Inhibition of Electron Transport in Photosystem II by NH₂OH: Further Evidence for Two Binding Sites

The reaction of hydroxylamine, a substrate analogue of the water-oxidizing complex (WOC), with spinach photosystem II (PSII) membranes has been further studied by using EPR spectroscopy to monitor the stepwise oxidation of donors and reduction of electron acceptors during successive low-temperature illuminations [Sivaraja, M., & Dismukes, G. C. (1988) Biochemistry 27, 3467–3475]. In addition to its well-known binding on the donor side of PSII, hydroxylamine binds in the dark with high affinity (K_D< 10 μM; <7 NH₂OH/PSII) to a site that structurally interacts with the primary electron acceptor FeQ_A^-. Binding in the dark to this acceptor site causes conversion of the normal g = 1.9 EPR signal for FeQ_A^-to g = 2.1 on the first turnover. This light-induced signal forms with or without exogenous electron acceptors and is maximized when Q_Ais oxidized in the dark. The original g = 1.9 form recovers upon successive turnovers as the NH₂OH is consumed. The binding is blocked by addition of DCMU, which displaces the secondary electron acceptor Q_B, or by the presence of excess quinol and NH₂OH. These results indicate that the binding site for NH₂OH overlaps with or interacts with the binding site for Q_B. The EPR microwave power saturation of the g = 2.1 signal at 5.5 K is similar to that found for the endogenous ferrosemiquinone acceptors. These results indicate a structural change in the primary acceptor site upon binding NH₂OH, with no change in oxidation state of the iron or the semiquinone. In contrast, NH₂OH does not bind in the dark to PSII centers exhibiting the other major form of the primary acceptor, which exhibit the g = 1.82 EPR signal, since no change in the EPR signal is observed. We also find that the high-affinity binding of NH₂OH within the WOC produces no observable EPR-active products in the dark. Following illumination, this site is characterized by loss of formation of both of the EPR signals for photooxidized manganese in the S₂state, without loss of photoreduction of the primary acceptor. We conclude that this binding site is closely associated with manganese, since there exists no blockage in the photooxidation of other donors like high-potential cytochrome b₅₅₉or signal II (¹⁶⁰Tyr-D₁protein). A new EPR signal can be observed in both untreated and NH₂OH-treated PSII membranes extending over a 1000-G line width and centered at g = 2. It forms in the presence of an exogenous quinone acceptor upon multiple turnovers at 255 K in PSII membranes, or by 200 K illumination of NH₂OH-inhibited membranes. It is eliminated by DCMU and by removal of manganese upon treatment with excess NH₂OH. The possible identity of the species responsible for this new signal is discussed.