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

Overview

Journal Protein Sci

Specialty Biochemistry

Date 1994 Nov 1

PMID 7703853

Citations 8

Authors

L C Seefeldt

Affiliations

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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)

Citing Articles

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References

Lowery R, Chang C, Davis L, McKenna M, Stephens P, Ludden P . Substitution of histidine for arginine-101 of dinitrogenase reductase disrupts electron transfer to dinitrogenase. Biochemistry. 1989; 28(3):1206-12. DOI: 10.1021/bi00429a038. View

Chen L, Gavini N, Tsuruta H, Eliezer D, Burgess B, Doniach S . MgATP-induced conformational changes in the iron protein from Azotobacter vinelandii, as studied by small-angle x-ray scattering. J Biol Chem. 1994; 269(5):3290-4. View

Jacobson M, Cash V, Weiss M, Laird N, Newton W, Dean D . Biochemical and genetic analysis of the nifUSVWZM cluster from Azotobacter vinelandii. Mol Gen Genet. 1989; 219(1-2):49-57. DOI: 10.1007/BF00261156. View

Deits T, Howard J . Effect of salts on Azotobacter vinelandii nitrogenase activities. Inhibition of iron chelation and substrate reduction. J Biol Chem. 1990; 265(7):3859-67. View

Willing A, Howard J . Cross-linking site in Azotobacter vinelandii complex. J Biol Chem. 1990; 265(12):6596-9. View

Wolle D, Kim C, Dean D, Howard J . Ionic interactions in the nitrogenase complex. Properties of Fe-protein containing substitutions for Arg-100. J Biol Chem. 1992; 267(6):3667-73. View

Seefeldt L, Morgan T, Dean D, Mortenson L . 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; 267(10):6680-8. View

Smith B, Eady R . Metalloclusters of the nitrogenases. Eur J Biochem. 1992; 205(1):1-15. DOI: 10.1111/j.1432-1033.1992.tb16746.x. View

Kim J, Rees D . Structural models for the metal centers in the nitrogenase molybdenum-iron protein. Science. 1992; 257(5077):1677-82. DOI: 10.1126/science.1529354. View

10.

Wolle D, Dean D, Howard J . Nucleotide-iron-sulfur cluster signal transduction in the nitrogenase iron-protein: the role of Asp125. Science. 1992; 258(5084):992-5. DOI: 10.1126/science.1359643. View

11.

Bolin J, Ronco A, Morgan T, Mortenson L, Xuong N . 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; 90(3):1078-82. PMC: 45814. DOI: 10.1073/pnas.90.3.1078. View

12.

Seefeldt L, Mortenson L . 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; 2(1):93-102. PMC: 2142304. DOI: 10.1002/pro.5560020110. View

13.

Lowe D, Fisher K, Thorneley R . 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; 292 ( Pt 1):93-8. PMC: 1134273. DOI: 10.1042/bj2920093. View

14.

Qin L, Kostic N . Importance of protein rearrangement in the electron-transfer reaction between the physiological partners cytochrome f and plastocyanin. Biochemistry. 1993; 32(23):6073-80. DOI: 10.1021/bi00074a019. View

15.

Mortenson L, Seefeldt L, Morgan T, Bolin J . 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. View

16.

Kim J, Woo D, Rees D . X-ray crystal structure of the nitrogenase molybdenum-iron protein from Clostridium pasteurianum at 3.0-A resolution. Biochemistry. 1993; 32(28):7104-15. DOI: 10.1021/bi00079a006. View

17.

Jeng D, Morris J, Mortenson L . The effect of reductant in inorganic phosphate release from adenosine 5'-triphosphate by purified nitrogenase of Clostridium pasteurianum. J Biol Chem. 1970; 245(11):2809-13. View

18.

Zumft W, Cretney W, Huang T, Mortenson L, Palmer G . On the structure and function of nitrogenase from Clostridium pasteurianum W5. Biochem Biophys Res Commun. 1972; 48(6):1525-32. DOI: 10.1016/0006-291x(72)90887-x. View

19.

Orme-Johnson W, Hamilton W, Jones T, Tso M, BURRIS R, Shah V . Electron paramagnetic resonance of nitrogenase and nitrogenase components from Clostridium pasteurianum W5 and Azotobacter vinelandii OP. Proc Natl Acad Sci U S A. 1972; 69(11):3142-5. PMC: 389722. DOI: 10.1073/pnas.69.11.3142. View

20.

Chromy V, Fischer J, KULHANEK V . Re-evaluation of EDTA-chelated biuret reagent. Clin Chem. 1974; 20(10):1362-3. View