The photosystem II subunit PsbS is essential for excess energy dissipation (qE); however, both lutein and zeaxanthin are needed for its full activation. Based on previous work, two models can be proposed in which PsbS is either 1) the gene product where the quenching activity is located or 2) a proton-sensing trigger that activates the quencher molecules. The first hypothesis requires xanthophyll binding to two PsbS-binding sites, each activated by the protonation of a dicyclohexylcarbodiimide-binding lumen-exposed glutamic acid residue. To assess the existence and properties of these xanthophyll-binding sites, PsbS point mutants on each of the two Glu residues PsbS E122Q and PsbS E226Q were crossed with the npq1/npq4 and lut2/ npq4 mutants lacking zeaxanthin and lutein, respectively. Double mutants E122Q/npq1 and E226Q/npq1 had no qE, whereas E122Q/lut2 and E226Q/lut2 showed a strong qE reduction with respect to both lut2 and single glutamate mutants. These findings exclude a specific interaction between lutein or zeaxanthin and a dicyclohexylcarbodiimide-binding site and suggest that the dependence of nonphotochemical quenching on xanthophyll composition is not due to pigment binding to PsbS. To verify, in vitro, the capacity of xanthophylls to bind PsbS, we have produced recombinant PsbS refolded with purified pigments and shown that Raman signals, previously attributed to PsbS-zeaxanthin interactions, are in fact due to xanthophyll aggregation. We conclude that the xanthophyll dependence of qE is not due to PsbS but to other pigment-binding proteins, probably of the Lhcb type.
Nonphotochemical quenching (NPQ)2 is a protective mechanism against overexcitation of photosystem II, consisting of heat dissipation of excess energy. This process, although present in all photosynthetic organisms, has been particularly studied in higher plants and green algae. Early work identified thylakoid lumen acidification as an essential factor for NPQ (1). Excess light increases proton pumping into the thylakoid lumen, which elicits chlorophyll fluorescence quenching dependent on protein protonation events. This is shown by the inhibitory effects of DCCD, a protein-modifying agent that covalently binds to protonatable residues in hydrophobic environments (2). It has been found that the photosystem II subunit PsbS, a DCCD-binding protein (3), is required for NPQ and, in particular, for its rapid and reversible component qE (4). In fact, the capacity for qE depends on the stoichiometric presence of PsbS polypeptide (5). Some insight into the importance of xanthophylls for the NPQ mechanism can be gained from the xanthophyll biosynthesis mutants found in NPQ-depleted plants. In the npq1 mutant, which is unable to convert violaxanthin into zeaxanthin upon lumen acidification, qE is reduced, whereas npq2, which constitutively accumulates zeaxanthin, has faster NPQ induction and slower relaxation kinetics (6). In the lut2 mutant, lacking lutein (7), and in the lut2/npq2 double mutant, which has zeaxanthin as the only xanthophyll (8, ...