Nitrite reductases are redox enzymes catalysing the one electron
reduction
of nitrite to nitrogen monoxide (NO) within the bacterial
denitrification
process. We have cloned the gene for cd1 nitrite
reductase
(Pa-nirS) from Pseudomonas aeruginosa into the NiRS-
strain MK202 of Pseudomonas stutzeri and expressed the enzyme
under
denitrifying conditions. In the MK202 strain, denitrification is
abolished
by the disruption of the endogenous nitrite reductase gene; thus, cells
can be grown only in the presence of oxygen. After complementation with
Pa-nirS gene, cells supplemented with nitrate can be grown in the
absence
of oxygen. The presence of nitrite reductase was proven in vivo by the
demonstration of NO production, showing that the enzyme was expressed
in
the active form, containing both heme c and d1.
A
purification procedure for the recombinant PaNir has been developed,
based
on the P. aeruginosa purification protocol; spectroscopic
analysis
of the purified protein fully confirms the presence of the d1
heme cofactor. Moreover, the functional characterisation of the
recombinant
NiR has been carried out by monitoring the production of NO by the
purified
NiR enzyme in the presence of nitrite by an NO electrode. The full
recovery
of the denitrification properties in the P. stutzeri MK202
strain
by genetic complementation with Pa-NiR underlines the high homology
between
enzymes of nitrogen oxianion respiration. Our work provides an
expression
system for cd1 nitrite reductase and its
site-directed
mutants in a non-pathogenic strain and is a starting point for the in
vivo
study of recombinant enzyme variants.