Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 1988 Oct;88(2):389–395. doi: 10.1104/pp.88.2.389

The Conversion of Nitrite to Nitrogen Oxide(s) by the Constitutive NAD(P)H-Nitrate Reductase Enzyme from Soybean 1

John V Dean 1,2,2, James E Harper 1,2
PMCID: PMC1055587  PMID: 16666314

Abstract

A two-step purification protocol was used in an attempt to separate the constitutive NAD(P)H-nitrate reductase [NAD(P)H-NR, pH 6.5; EC 1.6.6.2] activity from the nitric oxide and nitrogen dioxide (NO(x)) evolution activity extracted from soybean (Glycine max [L.] Merr.) leaflets. Both of these activities were eluted with NADPH from Blue Sepharose columns loaded with extracts from either wild-type or LNR-5 and LNR-6 (lack constitutive NADH-NR [pH 6.5]) mutant soybean plants regardless of nutrient growth conditions. Fast protein liquid chromatography-anion exchange (Mono Q column) chromatography following Blue Sepharose affinity chromatography was also unable to separate the two activities. These data provide strong evidence that the constitutive NAD(P)H-NR (pH 6.5) in soybean is the enzyme responsible for NO(x) formation. The Blue Sepharose-purified soybean enzyme has a pH optimum of 6.75, an apparent Km for nitrite of 0.49 millimolar, and an apparent Km for NADPH and NADH of 7.2 and 7.4 micromolar, respectively, for the NO(x) evolution activity. In addition to NAD(P)H, reduced flavin mononucleotide (FMNH2) and reduced methyl viologen (MV) can serve as electron donors for NO(x) evolution activity. The NADPH-, FMNH2-, and reduced MV-NO(x) evolution activities were all inhibited by cyanide. The NADPH activity was also inhibited by p-hydroxymer-curibenzoate, whereas, the FMNH2 and MV activities were relatively insensitive to inhibition. These data indicate that the terminal molybdenum-containing portion of the enzyme is involved in the reduction of nitrite to NO(x). NADPH eluted both NR and NO(x) evolution activities from Blue Sepharose columns loaded with extracts of either nitrate- or zero N-grown winged bean (Psophocarpus tetragonolobus [L.]), whereas NADH did not elute either type of activity. Winged bean appears to contain only one type of NR enzyme that is similar to the constitutive NAD(P)H-NR (pH 6.5) enzyme of soybean.

Full text

PDF
389

Selected References

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

  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  2. Dean J. V., Harper J. E. Nitric Oxide and Nitrous Oxide Production by Soybean and Winged Bean during the in Vivo Nitrate Reductase Assay. Plant Physiol. 1986 Nov;82(3):718–723. doi: 10.1104/pp.82.3.718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Harper J. E. Evolution of Nitrogen Oxide(s) during In Vivo Nitrate Reductase Assay of Soybean Leaves. Plant Physiol. 1981 Dec;68(6):1488–1493. doi: 10.1104/pp.68.6.1488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. IWASAKI H., SHIDARA S., SUZUKI H., MOR T. Studies on denitrification. VII. Further purification and properties of denitrifying enzyme. J Biochem. 1963 Apr;53:299–303. [PubMed] [Google Scholar]
  5. Iwasaki H., Matsubara T. A nitrite reductase from Achromobacter cycloclastes. J Biochem. 1972 Apr;71(4):645–652. [PubMed] [Google Scholar]
  6. Iwasaki H., Matsubara T. Cytochrome c-557 (551) and cytochrome cd of Alcaligenes faecalis. J Biochem. 1971 May;69(5):847–857. doi: 10.1093/oxfordjournals.jbchem.a129536. [DOI] [PubMed] [Google Scholar]
  7. Nelson R. S., Ryan S. A., Harper J. E. Soybean mutants lacking constitutive nitrate reductase activity : I. Selection and initial plant characterization. Plant Physiol. 1983 Jun;72(2):503–509. doi: 10.1104/pp.72.2.503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Nelson R. S., Streit L., Harper J. E. Nitrate Reductases from Wild-Type and nr(1)-Mutant Soybean (Glycine max [L.] Merr.) Leaves : II. Partial Activity, Inhibitor, and Complementation Analyses. Plant Physiol. 1986 Jan;80(1):72–76. doi: 10.1104/pp.80.1.72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Newton N. The two-haem nitrite reductase of Micrococcus denitrificans. Biochim Biophys Acta. 1969;185(2):316–331. doi: 10.1016/0005-2744(69)90425-2. [DOI] [PubMed] [Google Scholar]
  10. Ryan S. A., Nelson R. S., Harper J. E. Soybean Mutants Lacking Constitutive Nitrate Reductase Activity : II. Nitrogen Assimilation, Chlorate Resistance, and Inheritance. Plant Physiol. 1983 Jun;72(2):510–514. doi: 10.1104/pp.72.2.510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Streit L., Harper J. E. Biochemical Characterization of Soybean Mutants Lacking Constitutive NADH:Nitrate Reductase. Plant Physiol. 1986 Jun;81(2):593–596. doi: 10.1104/pp.81.2.593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Streit L., Martin B. A., Harper J. E. A method for the separation and partial purification of the three forms of nitrate reductase present in wild-type soybean leaves. Plant Physiol. 1987 Jul;84(3):654–657. doi: 10.1104/pp.84.3.654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Streit L., Nelson R. S., Harper J. E. Nitrate Reductases from Wild-Type and nr(1)-Mutant Soybean (Glycine max [L.] Merr.) Leaves : I. Purification, Kinetics, and Physical Properties. Plant Physiol. 1985 May;78(1):80–84. doi: 10.1104/pp.78.1.80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. YAMANAKA T., OKUNUKI K. Crystalline Pseudomonas cytochrome oxidase. I. Enzymic properties with special reference to the biological specificity. Biochim Biophys Acta. 1963 Mar 12;67:379–393. doi: 10.1016/0006-3002(63)91844-4. [DOI] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES