title: Characterization of a novel copper-haem c dissimilatory nitrite reductase from Ralstonia pickettii.
abstract: 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 Cu(2+) 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 K(m) 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 R(g) (gyration radius) and D(max) (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.
authors.affiliation: Molecular Biophysics Group, Institute of Integrative Biology, Faculty of Health and Life Sciences, University of Liverpool, UK.
authors.forename: Cong
authors.initials: C
authors.lastname: Han
authors.forename: Gareth S A
authors.initials: GS
authors.lastname: Wright
authors.forename: Karl
authors.initials: K
authors.lastname: Fisher
authors.forename: Stephen E J
authors.initials: SE
authors.lastname: Rigby
authors.forename: Robert R
authors.initials: RR
authors.lastname: Eady
authors.forename: S Samar
authors.initials: SS
authors.lastname: Hasnain
mesh_terms: D000595:Amino Acid Sequence; D003300:Copper / Q000737:chemistry*; D006418:Heme / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D009572:Nitrite Reductases / Q000737:chemistry* / Q000235:genetics; D049920:Ralstonia pickettii / Q000201:enzymology*; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction / Q000379:methods
chemical_list: C031690:heme C; D006418:Heme; D003300:Copper; D009572:Nitrite Reductases; C052695:nitrite reductase, copper-containing