Correlation between membrane-localized protons and flash-driven ATP formation in chloroplast thylakoids.
Flash-driven ATP formation by spinach chloroplast thylakoids, using the luciferin luminescence assay to detect ATP formed in single turnover flashes, was studied under conditions where a membrane protein amine buffering pool was either protonated or deprotonated before the beginning of the flash trains. The flash number for the onset of ATP formation was delayed by about 10 flashes (from 15 to about 25) when the amine pool was deprotonated as compared to the protonated state. The delay was substantially reversed again by reprotonating the pool upon application of 20-30 single-turnover flashes and 8 min of dark before addition of ADP, Pi, and the luciferin system. In the case of deprotonation by desaspidin, the uncoupler was removed by binding to BSA before the reprotonating flashes were given. Reprotonation was carried out before addition of ADP and Pi, to avoid a possible interference by the ATP-ase, which can energize the system by pumping protons. The reprotonated state, as indicated by an onset lag of about 15 flashes rather than 25 for the deprotonated state, was stable in the dark over extended dark times. The number of protons released by 10 flashes is approximately 30 nmol H+ (mg chl)-1, an amount similar to the size of the reversibly protonated amine group buffering pool. The data are consistent with the hypothesis that the amine buffering groups must be in the protonated state before any protons proceed to the coupling complex and energize ATP formation. Other work has suggested that the amine buffering pool is sequestered within membrane proteins rather than being exposed directly to the inner aqueous bulk phase. Therefore, it is possible that the sequestered amine group array may provide localized association-dissociation sites for proton movement to the coupling complex.
D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D002090:Butyrophenones / Q000494:pharmacology; D002736:Chloroplasts / Q000502:physiology*; D004579:Electron Transport / Q000187:drug effects; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008027:Light; D008564:Membrane Potentials; D010785:Photophosphorylation; D010944:Plants; D011522:Protons; D014475:Uncoupling Agents / Q000494:pharmacology
D002090:Butyrophenones; D011522:Protons; D014475:Uncoupling Agents; D000255:Adenosine Triphosphate; C004715:desaspidin