SPECTROSCOPIC AND CHEMICAL STUDIES OF THE LACCASE TRINUCLEAR COPPER ACTIVE-SITE - GEOMETRIC AND ELECTRONIC-STRUCTURE

Laccase contains four Cu atoms: a type 1, a type 2, and a coupled binuclear type 3 center. The type 2 and type 3 centers comprise a trinuclear Cu cluster which is thought to represent the active site for the binding and multielectron reduction of dioxygen. A combination of electronic spectroscopy, magnetic susceptibility, and exogenous ligand perturbation has been used to probe the geometric and electronic structure of the trinuclear site. A type 1 Hg2+ -substituted laccase derivative was employed in order to remove the overlapping spectral contributions from the type 1 Cu2+. The ligand-field and charge-transfer transitions of the type 2 and type 3 coppers were assigned by use of absorption, circular dichroism, and low-temperature magnetic circular dichroism spectroscopies. The ligand-field transition energies indicate that all three coppers have tetragonal geometries and that the two type 3 coppers are inequivalent. Magnetic susceptiblity measurements have defined the lower limit for the magnitude of the exchange interaction between the type 3 coppers and have probed type 2-type 3 interactions. Binding of the exogenous ligand azide to the trinuclear site produces characteristic azide --> Cu2+ charge-transfer features and also perturbs the type 2 and type 3 ligand-field transitions. Analysis of these spectral features demonstrates that azide binds as a bridging ligand between the type 2 site and one of the type 3 coppers. In addition, a second azide coordinates to the type 3 site with a lower binding constant, and this second azide also strongly interacts with the type 2 site. The type 2-type 3 bridged binding of azide suggests that a similar coordination mode is active in the irreversible binding and four-electron reduction of dioxygen. The second azide binding provides a further demonstration of the differences between the laccase type 3 site and the coupled binuclear sites in hemocyanin and tyrosinase. A model for the magnetic interactions among the three coppers in the resting and ligand bound forms of the trinuclear site is presented.