Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1994 Nov;3(11):2073–2081. doi: 10.1002/pro.5560031120

Docking of nitrogenase iron- and molybdenum-iron proteins for electron transfer and MgATP hydrolysis: the role of arginine 140 and lysine 143 of the Azotobacter vinelandii iron protein.

L C Seefeldt 1
PMCID: PMC2142651  PMID: 7703853

Abstract

Docking of the nitrogenase component proteins, the iron protein (FeP) and the molybdenum-iron protein (MoFeP), is required for MgATP hydrolysis, electron transfer between the component proteins, and substrate reductions catalyzed by nitrogenase. The present work examines the function of 3 charged amino acids, Arg 140, Glu 141, and Lys 143, of the Azotobacter vinelandii FeP in nitrogenase component protein docking. The function of these amino acids was probed by changing each to the neutral amino acid glutamine using site-directed mutagenesis. The altered FePs were expressed in A. vinelandii in place of the wild-type FeP. Changing Glu 141 to Gln (E141Q) had no adverse effects on the function of nitrogenase in whole cells, indicating that this charged residue is not essential to nitrogenase function. In contrast, changing Arg 140 or Lys 143 to Gln (R140Q and K143Q) resulted in a significant decrease in nitrogenase activity, suggesting that these charged amino acid residues play an important role in some function of the FeP. The function of each amino acid was deduced by analysis of the properties of the purified R140Q and K143Q FePs. Both altered proteins were found to support reduced substrate reduction rates when coupled to wild-type MoFeP. Detailed analysis revealed that changing these residues to Gln resulted in a dramatic reduction in the affinity of the altered FeP for binding to the MoFeP. This was deduced in FeP titration, NaCl inhibition, and MoFeP protection from Fe2+ chelation experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Full Text

The Full Text of this article is available as a PDF (2.6 MB).

