Biochem. J. (2012) 444, 219–226 (Printed in Great Britain) doi:10.1042/BJ20111623 219 Characterization of a novel copper-haem c dissimilatory nitrite reductase from Ralstonia pickettii Cong HAN*, Gareth S. A. WRIGHT*, Karl FISHER†, Stephen E. J. RIGBY†, Robert R. EADY* and 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., and †Manchester Interdisciplinary Biocentre and Faculty of Life Sciences, University of Manchester, Manchester M1 7DN, U.K. 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. Key words: autoinduction, c-type haem, gel permeation chromatography, nitrite reductase (NiR), Ralstonia pickettii, small angle X-ray scattering (SAXS). INTRODUCTION In the nitrogen cycle, the biological reduction of NO 3 − to N 2 , via the intermediates NO 2 − , NO and N 2 O, is called denitrification [1,2]. It is an anaerobic respiratory process coupled to ATP generation. This pathway is found widespread in organisms ranging from bacteria to archaea and even fungi. In some organisms, this reaction sequence is truncated and N 2 O is the major product [3]. Denitrification is of major significance in terrestrial and oceanic nitrogen cycling, and is not only important from a microbial bioenergetics perspective, but also has an agricultural and environmental impact. Benefits are the removal of excess nitrogen from the environment, counteracting NO 3 − pollution of ground and drinking water. However, negative economic and environmental consequences arise from agricultural productivity being adversely affected by the loss of available fixed nitrogen applied as a fertilizer for plant growth, and the increasing contribution to global warming and destruction of the ozone layer, because N 2 O is not only a major greenhouse gas, but also a natural catalyst of stratospheric ozone degradation. The denitrification process is also considered to be of medical importance as many of the infectious pathogens, including Brucella melitensis, Neisseria gonorrhoeae and Neisseria meningitidis, use denitrification to grow under low-oxygen conditions by respiration of nitrate. In the denitrification pathway, the first committed step is the one-electron reduction of nitrite to the gaseous product NO catalysed by NiR (nitrite reductase). There are two main categories of dissimilatory NiRs containing either haem cd 1 (cytochrome cd 1 NiR) or two types of Cu centres (CuNiR) as prosthetic groups, encoded by nirS and nirK respectively. In general, CuNiRs are a highly conserved enzyme family and reflecting this, the enzymes isolated from a wide range of organisms have very similar properties containing only two types of metal centres, an electron-accepting type 1 Cu site and a catalytic type 2 Cu centre. Crystallographic structures of AcNiR (Achromobacter cycloclastes CuNiR) (PDB entry 2NRD) [4,5], Af NiR (Alcaligenes faecalis S-6 CuNiR) (PDB entry 1AS7) [6,7], AxNiR (Alcaligenes xylosoxidans CuNiR) (PDB entry 1BQ5) [8,9] and RsNiR (Rhodobacter sphaeroides CuNiR) (PDB entry ZA3T) [10,11] reveal that they are homotrimers with very similar overall structures. The type 1 Cu centres, which function as an acceptor of electrons from the physiological electron donor, are located within each subunit and co-ordinated by two histidines, a cysteine and a methionine residue. The type 2 Cu centres are situated at the interface between subunits and co-ordinated by three histidine residues provided by two different subunits and a water molecule. Spectroscopic and structural studies, including time-resolved structures, show that the type 2 Cu site is involved in the binding and reduction of nitrite to NO [7,12]. The distance between two Cu sites within a subunit is ∼ 13 Å (1Å = 0.1 nm), and the Cu 2+ ions are linked by a histidine–cysteine bridge that promotes an efficient electron transfer from the type 1 to the type 2 Cu centre during catalysis. Small Cu-proteins, such as Abbreviations used: Cu1, type 1 Cu; Cu2, type 2 Cu; deoxyHb, deoxyhaemoglobin; ESI–MS, electrospray ionization MS; NiR, nitrite reductase; AcNiR, Achromobacter cycloclastes NiR; Af NiR, Alcaligenes faecalis NiR; Ax NiR, Alcaligenes xylosoxidans NiR; CuNiR, Cu-centre NiR; HdNiR, Hyphomicrobium denitrificans NiR; PhNiR, Pseudoalteromonas haloplanktis NiR; RpNiR, Ralstonia pickettii NiR; RsNiR, Rhodobacter sphaeroides NiR; SEC, size-exclusion chromatography; SAXS, small angle X-ray scattering. The nucleotide sequence data reported will appear in GenBank ® , EMBL, DDBJ and GSDB Nucleotide Sequence Databases under the accession number JN227882. 1 To whom correspondence should be addressed (email s.s.hasnain@liverpool.ac.uk). c  The Authors Journal compilation c  2012 Biochemical Society