Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1991 Aug 1;277(Pt 3):735–741. doi: 10.1042/bj2770735

Nitrogenase of Klebsiella pneumoniae. Reversibility of the reductant-independent MgATP-cleavage reaction is shown by MgADP-catalysed phosphate/water oxygen exchange.

R N Thorneley 1, G A Ashby 1, C Julius 1, J L Hunter 1, M R Webb 1
PMCID: PMC1151306  PMID: 1872810

Abstract

The steady-state kinetics of reductant-independent ATP hydrolysis by Klebsiella pneumoniae nitrogenase at 23 degrees C at pH 7.4 were determined as a function of component protein ratio (optimal at an oxidized Fe protein/MoFe protein ratio of 3:1) and MgATP concentration (Km 400 microM). Competitive inhibition was observed for MgADP (Ki 145 microM), [beta gamma-methylene]ATP (Mgp[CH2]ppA) (Ki 115 microM), [beta gamma-monofluoromethylene]ATP (Mgp[CHF]ppA) (Ki 53 microM) and [beta gamma-difluoromethylene]ATP (Mgp[CF2]ppA) (Ki 160 microM). The tighter binding of MgADP to free oxidized Fe protein (KD less than 10 microM) than to the oxidized Fe protein-MoFe protein complex (Ki 145 microM) is proposed as the driving force that induces rate-limiting protein dissociation in the catalytic cycle of nitrogenase. The reversible nature of the reductant-independent MgATP-cleavage reaction was demonstrated by an MgADP-induced enhancement of the rate of the phosphate/water oxygen exchange reaction with 18O-labelled phosphate ion. This enhancement, like the reductant-independent ATPase reaction, only occurred with the complex formed by oxidized Fe protein and MoFe protein and not with the individual proteins. The results are discussed in terms of the mechanism of ATP hydrolysis by nitrogenase and other systems involving protein-protein interactions.