Selected References

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

  1. Bolin J. T., Ronco A. E., Morgan T. V., Mortenson L. E., Xuong N. H. The unusual metal clusters of nitrogenase: structural features revealed by x-ray anomalous diffraction studies of the MoFe protein from Clostridium pasteurianum. Proc Natl Acad Sci U S A. 1993 Feb 1;90(3):1078–1082. doi: 10.1073/pnas.90.3.1078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chang C. L., Davis L. C., Rider M., Takemoto D. J. Characterization of nifH mutations of Klebsiella pneumoniae. J Bacteriol. 1988 Sep;170(9):4015–4022. doi: 10.1128/jb.170.9.4015-4022.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chen L., Gavini N., Tsuruta H., Eliezer D., Burgess B. K., Doniach S., Hodgson K. O. MgATP-induced conformational changes in the iron protein from Azotobacter vinelandii, as studied by small-angle x-ray scattering. J Biol Chem. 1994 Feb 4;269(5):3290–3294. [PubMed] [Google Scholar]
  4. Chromý V., Fischer J., Kulhánek V. Re-evaluation of EDTA-chelated biuret reagent. Clin Chem. 1974 Oct;20(10):1362–1363. [PubMed] [Google Scholar]
  5. Dean D. R., Bolin J. T., Zheng L. Nitrogenase metalloclusters: structures, organization, and synthesis. J Bacteriol. 1993 Nov;175(21):6737–6744. doi: 10.1128/jb.175.21.6737-6744.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Deits T. L., Howard J. B. Effect of salts on Azotobacter vinelandii nitrogenase activities. Inhibition of iron chelation and substrate reduction. J Biol Chem. 1990 Mar 5;265(7):3859–3867. [PubMed] [Google Scholar]
  7. Emerich D. W., Burris R. H. Complementary functioning of the component proteins of nitrogenase from several bacteria. J Bacteriol. 1978 Jun;134(3):936–943. doi: 10.1128/jb.134.3.936-943.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Jacobson M. R., Cash V. L., Weiss M. C., Laird N. F., Newton W. E., Dean D. R. Biochemical and genetic analysis of the nifUSVWZM cluster from Azotobacter vinelandii. Mol Gen Genet. 1989 Oct;219(1-2):49–57. doi: 10.1007/BF00261156. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Kim J., Rees D. C. Nitrogenase and biological nitrogen fixation. Biochemistry. 1994 Jan 18;33(2):389–397. doi: 10.1021/bi00168a001. [DOI] [PubMed] [Google Scholar]
  11. Kim J., Rees D. C. Structural models for the metal centers in the nitrogenase molybdenum-iron protein. Science. 1992 Sep 18;257(5077):1677–1682. doi: 10.1126/science.1529354. [DOI] [PubMed] [Google Scholar]
  12. Kim J., Woo D., Rees D. C. X-ray crystal structure of the nitrogenase molybdenum-iron protein from Clostridium pasteurianum at 3.0-A resolution. Biochemistry. 1993 Jul 20;32(28):7104–7115. doi: 10.1021/bi00079a006. [DOI] [PubMed] [Google Scholar]
  13. Lowe D. J., Fisher K., Thorneley R. N. Klebsiella pneumoniae nitrogenase: pre-steady-state absorbance changes show that redox changes occur in the MoFe protein that depend on substrate and component protein ratio; a role for P-centres in reducing dinitrogen? Biochem J. 1993 May 15;292(Pt 1):93–98. doi: 10.1042/bj2920093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lowery R. G., Chang C. L., Davis L. C., McKenna M. C., Stephens P. J., Ludden P. W. Substitution of histidine for arginine-101 of dinitrogenase reductase disrupts electron transfer to dinitrogenase. Biochemistry. 1989 Feb 7;28(3):1206–1212. doi: 10.1021/bi00429a038. [DOI] [PubMed] [Google Scholar]
  15. Meyer J., Gaillard J., Moulis J. M. Hydrogen-1 nuclear magnetic resonance of the nitrogenase iron protein (Cp2) from Clostridium pasteurianum. Biochemistry. 1988 Aug 9;27(16):6150–6156. doi: 10.1021/bi00416a048. [DOI] [PubMed] [Google Scholar]
  16. Mortenson L. E., Seefeldt L. C., Morgan T. V., Bolin J. T. The role of metal clusters and MgATP in nitrogenase catalysis. Adv Enzymol Relat Areas Mol Biol. 1993;67:299–374. doi: 10.1002/9780470123133.ch4. [DOI] [PubMed] [Google Scholar]
  17. Orme-Johnson W. H., Hamilton W. D., Jones T. L., Tso M. Y., Burris R. H., Shah V. K., Brill W. J. Electron paramagnetic resonance of nitrogenase and nitrogenase components from Clostridium pasteurianum W5 and Azotobacter vinelandii OP. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3142–3145. doi: 10.1073/pnas.69.11.3142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Qin L., Kostić N. M. Importance of protein rearrangement in the electron-transfer reaction between the physiological partners cytochrome f and plastocyanin. Biochemistry. 1993 Jun 15;32(23):6073–6080. doi: 10.1021/bi00074a019. [DOI] [PubMed] [Google Scholar]
  19. Seefeldt L. C., Morgan T. V., Dean D. R., Mortenson L. E. Mapping the site(s) of MgATP and MgADP interaction with the nitrogenase of Azotobacter vinelandii. Lysine 15 of the iron protein plays a major role in MgATP interaction. J Biol Chem. 1992 Apr 5;267(10):6680–6688. [PubMed] [Google Scholar]
  20. Seefeldt L. C., Mortenson L. E. Increasing nitrogenase catalytic efficiency for MgATP by changing serine 16 of its Fe protein to threonine: use of Mn2+ to show interaction of serine 16 with Mg2+. Protein Sci. 1993 Jan;2(1):93–102. doi: 10.1002/pro.5560020110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Smith B. E., Eady R. R. Metalloclusters of the nitrogenases. Eur J Biochem. 1992 Apr 1;205(1):1–15. doi: 10.1111/j.1432-1033.1992.tb16746.x. [DOI] [PubMed] [Google Scholar]
  22. Smith B. E., Thorneley R. N., Eady R. R., Mortenson L. E. Nitrogenases from Klebsiella pneumoniae and Clostridium pasteurianum. Kinetic investigations of cross-reactions as a probe of the enzyme mechanism. Biochem J. 1976 Aug 1;157(2):439–447. doi: 10.1042/bj1570439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Stephens P. J., McKenna C. E., Smith B. E., Nguyen H. T., McKenna M. C., Thomson A. J., Devlin F., Jones J. B. Circular dichroism and magnetic circular dichroism of nitrogenase proteins. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2585–2589. doi: 10.1073/pnas.76.6.2585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Willing A. H., Georgiadis M. M., Rees D. C., Howard J. B. Cross-linking of nitrogenase components. Structure and activity of the covalent complex. J Biol Chem. 1989 May 25;264(15):8499–8503. [PubMed] [Google Scholar]
  25. Willing A., Howard J. B. Cross-linking site in Azotobacter vinelandii complex. J Biol Chem. 1990 Apr 25;265(12):6596–6599. [PubMed] [Google Scholar]
  26. Wolle D., Dean D. R., Howard J. B. Nucleotide-iron-sulfur cluster signal transduction in the nitrogenase iron-protein: the role of Asp125. Science. 1992 Nov 6;258(5084):992–995. doi: 10.1126/science.1359643. [DOI] [PubMed] [Google Scholar]
  27. Wolle D., Kim C., Dean D., Howard J. B. Ionic interactions in the nitrogenase complex. Properties of Fe-protein containing substitutions for Arg-100. J Biol Chem. 1992 Feb 25;267(6):3667–3673. [PubMed] [Google Scholar]
  28. Zumft W. G., Cretney W. C., Huang T. C., Mortenson L. E., Palmer G. On the structure and function of nitrogenase from Clostridium pasteurianum W5. Biochem Biophys Res Commun. 1972 Sep 26;48(6):1525–1532. doi: 10.1016/0006-291x(72)90887-x. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES