Metal Cluster Active Sites in Proteines

Abstract

Binuclear and multimetal cluster centers are found in a wide variety of enzymes involved in electron transfer and small molecule activation. It is important to define the contributions of interactions between the metal ions in an active site to its structure and function. Attention is focussed on the coupled binuclear copper sites which occur in hemocyanin (reversible O2 binding), tyrosinase (O2 activation and hydroxylation) and laccase (O2 reduction to water). Excited state spectral studies on active site derivatives are used to define exogenous ligand bridging between the two coppers in the active sites of hemocyanin and tyrosinase. A chemical and spectroscopic comparison of the coupled site in hemocyanin to the binuclear center (T3) in laccase indicates that the sites are similar with respect to an endogenous bridge responsible for antiferromagnetic coupling but differ in that exogenous ligands bind to only one copper at the T3 site. In addition to the T3 coupled binuclear copper site, laccase contains two other coppers, the Type 1 and Type 2 centers. X-ray absorption edge spectral studies of the 1s→4p transition at 8984 eV are used to demonstrate that in the absence of the T2 copper the T3 site is reduced and will not react with O2. The role of the T2 and T3 coppers in exogenous ligand binding has been probed through low temperature MCD which allows a correlation between the excited state and ground state spectral features. Through these low temperature MCD studies it is demonstrated that one azide produces charge transfer intensity to both the paramagnetic T2 and the antiferromagnetic T3 center defining a new trinuclear copper active site which appears to be important in the irreversible multi-electron reduction of dioxygen to water.