Biochimica et Biophysica Acta 848 (1986) 41-47 41 Elsevier BBA 41896 Reversible and irreversible effects of alkaline pH on Photosystem II electron-transfer reactions James Cole, Michael Boska, Neil V. Blough * and Kenneth Sauer Department of Chemistry and Chemical Biodynamics Laboratory, Lawrence Berkeley Laboratory, University of California, Berkeley, CA 94720 (U.S.A.) (Received June 12th, 1985) Key words: Oxygen evolution; ESR; Photosystem II; pH effect; Electron transport; (Spinach chloroplast) Incubation of highly active, O2-evolving PS II preparations at alkaline pH inhibits donor side electron-trans- fer reactions in two distinct fashions, one reversible the other irreversible. In both cases, 0 2 evolution is inhibited, with concomitant loss of the light-induced multiline and g = 4.1 EPR signals and an increased steady-state level of EPR Signal II induced by continuous illumination. However, the inhibition that is observed between pH 7.0 and 8.0 is readily reversible by resuspension at low pH, while above pH 8.0 the effect is irreversible. In addition, under repetitive flash conditions the ms decay kinetics remains largely unchanged at pH < 8.0 but shows about a 2-fold increase in amplitude and is slowed at pH above 8.0. The irreversible component of inhibition most likely can be attributed to the loss of Mn and the 16, 24 and 33 kDa proteins. The reversible component may be mediated by displacement of CI - from an anion-binding site by OH - or by titration of ionizable groups on the protein(s) associated with water-splitting. We propose that the reversible inhibition blocks electron transfer between the O2-evolving complex and an intermediate which serves as the direct donor to Signal II, while the irreversible inhibition blocks the reduction of Signal II by this intermediate donor species. Introduction The loss of the water-splitting activity of chlo- roplast membrane preparations at alkaline pH in the presence of uncouplers has been observed by a number of investigators [1-4]. From these experi- ments it has been concluded that it is the internal thylakoid pH which controls water-splitting activ- ity and photosynthetic electron flow through PS II by its effect on the O2-evolving apparatus located * Present address: Department of Chemistry, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, U.S.A. Abbreviations: Mes, 4-morpholineethanesulfonic acid; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; Tricine, N-[2-hydroxy-l,l-bis(hydroxymethyl)ethyl]glycine; Chl, chloro- phyll; DCBQ, 2,5-dichlorobenzoquinone; PS, Photosystem. on the lumenal side of the membrane [1]. The flash illumination study of Briantais et al. [3] indicates that the target for alkaline inactivation is the S2 state of the oxygen-evolving complex. Highly active PS II preparations [5-7] offer an excellent means of examining the effect of pH on the water-splitting reactions. Because the lumenal surface of the membrane is exposed to the external medium in these preparations [7], the direct in- fluence of pH on the water-splitting apparatus can be investigated without interference from an inter- vening membrane barrier. Kuwabara and Murata [6,8] found that suspension of PS II preparations in Tricine buffers at pH > 8.0 produced a loss of 02 evolution [6], the release of the 33, 24 and 16 kDa proteins [8] and a significant decrease in the Mn content [8], indicating a substantial perturba- 0005-2728/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)