CHEMICAL AND SPECTROSCOPIC DEFINITION OF THE PEROXIDE-LEVEL INTERMEDIATE IN THE MULTICOPPER OXIDASES - RELEVANCE TO THE CATALYTIC MECHANISMOF 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 ty pe 3 copper centers together forming a trinuclear copper cluster. The type 1 mercury derivative of laccase (T1Hg Lc) has the type 1 center s ubstituted with a redox inactive Hg2+ ion and an intact trinuclear cop per cluster. Reaction of reduced T1Hg Lc with dioxygen produces an oxy gen intermediate which has now been studied in detail. Isotope ratio m ass spectrometry (IRMS) has shown that both oxygen atoms of O-2 are bo und 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 contai ns a bound peroxide and that the two electrons have derived from the t ype 3 center which is antiferromagnetically coupled. EXAFS data show t hat there is no short Cu-oxo bond in the intermediate and that there i s a new bridging interaction in the intermediate, with two coppers bei ng separated by 3.4 Angstrom, that is not present in the resting enzym e. Circular dichroism (CD) and magnetic circular dichroism (MCD) studi es 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 mu-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 analogo us to that of the oxygen intermediate in the native enzyme which is at least one electron further reduced. These studies lead to a spectrosc opically effective model for peroxide bound to the trinuclear copper c luster site in the intermediate, and provide significant insight into the molecular mechanism of the catalytic reduction of dioxygen to wate r by the multicopper oxidases.