title: Heterologous expression of Halomonas halodenitrificans nitric oxide reductase and its N-terminally truncated NorC subunit in Escherichia coli.
abstract: Halomonas halodenitrificans nitric oxide reductase (NOR) is the membrane-bound heterodimer complex of NorC, which contains a low-spin heme c center, and NorB, which contains a low-spin heme b center, a high-spin heme b3center, and a non-heme FeBcenter. The soluble domain of NorC, NorC* (ΔMet1-Val37) was heterologously expressed in Escherichia coli using expression plasmids harboring the truncated norC gene deleted of its 84 5'-terminal nucleotides. Analogous scission of the N-terminal helix as the membrane anchor took place when the whole norC gene was used. NorC* exhibited spectra typical of a low-spin heme c. In addition, NorC* functioned as the acceptor of an electron from a cytochrome c isolated from the periplasm of H. halodenitrificans and small reducing reagents. The redox potential of NorC* shifted ca. 40mV in the negative direction from that of NorC. Unlike NorC, recombinant NorB was not heterologously expressed. However, recombinant NOR (rNOR) could be expressed in E. coli by using a plasmid harboring all genes in the nor operon, norCBQDX, from which the three hairpin loops (mRNA) were deleted, and by using the ccm genes for the maturation of C-type heme. rNOR exhibited the same spectroscopic properties and reactivity to NO and O2as NOR, although its enzymatic activity toward NO was considerably decreased. These results on the expression of rNOR and NorC* will allow us to develop more profound studies on the properties of the four Fe centers and the reaction mechanism of NOR from this halophilic denitrifying bacterium.
authors.affiliation: Division of Biological Science, Graduate School of Natural Sciences, Nagoya City University, Yamanohata 1, Mizuho, Nagoya 467-8501, Japan. Electronic address: sakurai@nsc.nagoya-cu.ac.jp.
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Sakurai
authors.affiliation: Division of Material Science, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan.
authors.forename: Kunishige
authors.initials: K
authors.lastname: Kataoka
authors.affiliation: Division of Material Science, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Sugaya
authors.affiliation: Division of Material Science, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan.
authors.forename: Takaki
authors.initials: T
authors.lastname: Shimodaira
authors.affiliation: Division of Material Science, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan.
authors.forename: Mie
authors.initials: M
authors.lastname: Iwamoto
authors.affiliation: Division of Material Science, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan.
authors.forename: Munehiro
authors.initials: M
authors.lastname: Shoda
authors.affiliation: Division of Material Science, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan.
authors.forename: Hajime
authors.initials: H
authors.lastname: Horiuchi
authors.affiliation: Division of Material Science, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan.
authors.forename: Miyuki
authors.initials: M
authors.lastname: Kiyono
authors.affiliation: Division of Biological Science, Graduate School of Natural Sciences, Nagoya City University, Yamanohata 1, Mizuho, Nagoya 467-8501, Japan.
authors.forename: Yasuke
authors.initials: Y
authors.lastname: Ohta
authors.affiliation: Division of Biological Science, Graduate School of Natural Sciences, Nagoya City University, Yamanohata 1, Mizuho, Nagoya 467-8501, Japan.
authors.forename: Bambang
authors.initials: B
authors.lastname: Triwiyono
authors.affiliation: Division of Material Science, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Seo
authors.affiliation: Division of Material Science, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Sakurai
mesh_terms: D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D020632:Halomonas / Q000201:enzymology*; D009876:Operon / Q000235:genetics; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D021122:Protein Subunits; D010088:Oxidoreductases; C034747:nitric-oxide reductase
keywords: Heterologous expression; Membrane protein; Nitric oxide reductase (NOR); NorB; NorC