Biochemical characterization and solution structure of nitrous oxide reductase from Alcaligenes xylosoxidans (NCIMB 11015)

Nitrous oxide reductase (N2OR) is the terminal enzyme involved in denitrifi cation by microbes. No three-dimensional structural information has been pu blished for this enzyme. We have isolated and characterised N2OR from Alcal igenes xylosoxidans (AxN(2)OR) as a homodimer of M-r 134 000 containing sev en to eight copper atoms per dimer. Comparison of sequence and compositiona l data with other N(2)ORs suggests that AxN2OR is typical and can be expect ed to have similar domain folding and subunit structure to other members of this family of enzymes. We present synchrotron X-ray-scattering data, anal ysed using a model-independent method for shape restoration, which gave a a pproximate to 20 Angstrom resolution structure of the enzyme in solution, p roviding a glimpse of the structure of any N2OR and shedding light on the m olecular architecture of the molecule. The specific activity of AxN(2)OR wa s approximate to 6 mu mol 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 oxi dation 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 absor ption maximum at 650 nm. As isolated, the enzyme shows an almost featureles s EPR spectrum, which changes on oxidation to give an almost completely res olved seven-line hyperfine signal in the g(II) region, g = 2.18, with A(II) = 40 G, consistent with the enzyme being partially reduced as isolated. Bo th the optical and EPR spectra of the oxidized enzyme are characteristic of the presence of a Cu-A centre.