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. 1985 Apr;82(7):1886–1890. doi: 10.1073/pnas.82.7.1886

Evidence that the Mg-dependent low-affinity binding site for ATP and Pi demonstrated on the isolated beta subunit of the F0.F1 ATP synthase is a catalytic site.

D Khananshvili, Z Gromet-Elhanan
PMCID: PMC397436  PMID: 2858854

Abstract

Binding sites for one Pi and two ATP or ADP molecules have been identified on the isolated, reconstitutively active beta subunit from the Rhodospirillum rubrum F0.F1 ATP synthase. Chemical modification of this beta subunit by the histidine reagent diethyl pyrocarbonate or by the carboxyl group reagent Woodword's reagent K results in complete inhibition of Pi binding to beta. The same reagents inhibit the binding of ATP to a Mg-dependent low-affinity site but not to a Mg-independent high-affinity site on this beta subunit. The binding stoichiometry of ADP to either site is not affected by these modifications. The beta subunit modified by either one of these reagents retains its capacity to rebind to beta-less chromatophores but not its ability to restore their photo-phosphorylation. These results indicate that the low-affinity Pi binding site on beta is located at the binding site of the gamma-phosphate group of ATP in the Mg-dependent low-affinity nucleotide binding site. This site contains histidine and carboxyl group residues, both of which are required for the binding of Pi and of the gamma-phosphate group of ATP. The same residues must also be involved in the capacity of the isolated beta subunit to restore the catalytic activity of the beta-less ATP synthase. It is therefore concluded that the low-affinity Mg-dependent substrate binding site identified on the isolated beta subunit of the R. rubrum F0.F1 ATP synthase is the catalytic site of this enzyme complex.

