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 $\text{pK}_{\text{a}}\text{s}$, 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 [$\text{DQH}_{2}$] 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 $\text{Chl}^{-1}$, 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 $\text{CF}_{0}$ H+ channel. The sequestered, low-pKa Lys groups appear to have a role in the H+ diffusion process and chemically modifying them blocks the putative H+ relay system.