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 M NO 2 ؊ ⅐ min ؊1 ⅐ mg ؊1 .

Anaerobic Growth of Haloarchaeon Haloferax volcanii by Denitrification Is Controlled by the Transcription Regulator NarO

“…5, the transcription activity of the narO gene promoter was higher than that of the genes of the catabolic enzymes nitrate reductase and nitrite reductase, which are expressed in the microbial cells in large amounts (11,30). However, until now, identification of the recombinant NarO expressed in the H. volcanii cells has not been followed by immunoblotting experiments using anti-His 6 antibody.…”

“…The suspension thus treated was centrifuged at 12,000 ϫ g for 10 min, and the enzymatic activity of the cell extract supernatant was measured. The enzymatic activities of nitrate and nitrite reductions in the cell extract were measured according to the methods of Yoshimatsu et al (30) and Ichiki et al (11), respectively, with a slight modification. The protein concentration was measured by using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL) using bovine serum albumin as the standard.…”

“…1). The copper-containing nitrite reductase NirK has been isolated from Haloarcula marismortui (11). The norB and nosZ genes, which encode nitric oxide reductase and nitrous oxide reductase, respectively, were identified in the H. marismortui genome (12), although neither enzyme has been purified and characterized.…”

Genome sequence of Haloarcula marismortui: A halophilic archaeon from the Dead Sea

“…Organisms from all three domains of life are capable of carrying out this process. In addition to the experimentally characterized nitrite reductase (NirK) and nitrate reductase (NarGH) in H. marismortui (Yoshimatsu et al 2000(Yoshimatsu et al , 2002Ichiki et al 2001), the genome analysis identified at least four additional proteins with functions important for denitrification, viz. a nitrilase, a carbon-nitrogen hydrolase involved in reduction of organic nitrogen compounds and ammonia production; an ammonia-dependent NAD synthetase; a dissimilatory nitrate reductase; and an ammonium transporter.…”

A Eukaryotic Copper-Containing Nitrite Reductase Derived from a NirK Homolog Gene ofAspergillus oryzae

“…These kinetic parameters are plausible as compared with those due to bacterial NirKs. 2,25) The optimal pH and temperature were 5.5 and 25-30 C according with those for the growth of A. oryzae. Activity was strongly inhibited by diethyldithiocarbamate (DDC), cyanide, and carbon monoxide (Fig.…”