Structure-function correlations in copper clusters in proteins

A coupled binuclear copper active site is present in a wide variety of proteins and enzymes which perform different biological functions utilizing 0,. In the multicopper oxidases, the Type 2 and Type 3 centers comprise a trinuclear Cu cluster which represents the active site for the multielectron reduction of 0,. A coupled binuclear copper active site is present in a wide variety of proteins and enzymes which perform different biological functions utilizing 02. The hemocyanins (Hc) reversibly bind 02, the tyrosinases (Ty) are monoxygenases which hydroxylate monophenols to o-diphenols and oxidize these to o-quinones, and the multicopper oxidases (laccase (Lc), ascorbate oxidase and ceruloplasmin), which contain additional copper centers (Type 1 and Type 2) catalyze the four electron reduction of 02 to water. Our original chemical and spectroscopic studies over a series of protein active site derivatives of Hc and Ty demonstrated (ref. 1,2) that these proteins have very similar active sites. The key features of this spectroscopically effective model for the coupled binuclear copper active site, reproduced in Fig. 1, are that dioxygen binds as peroxide, bridging two tetragonal Cu(1I)'s in a cis p-l,2 fashion, with an additional endogenous bridge between the coppers (RO-). The endogenous bridge is likely hydroxide based on the crystal structure (ref. 3 ) of deoxy Hc. The major difference between the Hc and Ty sites is the high accessibility of the Ty active site to exogenous ligands (ref. 4 ) . We found that substrate analogues bind directly to the copper in Ty and compete with peroxide for the same binding site (ref, 5 ) . These studies resulted in the proposed structural mechanism for hydroxylation and oxidation catalysis given in Fig. 2. Alternatively, chemical and spectroscopic studies of a series of protein active site derivatives of Lc (which is the simplest multicopper oxidase, containing one Type 1, one Type 2 , and one coupled binuclear Type 3 center) showed the Type 3 site to be strikingly different from the coupled binuclear site in Hc and Ty (ref. 6 ) . Low-temperature magnetic circular dichroism (LTMCD) spectroscopy was found to be a powerful probe of the different copper centers in the multicopper oxidases, allowing a correlation of excited state spectral features with the ground state magnetic properties. From LTMCD studies, the Type 3 site was in fact found to be part of a trinuclear comer cluster with the Type 2 center (ref. 7 ) . This new class of copper site has recently been supported by crystallographic studies on ascorbate oxidase (ref. 8). Our recent studies have focussed on I) Spectroscopy of model complexes to further develop our understanding of the unique spectral features of oxy Hc; 11) A detailed comparison of the coupled binuclear site in Hc and the Type 3 site in Lc; 111) Evaluation of the metal centers required for the 02 reactivity of the multicopper oxidases; and IV) Detailed LTMCD spectral studies of the trinuclear copper cluster to establish its geometric and electronic structure and interactions with exogenous ligands as related to the mechanism of multielectron reduction of 02. These studies are summarized below. Fig. 1. The Spectroscopically Effective Fig. 2. Active site structural mechanism Active Site of Hemocyanin and Tyrosinase. for hydroxlyation and oxidation of phenols to form o-quinones by tyrosinase. I. ELECTRONIC STRUCTURE OF O X Y HEMOCYANIN In oxy Hc, the two Cu(I1) ions show strong antiferromagnetic coupling in the ground state, and are consequently inaccessible to ground state spectral study. spectroscopic features are therefore the most useful probe of the electronic structure of The excited state