NiRs (nitrite reductases) convert nitrite into NO in the denitrification process. RpNiR (Ralstonia pickettii NiR), a new type of dissimilatory Cu-containing NiR with a C-terminal haem c domain from R. pickettii, has been cloned, overexpressed in Escherichia coli and purified to homogeneity. The enzyme has a subunit molecular mass of 50515 Da, consistent with sequence data showing homology to the well-studied two-domain Cu NiRs, but with an attached C-terminal haem c domain. Gel filtration and combined SEC (size-exclusion chromatography)-SAXS (small angle X-ray scattering) analysis shows the protein to be trimeric. The metal content of RpNiR is consistent with each monomer having a single haem c group and the two Cu sites being metallated by Cu2+ ions. The absorption spectrum of the oxidized as-isolated recombinant enzyme is dominated by the haem c. X-band EPR spectra have clear features arising from both type 1 Cu and type 2 Cu centres in addition to those of low-spin ferric haem. The requirements for activity and low apparent Km for nitrite are similar to other CuNiRs (Cu-centre NiRs). However, EPR and direct binding measurements of nitrite show that oxidized RpNiR binds nitrite very weakly, suggesting that substrate binds to the reduced type 2 Cu site during turnover. Analysis of SEC-SAXS data suggests that the haem c domains in RpNiR form extensions into the solvent, conferring a high degree of conformational flexibility in solution. SAXS data yield Rg (gyration radius) and Dmax (maximum particle diameter) values of 43.4 Å (1 Å=0.1 nm) and 154 Å compared with 28 Å and 80 Å found for the two-domain CuNiR of Alcaligenes xylosoxidans.
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Research Article|
May 11 2012
Characterization of a novel copper-haem c dissimilatory nitrite reductase from Ralstonia pickettii
Cong Han;
Cong Han
*Molecular Biophysics Group, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Liverpool L69 7ZB, U.K.
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Gareth S. A. Wright;
Gareth S. A. Wright
*Molecular Biophysics Group, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Liverpool L69 7ZB, U.K.
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Karl Fisher;
Karl Fisher
†Manchester Interdisciplinary Biocentre and Faculty of Life Sciences, University of Manchester, Manchester M1 7DN, U.K.
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Stephen E. J. Rigby;
Stephen E. J. Rigby
†Manchester Interdisciplinary Biocentre and Faculty of Life Sciences, University of Manchester, Manchester M1 7DN, U.K.
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Robert R. Eady;
Robert R. Eady
*Molecular Biophysics Group, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Liverpool L69 7ZB, U.K.
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S. Samar Hasnain
S. Samar Hasnain
1
*Molecular Biophysics Group, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, Liverpool L69 7ZB, U.K.
1To whom correspondence should be addressed (email s.s.hasnain@liverpool.ac.uk).
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Publisher: Portland Press Ltd
Received:
September 09 2011
Revision Received:
March 12 2012
Accepted:
March 13 2012
Accepted Manuscript online:
March 13 2012
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2012 Biochemical Society
2012
Biochem J (2012) 444 (2): 219–226.
Article history
Received:
September 09 2011
Revision Received:
March 12 2012
Accepted:
March 13 2012
Accepted Manuscript online:
March 13 2012
Citation
Cong Han, Gareth S. A. Wright, Karl Fisher, Stephen E. J. Rigby, Robert R. Eady, S. Samar Hasnain; Characterization of a novel copper-haem c dissimilatory nitrite reductase from Ralstonia pickettii. Biochem J 1 June 2012; 444 (2): 219–226. doi: https://doi.org/10.1042/BJ20111623
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