Spectral and kinetic properties of the Fet3 protein from Saccharomyces cerevisiae, a multinuclear copper ferroxidase enzyme

High affinity iron uptake in Saccharomyces cerevisiae requires Fet3p. Fet3p is proposed to facilitate iron uptake by catalyzing the oxidation of Fe(II) to Fe(III) by O₂; in this model, Fe(III) is the substrate for the iron permease, encoded by FTR1. Here, a recombinant Fet3p has been produced in yeast that, lacking the C-terminal membrane-spanning domain, is secreted directly into the growth medium. Solutions of this Fet3p at >1 mg/ml have the characteristic blue color of a type 1 Cu(II)-containing protein, consistent with the sequence homology that placed this protein in the class of multinuclear copper oxidases that includes ceruloplasmin. Fet3p has an intense absorption at 607 nm (ε = 5500 M^-1 cm^-1) due to this type 1 Cu(II) and a shoulder in the near UV at 330 nm (ε = 5000 M^-1 cm^-1) characteristic of a type 3 binuclear Cu(II) cluster. The EPR spectrum of this Fet3p showed the presence of one type 1 Cu(II) and one type 2 Cu(II) (A is perpendicular with = 91 and 190 x 10^-4 cm^-1, respectively). Copper analysis showed this protein to have 3.85 g atom copper/mol, consistent with the presence of one each of the three types of Cu(II) sites found in multinuclear copper oxidases. N-terminal analysis demonstrated that cleavage of a signal peptide occurred after Ala-21 in the primary translation product. Mass spectral and carbohydrate analysis of the protein following Endo H treatment indicated that the preparation was still 15% (w/w) carbohydrate, probably O-linked. Kinetic analysis of the in vitro ferroxidase reaction catalyzed by this soluble Fet3p yielded precise kinetic constants. The K(m) values for Fe(II) and O₂ were 4.8 and 1.3 μM, respectively, while k(cat) values for Fe(H) and O₂ turnover were 9.5 and 2.3 min^-1, consistent with an Fe(II):O₂ reaction stoichiometry of 4:1.