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 (epsilon = 5500 M-1 cm-1) due to this type 1 Cu(II) and a shoulder in the near 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 = 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 Km values for Fe(II) and O2 were 4.8 and 1.3 microM, respectively, while kcat values for Fe(II) and O2 turnover were 9.5 and 2.3 min-1, consistent with an Fe(II):O2 reaction stoichiometry of 4:1.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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