The nosZ genes encoding
the
multicopper enzyme nitrous oxide reductase of Alcaligenes eutrophus
H16 and the type strain of Pseudomonas aeruginosa were cloned
and
sequenced for structural comparison of their gene products with the
homologous
product of the nosZ gene from Pseudomonas stutzeri [Viebrock,
A. & Zumft, W. G. (1988) J. Bacteriol.170, 4658-4668] and
the
subunit II of cytochrome-c oxidase (COII). Both types of enzymes
possess the CuA binding site. The nosZ genes were identified in
cosmid libraries by hybridization with an internal 1.22-kb PstI
fragment (NS220) of nosZ from P. stutzeri. The derived
amino
acid sequences indicate unprocessed gene products of 70084 Da (A.
eutrophus)
and 70695 Da (P. aeruginosa). The N-terminal sequences of the
NosZ
proteins have the characteristics of signal peptides for transport.
A homologous domain, extending over
at least 50 residues, is shared among the three derived NosZ sequences
and the CuA binding region of 32 COII sequences.
Only
three out of nine cysteine residues of the NosZ protein (P. stutzeri)
are invariant. Cys618 and Cys622 are assigned to a binuclear center, A,
which is thought to represent the CuA site of
NosZ
and is located close to the C terminus. Two conserved histidines, one
methionine,
one aspartate, one valine and two aromatic residues are also part of
the
CuA consensus sequence, which is the domain
homologous
between the two enzymes. The CuA consensus
sequence,
however, lacks four strictly conserved residues present in all COII
sequences.
Cys165 is likely to be a ligand of a second binuclear center,
Z,
for which we assume mainly histidine coordination. Of 23 histidine
residues
in NosZ (P. stutzeri), 14 are invariant, 7 of which are in
regions
with a degree of conservation well above the 50% positional identity
between
the Alcaligenes and Pseudomonas sequences. Conserved
tryptophan
residues are located close to several potential copper ligands. Trp615
may contribute to the observed quenching of fluorescence when the CuA
site is occupied.