SPECTROSCOPIC INVESTIGATION OF PEROXIDE BINDING TO THE TRINUCLEAR COPPER CLUSTER SITE IN LACCASE - CORRELATION WITH THE PEROXY-LEVEL INTERMEDIATE AND RELEVANCE TO CATALYSIS

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 I 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 H2O2 with fully oxidized T1Hg Le produces a p eroxide adduct which has now been studied in detail. Peroxide is shown to bind to the trinuclear cluster with low affinity, producing spectr al and geometric features similar to the intermediate formed in the re duction of O-2 to H2O which had been shown to have the type 2 copper r educed, the type 3 pair oxidized and antiferromagnetically coupled, an d two coppers bridged at a distance of 3.4 Angstrom. The peroxide addu ct and the intermediate have similar geometric and electronic features with the type 2 oxidized in the adduct. This center is paramagnetic a nd has been studied in detail. Peroxide binds to the type 2 center. EP R and ligand field (NiR MCD) coupled with CT (absorption and MCD) data demonstrate that peroxide binds to the type 2 Cu which goes from bein g 3-coordinate in the resting protein to 4-coordinate in the peroxide adduct. Peroxide also binds to the type 3 site from a comparison of li gand field absorption and CD and the presence of more than one intense O-2(2-) --> Cu(II) band in the CT absorption spectrum. A bridging int eraction between coppers at 3.4 Angstrom is seen from the EXAFS data. Possible geometric structures for the peroxide adduct and intermediate are proposed, with the electronic structural difference between the a dduct and the intermediate being related to the type 2 copper being re duced in the latter. This study (i) firmly establishes the role of the type 2 in catalysis, (ii) demonstrates a type 2/type 3 bridging mode of binding that promote further 2e(-) reduction of peroxide to water, and (iii) provides further support for a peroxide-level intermediate i n the catalytic cycle of the multicopper oxidases which involve two 2e (-) steps in the reduction of O-2 to H2O.