Photoproduction of Catalase-Insensitive Peroxides on the Donor Side of Manganese-Depleted Photosystem II: Evidence with a Specific Fluorescent Probe

Abstract

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.

Involvement of molecular oxygen in the donor-side photoinhibition of Mn-depleted photosystem II membranes

“…and Ca 2? at catalytic concentrations (Yanykin et al 2010(Yanykin et al , 2013Khorobrykh et al 2011). In this study, we experimentally verified the suggestion that molecular oxygen (rather than superoxide anion radical formed on the acceptor side of PSII) is directly involved in the donor-side photoinhibition of Mn-depleted PSII membranes.…”

“…It is accompanied by the formation of hydroperoxides, and it has been suggested that it may lead to the donor-side photoinhibition of PSII Yanykin et al 2010;Khorobrykh et al 2011).…”

“…Using a lipophilic fluorescence probe specific to peroxides, we demonstrated the light-induced formation of peroxides on the donor side of PSII in Mn-depleted PSII membranes, and the peroxides were insensitive to exogenously added catalase (Khorobrykh et al 2011).…”

Lipid Peroxide-Derived Short-Chain Carbonyls Mediate Hydrogen Peroxide-Induced and Salt-Induced Programmed Cell Death in Plants

“…The reactions of Spy-LHP with O 2 -c , alkyl hydroperoxyl radical, nitric oxide, and peroxinitrite are insignificant (Soh et al, 2007). Its reaction with H 2 O 2 is very slow and can be distinguished from that with LOOH by the measurement of fluorescence increase kinetics (Khorobrykh et al, 2011).…”

“…The supernatant was collected, and its fluorescence was measured with a spectrofluorometer (FP 8300; JASCO) with excitation at 524 nm and emission at 538 nm. To determine the amount of peroxides, a standard curve was prepared with the model LOOH m-chloroperbenzoic acid (Khorobrykh et al, 2011).…”