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
. 1978 Nov;75(11):5296–5300. doi: 10.1073/pnas.75.11.5296

Stoichiometry of vectorial H+ movements coupled to electron transport and to ATP synthesis in mitochondria

Adolfo Alexandre 1,*, Baltazar Reynafarje 1, Albert L Lehninger 1,†
PMCID: PMC392949  PMID: 31621

Abstract

In order to verify more directly our earlier measurements showing that, on the average, close to four vectorial H+ are rejected per pair of electrons passing each of the three energy-conserving sites of the mitochondrial electron transport chain, direct tests of the H+/2e- ratio for sites 2 and 3 were carried out in the presence of permeant charge-compensating cations. Site 2 was examined by utilizing succinate as electron donor and ferricyanide as electron acceptor from mitochondrial cytochrome c; the directly measured H+/2e- ratio was close to 4. Energy-conserving site 3 was isolated for study with ferrocyanide or ascorbate plus tetramethylphenylenediamine as electron donors to cytochrome c and with oxygen as electron acceptor. The directly measured H+/2e- ratio for site 3 was close to 4. The H+/ATP ratio (number of vectorial H+ ejected per ATP hydrolyzed) was determined with a new method in which the steady-state rates of both H+ ejection and ATP hydrolysis were measured in the presence of K+ + valinomycin. The H+/ATP ratio was found to approach 3.0. A proton cycle for oxidative phosphorylation is proposed, in which four electrochemical H+ equivalents are ejected per pair of electrons passing each energy-conserving site; three of the H+ equivalents pass inward to derive ATP synthesis from ADP and phosphate and the fourth H+ is used to bring about the energy-requiring electrogenic expulsion of ATP4- in exchange for extramitochondrial ADP3-, via the H+/H2PO4- symporter.

Keywords: electron transport, ATP hydrolysis, oxidative phosphorylation, H+ transport

