title: Purification, characterization, and genetic analysis of Cu-containing dissimilatory nitrite reductase from a denitrifying halophilic archaeon, Haloarcula marismortui.
abstract: 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 (g(II) = 2.232; A(II) = 4.4 mT) and type 2 Cu centers (g(II) = 2.304; A(II) = 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 microM NO(2)(-) x min(-1) x mg(-1).
authors: Department of Biology and Geosciences, Faculty of Science, Shizuoka University, Shizuoka 422-8529, Japan.
authors: H
authors: H
authors: Ichiki
authors: Y
authors: Y
authors: Tanaka
authors: K
authors: K
authors: Mochizuki
authors: K
authors: K
authors: Yoshimatsu
authors: T
authors: T
authors: Sakurai
authors: T
authors: T
authors: Fujiwara
mesh_terms: D000042:Absorption; D000595:Amino Acid Sequence; D001483:Base Sequence; D019641:DNA, Archaeal; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D019614:Haloarcula marismortui / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009572:Nitrite Reductases / Q000032:analysis* / Q000145:classification / Q000235:genetics; D010802:Phylogeny; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D019641:DNA, Archaeal; D009572:Nitrite Reductases; C052695:nitrite reductase, copper-containing