__x . : ;, ELSEVIER Biochimica et Biophysica Acta 1188 (1994) 260-270 BB Biochi~ic~a etBiophysica A~ta Binding of carboxylate anions at the non-heme Fe(II) of PS II. I. Effects on the QAFe 2÷ and QAFe 3+ EPR spectra and the redox properties of the iron Y. Deligiannakis a V. Petrouleas a,* B.A. Diner b a Institute of Materials Science, NCSR Democritos, 15310 Aghia Paraskeui A ttiki, Athens, Greece b Central Research and Development Department, Experimental Station, E.I. DuPont de Nemours Co., Wilmington, DE 19880-0173, USA Received 10 December 1993; revised 7 July 1994 Abstract We have examined the effects of a number of carboxylate anions on the iron-quinone complex of Photosystem II (PS II). Typical effects are the following. In the state QAFe 2+ oxalate enhances significantly the g = 1.84 EPR resonance while, for example, glycolate and glyoxylate suppress it. The anions have variable effects on the iron midpoint potential. Formate and oxalate raise significantly the E m of the iron. Glycolate lowers the E m significantly and the E m shows a weak pH dependence. In the presence of glycolate the native plastosemiquinone (Q~/QBH2 couple) can oxidise the iron. Glyoxylate also lowers the Em, but the E m shows a greater pH dependence than with glycolate but still weaker than the - 60 mV/pH unit of the untreated iron. The Fe 3+ EPR spectra are characterised by small but distinct shifts, while in addition an unusual resonance at close to g = 4.3 is observed. These as well as the temperature dependence of the spectra are analysed by a spin-Hamiltonian model. Comparison with competition studies in the companion paper indicates that the anions bind as iron ligands displacing bicarbonate. Keywords: Photosystem II; Iron-quinone complex; EPR; Carboxylate anion 1. Introduction The non-heme iron of Photosystem II (PS II) has a number of properties which differentiate it from its analogue in the purple-photosynthetic bacteria. Be- sides spectroscopic differences and its redox activity the iron of PS II can reversibly bind small molecules such as NO [1] or CN [2]. Bicarbonate-NO competition studies have suggested that bicarbonate is the natural labile ligand to the non-heme iron [3]. Normal rates of electron flow require the presence of this anion (see, e.g., Refs. [4,5]. The mode of bicarbonate binding and action, however, is not fully understood at present. Abbreviations: PS II, Photosystem II; BBY, thylakoid membrane fragments isolated according to Refs. [10-12]; QA, primary quinone electron acceptor; QB, secondary quinone electron acceptor; Mes, 4-morpholinethanesulfonic acid; Hepes, 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid; Tris, tris(hydroxymethyl)amino- methane. * Corresponding author. Fax: + 30 1 6519430. 0005-2728/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0005-2728(94)00132-4 A well known class of proteins which require CO 2 for their functioning are the transferrins. In the trans- ferrin proteins CO 2 in the form of carbonate is re- quired as a synergistic anion for iron binding. An X-ray structure determination of lactoferrin has shown that the protein provides four ligands to the iron (two tyrosines one histidine and one aspartate) while car- bonate is bound to the iron as a bidentate ligand [6]. Schlabach and Bates [7] have examined an extensive list of carboxylate anions which can substitute for car- bonate as the synergistic anion in Fe-transferrin. More recently Dubach et al. [8] have reported on EPR spec- tra of iron-transferrin anion complexes and have pro- vided insights into the mode of binding of these anions as well as of carbonate to the iron. Prompted by these studies, we examined in a prelim- inary report [9] the effect of these anions upon their substitution of bicarbonate in Photosystem II. We elab- orate here on the effects of these anions on the QAFe 2+ and QAFe 3+ EPR spectra and the redox properties of the Fe3+/Fe 2+ couple.