in triazine-resistant Brassica napus L. Plant Physiology, 103(1), 105–113.

Tischer, W. and Strotmann, H. (1977). Relationship between inhibitor binding by chloroplasts and inhibition of photosynthetic electron transport. Biochimica et Biophysica Acta (Bioenergetics), 460(1), 113–125.

Vermaas, W.F., Renger, G. and Arntzen, C.J. (1984). Herbicide/quinone binding interactions in photosystem II. Zeitschrift für Naturforschung C, 39(5), 368–373.

Zimmermann, K., Heck, M., Frank, J., Kern, J., Vass, I. and Zouni, A. (2006). Herbicide binding and thermal stability of photosystem II isolated from Thermosynechococcus elongatus. Biochimica et Biophysica Acta – Bioenergetics, 1757(2), 106–114.