EVIDENCE FOR CN- BINDING AT THE PS-II NONHEME FE2+ - EFFECTS ON THE EPR SIGNAL FOR Q(A)-FE2+ AND ON Q(A)/Q(B) ELECTRON-TRANSFER

Earlier studies have demonstrated that NO binds to the non-heme iron of the PS II ferroquinone complex in competition with the physiological ligand CO2/HCO3- (Petrouleas, V. and Diner, B.A. (1990) Biochim. Biophys. Acta 1015,131-140; Diner, B.A. and Petrouleas, V. (1990) Biochim. Biophys. Acta 1015, 141-149). We examine in this paper the effect of cyanide, also a potential iron chelator. Competition experiments involving CN- and NO show that 50 mM CN- at pH 6.5 eliminates the EPR signal at g = 4 arising from the Fe2+-NO adduct. Illumination of CN--treated PS II preparations under conditions which induce single charge separation produces a new EPR signal at g = 1.98. The temperature and power dependence indicate that this signal is directly or indirectly associated with a transition metal species. The signal is produced with undiminished intensity upon illumination in the presence of hydroxylamine as an exogenous electron donor and of DCMU, indicating that it originates from an acceptor side species prior to the DCMU block. Upon successive one-electron charge separations the g = 1.98 signal oscillates with period of two. These results strongly suggest that the g = 1.98 signal originates from the state Q(A)-Fe2+. An approximate titration of the g = 1.98 signal development as a function of the total cyanide concentration at pH 6.5 indicates a k(d) of 50-80 mM, significantly higher than the k(d) for cyanide-NO competition, estimated to be in the range of 10 mM. It is likely that, while displacement of NO requires the binding of one cyanide molecule, development of the modified Q(A)-Fe2+ signal at g = 1.98 requires the binding of more than one cyanide molecules. The kinetics of the fluorescence relaxation following saturating flashes show only subtle differences over the concentration range at which cyanide displaces NO and probably bicarbonate as well. Treatment with higher concentrations of cyanide at pH 6.5 causes an inversion in the phase of the oscillation pattern that characterizes the decay of the fluorescence yield. The latter effect becomes more pronounced at higher pH levels. The absence of a slowing of the fluorescence relaxation in the presence of cyanide may indicate that CN- can fulfill the same role as bicarbonate, perhaps in promoting proton transfer coupled to interquinone electron transfer. Only the relative rates of the two interquinone electron transfer reactions are reversed.