Proton Translocations in Isolated Spinach Chloroplasts after Single-turnover Actinic Flashes

Abstract

Proton movements were monitored with cresol red and neutral red. Absorbance changes of these dyes following actinic flashes were measured both for the steady state and for sequences of 6-9 flashes given to separate dark-adapted suspensions of chloroplasts. The cresol red changes corresponded to a steady-state proton uptake (¿H*+ss) of 0.0015-0.0017 mol H+/mol Chl per flash (ferricyanide as electron acceptor) or 0.0031 mol H+/mol Chl per flash (methyl viologen). Assuming 600 Chl per electron transport chain, these uptakes corresponded to H+/e- = 1 or 2 respectively. In flash sequences with ferricyanide, a minimum of about 0.6 ¿H*+ss was noted after flash No. 3. Controls, and considerations of the partitioning of unprotonated neutral red into the thylakoid membrane phase, strongly indicated that neutral red was responding to an acidification of the intrathylakoid aqueous phase. The steady-state signal was interpreted as 2 H+ deposited per electron transport chain per flash (H+/e- = 2), one proton from the oxidation of water and one from the oxidation of plastoquinone. Patterns of proton deposition following sequences of flashes and their change upon adding dibromothymoquinone suggested that (a) protons are produced one at a time in the advance of the 'S-states' (Z*n+ ¿ Z(n+1)+ ); and (b) the precursor to the plastoquinone pool, B, is a special plastoquinone, requiring 1 e- and 1 H+ for its initial reduction but passing on to the plastoquinone pool 2 e- upon the next flash. The proton deposition on flash No. 1 corresponded to a small proportion of reduced B in dark-adapted chloroplasts, as previously postulated.