SUPEROXIDE CONTRIBUTES TO THE RAPID INACTIVATION OF SPECIFIC SECONDARY DONORS OF THE PHOTOSYSTEM-II REACTION-CENTER DURING PHOTODAMAGE OF MANGANESE-DEPLETED PHOTOSYSTEM-II MEMBRANES

The role of superoxide in the mechanism of photoinactivation of the se condary donors of the reaction center of photosystem II membranes depl eted of Mn by extraction with NH2OH plus EDTA (NH2OH/EDTA-PSII) was as sessed. EPR analyses (g = 2 region) in continuous light, optical kinet ic spectrophotometric analyses of P-680(+) and Car(+), and A(T)-band e mission measurements were made after various durations of weak and str ong light treatment of NH2OH/EDTA-PSII in the presence and absence of superoxide dismutase, or of PSII electron accepters to suppress supero xide formation. Additionally, flash-induced variable fluorescence of c hlorophyll a and the capabilities of the membranes of photooxidize Mn2 + (in the presence of H2O2) via a high-affinity site (K-m similar to 1 80 nM) and to carry out the photoactivation of the Mn-cluster were det ermined. In the absence of any additions to the NH2OH/EDTA-PSII membra nes which were highly depleted of Mn, weak Light treatment caused rapi d (t(1/2)similar to 20 s) and parallel losses of (a) the similar to 10 mu s phase of P-680(+) reduction, which reflects the TyrZ --> P-680() reaction, (b) the amplitude of chlorophyll a variable fluorescence, (c) the capability to accumulate the TyrZ(+)-radical in continuous lig ht, and (d) the capability to photooxidize Mn2+/H2O2 in continuous Lig ht. As reported previously [Blubaugh et al. (1991) Biochemistry 30, 75 86-7597], a dark-stable 12-G-wide featureless EPR signal centered at g = 2.004 was formed rapidly during illumination, This signal previousl y 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 --> P-680(+) reaction. However, we failed to detect Car(+) formation by sensitive optical spectrophotometry and obtained no definable evidence for either a quencher of fluorescence o ther than P-680(+) itself or a slowing of the TyrZ --> P-680(+) reacti on. Addition of a saturating concentration (96 units/mL) of superoxide dismutase diminished the rate of photodamage(s) by similar to 30-fold , but did not abolish it completely. Superoxide dismutase similarly su ppressed strong light-induced photodamages, causing the loss of capabi lity to photooxidize Mn2+/H2O2, to carry out photoactivation, and to g enerate the A(T)-band emission as well as TyrZ(+) EPR signal. In contr ast to others, we found no evidence that the initial target(s) of phot odamage 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/EDTA-PSII is a decoupling of the redox activity of TyrZ from P-680. This occurs slowly when medi ated by P-680(+)/TyrZ(+) hut much more rapidly in the presence of supe roxide.