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. 1993 Oct 15;295(Pt 2):587–593. doi: 10.1042/bj2950587

Purification and characterization of the dissimilatory nitrite reductase from Alcaligenes xylosoxidans subsp. xylosoxidans (N.C.I.M.B. 11015): evidence for the presence of both type 1 and type 2 copper centres.

Z H Abraham 1, D J Lowe 1, B E Smith 1
PMCID: PMC1134921  PMID: 8240262

Abstract

Dissimilatory nitrite reductase was isolated from extracts of Alcaligenes xylosoxidans subsp. xylosoxidans (N.C.I.M.B. 11015), after activation of crude extracts by the addition of copper(II) sulphate. The enzyme was purified by a combination of (NH4)2SO4 fractionation and cationic-exchange chromatography to 93% homogeneity as judged by SDS/PAGE. SDS/PAGE and spray m.s. showed that the enzyme had a subunit M(r) of 36.5 kDa. The copper content was 3.5 +/- 0.8 Cu atoms/trimer of M(r) 109,500. E.p.r. spectroscopy of nitrite reductase as isolated showed that both type 1 (g parallel = 2.208, A parallel = 6.3 mT) and type 2 (g parallel = 2.298, A parallel = 14.2 mT) Cu centres were present, in contrast with published data [Masuko, Iwasaki, Sakurai, Suzuki and Nakahara (1984) J. Biochem. (Tokyo) 96, 447-454], where only type 1 copper centres were reported. Our preparations had a specific activity of 150-300 mumol of NO2- reduced/min per mg of protein, 6-12-fold higher than reported previously. As isolated, the oxidized form of our preparations of the enzyme showed absorption maxima in the visible region at 460, 593 and 770 nm. The ratio of the absorption bands at 460 nm and 593 nm resulted in this protein having a strong blue colour, in contrast with the green colour of other purified copper-containing nitrite reductases. We conclude that, in contrast with previous reports, this 'blue' nitrite reductase requires both type 1 and type 2 copper centres for optimal activity.

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Selected References

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  1. CHANEY A. L., MARBACH E. P. Modified reagents for determination of urea and ammonia. Clin Chem. 1962 Apr;8:130–132. [PubMed] [Google Scholar]
  2. Fenderson F. F., Kumar S., Adman E. T., Liu M. Y., Payne W. J., LeGall J. Amino acid sequence of nitrite reductase: a copper protein from Achromobacter cycloclastes. Biochemistry. 1991 Jul 23;30(29):7180–7185. doi: 10.1021/bi00243a020. [DOI] [PubMed] [Google Scholar]
  3. Fenn J. B., Mann M., Meng C. K., Wong S. F., Whitehouse C. M. Electrospray ionization for mass spectrometry of large biomolecules. Science. 1989 Oct 6;246(4926):64–71. doi: 10.1126/science.2675315. [DOI] [PubMed] [Google Scholar]
  4. Godden J. W., Turley S., Teller D. C., Adman E. T., Liu M. Y., Payne W. J., LeGall J. The 2.3 angstrom X-ray structure of nitrite reductase from Achromobacter cycloclastes. Science. 1991 Jul 26;253(5018):438–442. doi: 10.1126/science.1862344. [DOI] [PubMed] [Google Scholar]
  5. Hochstein L. I., Tomlinson G. A. The enzymes associated with denitrification. Annu Rev Microbiol. 1988;42:231–261. doi: 10.1146/annurev.mi.42.100188.001311. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Iwasaki H., Matsubara T. A nitrite reductase from Achromobacter cycloclastes. J Biochem. 1972 Apr;71(4):645–652. [PubMed] [Google Scholar]
  8. Iwasaki H., Noji S., Shidara S. Achromobacter cycloclastes nitrite reductase. The function of copper, amino acid composition, and ESR spectra. J Biochem. 1975 Aug;78(2):355–361. doi: 10.1093/oxfordjournals.jbchem.a130915. [DOI] [PubMed] [Google Scholar]
  9. Kakutani T., Watanabe H., Arima K., Beppu T. Purification and properties of a copper-containing nitrite reductase from a denitrifying bacterium, Alcaligenes faecalis strain S-6. J Biochem. 1981 Feb;89(2):453–461. doi: 10.1093/oxfordjournals.jbchem.a133220. [DOI] [PubMed] [Google Scholar]
  10. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  11. Libby E., Averill B. A. Evidence that the type 2 copper centers are the site of nitrite reduction by Achromobacter cycloclastes nitrite reductase. Biochem Biophys Res Commun. 1992 Sep 30;187(3):1529–1535. doi: 10.1016/0006-291x(92)90476-2. [DOI] [PubMed] [Google Scholar]
  12. Liu M. Y., Liu M. C., Payne W. J., Legall J. Properties and electron transfer specificity of copper proteins from the denitrifier "Achromobacter cycloclastes". J Bacteriol. 1986 May;166(2):604–608. doi: 10.1128/jb.166.2.604-608.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lowe D. J. Electron paramagnetic resonance in biochemistry. Computer simulation of spectra from frozen aqueous samples. Biochem J. 1978 Jun 1;171(3):649–651. doi: 10.1042/bj1710649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. MacGregor C. H. Isolation and characterization of nitrate reductase from Escherichia coli. Methods Enzymol. 1978;53:347–355. doi: 10.1016/s0076-6879(78)53040-1. [DOI] [PubMed] [Google Scholar]
  15. Masuko M., Iwasaki H., Sakurai T., Suzuki S., Nakahara A. Characterization of nitrite reductase from a denitrifier, Alcaligenes sp. NCIB 11015. A novel copper protein. J Biochem. 1984 Aug;96(2):447–454. doi: 10.1093/oxfordjournals.jbchem.a134856. [DOI] [PubMed] [Google Scholar]
  16. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  17. Zumft W. G., Gotzmann D. J., Kroneck P. M. Type 1, blue copper proteins constitute a respiratory nitrite-reducing system in Pseudomonas aureofaciens. Eur J Biochem. 1987 Oct 15;168(2):301–307. doi: 10.1111/j.1432-1033.1987.tb13421.x. [DOI] [PubMed] [Google Scholar]

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