Nitric oxide reductase from Pseudomonas stutzeri

Abstract

Nitric oxide (NO) reductase is an integral membrane component of the anaerobic respiratory chain of Pseudomonas stutzeri that transforms nitrate to dinitrogen (denitrification). The enzyme catalyzes the reduction of NO to nitrous oxide. The structural genes for the NO reductase complex, norC and norB, were sequenced and their organization established by primer extension and Northern blot analysis. The norCB genes encoding the cytochrome c and cytochrome b subunits of the enzyme are contiguous and transcribed as a single 2.0-kb transcript. The promoter region has a canonical recognition motif for the transcriptional activator protein Fnr, centered at -40.5 nucleotides from the initiation site of transcription. No similarity of the derived gene products to known cytochromes of b-or c-type was found in a data bank search.Post-translational processing of the two subunits was limited to the removal of the terminal methionine to leave an N-terminal serine in either subunit. The mature cytochrome c subunit (16508Da, 145 residues) is predicted to be a bitopic protein with a single membrane anchor. The mature cytochrome b subunit (53 006Da, 473 residues) is a putatively polytopic, strongly hydrophobic membrane-bound protein with 12 potential transmembrane segments. Several histidine and proline residues were identified with potentially structural andlor functional importance.Mutational inactivation of NO reductase by deletion of norB or the norCB genes affected strongly the in vivo activity of respiratory nitrite reductase (cytochrome cd,), but to a much lesser extent the expression level of this enzyme. In turn, mutational inactivation of the structural gene for cytochrome cd,, nirS, or loss of in vivo nitrite reduction by mutation of the nirT gene, encoding a presumed tetraheme cytochrome, lowered the expression level of NO reductase to 5 -20%, but hardly its catalytic activity. The cellular concentration of NO reductase increased again on restoration of nitrite reduction in the nirS: :Tn5 mutant MK202 by complementation with nirS or with the heterologous nirK gene, encoding the Cu-containing nitrite reductase from Pseudomonas aureofaciens. Thus, NO may be required as an inducer for its own reductase. Our results show that the nitrite-reducing system and the NO-reducing system are not operating independently from each other but are interlaced by activity modulation and regulation of enzyme synthesis.

The transcription factor DNR from Pseudomonas aeruginosa specifically requires nitric oxide and haem for the activation of a target promoter in Escherichia coli

“…2), suggesting that the endogenous FNR protein is unable to recognize this promoter. A preliminary in silico analysis of the P. aeruginosa nor promoter (identifying a good candidate for a 210 sequence centred around base 119.5) and its comparison with the norC promoter of Pseudomonas stutzeri (in which the transcriptional start site has been mapped: Zumft et al, 1994) suggest that the FNR box could be located as in class II promoters; this hypothesis will require to be further confirmed experimentally. Interestingly, however, the region located between the palindromic TTGAT motif of the consensus FNR box in the nor promoter has a high GC content (Fig.…”

Characterization of the nitric oxide reductase-encoding region in Rhodobacter sphaeroides 2.4.3

“…norC and norB are structural genes. norC encodes a cytochrome c-containing subunit responsible for transferring electrons to the norB-encoded protein, which, by its homology to subunit I of cytochrome c oxidases, likely contains the active site (43). The physiological roles of the norQ and norD products have not been elucidated, but the phenotypic similarity of the norC and norB mutants to the norQ and norD mutants demonstrates that the latter two genes encode products whose function appears to be limited to the production of an active Nor complex.…”

“…In Pseudomonas stutzeri, the two structural genes form a distinct transcriptional unit (43). In Pseudomonas aeruginosa, the two structural genes and a third gene, encoding a product of unknown function, apparently form an operon (3).…”

Regulation of oxidative phosphorylation: The flexible respiratory network ofParacoccus denitrificans

“…The third step in denitrification is the reduction of nitric oxide to nitrous oxide (2NO + 2H § + 2e-N20 + H20), catalyzed by the membrane-bound enzyme nitric oxide reductase (Nor) (Heiss et al, 1989;Carr and Ferguson, 1990;Zumft et al, 1994). Nor receives electrons via the bCl complex but it is not known which electron carriers, if any, mediate this transport.…”