Chemical and Spectroscopic Definition of the Peroxide-Level Intermediate in the Multicopper Oxidases: Relevance to the Catalytic Mechanism of Dioxygen Reduction to Water

Laccase is a multicopper oxidase which contains four coppers, one
type 1, one type 2, and a coupled
binuclear type 3 pair, the type 2 and type 3 copper centers together
forming a trinuclear copper cluster. The type
1 mercury derivative of laccase (T1Hg Lc) has the type 1 center
substituted with a redox inactive Hg2+ ion and
an
intact trinuclear copper cluster. Reaction of reduced T1Hg Lc with
dioxygen produces an oxygen intermediate
which has now been studied in detail. Isotope ratio mass
spectrometry (IRMS) has shown that both oxygen atoms
of O2 are bound in the intermediate. EPR and SQUID
magnetic susceptibility studies have shown that the
intermediate
is diamagnetic. The results combined with X-ray absorption edge
data indicate that the intermediate contains a
bound peroxide and that the two electrons have derived from the type 3
center which is antiferromagnetically coupled.
EXAFS data show that there is no short Cu−oxo bond in the
intermediate and that there is a new bridging interaction
in the intermediate, with two coppers being separated by 3.4 Å, that
is not present in the resting enzyme. Circular
dichroism (CD) and magnetic circular dichroism (MCD) studies in the
ligand field region confirm that the two type
3 coppers are oxidized and antiferromagnetically coupled and that the
type 2 copper is reduced. In addition, the
charge transfer (CT) absorption spectrum of the intermediate supports a
μ-1, 1 hydroperoxide description based on
a comparison to Cu(II)−peroxo model spectra. The decay of
the T1Hg Lc oxygen intermediate is pH dependent,
slow, and proceeds through an additional intermediate with an MCD
spectrum in the CT region analogous to that of
the oxygen intermediate in the native enzyme which is at least one
electron further reduced. These studies lead to
a spectroscopically effective model for peroxide bound to the
trinuclear copper cluster site in the intermediate, and
provide significant insight into the molecular mechanism of the
catalytic reduction of dioxygen to water by the
multicopper oxidases.