Under high light, oxygenic photosynthetic organisms avoid photodamage by thermally dissipating absorbed energy, which is called nonphotochemical quenching. In green algae, a chlorophyll and carotenoid-binding protein, light-harvesting complex stress-related (LHCSR3), detects excess energy via a pH drop and serves as a quenching site. Using a combined in vivo and in vitro approach, we investigated quenching within LHCSR3 from Chlamydomonas reinhardtii. In vitro two distinct quenching processes, individually controlled by pH and zeaxanthin, were identified within LHCSR3. The pH-dependent quenching was removed within a mutant LHCSR3 that lacks the residues that are protonated to sense the pH drop. Observation of quenching in zeaxanthin-enriched LHCSR3 even at neutral pH demonstrated zeaxanthin-dependent quenching, which also occurs in other light-harvesting complexes. Either pH- or zeaxanthin-dependent quenching prevented the formation of damaging reactive oxygen species, and thus the two quenching processes may together provide different induction and recovery kinetics for photoprotection in a changing environment.
