Characterization of Nitrous Oxide Reductase from a Methylotrophic Denitrifying Bacterium, Hyphomicrobium denitrificans A3151

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A Cu-containing nitrous oxide reductase (HdN₂OR) from a methylotrophic denitrifying bacterium, Hyphomicrobium denitrificans A3151, has been aerobically prepared and spectroscopically characterized. Purple and blue forms of HdN₂OR have been isolated. Each form is a homodimer comprising monomers with a molecular mass of 65 kDa. The visible absorption spectrum of the purple form (designated as form A) exhibits three absorption bands at 480 nm, 540 nm, and 650 nm, with a shoulder near 780 nm, and that of the blue form (designated as form B) shows only one absorption band at 650 nm. Reversible spectral changes, between those of forms A and B, are observed on treatment of these forms with redox reagents. Forms A and B are oxidized and reduced forms, respectively. The 77-K EPR spectrum of form A indicates a seven-line copper hyperfine structure centered at g_??_ (g_??_=2.18, A_??_=4.5 mT), which is characteristic of a mixed-valence binuclear Cu_A site (A_mv), and that of form B exhibits a broad featureless signal (g=2.06). The various spectral data of HdN₂OR suggest that form A contains A_mv, and a mixed-valence tetranuclear Cu_z site (Z_mv^*), while form B includes reduced Cu_A (A_red) and Z_mv^*. The pH profiles of N₂OR activity of the two forms are similar to each other, and the specific activity at optimum pH 8.8 was estimated to be 45±5 and 29±3 μunol•min^-1•mg^-1 for forms A and B, respectively.