Nitrous oxide reductase from the
denitrifying bacterium Pseudomonas perfectomarina has been
isolated
and purified to homogeneity. The enzyme contained about eight copper
atoms/120
kDa and was composed of two presumably identical subunits. The
isoelectric
point was 5.1. Several spectroscopically distinct forms of the enzyme
were
identified. A 'pink' form of the enzyme was obtained when the
purification
was done aerobically. The specific activity of this species was around
30 nkat/mg protein as measured by the nitrous-oxide-dependent oxidation
of photochemically reduced benzyl viologen. A 'purple' form of the
enzyme,
whose catalytic acticity was 2-5-fold higher, was obtained when the
purification
was done anaerobically. The activity of both forms of the enzyme was
substantially
increased by dialyzing the protein against
2-(N-cyclohexylamino)ethanesulfonate
buffer at pH »10.
A maximal activity of 1000 nkat/mg protein has been obtained for the
purple
form using this procedure. A 'blue', enzymatically inactive form of the
enzyme resulted when either the pink or the purple species was exposed
to excess dithionite or ascorbate. Anaerobic, potentiometric titrations
of both the purple and the pink form of the enzyme gave a Nernst
factor,
n540,
of 0.95 and a midpoint potential, E'0,540 of +260 mV (vs SHE, 25°C,
Tris/HCl buffer, pH 7.5). Electron paramagnetic resonance (EPR) and
optical
spectra of N2O reductase suggested the presence
of
an unusual type 1 copper center. Type 2 copper was absent. The
hyperfine
splitting in the g|| region consisted of a seven-line pattern.
In
the presence of excess of reductant, a broad EPR signal with g values
at 2.18 and 2.06 was observed. The EPR spectra of the pink and purple
forms
of the enzyme were similar; however, the spectrum of the purple form
was
better resolved with
g|| = 2.18 (A||
= 3.83 mT) and g^
= 2.03 (A^=
2.8 mT). Most of the copper in N2O reductase was
removed
by anaerobic dialysis against KCN. Reaction of the apoprotein with
Cu(en)2SO4
partially regenerated the optical and EPR spectra of the holoprotein;
the
resulting protein was enzymatically inactive. Monospecific antibodies
against
the copper protein strongly inhibited the N2O
reductase
activity of purified samples and cell-free extracts.