1D- and 2D-ESEEM Study of the Semiquinone Radical QA⁻ of Photosystem II

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 14 N nuclei (N I and N II ) were found to be magnetically coupled with the Q A - spin, and on the basis of 14 N-NQR and 14 N-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, Nδ, of an imidazole. A physical explanation for such couplings is suggested where the coupling occurs through H-bonds from the protein to the carbonyls of the semiquinone. In PSII membranes treated with CN -, only the N I 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 on 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 p K 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 H-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.