Distinguishing between Luminal and Localized Proton Buffering Pools in Thylakoid Membranes

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(+) mg(−1) chlorophyll (Chl). A total of 30 to 40 nmol mg(−1) Chl of the total sequestered domain buffering capacity is contributed by lysines with anomolously low pK(a)s, 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 [DQH(2)] 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 Chl(−1), 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 CF(0) 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.