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. 1986 May;166(2):513–518. doi: 10.1128/jb.166.2.513-518.1986

Reversible regulation of the nitrogenase iron protein from Rhodospirillum rubrum by ADP-ribosylation in vitro.

R G Lowery, L L Saari, P W Ludden
PMCID: PMC214634  PMID: 3084451

Abstract

Nitrogenase activity in the photosynthetic bacterium Rhodospirillum rubrum is reversibly regulated by interconversion of the Fe protein between a modified and an unmodified form. Since the discovery of the activation process in 1976, investigators have been unable to demonstrate the inactivation (modification) reaction in vitro. In this study, NAD-dependent modification and concomitant inactivation of the Fe protein were demonstrated in crude extracts of R. rubrum. Activation of the in vitro-modified Fe protein by activating enzyme and structural similarity between the in vivo and in vitro modifications are presented as evidence that the in vitro modification is the physiologically relevant ADP-ribosylation reaction. Using a partially purified preparation, we showed that the inactivating enzyme activity is stimulated by divalent metal ions and ADP, that O2-denatured Fe protein will not serve as a substrate, and that dithionite inhibits the modification reaction.

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Selected References

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  1. Bognar A., Desrosiers L., Libman M., Newman E. B. Control of nitrogenase in a photosynthetic autotrophic bacterium, Ectothiorhodospira sp. J Bacteriol. 1982 Nov;152(2):706–713. doi: 10.1128/jb.152.2.706-713.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Caban C. E., Ginsburg A. Glutamine synthetase adenylyltransferase from Escherichia coli: purification and physical and chemical properties. Biochemistry. 1976 Apr 6;15(7):1569–1580. doi: 10.1021/bi00652a030. [DOI] [PubMed] [Google Scholar]
  3. Carithers R. P., Yoch D. C., Arnon D. I. Two forms of nitrogenase from the photosynthetic bacterium Rhodospirillum rubrum. J Bacteriol. 1979 Feb;137(2):779–789. doi: 10.1128/jb.137.2.779-789.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chock P. B., Stadtman E. R. Superiority of interconvertible enzyme cascades in metabolite regulation: analysis of multicyclic systems. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2766–2770. doi: 10.1073/pnas.74.7.2766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. El-Maghrabi M. R., Claus T. H., Pilkis J., Fox E., Pilkis S. J. Regulation of rat liver fructose 2,6-bisphosphatase. J Biol Chem. 1982 Jul 10;257(13):7603–7607. [PubMed] [Google Scholar]
  6. Goldbeter A., Koshland D. E., Jr An amplified sensitivity arising from covalent modification in biological systems. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6840–6844. doi: 10.1073/pnas.78.11.6840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gotto J. W., Yoch D. C. Regulation of nitrogenase activity by covalent modification in Chromatium vinosum. Arch Microbiol. 1985 Feb;141(1):40–43. doi: 10.1007/BF00446737. [DOI] [PubMed] [Google Scholar]
  8. Hallenbeck P. C., Meyer C. M., Vignais P. M. Nitrogenase from the photosynthetic bacterium Rhodopseudomonas capsulata: purification and molecular properties. J Bacteriol. 1982 Feb;149(2):708–717. doi: 10.1128/jb.149.2.708-717.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Howard K. S., Hales B. J., Socolofsky M. D. Nitrogen fixation and ammonia switch-off in the photosynthetic bacterium Rhodopseudomonas viridis. J Bacteriol. 1983 Jul;155(1):107–112. doi: 10.1128/jb.155.1.107-112.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jones B. L., Monty K. J. Glutamine as a feedback inhibitor of the Rhodopseudomonas sphaeroides nitrogenase system. J Bacteriol. 1979 Sep;139(3):1007–1013. doi: 10.1128/jb.139.3.1007-1013.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kamen M. D., Gest H. Evidence for a Nitrogenase System in the Photosynthetic Bacterium Rhodospirillum rubrum. Science. 1949 Jun 3;109(2840):560–560. doi: 10.1126/science.109.2840.560. [DOI] [PubMed] [Google Scholar]
  12. Kanemoto R. H., Ludden P. W. Effect of ammonia, darkness, and phenazine methosulfate on whole-cell nitrogenase activity and Fe protein modification in Rhodospirillum rubrum. J Bacteriol. 1984 May;158(2):713–720. doi: 10.1128/jb.158.2.713-720.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Koshland D. E., Jr, Goldbeter A., Stock J. B. Amplification and adaptation in regulatory and sensory systems. Science. 1982 Jul 16;217(4556):220–225. doi: 10.1126/science.7089556. [DOI] [PubMed] [Google Scholar]
  14. Krebs E. G., Beavo J. A. Phosphorylation-dephosphorylation of enzymes. Annu Rev Biochem. 1979;48:923–959. doi: 10.1146/annurev.bi.48.070179.004423. [DOI] [PubMed] [Google Scholar]
  15. LaPorte D. C., Koshland D. E., Jr A protein with kinase and phosphatase activities involved in regulation of tricarboxylic acid cycle. Nature. 1982 Dec 2;300(5891):458–460. doi: 10.1038/300458a0. [DOI] [PubMed] [Google Scholar]
