The role of superoxide in the mechanism of photoinactivation of the secondary donors of the reaction center of photosystem II membranes depleted of Mn by extraction with NH2OH plus EOT A (NH2OH/EDTA-PSII) was assessed. EPR analyses (g = 2 region) in continuous light, optical kinetic spectrophotometric analyses of P680+ and Car+, and AT-band emission measurements were made after various durations of weak and strong light treatment of NH2OH/EDTA-PSII in the presence and absence of superoxide dismutase, or of PSII electron acceptors to suppress superoxide formation. Additionally, flash-induced variable fluorescence of chlorophyll a and the capabilities of the membranes of photooxidize Mn2+ (in the presence of H2O2) via a high-affinity site (Km ~180 nM) and to carry out the photoactivation of the Mn-cluster were determined. In the absence of any additions to the NH2OH/EDTA-PSII membranes which were highly depleted of Mn, weak light treatment caused rapid (tm ~20 s) and parallel losses of (a) the ~10 µs phase of P680+ reduction, which reflects the TyrZ → P680+ reaction, (b) the amplitude of chlorophyll a variable fluorescence, (c) the capability to accumulate the TyrZ+-radical in continuous light, and (d) the capability to photooxidize Mn2+/H2O2 in continuous light. As reported previously [Blubaugh et al. (1991) Biochemistry 30, 7586-7597], a dark-stable 12-G-wide featureless EPR signal centered at g = 2.004 was formed rapidly during illumination. This signal previously was tentatively identified as a Car+ radical and was suggested to contribute to the quenching of chlorophyll a variable fluorescence and to the slowing of the TyrZ → P680+ reaction. However, we failed to detect Car+ formation by sensitive optical spectrophotometry and obtained no definable evidence for either a quencher of fluorescence other than P680+ itself or a slowing of the TyrZ → P680+ reaction. Addition of a saturating concentration (96 units/mL) of superoxide dismutase diminished the rate of photodamage(s) by ~30-fold, but did not abolish it completely. Superoxide dismutase similarly suppressed strong light-induced photodamages, causing the loss of capability to photooxidize MN2+/H2O2, to carry out photoactivation, and to generate the AT-band emission as well as TyrZ+ EPR signal. In contrast to others, we found no evidence that the initial target(s) of photodamage is (are) different in weak versus strong light treatment. The totality of the results suggests that the initial event in either weak light or strong light photodamage of NH2OH/EDTAPSII is a decoupling of the redox activity of TyrZ from P680. This occurs slowly when mediated by P680+/TyrZ+ but much more rapidly in the presence of superoxide.
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