N2O
reductase
(N2O ®
N2) is the terminal enzyme in the
energy-conserving
denitrification pathway of soil and marine denitrifying bacteria. The
protein
is composed of two identical subunits and contains eight copper ions
per
enzyme molecule. The magnetic circular dichroism spectrum of resting
(oxidized)
N2O reductase is strikingly similar to the
magnetic
circular dichroism spectrum of the CuA site in
mammalian
cytochrome c oxidase [Greenwood, C., Hull, B. C., Barber, D.,
Eglinton,
D. G. & Thomson, A. J. (1983) Biochem. J. 215,303-316] and
is
unlike the magnetic circular dichroism spectra of all other biological
copper chromophores obtained to date. Sulfur (or chlorine) scatterers
are
required to fit the copper extended x-ray absorption fine structure
data
of both the oxidized and reduced forms of N2O
reductase.
Satisfactory fits require a Cu-N or Cu-O [denoted Cu-(N, O)]
interaction
at 2.0 Å, a Cu-(S, Cl) interaction at 2.3 Å and an
additional
Cu-(S, Cl) interaction at »2.6
Å (oxidized) or »2.7
Å (reduced). Approximately eight sulfur ions (per eight copper
ions)
at »2.3
Å are required to fit the extended x-ray absorption fine
structure
data for both the oxidized and reduced N2O
reductase.
The 2.3-Å Cu-(S, Cl) distance is nearly identical to that
previously
determined for the CuA site in cytochrome c
oxidase. A 2.6-2.7 Å Cu-(S, Cl) interaction is also present in
resting
and fully reduced cytochrome
c oxidase. Comparison of the N2O
reductase sequence, determined by translating the structural NosZ
gene, with cytochrome c oxidase subunit II sequences from
several
sources indicates that a
Gly-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Ser-Xaa-Xaa-Cys-Xaa-
Xaa-Xaa-His stretch is highly conserved. This sequence contains three
of
the probable ligands (two cysteines and one histidine) in a CuA-type
site. Collectively these data establish that Pseudomonas stutzeri
N2O reductase contains CuA-type
sites.