  16. LaPorte D. C., Koshland D. E., Jr Phosphorylation of isocitrate dehydrogenase as a demonstration of enhanced sensitivity in covalent regulation. Nature. 1983 Sep 22;305(5932):286–290. doi: 10.1038/305286a0. [DOI] [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Ludden P. W., Burris R. H. Activating factor for the iron protein of nitrogenase from Rhodospirillum rubrum. Science. 1976 Oct 22;194(4263):424–426. doi: 10.1126/science.824729. [DOI] [PubMed] [Google Scholar]
  19. Mortenson L. E., Thorneley R. N. Structure and function of nitrogenase. Annu Rev Biochem. 1979;48:387–418. doi: 10.1146/annurev.bi.48.070179.002131. [DOI] [PubMed] [Google Scholar]
  20. Mura U., Chock P. B., Stadtman E. R. Allosteric regulation of the state of adenylylation of glutamine synthetase in permeabilized cell preparations of Escherichia coli. Studies of monocyclic and bicyclic interconvertible enzyme cascades, in situ. J Biol Chem. 1981 Dec 25;256(24):13022–13029. [PubMed] [Google Scholar]
  21. Neilson A. H., Nordlund S. Regulation of nitrogenase synthesis in intact cells of Rhodospirillum rubrum: inactivation of nitrogen fixation by ammonia, L-glutamine and L-asparagine. J Gen Microbiol. 1975 Nov;91(1):53–62. doi: 10.1099/00221287-91-1-53. [DOI] [PubMed] [Google Scholar]
  22. Nordlund S., Eriksson U., Baltscheffsky H. Necessity of a membrane component for nitrogenase activity in Rhodospirillum rubrum. Biochim Biophys Acta. 1977 Oct 12;462(1):187–195. doi: 10.1016/0005-2728(77)90201-8. [DOI] [PubMed] [Google Scholar]
  23. ORMEROD J. G., ORMEROD K. S., GEST H. Light-dependent utilization of organic compounds and photoproduction of molecular hydrogen by photosynthetic bacteria; relationships with nitrogen metabolism. Arch Biochem Biophys. 1961 Sep;94:449–463. doi: 10.1016/0003-9861(61)90073-x. [DOI] [PubMed] [Google Scholar]
  24. Pope M. R., Murrell S. A., Ludden P. W. Covalent modification of the iron protein of nitrogenase from Rhodospirillum rubrum by adenosine diphosphoribosylation of a specific arginine residue. Proc Natl Acad Sci U S A. 1985 May;82(10):3173–3177. doi: 10.1073/pnas.82.10.3173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Preston G. G., Ludden P. W. Change in subunit composition of the iron protein of nitrogenase from Rhodospirillum rubrum during activation and inactivation of iron protein. Biochem J. 1982 Sep 1;205(3):489–494. doi: 10.1042/bj2050489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rhee S. G., Park R., Chock P. B., Stadtman E. R. Allosteric regulation of monocyclic interconvertible enzyme cascade systems: use of Escherichia coli glutamine synthetase as an experimental model. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3138–3142. doi: 10.1073/pnas.75.7.3138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Saari L. L., Triplett E. W., Ludden P. W. Purification and properties of the activating enzyme for iron protein of nitrogenase from the photosynthetic bacterium Rhodospirillum rubrum. J Biol Chem. 1984 Dec 25;259(24):15502–15508. [PubMed] [Google Scholar]
  28. Schick H. J. Substrate and light dependent fixation of molecular nitrogen in Rhodospirillum rubrum. Arch Mikrobiol. 1971;75(2):89–101. doi: 10.1007/BF00407997. [DOI] [PubMed] [Google Scholar]
  29. Shacter E., Chock P. B., Stadtman E. R. Regulation through phosphorylation/dephosphorylation cascade systems. J Biol Chem. 1984 Oct 10;259(19):12252–12259. [PubMed] [Google Scholar]
  30. Simpson F. B., Burris R. H. A nitrogen pressure of 50 atmospheres does not prevent evolution of hydrogen by nitrogenase. Science. 1984 Jun 8;224(4653):1095–1097. doi: 10.1126/science.6585956. [DOI] [PubMed] [Google Scholar]
  31. Stadtman E. R., Chock P. B. Interconvertible enzyme cascades in metabolic regulation. Curr Top Cell Regul. 1978;13:53–95. doi: 10.1016/b978-0-12-152813-3.50007-0. [DOI] [PubMed] [Google Scholar]
  32. Stadtman E. R., Chock P. B. Superiority of interconvertible enzyme cascades in metabolic regulation: analysis of monocyclic systems. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2761–2765. doi: 10.1073/pnas.74.7.2761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sweet W. J., Burris R. H. Inhibition of nitrogenase activity by NH+4 in Rhodospirillum rubrum. J Bacteriol. 1981 Feb;145(2):824–831. doi: 10.1128/jb.145.2.824-831.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Triplett E. W., Wall J. D., Ludden P. W. Expression of the activating enzyme and Fe protein of nitrogenase from Rhodospirillum rubrum. J Bacteriol. 1982 Nov;152(2):786–791. doi: 10.1128/jb.152.2.786-791.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Yoch D. C., Cantu M. Changes in the regulatory form of Rhodospirillum rubrum nitrogenase as influenced by nutritional and environmental factors. J Bacteriol. 1980 Jun;142(3):899–907. doi: 10.1128/jb.142.3.899-907.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yoch D. C., Gotto J. W. Effect of light intensity and inhibitors of nitrogen assimilation on NH4+ inhibition of nitrogenase activity in Rhodospirillum rubrum and Anabaena sp. J Bacteriol. 1982 Aug;151(2):800–806. doi: 10.1128/jb.151.2.800-806.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Zumft W. G., Castillo F. Regulatory properties of the nitrogenase from Rhodopseudomonas palustris. Arch Microbiol. 1978 Apr 27;117(1):53–60. doi: 10.1007/BF00689351. [DOI] [PubMed] [Google Scholar]

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