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 (53006 Da, 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 and/or 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 cd1), but to a much lesser extent
the expression level of this enzyme. In turn, mutational inactivation
of
the structural gene for cytochrome cd1,
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.