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

Heterologous expression of Halomonas halodenitrificans nitric oxide reductase and its N-terminally truncated NorC subunit in Escherichia coli. J Inorg Biochem. 2017 Jan 21;169:61-67 Authors: Sakurai N, Kataoka K, Sugaya N, Shimodaira T, Iwamoto M, Shoda M, Horiuchi H, Kiyono M, Ohta Y, Triwiyono B, Seo D, Sakurai T 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 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. 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...
Source: Journal of Inorganic Biochemistry - Category: Biochemistry Authors: Tags: J Inorg Biochem Source Type: research