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 (t1/2 = 2-3 min) and slow (t1/2 = 3-4 h) losses of formation of the tyrosyl radicals Y(Z)+ and Y(D)+, respectively, and the rapid appearance (t1/2 = 0.8 min) of a 12-G-wide signal, centered at g = 2.004, which persisted at 4-degrees-C in subsequent darkness in rather constant abundance (approximately 1/2 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 greater-than-or-equal-to 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-mu-s decay of P680+, reflecting Y(Z) --> P680+, was rapidly (t1/2 = 0.8 min) replaced by an 80-140-mu-s decay, presumably reflecting Q(A)-/P680+ back-reaction. Photoinhibition 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 02 evolution is impaired is Chl/Car > Y(Z) > Y(D) >> P680, Pheo, Q(A).