The single conserved Cys165
outside
of the CuA domain of nitrous oxide reductase (N2OR)
from Pseudomonas stutzeri was mutated to glycine to test its
presumed
function in metal coordination of the catalytic site, CuZ.
The point mutation reduced the cellular level of N2OR
5-10-fold compared to the level of the control strain. In the mutant,
the
activity and the Cu content of the enzyme, as well as the transcript
level
of the N2OR structural gene,
nosZ, remained
unaffected. The mutant enzyme was processed and exported into the
periplasm
like the wild-type enzyme. Chemical analysis for sulfhydryl groups gave
about nine -SH groups/monomer of the apoenzyme prepared from the
wild-type
enzyme, in accordance with the nine cysteine residues of the derived
amino
acid sequence. Eight -SH groups were found to form disulfide bridges in
the holoenzyme dimer. We propose that in the native state of the enzyme
Cys165 does not bind to CuZ, but may be part of
a
disulfide bridge essential for the stability of N2OR.
Immediately downstream of the genes nosDFY, encoding the
components
for Cu incorporation into the reductase, we have identified the open
reading
frame, ORFL, whose derived product has the signature of a protein
disulfide
isomerase.