244 Biochbnica et Biophysica Acta, 1101 (1992) 244-248 © 1992 Elsevier Science Publishers B.V. All rights reserved 0005-2728/92/$05.00 BBABIO 43660 Cooperative transient trapping of protons by structurally distorted chloroplast ATPase: Evidence for the proton well? Cord Griwatz J and Wolfgang Junge Biophysik, Fachbereich Biologic/Chemic, Unicersitiit Osnabriick, D-4500 Osnabriick (Germany) (Received 14 April 1992) Key words: ATPase; Proton; Protonmotive force; Photosynthesis; (Chloroplast) The structure of membrane-bound ATPase, CFoCF1, was distorted by mild EDTA treatment, but without solubilization of CF I. Distorted CFoCF l rapidly trapped protons from the lumen side without transport. Trapping required electric driving force, showed very high cooperativity as function of pH and hysteresis as function of time. It is discussed under the concept of a 'proton well'. Introduction The proton-translocating ATPase in thylakoid mem- branes of chloroplasts, CFoCF t, belongs to the ubiqui- tous class of F-type ATPases with bipartite structure. The extrinsic portion, CF~, binds nucleotides and phos- phate. When dissociated from its intrinsic membrane counterpart, it exposes CF 0, a proton channel with unparalleled selectivity. CF 0 discriminates protons against other cations from pH 6 to 8 and even in the presence of 300 mM KCI in the suspending medium. This has been evaluated by flash spectrophotometry [1]. In such experiments both photosystems are stimu- lated for single turnover by a short flash of light. This generates a transmembrane voltage (by 30-60 mV more positive in the lumen) and a pH difference (about 0.05 units more acid in the lumen) [2]. Three observables have been monitored, namely: (a) pH transients in the lumen, (b) transients of the transmembrane voltage, and (c) pH transients in the suspending medium [1,3,4]. When more than 50% of total CFI is solubilized by treatment of thylakoids with some 100/zM EDTA, the extent and the time-course of proton intake from the lumen and of charge transfer across the membrane are equal [1]. Proton binding by CF 0 and transfer across Correspondence: W. Junge, Fachbereich 5, Universit~it Osnabriick, Postfach 4469, D-4500 OsnabrOck, Germany. J Present address: C. Griwatz, Institut fOr Klinische Chemic, Zen- tralklinikum, Universit~it MOnster, D-4400 MOnster, Germany. Abbreviations: DCCD, N',N'-dicyclohexylcarbodiimide; DCMU, 3- (3,4-dichlorophenyl)-l,l-dimethylurea; NTA0 nitrolotriacetic acid. the membrane dielectric appear to occur in synchrony. The same holds for the coupled transfer of protons during ATP synthesis through the holoenzyme, CFoCF ~ [5]. We report here on highly cooperative proton trap- ping by CFoCF 1without concomitant transfer of charges across the membrane. This effect is elicited by treat- ment of thylakoids with very low concentrations of EDTA, say 10/~M. It was first described several years ago [6]. The experimental results of the previous work were fully reproduced in this work. Further data led to better understanding of the mechanism of this effect, which is sketched in this article. Materials and Methods Thylakoids were prepared from spinach or pea by standard procedures [1], except for the absence of magnesium in the media (destacked thylakoids). The ionic milieu in preparation and storage media was domiriated by 10 mM NaCI. The procedure to evoke transient trapping of protons was as follows. Concen- trated stock (2-3 mg chlorophyll/ml) was diluted in distilled water to yield a chlorophyll concentration of 10 /zM, the pH was adjusted to 7.5 at room tempera- ture. EDTA was added, typically at 10 p.M (for neces- sary variations, see below). After 2 rain, NaCI was added at 10 mM to stop exposure to low ionic strength. Longer incubation and higher concentration of EDTA resulted in CF~ solubilization with the drastic increase of the proton conductance by exposed CF 0. After preincubation, methyl viologen (10 /zM) was added as electron acceptor.