Biochemical characterization and solution structure of nitrous oxide reductase from Alcaligenes xylosoxidans (NCIMB 11015).
Nitrous oxide reductase (N2OR) is the terminal enzyme involved in denitrification by microbes. No three-dimensional structural information has been published for this enzyme. We have isolated and characterised N2OR from Alcaligenes xylosoxidans (AxN2OR) as a homodimer of M(r) 134,000 containing seven to eight copper atoms per dimer. Comparison of sequence and compositional data with other N2ORs suggests that AxN2OR is typical and can be expected to have similar domain folding and subunit structure to other members of this family of enzymes. We present synchrotron X-ray-scattering data, analysed using a model-independent method for shape restoration, which gave a approximately 20 A resolution structure of the enzyme in solution, providing a glimpse of the structure of any N2OR and shedding light on the molecular architecture of the molecule. The specific activity of AxN2OR was approximately 6 mumol of N2O reduced.min-1. (mg of protein)-1; N2OR activity showed both base and temperature activation. The visible spectrum exhibited an absorption maximum at 550 nm with a shoulder at 635 nm. On oxidation with K3Fe(CN)6, the absorption maximum shifted to 540 nm and a new shoulder at 480 nm appeared. Reduction under anaerobic conditions resulted in the formation of an inactive blue form of the enzyme with a broad absorption maximum at 650 nm. As isolated, the enzyme shows an almost featureless EPR spectrum, which changes on oxidation to give an almost completely resolved seven-line hyperfine signal in the gII region, g = 2.18, with AII = 40 G, consistent with the enzyme being partially reduced as isolated. Both the optical and EPR spectra of the oxidized enzyme are characteristic of the presence of a CuA centre.
Ferretti
S
S
Nitrogen Fixation Laboratory, John Innes Centre, Norwich, UK.
Grossmann
J G
JG
Hasnain
S S
SS
Eady
R R
RR
Smith
B E
BE
D000421:Alcaligenes / Q000201:enzymology*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000737:chemistry*; D003300:Copper / Q000032:analysis; D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D007700:Kinetics; D008667:Metalloproteins / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry*; D011487:Protein Conformation; D012542:Scattering, Radiation; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013696:Temperature
D000596:Amino Acids; D001426:Bacterial Proteins; D008667:Metalloproteins; D003300:Copper; D010088:Oxidoreductases; C022763:nitrous oxide reductase