Isolation of a high specific activity pink, monomeric nitrous oxide reductase from Achromobacter cycloclastes

Nitrous oxide reductase from Achromobacter cycloclastes has been purified to homogeneity under aerobic conditions via DEAE-cellulose, phenyl-Sepharose, hydroxyapatite, and Sephacryl S-200 chromatography. It consists of a single polypeptide of MW 72 kDa, and contains 3.8 +/- 0.1 copper atoms per molecule. The enzyme is pink as isolated, yet exhibits a specific activity (86 U/mg) that is ca. 40 times greater than that observed for other N2O reductases under similar conditions. Double integration of the anomalous EPR spectrum at 77K showed the presence of 2.0 +/- 0.1 spins per molecule, implying the presence of EPR-silent copper atoms and/or spin-coupled mixed-valent centers.