Plant Physiol, February 2000, Vol. 122, pp. 583-596

Distinguishing between Luminal and Localized Proton Buffering Pools in Thylakoid Membranes¹

Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.

The dual gradient energy coupling hypothesis posits that chloroplast thylakoid membranes are energized for ATP formation by either a delocalized or a localized proton gradient geometry. Localized energy coupling is characterized by sequestered domains with a buffering capacity of approximately 150 nmol H⁺ mg¹ chlorophyll (Chl). A total of 30 to 40 nmol mg¹ Chl of the total sequestered domain buffering capacity is contributed by lysines with anomolously low pK_as, which can be covalently derivatized with acetic anhydride. We report that in thylakoid membranes treated with acetic anhydride, luminal acidification by a photosystem I (duraquinol [DQH₂] to methyl viologen [MV]) proton pumping partial reaction was nearly completely inhibited, as measured by three separate assays, yet surprisingly, H⁺ accumulation still occurred to the significant level of more than 100 nmol H⁺ mg Chl¹, presumably into the sequestered domains. The treatment did not increase the observed rate constant of dark H⁺ efflux, nor was electron transport significantly inhibited. These data provide support for the existence of a sequestered proton translocating pathway linking the redox reaction H⁺ ion sources with the CF₀ H⁺ channel. The sequestered, low-pK_a Lys groups appear to have a role in the H⁺ diffusion process and chemically modifying them blocks the putative H⁺ relay system.