Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1987 Oct;84(20):7084–7088. doi: 10.1073/pnas.84.20.7084

Complete tracking of transient proton flow through active chloroplast ATP synthase

Wolfgang Junge 1
PMCID: PMC299234  PMID: 16593881

Abstract

Proton pumping in thylakoid membranes and backflow of protons through the active ATP synthase CF0-CF1 (where CF0 is the proton channel and CF1 is the catalytic portion) were investigated by flash spectrophotometry. A steady pH difference across the membrane was generated by continuous measuring light, supplemented by voltage transients that were generated by flashing light. In the presence of Pi and ADP, the electric potential transients elicited transients of proton flow via CF0-CF1, typically 1.3 H+ per CF1 and flash group. Proton flow was blocked by CF0-CF1 inhibitors: N,N′-dicyclohexylcarbodiimide, acting on the channel component CF0, and tentoxin, acting on the catalytic component CF1. The half-rise time was 40 ms in 1H2O and 78 ms in 2H2O. ATP synthesis under conditions of flashing light and transient proton flow was characterized by a Km(Pi) of only 14 μM, contrasting with a Km of several hundred micromolar for continuous ATP synthesis at high rate. This might reflect a resistance to Pi diffusion. The degree of proton delocalization in the chemiosmotic coupling between redox reactions and ATP synthesis is under debate. In thylakoids, it has been proposed that intramembrane proton buffering domains act as ducts for protons between pumps and ATP synthases. In this work, transient proton flow by way of CF0-CF1 was completely tracked from the lumen, across the membrane, and into the suspending medium. Proton uptake from the lumen and charge flow across the membrane occurred synchronously and in stoichiometric proportion. The uptake of protons from the lumen by CF0-CF1, half completed in 40 ms, was preceded by release of protons from water oxidation into the lumen, half completed in <1 ms. Hence, pumps and ATP synthases were coupled through the lumen without involvement of intramembrane domains.

Keywords: photosynthesis, ATPase, phosphorylation, chemiosmotic mechanism

Full text

PDF
7085

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andersson B., Anderson J. M. Lateral heterogeneity in the distribution of chlorophyll-protein complexes of the thylakoid membranes of spinach chloroplasts. Biochim Biophys Acta. 1980 Dec 3;593(2):427–440. doi: 10.1016/0005-2728(80)90078-x. [DOI] [PubMed] [Google Scholar]
  2. Dilley R. A., Schreiber U. Correlation between membrane-localized protons and flash-driven ATP formation in chloroplast thylakoids. J Bioenerg Biomembr. 1984 Jun;16(3):173–193. doi: 10.1007/BF00751048. [DOI] [PubMed] [Google Scholar]
  3. Gräber P., Schlodder E., Witt H. T. Conformational change of the chloroplast ATPase induced by a transmembrane electric field and its correlation to phosphorylation. Biochim Biophys Acta. 1977 Sep 14;461(3):426–440. doi: 10.1016/0005-2728(77)90231-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Haines T. H. Anionic lipid headgroups as a proton-conducting pathway along the surface of membranes: a hypothesis. Proc Natl Acad Sci U S A. 1983 Jan;80(1):160–164. doi: 10.1073/pnas.80.1.160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hoppe J., Sebald W. The proton conducting F0-part of bacterial ATP synthases. Biochim Biophys Acta. 1984 Apr 9;768(1):1–27. doi: 10.1016/0304-4173(84)90005-3. [DOI] [PubMed] [Google Scholar]
  6. Junge W., Ausländer W., McGeer A. J., Runge T. The buffering capacity of the internal phase of thylakoids and the magnitude of the pH changes inside under flashing light. Biochim Biophys Acta. 1979 Apr 11;546(1):121–141. doi: 10.1016/0005-2728(79)90175-0. [DOI] [PubMed] [Google Scholar]
  7. Junge W., Rumberg B., Schröder H. The necessity of an electric potential difference and its use for photophosphorylation in short flash groups. Eur J Biochem. 1970 Jul;14(3):575–581. doi: 10.1111/j.1432-1033.1970.tb00326.x. [DOI] [PubMed] [Google Scholar]
  8. Junge W. The critical electric potential difference for photophosphorylation. Its relation to the chemiosmotic hypothesis and to the triggering requirements of the ATPase system. Eur J Biochem. 1970 Jul;14(3):582–592. doi: 10.1111/j.1432-1033.1970.tb00327.x. [DOI] [PubMed] [Google Scholar]
  9. Junge W., Witt H. T. On the ion transport system of photosynthesis--investigations on a molecular level. Z Naturforsch B. 1968 Feb;23(2):244–254. doi: 10.1515/znb-1968-0222. [DOI] [PubMed] [Google Scholar]
  10. MITCHELL P. Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism. Nature. 1961 Jul 8;191:144–148. doi: 10.1038/191144a0. [DOI] [PubMed] [Google Scholar]
  11. Magnusson R. P., Portis A. R., Jr, McCarty R. E. Quantitative, analytical separation of adenine nucleotides by column chromatography on polyethyleneimine-coated cellulose. Anal Biochem. 1976 May 7;72:653–657. doi: 10.1016/0003-2697(76)90580-7. [DOI] [PubMed] [Google Scholar]
  12. Mitchell P. Chemiosmotic coupling in oxidative and photosynthetic phosphorylation. Biol Rev Camb Philos Soc. 1966 Aug;41(3):445–502. doi: 10.1111/j.1469-185x.1966.tb01501.x. [DOI] [PubMed] [Google Scholar]
  13. Nagle J. F., Morowitz H. J. Molecular mechanisms for proton transport in membranes. Proc Natl Acad Sci U S A. 1978 Jan;75(1):298–302. doi: 10.1073/pnas.75.1.298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Pietrobon D., Zoratti M., Azzone G. F., Caplan S. R. Intrinsic uncoupling of mitochondrial proton pumps. 2. Modeling studies. Biochemistry. 1986 Feb 25;25(4):767–775. doi: 10.1021/bi00352a005. [DOI] [PubMed] [Google Scholar]
  15. Steele J. A., Uchytil T. F., Durbin R. D., Bhatnagar P., Rich D. H. Chloroplast coupling factor 1: A species-specific receptor for tentoxin. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2245–2248. doi: 10.1073/pnas.73.7.2245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. WILLIAMS R. J. Possible functions of chains of catalysts. J Theor Biol. 1961 Jan;1:1–17. doi: 10.1016/0022-5193(61)90023-6. [DOI] [PubMed] [Google Scholar]
  17. Westerhoff H. V., Melandri B. A., Venturoli G., Azzone G. F., Kell D. B. A minimal hypothesis for membrane-linked free-energy transduction. The role of independent, small coupling units. Biochim Biophys Acta. 1984 Dec 17;768(3-4):257–292. doi: 10.1016/0304-4173(84)90019-3. [DOI] [PubMed] [Google Scholar]
  18. Witt H. T. Coupling of quanta, electrons, fields, ions and phosphrylation in the functional membrane of photosynthesis. Results by pulse spectroscopic methods. Q Rev Biophys. 1971 Nov;4(4):365–477. doi: 10.1017/s0033583500000834. [DOI] [PubMed] [Google Scholar]
  19. Woelders H., van der Zande W. J., Colen A. M., Wanders R. J., van Dam K. The phosphate potential maintained by mitochondria in State 4 is proportional to the proton-motive force. FEBS Lett. 1985 Jan 7;179(2):278–282. doi: 10.1016/0014-5793(85)80534-2. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES