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The substrate-binding site in Cu nitrite reductase and its similarity to Zn carbonic anhydrase

  • A Corrigendum to this article was published on 01 October 1995


Here we investigate the structure of the two types of copper site in nitrite reductase from Alcaligenes xylosoxidans, the molecular organisation of the enzyme when the type-2 copper is absent, and its mode of substrate binding. X-ray absorption studies provide evidence for a fourth ligand at the type-2 Cu, that substrate binds to this site and indicates that this binding does not change the type-1 Cu centre. The substrate replaces a putative water ligand and is accommodated by a lengthening of the Cu–histidine bond by approximately 0.08 Å. Modelling suggests a similarity between this unusual type-2 Cu site and the Zn site in carbonic anhydrase and that nitrite is anchored by hydrogen bonds to an unligated histidine present in the type-2 Cu cavity.

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  1. 1

    Payne, W.J. in Denitrification in the Nitrogen Cycle (ed. Golterman, H.L.) 47–65 (Plenum, New York; 1985)

  2. 2

    Hochstein, L.I. & Tomlinson, G.A. A. Rev. Microbiol. 42, 231–261 (1989).

  3. 3

    Zumft, W.G. in The Prokaryotes (eds Barlows, A. et al.) 534–553 (Springer-verlag, New York; 1991)

  4. 4

    Ye, R.W., Fries, M.R., Bezborodnikov, S.G., Averill, B.A. & Tiedje, J.M. Characterization of the structural gene encoding a copper-containing nitrite reductase and homology of this gene to DNA of other denitrifiers. Appl. environ. Microbiol. 59, 250–254 (1993).

  5. 5

    Zumft, W.G., Gotzmann, D.J. & Kroneck, P.M.H. Type 1, blue copper protein constitute a respiratory nitrite reducing System in Pseudomonas aureofaciens. E. J. Biochem. 168, 301–307 (1987).

  6. 6

    Fenderson, F.F., Kumar, S., Adman, E.T., Lui, M.Y., Payne, W.J. & LeGall, J. Amino acid sequence of nitrite reductase: A copper protein from Achromobacter cycloclastes. Biochemistry 30, 7180–7185 (1986).

  7. 7

    Nishiyama, M. et al. Cloning and characterisation of a nitrite reductase gene from Alcaligenes faecalis and its expression in Escherichia coli.. J. gen. Microbiol. 139, 725–733 (1993).

  8. 8

    Godden, J.W. et al. The 2.3Å X-ray structure of nitrite reductase from Anchromobactercycloclastes. Science 253, 438–442 (1991).

  9. 9

    Grossmann, J.G. et al. X-ray scattering using synchrotron radiation shows nitrite reductase from Achromabactor xylosoxidans to be a trimer in solution. Biochemistry 32, 7360–7366 (1993).

  10. 10

    Abraham, Z.H.L., Lowe, D.J. & Smith, B.E. Purification and characterisation of the dissimilatory nitrite reductase from Alcaligenes xylosoxidans subsp xylosoxidans (NCIMB 11015): Evidence for the presence of both type 1 and type 2 copper centres. Biochem. J. 296, 885(1993).

  11. 11

    Dodd, F.E. et al. Structural studies of copper proteins. Daresbury Annual Report 221 (1993).

  12. 12

    Kokimota, M. et al. X-ray structure and site directed mutagenesis of a nitrite reductase from alcaligenes faecalis-S6 - roles of 2 copper atoms in nitrite reduction. Biochemistry 33, 5246–5252 (1994).

  13. 13

    Spiro, T.G. Copper Proteins. (Wiley, New York; 1981)

  14. 14

    Adman, E. Copper protein structures in Advances in Protein Chemistry (eds Anfinsen, C.B. et al.) 145–197 (Academic Press Ltd, London; 1991)

  15. 15

    Howes, B.D. et al. EPR and electron nuclear double resonance (ENDOR) studies show nitrite binding to the Type 2 copper centres of the dissimilatory nitrite reductase of Alcaligenes xylosoxidans (NCIMB 11015). Biochemistry 33, 3171–3177(1994).

  16. 16

    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. 187, 1529–1535 (1992).

  17. 17

    Sano, M. & Matsubara, T. Copper site of nitrite reductase from AlcaligenesSp. Structural evidence from X-ray absorption spectroscopy. Chem. Letts 2121–2124 (1984).

  18. 18

    Sano, M. & Matsubara, T. Structural change in the one-electron oxidation -reduction at the copper site in nitrite reductase. Evidence from EXAFS. Inorg. chim. Acta. 152, 53–54 (1988).

  19. 19

    Suzuki, S. et al. Spectroscopic evidence for a copper-nitrosyl intermediate in nitrite reduction by blue copper-containing nitrite reductase. Biochem. biophys. Res. Commun. 164, 1366–1372 (1989).

  20. 20

    Hasnain, S.S. Application of EXAFS to biochemical systems. Topics Curr. Chem. 147, 7 3 (1988).

  21. 21

    Lindley, P.F., Garratt, R.C. & Hasnain, S.S. EXAFS and crystallographic studies of metalloproteins. in Synchrotron Radiation and Biophysics (ed. Hasnain, S.S.) 63 (Ellis Norwood Ltd., Chichester, U.K.; 1990)

  22. 22

    Dent, A.J., Beyersmann, D., Block, C. & Hasnain, S.S. Two different zinc sites in bovine 5-aminolevulinate dehdratase distinguised by EXAFS. Biochemistry 29, 7822–7828 (1990).

  23. 23

    Tullius, T.D., Frank, P. & Hodgson, K.O. Characterisation of the blue copper site in oxidised azurin by extended X-ray absorption fine structure: determination of a short Cu-S distance. Proc. natn Acad. Sci. U.S.A. 75, 4069–4073 (1978).

  24. 24

    Peisach, J., Powers, L., Blumberg, W.E. & Chance, B. Stellacyanin: studies on the metal binding site using X-ray absorption spectroscopy. Biophys. J. 38, 277–285(1982).

  25. 25

    Lommen, A., Pandya, K.I., Koningsberger, D.C. & Canters, G.W. EXAFS analysis of the pH dependence of the blue-copper site in amicyanin from Thiobadllus versutus. Biochim. biophys. Acta 1076, 439–447. (1991).

  26. 26

    Murphy, L.M. et al. Structural characterisation of azurin from Pseudomonas aeruginosa and some of its methionine-121 mutants. Biochemistry 32, 1965–1975 (1993).

  27. 27

    Gurman, S.J., Binsted, N. & Ross, I. A rapid, exact curved-wave theory for EXAFS calculations: II. The multiple scattering contributions. J. Phys. Chem. C19, 1845–1861 (1986).

  28. 28

    Pantoliano, M.W., Valentine, J.S. & Nafie, L.A. Spectroscopic studies of copper(II) bound at the native copper site or substituted at the native zinc site of bovine erythrocuprein (superoxide dismutase). J. Am. chem. Soc. 104, 6310–6317 (1982).

  29. 29

    Solomon, E.I., Penfield, K.W. & Wilcox, D.E. Active sites in copper proteins: An electronic-structure overview. Struct. Bonding 53, 1–57 (1983).

  30. 30

    Eriksson, A.E., Jones, T.A. & Liljas, A. A refined structure of human carbonic anhydrase II at 2.0 Å resolution. Proteins 4, 274–282 (1988).

  31. 31

    Holmes, M.A. & Matthews, B.W. Structure of thermolysin refined at 1.6 Å Resolution. J. molec. Biol. 160, 623–629 (1982).

  32. 32

    Liljas, A., Håkansson, K., Jonsson, B.H. & Xue, Y. Inhibition and catalysis of carbonic anhydrase - recent crystallographic analyses. E. J. Biochem. 219, 110 (1994).

  33. 33

    Håkansson, K., Wehnert, A. & Liljas, A. X-ray analysis of metal-substituted human carbonic anhydrase II derivatives. Acta crystallogr. D 50, 93–100 (1994).

  34. 34

    Tainer, J.A., Getzoff, E.D., Richardson, J.S. & Richardson, D.C. Structure and mechanism of copper-zinc superoxide dismutase. Nature 306, 284–287(1983).

  35. 35

    Adman, E.T. & Turley, S.T. Two crystal forms of A. cycloclastes nitrite reductase. in Bioinorganic Chemistry of Copper(eds Karlin, K.D. and Tyeklar, Z.) (Chapman & Hall, New York-London; 1993)

  36. 36

    Hasnain, S.S. & Strange, R.W. Recent advances in XAFS data analysis in Biophysics and Synchrotron Radiation (eds Hasnain, S. S.) 104–122 (Ellis Horwood Ltd, Chichester, U.K.; 1990)

  37. 37

    Binsted, N., Strange, R.W. & Hasnain, S.S. Constrained and restrained refinement in EXAFS data analysis with curved wave theory. Biochemistry 31, 12117–12125(1992).

  38. 38

    Perutz, M.F., Hasnain, S.S., Duke, P.J., Sessler, J.L. & Hahn, J.E. Stereochemistry of iron in deoxyhaemoglobin. Nature 295, 535 (1982).

  39. 39

    Strange, R.W., Blackburn, N.J., Knowles, P.F. & Hasnain, S.S. X-ray absorption spectroscopy of metal-histidine coordination in metalloproteins. Exact simulation of the EXAFS of tetrakis(imidazole)copper(II) nitrate and other copper-imidazole complexes by the use of a multiple dcattering treatment. J. Am. chem. Soc. 109, 7157–7162(1987).

  40. 40

    Bernstein, F.E. et al. The protein data bank: a computer based archival file for macromolecular structures. J. molec. Biol. 112, 535–542. (1977).

  41. 41

    Iwasaki, H. & Matsubara, T. A nitrite reductase from Achromabactor cycloclastes. Biochem. J. 71, 645–652 (1972).

  42. 42

    Kraulis, P.J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. appl. Crystallogr. 24, 946–950 (1991).

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