title: Spectroscopy and reactivity of the type 1 copper site in Fet3p from Saccharomyces cerevisiae: correlation of structure with reactivity in the multicopper oxidases.
abstract: Fet3p is a multicopper oxidase recently isolated from the yeast, Saccharomyces cerevisiae. Fet3p is functionally homologous to ceruloplasmin (Cp) in that both are ferroxidases. However, by sequence homology Fet3p is more similar to fungal laccase, and both contain a type 1 Cu site that lacks the axial methionine ligand present in the functional type 1 sites of Cp. To determine the contribution of the electronic structure of the type 1 Cu site of Fet3p to the ferroxidase mechanism, we have examined the absorption, circular dichroism, magnetic circular dichroism, electron paramagnetic resonance, and resonance Raman spectra of wild-type Fet3p and type 1 and type 2 Cu-depleted mutants. The spectroscopic features of the type 1 Cu site of Fet3p are nearly identical to those of fungal laccase, indicating a very similar three-coordinate geometry. We have also examined the reactivity of the type 1 Cu site by means of redox titrations and stopped-flow kinetics. From poised potential redox titrations, the E degrees of the type 1 Cu site is 427 mV, which is low for a three-coordinate type 1 Cu site. The kinetics of reduction of the type 1 Cu sites of four different multicopper oxidases with two different substrates were compared. The type 1 site of a plant laccase (Rhus vernicifera) is reduced moderately slowly by both Fe(II) and a bulky organic substrate, 1,4-hydroquinone (with 6 equiv of substrate, k(obs) = 0.029 and 0.013 s(-)(1), respectively). On the other hand, the type 1 site of a fungal laccase (Coprinus cinereus) is reduced very rapidly by both substrates (k(obs) > 23 s(-)(1)). In contrast, both Fet3p and Cp are rapidly reduced by 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.
authors.affiliation: Department of Chemistry, Stanford University, Stanford, California 94305, USA.
authors.forename: T E
authors.initials: TE
authors.lastname: Machonkin
authors.forename: L
authors.initials: L
authors.lastname: Quintanar
authors.forename: A E
authors.initials: AE
authors.lastname: Palmer
authors.forename: R
authors.initials: R
authors.lastname: Hassett
authors.forename: S
authors.initials: S
authors.lastname: Severance
authors.forename: D J
authors.initials: DJ
authors.lastname: Kosman
authors.forename: E I
authors.initials: EI
authors.lastname: Solomon
mesh_terms: D002570:Ceruloplasmin / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D042845:Laccase; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D010940:Plant Proteins; D029701:Saccharomyces cerevisiae Proteins; D003300:Copper; D010088:Oxidoreductases; D042845:Laccase; D002570:Ceruloplasmin; C085513:FET3 protein, S cerevisiae