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J. Biochem, 2003, Vol. 134, No. 6 853-858
© 2003 Japanese Biochemical Society

BIOCHEMISTRY

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

Kazuya Yamaguchi, Akiro Kawamura, Hideyuki Ogawa and Shinnichiro Suzuki*

Department of Chemistry, Graduate School of Science, Osaka University, 1-16 Machikaneyama, Toyonaka, Osaka 560-0043

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 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 µmol·min–1·mg–1 for forms A and B, respectively.

* To whom correspondence should be addressed. Tel: +81-6-6850-5767, Fax: +81-6-6850-5785, E-mail: bic{at}ch.wani.osaka-u.ac.jp


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D. Hira, M. Nojiri, K. Yamaguchi, and S. Suzuki
Identification of a Blue Copper Protein from Hyphomicrobium denitrificans and its Functions in the Periplasm
J. Biochem., September 1, 2007; 142(3): 335 - 341.
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