Photoinhibition of Hydroxylamine-Extracted Photosystem II Membranes: Identification of the Sites of Photodamage

Electron paramagnetic resonance (EPR) analyses (g = 2 region) and optical spectrophotometric analyses of P₆₈₀⁺ were made of NH₂OH-extracted photosystem II (PSII) membranes after various durations of weak-light photoinhibition, in order to identify the sites of damage responsible for the observed kinetic components of the loss of electron transport [Blubaugh, D. J., & Cheniae, G. M. (1990) Biochemistry 29, 5109-5118]. The EPR spectra, recorded in the presence of K₃Fe(CN)₆, gave evidence for rapid (t_1/2 = 2–3 min) and slow (t_1/2 = 3–4 h) losses of formation of the tyrosyl radicals Y_z⁺ and Y_D⁺, respectively, and the rapid appearance (t_1/2 = 0.8 min) of a 12-G-wide signal, centered at g = 2.004, which persisted at 4 °C in subsequent darkness in rather constant abundance (ç½ spin Per PSII). This latter EPR signal is correlated with quenching of the variable chlorophyll a fluorescence yield and is tentatively attributed to a carotenoid (Car) cation. Exogenous reductants (NH₂OH ≥ NH₂NH₂ > DPC ≫ Mn²⁺) were observed to reduce the quencher, but did not reverse other photoinhibition effects. An additional 10-G-wide signal, tentatively attributed to a chlorophyll (Chl) cation, is observed during illumination of photoinhibited membranes and rapidly decays following illumination. The amplitude of formation of the oxidized primary electron donor, P₆₈₀⁺, was unaffected throughout 120 min of photoinhibition, indicating no impairment of charge separation from P₆₈₀, via pheophytin (Pheo), to the first stable electron acceptor, Q_A. However, a 4-μs decay of P₆₈₀⁺, reflecting Y_z → P₆₈₀⁺, was rapidly (t_½ = 0.8 min) replaced by an 80–140-μs decay, presumably reflecting Q_A⁻/P₆₈₀⁺ back-reaction. Photoinhioition caused no discernible decoupling of the antenna chlorophyll from the reaction center complex. We conclude that the order of susceptibility of PSII components to photodamage when O₂ evolution is impaired is Chl/Car > Y_z > Y_D ≫ P₆₈₀, Pheo, Q_A.