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

Singlet oxygen ((1)O(2)) is a harmful species that functions also as a signaling molecule. In chloroplasts, (1)O(2) is produced via charge recombination reactions in photosystem II, but which recombination pathway(s) produce triplet Chl and (1)O(2) remains open. Furthermore, the role of (1)O(2) in photoinhibition is not clear. We compared temperature dependences of (1)O(2) production, photoinhibition, and recombination pathways. (1)O(2) production by pumpkin thylakoids increased from −2 to +35°C, ruling out recombination of the primary charge pair as a main contributor. S(2)Q(A) (−) or S(2)Q(B) (−) recombination pathways, in turn, had too steep temperature dependences. Instead, the temperature dependence of (1)O(2) production matched that of misses (failures of the oxygen (O(2)) evolving complex to advance an S‐state). Photoinhibition in vitro and in vivo (also in Synechocystis), and in the presence or absence of O(2), had the same temperature dependence, but ultraviolet (UV)‐radiation‐caused photoinhibition showed a weaker temperature response. We suggest that the miss‐associated recombination of P(680) (+)Q(A) (−) is the main producer of (1)O(2). 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)O(2) or long‐lived P(680) (+) as damaging agents, dominate in red light.