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Photosynthesis Research

, Volume 103, Issue 2, pp 67–77 | Cite as

Enzymatic properties of the ferredoxin-dependent nitrite reductase from Chlamydomonas reinhardtii. Evidence for hydroxylamine as a late intermediate in ammonia production

  • Masakazu Hirasawa
  • Jatindra N. Tripathy
  • Frederik Sommer
  • Ramasamy Somasundaram
  • Jung-Sung Chung
  • Matthew Nestander
  • Mahima Kruthiventi
  • Masoud Zabet-Moghaddam
  • Michael K. Johnson
  • Sabeeha S. Merchant
  • James P. Allen
  • David B. KnaffEmail author
Regular Paper

Abstract

The ferredoxin-dependent nitrite reductase from the green alga Chlamydomonas reinhardtii has been cloned, expressed in Escherichia coli as a His-tagged recombinant protein, and purified to homogeneity. The spectra, kinetic properties and substrate-binding parameters of the C. reinhardtii enzyme are quite similar to those of the ferredoxin-dependent spinach chloroplast nitrite reductase. Computer modeling, based on the published structure of spinach nitrite reductase, predicts that the structure of C. reinhardtii nitrite reductase will be similar to that of the spinach enzyme. Chemical modification studies and the ionic-strength dependence of the enzyme’s ability to interact with ferredoxin are consistent with the involvement of arginine and lysine residues on C. reinhardtii nitrite reductase in electrostatically-stabilized binding to ferredoxin. The C. reinhardtii enzyme has been used to demonstrate that hydroxylamine can serve as an electron-accepting substrate for the enzyme and that the product of hydroxylamine reduction is ammonia, providing the first experimental evidence for the hypothesis that hydroxylamine, bound to the enzyme, can serve as a late intermediate during the reduction of nitrite to ammonia catalyzed by the enzyme.

Keywords

Nitrite reductase NII1 PETF Ferredoxin Hydroxylamine reduction Tertiary structure 

Abbreviations

EPR

Electron paramagnetic resonance

EST

Expressed sequence tag

FNR

Ferredoxin:NADP+ oxidoreductase

MALDI-TOF/TOF

Matrix-assisted laser desorption ionization-time of flight mass spectrometry

PVDF

Polyvinylidene fluoride

SDS-PAGE

Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate

Notes

Acknowledgments

This research was supported by contracts from the Chemical Sciences, Geosciences and Biosciences Division, Office of the Basic Energy Sciences, Office of Sciences, U.S. Department of Energy (Contract Numbers DE-FG03-99ER20346 to D.B.K. and DE-FG02-04ER15529 to S.S.M.). F.S. was supported by a fellowship from the DFG (SO706/1-1). The EPR spectroscopy characterizations were funded by a grant from the U.S. National Institutes of Health (GM62524 to M.K.J.). The authors would like to thank Ms. Shu Fen Lu for her assistance in purifying the C. reinhardtii ferredoxin used in this study, Prof. Toshiharu Hase (Osaka University, Japan) for his suggestion that hydroxylamine be tested as a potential substrate and his advice on the assay system for hydroxylamine reduction, Prof. Emilio Fernández (University of Córdoba, Spain) for his assistance and advice on the cloning of the gene for C. reinhardtii nitrite reductase and Dr. Arthur Grossman (Carnegie Institution) for his gift of the cDNA clone for NII1.

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Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Masakazu Hirasawa
    • 1
  • Jatindra N. Tripathy
    • 2
  • Frederik Sommer
    • 3
    • 6
  • Ramasamy Somasundaram
    • 4
  • Jung-Sung Chung
    • 1
  • Matthew Nestander
    • 1
  • Mahima Kruthiventi
    • 2
  • Masoud Zabet-Moghaddam
    • 2
  • Michael K. Johnson
    • 4
  • Sabeeha S. Merchant
    • 3
  • James P. Allen
    • 5
  • David B. Knaff
    • 1
    • 2
    Email author
  1. 1.Department of Chemistry and BiochemistryTexas Tech UniversityLubbockUSA
  2. 2.Center for Biotechnology and GenomicsTexas Tech UniversityLubbockUSA
  3. 3.Department of Chemistry and Biochemistry and the Institute of Genomics and ProteomicsUniversity of California, Los AngelesLos AngelesUSA
  4. 4.Department of ChemistryUniversity of GeorgiaAthensUSA
  5. 5.Department of Chemistry and BiochemistryArizona State UniversityTempeUSA
  6. 6.Max Planck Institute of Molecular Plant PhysiologyPotsdam-GolmGermany

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