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 oxidatio n of Fe(II) to Fe(III) by O-2; 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 thi s Fet3p at >1 mg/ml have the characteristic blue color of a type 1 Cu( II)-containing protein, consistent with the sequence homology that pla ced this protein in the class of multinuclear copper oxidases that inc ludes ceruloplasmin. Fet3p has an intense absorption at 607 nm (epsilo n = 5500 M-1 cm(-1)) due to this type 1 Cu(II) and a shoulder in the n ear UV at 330 nm (epsilon = 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(parallel to) = 91 and 190 x 10(-4) cm(-1), respectively). Copper analysis showed thi s 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 s ignal peptide occurred after Ala-21 in the primary translation product . Mass spectral and carbohydrate analysis of the protein following End o H treatment indicated that the preparation was still 15% (w/w) carbo hydrate, probably O-linked. Kinetic analysis of the in vitro ferroxida se reaction catalyzed by this soluble Fet3p yielded precise kinetic co nstants. The K-m values for Fe(II) and O-2 were 4.8 and 1.3 mu M, resp ectively, while k(cat) values for Fe(II) and O-2 turnover were 9.5 and 2.3 min(-1), consistent with an Fe(II):O-2 reaction stoichiometry of 4:1.