Photoproduction of catalase-insensitive peroxides on the donor side of manganese-depleted photosystem II: evidence with a specific fluorescent probe.

The photoproduction of organic peroxides (ROOH) in photosystem II (PSII) membranes was studied using the fluorescent probe Spy-HP. Two types of peroxide, highly lipophilic ones and relatively hydrophilic ones, were distinguished by the rate of reaction with Spy-HP; the former oxidized Spy-HP to the higher fluorescent form Spy-HPOx within 5 min, while the latter did so very slowly (the reaction was still not completed after 180 min). The level of photoproduction of these peroxides was significantly larger in the alkaline-treated, Mn-depleted PSII membranes than that in the untreated membranes, and it was suppressed by an artificial electron donor (diphenylcarbazide or ferrocyanide) and by the electron transport inhibitor diuron. Postillumination addition of Fe(2+) ions, which degrade peroxides by the Fenton mechanism, abolished the accumulation of Spy-HPOx, but catalase did not change the peroxide level, indicating that the detected species were organic peroxides, excluding H(2)O(2). These results agreed with our previous observation of an electron transport-dependent O(2) consumption on the PSII donor side and indicated that ROOH accumulated via a radical chain reaction that started with the formation of organic radicals on the donor side. Illumination (λ > 600 nm; 1500 µmol of photons m(-2) s(-1)) of the Mn-depleted PSII membranes for 3 min resulted in the formation of nearly 200 molecules of hydrophilic ROOH per reaction center, but only four molecules of highly lipophilic ROOH. The limited formation of the latter was due to the limited supply of its precursor to the reaction, suggesting that it represented structurally fixed peroxides, i.e., either protein peroxides or peroxides of the lipids tightly bound to the core complex. These ROOH forms, likely including several species derived from lipid peroxides, may mediate the donor side-induced photoinhibition of PSII via protein modification.

Photoconsumption of molecular oxygen on both donor and acceptor sides of photosystem II in Mn-depleted subchloroplast membrane fragments.

Oxygen consumption in Mn-depleted photosystem II (PSII) preparations under continuous and pulsed illumination is investigated. It is shown that removal of manganese from the water-oxidizing complex (WOC) by high pH treatment leads to a 6-fold increase in the rate of O(2) photoconsumption. The use of exogenous electron acceptors and donors to PSII shows that in Mn-depleted PSII preparations along with the well-known effect of O(2) photoreduction on the acceptor side of PSII, there is light-induced O(2) consumption on the donor side of PSII (nearly 30% and 70%, respectively). It is suggested that the light-induced O(2) uptake on the donor side of PSII is related to interaction of O(2) with radicals produced by photooxidation of organic molecules. The study of flash-induced O(2) uptake finds that removal of Mn from the WOC leads to O(2) photoconsumption with maximum in the first flash, and its yield is comparable with the yield of O(2) evolution on the third flash measured in the PSII samples before Mn removal. The flash-induced O(2) uptake is drastically (by a factor of 1.8) activated by catalytic concentration (5-10microM, corresponding to 2-4 Mn per RC) of Mn(2+), while at higher concentrations (>100microM) Mn(2+) inhibits the O(2) photoconsumption (like other electron donors: ferrocyanide and diphenylcarbazide). Inhibitory pre-illumination of the Mn-depleted PSII preparations (resulting in the loss of electron donation from Mn(2+)) leads to both suppression of flash-induced O(2) uptake and disappearance of the Mn-induced activation of the O(2) photoconsumption. We assume that the light-induced O(2) uptake in Mn-depleted PSII preparations may reflect not only the negative processes leading to photoinhibition but also possible participation of O(2) or its reactive forms in the formation of the inorganic core of the WOC.

Reconstitution of the water-oxidizing complex in manganese-depleted photosystem II preparations using synthetic Mn complexes: a fluorine-19 NMR study of the reconstitution process.

Reconstitution of Mn-depleted photosystem II (PSII) particles was examined with synthetic trinuclear Mn complexes of newly developed tripod ligands. Rates of the electron transfer and oxygen evolution were up to 74-86 and 52-56% of those measured in native PSII. These values are higher than those for the PSII reconstituted by MnCl(2). The role of the tripod ligands during the reconstitution process was examined by (19)F NMR. Due to the high NMR sensitivity of the (19)F nucleus and the low abundance of fluorine atoms in natural PSII, it was possible to selectively observe the fluorine atoms on the tripod ligand. It was shown that the tripod ligands were released from the Mn complex after the reconstitution. We propose that the primary step in the reconstitution process is the prebinding of the Mn complex to the hydrophobic part of the PSII particle.