The reaction of hydroxylamine with the O2-evolving center of photosystem II (PSII) in the S1state delays the advance of the H2O-oxidation cycle by two charge separations. In this paper, we compare and contrast the reactions of hydroxylamine and N-methyl-substituted analogues with the electron-donor side of PSII in both O2-evolving and inactivated [tris(hydroxymethyl)aminomethane- (Tris-) washed] spinach PSII membrane preparations. We have employed low-temperature electron paramagnetic resonance (EPR) spectroscopy in order to follow the oxidation state of the Mn complex in the O2-evolving center and to detect radical oxidation products of hydroxylamine. When the reaction of hydroxylamine with the S1state in O2-evolving membranes is allowed to proceed to completion, the S2-state multiline EPR signal is suppressed until after three charge separations have occurred. Chemical removal of hydroxylamine from treated PSII membrane samples prior to illumination fails to reverse the effects of the dark reaction, which argues against an equilibrium coordination of hydroxylamine to a site in the O2-evolving center. Instead, the results indicate that the Mn complex is reduced by two electrons by hydroxylamine, forming the S-1state. An additional two-electron reduction of the Mn complex to a labile “S-3” state probably occurs by a similar mechanism, accounting for the release of Mn(II) ions upon prolonged dark incubation of O2-evolving membranes with high concentrations of hydroxylamine. In N,N-dimethylhydroxylamine-treated, Tris-washed PSII membranes, which lack O2 evolution activity owing to loss of the Mn complex, a large yield of dimethyl nitroxide radical is produced immediately upon illumination at temperatures above 0 °C. The dimethyl nitroxide radical is not observed upon illumination under similar conditions in O2-evolving PSII membranes, suggesting that one-electron photooxidations of hydroxylamine do not occur in centers that retain a functional Mn complex. We suggest that the flash-induced N2 evolution observed in hydroxylamine-treated spinach thylakoid membrane preparations arises from recombination of hydroxylamine radicals formed in inactivated O2-evolving centers.
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