The effects of NO and fluoride on the iron-quinone complex of the acceptor side of Photosystem II (PSII) are examined by X-and Q-band EPR spectroscopy. It is found that the EPR signal of the iron-nitrosyl complex changes upon addition of F-. The change is determined to be due to a superhyperfine interaction between the electronic spin (S = 3/2) and the fluorine nuclear spin (I = 1/2), indicating that both F- acid NO are bound to the same nonheme iron. To the best of our knowledge, this is the first report of simultaneous binding of both F- and NO to the same iron of a mononuclear nonheme protein. On the basis of an analysis of the hyperfine interaction, a cis F-Fe-NO coordination is indicated. Upon illumination of (NO, Cl-)- or (NO, F-)-treated PSII membranes at 200 K, new X-and Q-band EPR signals are observed in B(1)parallel to B and B(1)perpendicular to B modes. Quantitative simulations of these signals provide an unambiguous assignment to the iron-quinone complex, Q(A)(-){FeNO},(7) of the acceptor site of PSII. The exchange interaction between the iron-nitrosyl complex (S = 3/2) and the semiquinone Q(A)(-) radical (S = 1/2) is determined to be J = +0.5 cm(-1) (F-) and +1.3 cm(-1) (Cl-) for H-ex = JS(1)S(2). A distribution in the exchange coupling is required to satisfactorily simulate the EPR spectra. This distribution is correlated to small structural variations of the iron-quinone acceptor side of PSII.