Characterization of nitrous oxide reductase from a methylotrophic denitrifying bacterium, Hyphomicrobium denitrificans A3151.
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 gparallel (gparallel=2.18, Aparallel=4.5 mT), which is characteristic of a mixed-valence binuclear CuA site (Amv), and that of form B exhibits a broad featureless signal (g=2.06). The various spectral data of HdN2OR suggest that form A contains Amv and a mixed-valence tetranuclear CuZ site (Zmv*), while form B includes reduced CuA (Ared) and Zmv*. 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 micromol.min(-1).mg(-1) for forms A and B, respectively.
Yamaguchi
Kazuya
K
Department of Chemistry, Graduate School of Science, Osaka University, 1-16 Machikaneyama, Toyonaka, Osaka 560-0043.
Kawamura
Akiro
A
Ogawa
Hideyuki
H
Suzuki
Shinnichiro
S
D000332:Aerobiosis; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002942:Circular Dichroism; D003300:Copper / Q000032:analysis; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D020582:Hyphomicrobium / Q000201:enzymology*; D010088:Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification
D001426:Bacterial Proteins; D003300:Copper; D010088:Oxidoreductases; C022763:nitrous oxide reductase