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Journal of Bacteriology, July 2001, p. 4149-4156, Vol. 183, No. 14
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.14.4149-4156.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Purification, Characterization, and Genetic Analysis of Cu-Containing Dissimilatory Nitrite Reductase from a Denitrifying Halophilic Archaeon, Haloarcula marismortui

Hirotaka Ichiki,1 Yoko Tanaka,1,dagger Kiyotaka Mochizuki,1 Katsuhiko Yoshimatsu,1 Takeshi Sakurai,2 and Taketomo Fujiwara1,*

Department of Biology and Geosciences, Faculty of Science, Shizuoka University, Shizuoka 422-8529,1 and Department of Chemistry, Faculty of Science, Kanazawa University, Kanazawa 920-1192,2 Japan

Received 16 January 2001/Accepted 6 April 2001

Cu-containing dissimilatory nitrite reductase (CuNiR) was purified from denitrifying cells of a halophilic archaeon, Haloarcula marismortui. The purified CuNiR appeared blue in the oxidized state, possessing absorption peaks at 600 and 465 nm in the visible region. Electron paramagnetic resonance spectroscopy suggested the presence of type 1 Cu (gII = 2.232; AII = 4.4 mT) and type 2 Cu centers (gII = 2.304; AII = 13.3 mT) in the enzyme. The enzyme contained two subunits, whose apparent molecular masses were 46 and 42 kDa, according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-terminal amino acid sequence analysis indicated that the two subunits were identical, except that the 46-kDa subunit was 16 amino acid residues longer than the 42-kDa subunit in the N-terminal region. A nirK gene encoding the CuNiR was cloned and sequenced, and the deduced amino acid sequence with a residual length of 361 amino acids was homologous (30 to 41%) with bacterial counterparts. Cu-liganding residues His-133, Cys-174, His-182, and Met-187 (for type 1 Cu) and His-138, His-173, and His-332 (for type 2 Cu) were conserved in the enzyme. As generally observed in the halobacterial enzymes, the enzymatic activity of the purified CuNiR was enhanced during increasing salt concentration and reached its maximum in the presence of 2 M NaCl with the value of 960 µM NO2- · min-1 · mg-1.

* Corresponding author. Mailing address: Department of Biology and Geosciences, Faculty of Science, Shizuoka University, Shizuoka 422-8529, Japan. Phone: 81-54-238-4776. Fax: 81-54-238-0986. E-mail: sbtfuji{at}ipc.shizuoka.ac.jp.

dagger Present address: Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama, Ikoma 630-0101, Japan.

Journal of Bacteriology, July 2001, p. 4149-4156, Vol. 183, No. 14
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.14.4149-4156.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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