Reduction of the Mn cluster of the water-oxidizing enzyme by nitric oxide:: Formation of an S-2 state

The manganese cluster of the oxygen-evolving enzyme of photosystem II is chemically reduced upon interaction with nitric oxide at -30 degreesC. The state formed gives rise to an S = 1/2 multiline EPR signal [Goussias, Ch., Ioannidis, N., and Petrouleas, V. (1997) Biochemistry 36, 9261] that is attributed to a Mn(II)-Mn(III) dimer [Sarrou, J., Ioannidis, N., Deligiannakis, Y., and Petrouleas, V. (1998) Biochemistry 37, 3581]. In this work, we sought to establish whether the state could be assigned to a specific, reduced S state by using flash oxymetry, chlorophyll a fluorescence, and electron paramagnetic resonance spectroscopy. With the Joliot-type O-2 electrode, the first maximum of oxygen evolution was observed on the sixth or seventh flash. Three saturating preflashes were required to convert the flash pattern characteristic of NO-reduced samples to that of the untreated control (i.e., O-2 evolution maximum on the third flash). Measurements of the S state-dependent level of chlorophyll fluorescence in NO-treated PSII showed a three-flash downshift compared to untreated controls. In the EPR study, the maximum S-2 multiline EPR signal was observed after the fourth flash. The results from all three methods are consistent with the Mn cluster being in a redox state corresponding to an S-2 state in a majority of centers after treatment with NO. We were unable to generate the Mn(H)-Mn(III) multiline signal using hydrazine as a reductant; it appears that the valence distribution and possibly the structure of the Mn cluster in the S-2 state are dependent on the nature of the reductant that is used.