Nitrous oxide reductase (N2OR),
Pseudomonas stutzeri, catalyses the 2 electron reduction of
nitrous
oxide to di-nitrogen. The enzyme has 2 identical subunits (Mr
~ 70000) of known amino acid sequence and contains ~ 4 Cu ions per
subunit.
By measurement of the optical absorption, electron paramagnetic
resonance
(EPR) and low-temperature magnetic circular dichroism (MCD) spectra of
the oxidised state, a semi-reduced form and the fully reduced state of
the enzyme it is shown that the enzyme contains 2 distinct copper
centres
of which one is assigned to an electron- transfer function, centre A,
and the other to a catalytic site, centre Z. The latter is a
binuclear
copper centre with at least 1 cysteine ligand and cycles between
oxidation
levels Cu(II)/Cu(II) and Cu(II)/Cu(I) in the absence of substrate or
inhibitors.
The state Cu(II)/Cu(I) is enzymatically inactive. The MCD spectra
provide
evidence for a second form of centre Z, which may be
enzymatically
active, in the oxidised state of the enzyme. Centre A is
structurally
similar to that of CuA in bovine and bacterial
cytochrome
c oxidase and also contains copper ligated by cysteine. This
centre
may also be a binuclear copper complex.