The semiquinone radical Q(A)(-) has been studied by Electron Spin-Echo Envelope Modulation (ESEEM) spectroscopy in Photosystem II membranes at various pH values. The observed nuclear modulations have been assigned by the use of two-dimensional Hyperfine Sublevel Correlation Spectroscopy (HYSCORE) and numerical simulations. Two protein N-14 nuclei (N-I and N-II) were found to be magnetically coupled with the Q(A)(-) spin, and on the basis of N-14-NQR and N-14-ESEEM data from the literature, N-I is assigned to an amide nitrogen from the protein backbone while N-II is assigned to the amino nitrogen, N delta, of an imidazole. A physical explanation for such couplings is suggested where the coupling occurs through II-bonds from the protein to the carbonyls of the semiquinone. In PSII membranes treated with CN-, only the Nr coupling is present above pH 8.5 while both N-I and N-II couplings are present at lower pH values. In samples treated at high pH to remove the iron, both N-I and N-II couplings are present throughout the pH range studied but at pH <6 these couplings strengthen. These results are interpreted in terms of a model based bn the structure of the bacterial reaction center and involving two determining factors. (1) The nonheme iron, when present, is liganded to the imidazole that H-bonds to one of the Q(A)(-) carbonyls. This physical attachment of the imidazole to the iron limits the strength of the H-bond to Q(A)(-) (2) A pH-dependent group on the protein-controls the strength of the H-bonds to Q(A)(-) The pK(a) of this group is influenced by the biochemical treatment used to uncouple the iron, being around pH 7.5 in CN--treated PSII but around pH 6 in high pH-treated PSII. It is proposed that such a pH effect on the II-bond strength exists in untreated PSII and that earlier observations of pH-induced changes in the EPR signal from the semiquinone iron may reflect this change.