Incubation of Fe(II) cations with Mn-depleted PSII membranes (PSII(-Mn)) under weak continuous light is accompanied by blocking of the high-affinity, Mn-binding (HAZ) site with ferric cations (Semin, B. K. et al. Biochemistry 2002, 41, 5854-5864). In this study we investigated the blocking yield under single-turnover flash conditions. The flash-probe fluorescence method was used to estimate the blocking efficiency. We found that the yield of blocking increases with flash number and reaches 50% after 7 flashes. When the dark interval between the flashes (Delta t) was varied, we found that the percentage of blocking decreases at Delta t < 100 ms (t(1/2), 4-10 ms). No inhibition of the blocking yield was found at longer time intervals ( as with photoactivation). This result shows the necessity of a dark rearrangement during the blocking process ( the dual-site hypothesis described in the text) and indicates the formation of a binuclear iron center. During the blocking experiments, we found a binary oscillation of the F-max elicited during a train of flashes. The oscillations were observed only in the presence of Fe( II) cations or other electron donors ( including Mn(II)) but not in the presence of Ca2+. Chelators had no effect on the oscillations. Our results indicate that the oscillations are due to processes on the acceptor side of PSII and to the appearance of "acceptor X" after odd flashes. Acceptor X is reduced by Q(A)(-) at very high rate (<< 2 ms), is not sensitive to DCMU, and is rather stable in the dark (t(1/2) approximate to 2 min). These properties are similar to those of nonheme Fe(III) (Fe(III)(NHI)). When Fe( II) NHI was oxidized with ferricyanide (Fe(CN)(6)), the fluorescence decay kinetics and yield of fluorescence were identical to those observed when the sample was exposed to 1 flash prior to the fluorescence measurement. We suggest that acceptor X is Fe(III) NHI, oxidized by the semiquinone form of Q(B)(-). This is similar to the mechanism of "reduction- induced oxidation of Fe(II)(NHI)" by exogenous quinones reported in the literature. We suggest that involvement of Q(B)(-) in the oxidation of Fe(II) NHI in PSII(-Mn) membranes is due to the modification of the QB- binding site and increase of its redox potential resulting from extraction of the functional Mn cluster.