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Selected References

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

  1. Amzel L. M., Pedersen P. L. Proton atpases: structure and mechanism. Annu Rev Biochem. 1983;52:801–824. doi: 10.1146/annurev.bi.52.070183.004101. [DOI] [PubMed] [Google Scholar]
  2. Baird B. A., Hammes G. G. Structure of oxidative- and photo-phosphorylation coupling factor complexes. Biochim Biophys Acta. 1979 Jul 3;549(1):31–53. doi: 10.1016/0304-4173(79)90017-x. [DOI] [PubMed] [Google Scholar]
  3. Bengis-Garber C., Gromet-Elhanan Z. Purification of the energy-transducing adenosine triphosphatase complex from Rhodospirillum rubrum. Biochemistry. 1979 Aug 7;18(16):3577–3581. doi: 10.1021/bi00583a022. [DOI] [PubMed] [Google Scholar]
  4. Ceccarelli E., Vallejos R. H. Two types of essential carboxyl groups in Rhodospirillum rubrum proton ATPase. Arch Biochem Biophys. 1983 Jul 1;224(1):382–388. doi: 10.1016/0003-9861(83)90224-2. [DOI] [PubMed] [Google Scholar]
  5. Cosson J. J., Guillory R. J. The use of arylazido-beta-alanyl-ATP as a photoaffinity label for the isolated and membrane-bound mitochondrial ATPase complex. J Biol Chem. 1979 Apr 25;254(8):2946–2955. [PubMed] [Google Scholar]
  6. Cross R. L. The mechanism and regulation of ATP synthesis by F1-ATPases. Annu Rev Biochem. 1981;50:681–714. doi: 10.1146/annurev.bi.50.070181.003341. [DOI] [PubMed] [Google Scholar]
  7. Dunn S. D., Futai M. Reconstitution of a functional coupling factor from the isolated subunits of Escherichia coli F1 ATPase. J Biol Chem. 1980 Jan 10;255(1):113–118. [PubMed] [Google Scholar]
  8. Futai M., Kanazawa H. Structure and function of proton-translocating adenosine triphosphatase (F0F1): biochemical and molecular biological approaches. Microbiol Rev. 1983 Sep;47(3):285–312. doi: 10.1128/mr.47.3.285-312.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Futai M. Reconstitution of ATPase activity from the isolated alpha, beta, and gamma subunits of the coupling factor, F1, of Escherichia coli. Biochem Biophys Res Commun. 1977 Dec 21;79(4):1231–1237. doi: 10.1016/0006-291x(77)91138-x. [DOI] [PubMed] [Google Scholar]
  10. Gromet-Elhanan Z. Differences in sensitivity to valinomycin and nonactin of various photophosphorylating and photoreducing systems of Rhodospirillum rubrum chromatpohores. Biochim Biophys Acta. 1970 Nov 3;223(1):174–182. doi: 10.1016/0005-2728(70)90142-8. [DOI] [PubMed] [Google Scholar]
  11. Gromet-Elhanan Z. Role of photophosphorylation coupling factor in energy conversion by depleted chromatophores of Rhodospirillum rubrum. J Biol Chem. 1974 Apr 25;249(8):2522–2527. [PubMed] [Google Scholar]
  12. Grubmeyer C., Cross R. L., Penefsky H. S. Mechanism of ATP hydrolysis by beef heart mitochondrial ATPase. Rate constants for elementary steps in catalysis at a single site. J Biol Chem. 1982 Oct 25;257(20):12092–12100. [PubMed] [Google Scholar]
  13. Harris D. A. The interactions of coupling ATPases with nucleotides. Biochim Biophys Acta. 1978 Mar 10;463(3-4):245–273. doi: 10.1016/0304-4173(78)90002-2. [DOI] [PubMed] [Google Scholar]
  14. Kasahara M., Penefsky H. S. High affinity binding of monovalent Pi by beef heart mitochondrial adenosine triphosphatase. J Biol Chem. 1978 Jun 25;253(12):4180–4187. [PubMed] [Google Scholar]
  15. Khananshvili D., Gromet-Elhanan Z. Chemical modification of the beta-subunit isolated from a membrane-bound Fo-F1-ATP synthase: modification by 4-chloro-7-nitrobenzofurazan does not inhibit restoration of ATP synthesis or hydrolysis. Biochem Biophys Res Commun. 1982 Sep 30;108(2):881–887. doi: 10.1016/0006-291x(82)90913-5. [DOI] [PubMed] [Google Scholar]
  16. Khananshvili D., Gromet-Elhanan Z. Isolation and purification of an active gamma-subunit of the F0.F1-ATP synthase from chromatophore membranes of Rhodospirillum rubrum. The role of gamma in ATP synthesis and hydrolysis as compared to proton translocation. J Biol Chem. 1982 Oct 10;257(19):11377–11383. [PubMed] [Google Scholar]
  17. Khananshvili D., Gromet-Elhanan Z. The interaction of 4-chloro-7-nitrobenzofurazan with Rhodospirillum rubrum chromatophores, their soluble F1-ATPase, and the isolated purified beta-subunit. J Biol Chem. 1983 Mar 25;258(6):3714–3719. [PubMed] [Google Scholar]
  18. Khananshvili D., Gromet-Elhanan Z. The interaction of carboxyl group reagents with the Rhodospirillum rubrum F1-ATPase and its isolated beta-subunit. J Biol Chem. 1983 Mar 25;258(6):3720–3725. [PubMed] [Google Scholar]
  19. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  20. Lauquin G., Pougeois R., Vignais P. V. 4-Azido-2-nitrophenyl phosphate, a new photoaffinity derivative of inorganic phosphate. Study of its interaction with the inorganic phosphate binding site of beef heart mitochondrial adenosine triphosphatase. Biochemistry. 1980 Sep 30;19(20):4620–4626. doi: 10.1021/bi00561a013. [DOI] [PubMed] [Google Scholar]
  21. Miles E. W. Modification of histidyl residues in proteins by diethylpyrocarbonate. Methods Enzymol. 1977;47:431–442. doi: 10.1016/0076-6879(77)47043-5. [DOI] [PubMed] [Google Scholar]
  22. O'Neal C. C., Boyer P. D. Assessment of the rate of bound substrate interconversion and of ATP acceleration of product release during catalysis by mitochondrial adenosine triphosphatase. J Biol Chem. 1984 May 10;259(9):5761–5767. [PubMed] [Google Scholar]
  23. Penefsky H. S. Reversible binding of Pi by beef heart mitochondrial adenosine triphosphatase. J Biol Chem. 1977 May 10;252(9):2891–2899. [PubMed] [Google Scholar]
  24. Philosoph S., Binder A., Gromet-Elhanan Z. Coupling factor ATPase complex of Rhodospirillum rubrum. Purification and properties of a reconstitutively active single subunit. J Biol Chem. 1977 Dec 10;252(23):8747–8752. [PubMed] [Google Scholar]
  25. Pougeois R., Lauquin G. J., Vignais P. V. Evidence that 4-azido-2-nitrophenylphosphate binds to the phosphate site on the beta-subunit of Escherichia coli BF1-ATPase. FEBS Lett. 1983 Mar 7;153(1):65–70. doi: 10.1016/0014-5793(83)80120-3. [DOI] [PubMed] [Google Scholar]
  26. Pougeois R., Lauquin G. J., Vignais P. V. Interaction of 4-azido-2-nitrophenyl phosphate, an inorganic phosphate photoreactive analogue, with chloroplast coupling factor 1. Biochemistry. 1983 Mar 1;22(5):1241–1245. doi: 10.1021/bi00274a039. [DOI] [PubMed] [Google Scholar]
  27. Rosenthal H. E. A graphic method for the determination and presentation of binding parameters in a complex system. Anal Biochem. 1967 Sep;20(3):525–532. doi: 10.1016/0003-2697(67)90297-7. [DOI] [PubMed] [Google Scholar]
  28. Schäfer H. J., Scheurich P., Rathgeber G., Dose K., Mayer A., Klingenberg M. 3'-Arylazido-8-azido ATP--a cross-linking photoaffinity label for ATP binding proteins. Biochem Biophys Res Commun. 1980 Jul 31;95(2):562–568. doi: 10.1016/0006-291x(80)90821-9. [DOI] [PubMed] [Google Scholar]
  29. Senior A. E., Wise J. G. The proton-ATPase of bacteria and mitochondria. J Membr Biol. 1983;73(2):105–124. doi: 10.1007/BF01870434. [DOI] [PubMed] [Google Scholar]
  30. Shavit N. Energy transduction in chloroplasts: structure and function of the ATPase complex. Annu Rev Biochem. 1980;49:111–138. doi: 10.1146/annurev.bi.49.070180.000551. [DOI] [PubMed] [Google Scholar]
  31. Slater E. C. Mechanism of oxidative phosphorylation. Annu Rev Biochem. 1977;46:1015–1026. doi: 10.1146/annurev.bi.46.070177.005055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Williams N., Coleman P. S. Exploring the adenine nucleotide binding sites on mitochondrial F1-ATPase with a new photoaffinity probe, 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate. J Biol Chem. 1982 Mar 25;257(6):2834–2841. [PubMed] [Google Scholar]
  33. Yoshida M., Sone N., Hirata H., Kagawa Y. Reconstitution of adenosine triphosphatase of thermophilic bacterium from purified individual subunits. J Biol Chem. 1977 May 25;252(10):3480–3485. [PubMed] [Google Scholar]

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