Singlet oxygen production by photosystem II is caused by misses of the oxygen evolving complex

Singlet oxygen (¹ O₂ ) is a harmful species that functions also as a signaling molecule. In chloroplasts, ¹ O₂ is produced via charge recombination reactions in photosystem II, but which recombination pathway(s) produce triplet Chl and ¹ O₂ remains open. Furthermore, the role of ¹ O₂ in photoinhibition is not clear. We compared temperature dependences of ¹ O₂ production, photoinhibition, and recombination pathways. ¹ O₂ production by pumpkin thylakoids increased from -2 to +35°C, ruling out recombination of the primary charge pair as a main contributor. S₂ Q_A ^- or S₂ Q_B ^- recombination pathways, in turn, had too steep temperature dependences. Instead, the temperature dependence of ¹ O₂ production matched that of misses (failures of the oxygen (O₂ ) evolving complex to advance an S-state). Photoinhibition in vitro and in vivo (also in Synechocystis), and in the presence or absence of O₂ , had the same temperature dependence, but ultraviolet (UV)-radiation-caused photoinhibition showed a weaker temperature response. We suggest that the miss-associated recombination of P₆₈₀ ⁺ Q_A ^- is the main producer of ¹ O₂ . Our results indicate three parallel photoinhibition mechanisms. The manganese mechanism dominates in UV radiation but also functions in white light. Mechanisms that depend on light absorption by Chls, having ¹ O₂ or long-lived P₆₈₀ ⁺ as damaging agents, dominate in red light.