Spectroscopy and reactivity of the type 1 copper site in Fet3p from Saccharomyces cerevisiae: Correlation of structure with reactivity in the multicopper oxidases

Fet3p is a multicopper oxidase recently isolated from the yeast, Saccharomy ces cerevisiae. Fet3p is functionally homologous to ceruloplasmin (Cp) in t hat both are ferroxidases. However, by sequence homology Fet3p is more simi lar to fungal laccase, and both contain a type I Cu site that lacks the axi al methionine ligand present in the functional type 1 sites of Cp. To deter mine the contribution of the electronic structure of the type 1 Cu site of Fet3p to the ferroxidase mechanism, we have examined the absorption, circul ar dichroism, magnetic circular dichroism, electron paramagnetic resonance, and resonance Raman spectra of wild-type Fet3p and type 1 and type 2 Cu-de pleted mutants. The spectroscopic features of the type 1 Cu site of Fet3p a re nearly identical to those of fungal laccase, indicating a very similar t hree-coordinate geometry. We have also examined the reactivity of the type 1 Cu site by means of redox titrations and stopped-flow kinetics. From pois ed potential redox titrations, the E degrees of the type 1 Cu site is 427 m V, which is low for a three-coordinate type 1 Cu site. The kinetics of redu ction of the type 1 Cu sites of four different multicopper oxidases with tw o different substrates were compared. The type 1 site of a plant laccase (R hus vernicifera) is reduced moderately slowly by both Fe(II) and a bulky or ganic substrate, 1,4-hydroquinone (with 6 equiv of substrate, k(obs) = 0.02 9 and 0.013 s(-1), respectively). On the other hand, the type 1 site of a f ungal laccase (Coprinus cinereus) is reduced very rapidly by both substrate s (k(obs) > 23 s(-1)). In contrast, both Fet3p and Cp are rapidly reduced b y Fe(II) (k(obs) > 23 s(-1)), but only very slowly by 1,4-hydroquinone (10- and 100-fold more slowly than plant laccase, respectively). Semiclassical theory is used to analyze the origin of these differences in reactivity in terms of type 1 Cu site accessibility to specific substrates.