0162-0134/00/$ - see front matter q2000 Elsevier Science Inc. All rights reserved. PII S0162-0134 ( 99 ) 00173-7 Friday Apr 07 11:05 AM StyleTag -- Journal: JIB (Journal of Inorganic Biochemistry) Article: 6271 www.elsevier.nl/locate/jinorgbio Journal of Inorganic Biochemistry 79 (2000) 339–345 Effect of pH on the semiquinone radical Q A y in CN-treated photosystem II: study by hyperfine sublevel correlation spectroscopy Yiannis Deligiannakis a, *, A. William Rutherford b a The Institute of Materials Science, NCSR ‘Democritos’, Aghia Paraskevi, 15310 Athens, Greece b Section de Bioenergetique (URA CNRS 2096), Departement de Biologie Cellulaire et Moleculaire, CEA Saclay, F-91191 Gif-sur-Yvette, France ´ ´ ´ ´ Received 24 April 1999; accepted 27 July 1999 Abstract The semiquinone radical Q A y has been studied by electron spin echo envelope modulation (ESEEM) spectroscopy in Photosystem II membranes treated with CN y at various pH values. Two protein 14 N nuclei (N I and N II ) were found to be magnetically coupled with the Q A y spin. N I is assigned to an amide nitrogen from the protein backbone while N II is assigned to the amino nitrogen, N ´ , of an imidazole. Above pH 8.5 only the N I coupling is present while both N I and N II couplings are present at lower pH values. These results are interpreted in terms of a model based on the structure of the bacterial reaction center and involving two determining factors. First, the non-heme iron, when present, is ligated to the imidazole that H-bonds to one of the Q A y carbonyls. This physical attachment of the imidazole to the iron limits the strength of the H-bond to Q A y . Second, a pH-dependent group on the protein controls the strength of the H-bonds to Q A y . The pK a of this group is around pH 7.5 in CN y -treated PSII. q2000 Elsevier Science Inc. All rights reserved. Keywords: Electron spin echo envelope modulation spectroscopy; Hyperfine sub-level correlation spectroscopy; Photosystem II; Q A y ; 14 N; H-bonds 1. Introduction In the Photosystem II (PSII) reaction centre a plastoqui- none-9 (Q A ) is magnetically coupled to a high spin non- heme Fe 2q atom (Ss2) when it is reduced to the semiquinone [1,2]. The Q A y spin can be decoupled from the non-heme iron using two different approaches. First, the removal of the iron can be done by various biochemical methods using LiClO 4 and o-phenanthroline [1,3], or treat- ment at high pH [4]. The second approach involves the conversion of the iron from its physiological high-spin form (Ss2) to the low-spin (Ss0) state by treatment of PSII with high concentrations of CN y at alkaline pH [5]. Unlike other methods the decoupling of the semiquinone from the iron is reversible in this case [5]. The decoupling of the semiquinone from the iron has allowed the use of electron magnetic resonance methods to investigate Q A y . Recently, electron spin echo envelope mod- ulation (ESEEM) spectroscopy [6] on Q A y in PSII [3,4,7], on both Q A y [8,9] and Q B y [9] in bacterial reaction centres, and on A 1 y in Photosystem I [10] showed that the electron spin interacted magnetically with protein nitrogens. In most cases it was assumed that these interactions occurred through * Corresponding author. H-bonds between the carbonyls of the semiquinone and the protein [3,4,7–9]. In LiClO 4 /Zn-treated PSII [3] and in PSII treated at pH 11[4], there are two 14 N nuclei interacting with Q A y and from their nuclear quadrupole interaction (NQI) parameters these were assigned to the backbone peptide nitro- gen (N I ) and the amino nitrogen N d of an imidazole (N II ) [3,4]. Based on amino acid sequence analysis and compari- son to the crystal structure of the bacterial reaction centre [11,12],N I and N II were proposed to belong to the residues Ala261 and His215 of the D2 protein, respectively [3,4]. Equivalent assignments were proposed to explain the cou- plings detected by ESEEM of Q A y in bacterial reaction cen- tres [8,9]. In ESEEM data for 14 N- and 15 N-labelled PSII treated with CN at pH 8 only one nitrogen coupling was detected [7]. The NQI for this nitrogen corresponded to those of a backbone nitrogen (N I ) and it was assigned to the Ala261 of the D2 protein [7]. Hyperfine sub-level correlation (HYSCORE)[13] spec- troscopy is a two-dimensional (2D) four-pulse ESEEM technique which provides correlations between nuclear frequencies originating from different Ms manifolds. This technique has proven to be a useful tool for the analysis of overlapping complicated ESEEM spectra [14,15]. The supe- riority of HYSCORE for resolving and assigning complicated ESEEM spectra has been demonstrated recently for the case