Studies on electron-transferring mechanism in an nitrite-reducing system containing Cu-proteins

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We planned to elucidate the denitrification mechanism ofAlcaligenes faecalis S-6 by characterizing enzymes and genes involved in the denitrification process. Since a cytochrome c is often served as an electron donor in this process, a protein which reacted with heme-staining reagent was partially purified from A.faecalis S-6, and its N-terminal amino acid sequence was determined. The sequence was highly similar to those of small subunits of nitric oxide reductases (NOR) from several denitrifying bacteria. DNA fragments containing the gene coding for the subunit was cloned by oligonucleotide-probing method. Nucleotide sequencing revealed that the cloned fragments contained nine open reading frames. Base on the homology analysis we tentatively designated the open reading frames as dnr, orf1, orf2, norO, norP, norC, norB, norQ, and norD.Just upstream of the norO, norC, and norQ, short palin-drome sequences similar to FNR-recognition sequence with a motif of TTGATxxxxATCAA were found. Considering that FNR was a transcriptional activater for genes expressed under the anaerobic conditions, this suggested, that expression of the genes was controled in the stage of transcription in the absence of oxygen, and these genes were transcribed as at least three different operons. We constructed several plasmids for the expression of nor genes and introduced them into Escherichia coli JM105. E.coli cells carrying the plasmids were cultured for 30 h after IPTG induction, and their membrane fractions were subjected to SDS-PAGE and heme staining. Although no accumulation of specific proteins was not seen by CBB staining, production of NORB (38 kDa) and NORC (17 kDa) was detected by heme staining.However, no NOR activity was detected in any fraction of E.coli cells. This suggested the presence of some other genes or their products involved in the activation of NOR proteins.