Full text

PDF
737

Selected References

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

  1. Ashby G. A., Thorneley R. N. Nitrogenase of Klebsiella pneumoniae. Kinetic studies on the Fe protein involving reduction by sodium dithionite, the binding of MgADP and a conformation change that alters the reactivity of the 4Fe-4S centre. Biochem J. 1987 Sep 1;246(2):455–465. doi: 10.1042/bj2460455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cordewener J., ten Asbroek A., Wassink H., Eady R., Haaker H., Veeger C. Binding of ADP and orthophosphate during the ATPase reaction of nitrogenase. Eur J Biochem. 1987 Jan 15;162(2):265–270. doi: 10.1111/j.1432-1033.1987.tb10594.x. [DOI] [PubMed] [Google Scholar]
  3. Cordewener J., ten Asbroek A., Wassink H., Eady R., Haaker H., Veeger C. Binding of ADP and orthophosphate during the ATPase reaction of nitrogenase. Eur J Biochem. 1987 Jan 15;162(2):265–270. doi: 10.1111/j.1432-1033.1987.tb10594.x. [DOI] [PubMed] [Google Scholar]
  4. Hackney D. D., Stempel K. E., Boyer P. D. Oxygen-18 probes of enzymic reactions of phosphate compounds. Methods Enzymol. 1980;64:60–83. doi: 10.1016/s0076-6879(80)64005-1. [DOI] [PubMed] [Google Scholar]
  5. Hageman R. V., Burris R. H. Kinetic studies on electron transfer and interaction between nitrogenase components from Azotobacter vinelandii. Biochemistry. 1978 Oct 3;17(20):4117–4124. doi: 10.1021/bi00613a002. [DOI] [PubMed] [Google Scholar]
  6. Hibberd M. G., Webb M. R., Goldman Y. E., Trentham D. R. Oxygen exchange between phosphate and water accompanies calcium-regulated ATPase activity of skinned fibers from rabbit skeletal muscle. J Biol Chem. 1985 Mar 25;260(6):3496–3500. [PubMed] [Google Scholar]
  7. Imam S., Eady R. R. Nitrogenase of Klebsiella pneumoniae: reductant-independent ATP hydrolysis and the effect of pH on the efficiency of coupling of ATP hydrolysis to substrate reduction. FEBS Lett. 1980 Jan 28;110(1):35–38. doi: 10.1016/0014-5793(80)80016-0. [DOI] [PubMed] [Google Scholar]
  8. Jeng D. Y., Morris J. A., Mortenson L. E. The effect of reductant in inorganic phosphate release from adenosine 5'-triphosphate by purified nitrogenase of Clostridium pasteurianum. J Biol Chem. 1970 Jun 10;245(11):2809–2813. [PubMed] [Google Scholar]
  9. KOSHLAND D. E., Jr, CLARKE E. Mechanism of hydrolysis of adenosinetriphosphate catalyzed by lobster muscle. J Biol Chem. 1953 Dec;205(2):917–924. [PubMed] [Google Scholar]
  10. LEVY H. M., KOSHLAND D. E., Jr Mechanism of hydrolysis of adenosinetriphosphate by muscle proteins and its relation to muscular contraction. J Biol Chem. 1959 May;234(5):1102–1107. [PubMed] [Google Scholar]
  11. Lienhard G. E., Secemski I. I. P 1 ,P 5 -Di(adenosine-5')pentaphosphate, a potent multisubstrate inhibitor of adenylate kinase. J Biol Chem. 1973 Feb 10;248(3):1121–1123. [PubMed] [Google Scholar]
  12. Lowe D. J., Thorneley R. N. The mechanism of Klebsiella pneumoniae nitrogenase action. Pre-steady-state kinetics of H2 formation. Biochem J. 1984 Dec 15;224(3):877–886. doi: 10.1042/bj2240877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Millar N. C., Howarth J. V., Gutfreund H. A transient kinetic study of enthalpy changes during the reaction of myosin subfragment 1 with ATP. Biochem J. 1987 Dec 15;248(3):683–690. doi: 10.1042/bj2480683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Neal S. E., Eccleston J. F., Webb M. R. Hydrolysis of GTP by p21NRAS, the NRAS protooncogene product, is accompanied by a conformational change in the wild-type protein: use of a single fluorescent probe at the catalytic site. Proc Natl Acad Sci U S A. 1990 May;87(9):3562–3565. doi: 10.1073/pnas.87.9.3562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ottolenghi P. The reversible delipidation of a solubilized sodium-plus-potassium ion-dependent adenosine triphosphatase from the salt gland of the spiny dogfish. Biochem J. 1975 Oct;151(1):61–66. doi: 10.1042/bj1510061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Thorneley R. N., Ashby G., Howarth J. V., Millar N. C., Gutfreund H. A transient-kinetic study of the nitrogenase of Klebsiella pneumoniae by stopped-flow calorimetry. Comparison with the myosin ATPase. Biochem J. 1989 Dec 15;264(3):657–661. doi: 10.1042/bj2640657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Thorneley R. N., Deistung J. Electron-transfer studies involving flavodoxin and a natural redox partner, the iron protein of nitrogenase. Conformational constraints on protein-protein interactions and the kinetics of electron transfer within the protein complex. Biochem J. 1988 Jul 15;253(2):587–595. doi: 10.1042/bj2530587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Thorneley R. N., Lowe D. J. Nitrogenase of Klebsiella pneumoniae. Kinetics of the dissociation of oxidized iron protein from molybdenum-iron protein: identification of the rate-limiting step for substrate reduction. Biochem J. 1983 Nov 1;215(2):393–403. doi: 10.1042/bj2150393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Thorneley R. N. Nitrogenase of Klebsiella pneumoniae. A stopped-flow study of magnesium-adenosine triphosphate-induce electron transfer between the compeonent proteins. Biochem J. 1975 Feb;145(2):391–396. doi: 10.1042/bj1450391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Watt G. D., Bulen W. A., Burns A., Hadfield K. L. Stoichiometry, ATP/2e values, and energy requirements for reactions catalyzed by nitrogenase from Azotobacter vinelandii. Biochemistry. 1975 Sep 23;14(19):4266–4272. doi: 10.1021/bi00690a019. [DOI] [PubMed] [Google Scholar]
  21. Webb M. R., Hibberd M. G., Goldman Y. E., Trentham D. R. Oxygen exchange between Pi in the medium and water during ATP hydrolysis mediated by skinned fibers from rabbit skeletal muscle. Evidence for Pi binding to a force-generating state. J Biol Chem. 1986 Nov 25;261(33):15557–15564. [PubMed] [Google Scholar]
  22. Webb M. R., McDonald G. G., Trentham D. R. Kinetics of oxygen-18 exchange between inorganic phosphate and water catalyzed by myosin subfragment 1, using the 18O shift in 31P NMR. J Biol Chem. 1978 May 10;253(9):2908–2911. [PubMed] [Google Scholar]
  23. Webb M. R., Trentham D. R. The stereochemical course of phosphoric residue transfer during the myosin ATPase reaction. J Biol Chem. 1980 Sep 25;255(18):8629–8632. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES