PLANT PHYSIOLOGY , Vol 101, Issue 1 89-96, Copyright © 1993 by American Society of Plant Biologists
Dissipation of the Proton Electrochemical Potential in Intact Chloroplasts (II. The pH Gradient Monitored by Cytochrome f Reduction Kinetics)
J. N. Nishio and J. Whitmarsh
Department of Botany, University of Wyoming, Laramie, Wyoming 82071-3165 (J.N.N.)
The potency of various uncouplers for collapsing the light-induced pH
gradient across thylakoid membranes in intact chloroplasts was investigated
by time-resolved optical spectroscopy. The thylakoid transmembrane pH
gradient ([delta]pH) was monitored indirectly by measuring the rate of
cytochrome (Cyt) f reduction following a light flash of sufficient duration
to create a sizable [delta]pH. The results show that the rate of Cyt f
reduction is controlled in part by the internal pH of the thylakoid inner
aqueous space. At pH values from 6.5 to 8.0, the Cyt f reduction rate was
maximal, whereas at lower pH values from 6.5 to 5.5 the reduction rate
decreased to 25% of the maximal rate. The ability of three uncouplers,
nigericin, carbonylcyanide m-chlorophenylhydrazone, and gramicidin, to
accelerate the rate of Cyt f reduction was determined for intact
chloroplasts isolated from spinach (Spinacia oleracea). The efficacy of the
uncouplers for collapsing the [delta]pH was determined using the empirical
relationship between the [delta]pH and the Cyt f reduction rate. For intact
chloroplasts, nigericin was the most effective uncoupler, followed by
carbonylcyanide m-chlorophenylhydrazone, which interacted strongly with
bovine serum albumin. Gramicidin D, even at high gramicidin:chlorophyll
ratios, did not completely collapse the pH gradient, probably because it
partitions in the envelope membranes and does not enter the intact
chloroplast.