Full text

PDF
5299

Selected References

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

  1. Azzi A., Azzone G. F. Swelling and shrinkage phenomena in liver mitochondria. VI. Metabolism-independent swelling coupled to ion movement. Biochim Biophys Acta. 1967 May 9;131(3):468–478. doi: 10.1016/0005-2728(67)90006-0. [DOI] [PubMed] [Google Scholar]
  2. Azzone G. F., Massari S. Thermodynamic and kinetic aspects of the interconversion of chemical and osmotic energies in mitochondria. Eur J Biochem. 1971 Mar 1;19(1):97–107. doi: 10.1111/j.1432-1033.1971.tb01292.x. [DOI] [PubMed] [Google Scholar]
  3. Brand M. D., Chen C. H., Lehninger A. L. Stoichiometry of H+ ejection during respiration-dependent accumulation of Ca2+ by rat liver mitochondria. J Biol Chem. 1976 Feb 25;251(4):968–974. [PubMed] [Google Scholar]
  4. Brand M. D., Lehninger A. L. H+/ATP ratio during ATP hydrolysis by mitochondria: modification of the chemiosmotic theory. Proc Natl Acad Sci U S A. 1977 May;74(5):1955–1959. doi: 10.1073/pnas.74.5.1955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brand M. D., Reynafarje B., Lehninger A. L. Re-evaluation of the H+/site ratio of mitochondrial electron transport with the oxygen pulse technique. J Biol Chem. 1976 Sep 25;251(18):5670–5679. [PubMed] [Google Scholar]
  6. Brand M. D., Reynafarje B., Lehninger A. L. Stoichiometric relationship between energy-dependent proton ejection and electron transport in mitochondria. Proc Natl Acad Sci U S A. 1976 Feb;73(2):437–441. doi: 10.1073/pnas.73.2.437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brierley G. P. Energy-linked alteration of the permeability of heart mitochondria to chloride and other anions. Biochemistry. 1970 Feb 17;9(4):697–707. doi: 10.1021/bi00806a001. [DOI] [PubMed] [Google Scholar]
  8. JACOBS E. E., SANADI D. R. Phosphorylation coupled to electron transport mediated by high potential electron carriers. Biochim Biophys Acta. 1960 Feb 12;38:12–34. doi: 10.1016/0006-3002(60)91192-6. [DOI] [PubMed] [Google Scholar]
  9. LaNoue K., Mizani S. M., Klingenberg M. Electrical imbalance of adenine nucleotide transport across the mitochondrial membrane. J Biol Chem. 1978 Jan 10;253(1):191–198. [PubMed] [Google Scholar]
  10. Lüstorff J., Schlimme E. Does an inhibitor of mitochondrial adenylate kinase also affect oxidative phosphorylation? Experientia. 1976 Mar 15;32(3):298–299. doi: 10.1007/BF01940801. [DOI] [PubMed] [Google Scholar]
  11. Mitchell P., Moyle J. Proton translocation coupled to ATP hydrolysis in rat liver mitochondria. Eur J Biochem. 1968 May;4(4):530–539. doi: 10.1111/j.1432-1033.1968.tb00245.x. [DOI] [PubMed] [Google Scholar]
  12. Mitchell P., Moyle J. Respiration-driven proton translocation in rat liver mitochondria. Biochem J. 1967 Dec;105(3):1147–1162. doi: 10.1042/bj1051147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Moyle J., Mitchell P. Cytochrome c oxidase is not a proton pump. FEBS Lett. 1978 Apr 15;88(2):268–272. doi: 10.1016/0014-5793(78)80190-2. [DOI] [PubMed] [Google Scholar]
  14. Moyle J., Mitchell P. Measurements of mitochondrial comes from H+/O quotients: effects of phosphate and N-ethylmaleimide. FEBS Lett. 1978 Jun 15;90(2):361–365. doi: 10.1016/0014-5793(78)80405-0. [DOI] [PubMed] [Google Scholar]
  15. Moyle J., Mitchell P. Proton translocation quotient for the adenosine triphosphatase of rat liver mitochondria. FEBS Lett. 1973 Mar 15;30(3):317–320. doi: 10.1016/0014-5793(73)80678-7. [DOI] [PubMed] [Google Scholar]
  16. Papa S. Proton translocation reactions in the respiratory chains. Biochim Biophys Acta. 1976 Apr 30;456(1):39–84. doi: 10.1016/0304-4173(76)90008-2. [DOI] [PubMed] [Google Scholar]
  17. Reynafarje B., Brand M. D., Lehninger A. L. Evaluation of the H+/site ratio of mitochondrial electron transport from rate measurements. J Biol Chem. 1976 Dec 10;251(23):7442–7451. [PubMed] [Google Scholar]
  18. Reynafarje B., Lehninger A. L. An alternative membrane transport pathway for phosphate and adenine nucleotides in mitochondria and its possible function. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4788–4792. doi: 10.1073/pnas.75.10.4788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Reynafarje B., Lehninger A. L. Electric charge stoichiometry of calcium translocation in mitochondria. Biochem Biophys Res Commun. 1977 Aug 22;77(4):1273–1279. doi: 10.1016/s0006-291x(77)80117-4. [DOI] [PubMed] [Google Scholar]
  20. Sorgato M. C., Ferguson S. J. Measurements of the components of the protonmotive force generated by cytochrome oxidase in submitochondrial particles. FEBS Lett. 1978 Jun 1;90(1):178–182. doi: 10.1016/0014-5793(78)80324-x. [DOI] [PubMed] [Google Scholar]
  21. Wikstrom M. K. Proton pump coupled to cytochrome c oxidase in mitochondria. Nature. 1977 Mar 17;266(5599):271–273. doi: 10.1038/266271a0. [DOI] [PubMed] [Google Scholar]
  22. Wikström M. K., Saari H. T. The mechanism of energy conservation and transduction by mitochondrial cytochrome c oxidase. Biochim Biophys Acta. 1977 Nov 17;462(2):347–361. doi: 10.1016/0005-2728(77)90133-5. [DOI] [PubMed] [Google Scholar]
  23. Wikström M., Krab K. Cytochrome c oxidase is a proton pump: a rejoinder to recent criticism. FEBS Lett. 1978 Jul 1;91(1):8–14. doi: 10.1016/0014-5793(78)80006-4. [DOI] [PubMed] [Google Scholar]
  24. Wulf R., Kaltstein A., Klingenberg M. H+ and cation movements associated with ADP, ATP transport in mitochondria. Eur J Biochem. 1978 Jan 16;82(2):585–592. doi: 10.1111/j.1432-1033.1978.tb12054.x. [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