論文

査読有り
2017年4月1日

Heterologous expression of Halomonas halodenitrificans nitric oxide reductase and its N-terminally truncated NorC subunit in Escherichia coli

Journal of Inorganic Biochemistry
  • Nobuhiko Sakurai
  • ,
  • Kunishige Kataoka
  • ,
  • Noriko Sugaya
  • ,
  • Takaki Shimodaira
  • ,
  • Mie Iwamoto
  • ,
  • Munehiro Shoda
  • ,
  • Hajime Horiuchi
  • ,
  • Miyuki Kiyono
  • ,
  • Yasuke Ohta
  • ,
  • Bambang Triwiyono
  • ,
  • Daisuke Seo
  • ,
  • Takeshi Sakurai

169
開始ページ
61
終了ページ
67
記述言語
英語
掲載種別
研究論文(学術雑誌)
DOI
10.1016/j.jinorgbio.2017.01.006
出版者・発行元
Elsevier Inc.

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 b3 center, and a non-heme FeB center. 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. 40 mV 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 O2 as 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.

リンク情報
DOI
https://doi.org/10.1016/j.jinorgbio.2017.01.006
PubMed
https://www.ncbi.nlm.nih.gov/pubmed/28131879

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