Structural Basis of the Ferrous Iron Specificity of the Yeast Ferroxidase, Fet3p†
journal contributionposted on 24.10.2006 by Christopher S. Stoj, Anthony J. Augustine, Lynn Zeigler, Edward I. Solomon, Daniel J. Kosman
Any type of content formally published in an academic journal, usually following a peer-review process.
Fet3p is a multicopper oxidase (MCO) that functions together with the iron permease, Ftr1p, to support high-affinity Fe uptake in yeast. Fet3p is a ferroxidase that, like ceruloplasmin and hephaestin, couples the oxidation of 4 equiv of FeII to the reduction of O2 to 2H2O. The ferrous iron specificity of this subclass of MCO proteins has not been delineated by rigorous structure−function analysis. Here the crystal structure of Fet3p has been used as a template to identify the amino acid residues that confer this substrate specificity and then to quantify the contributions they make to this specific reactivity by thermodynamic and kinetic analyses. In terms of the Marcus theory of outer-sphere electron transfer, we show here that D283, E185, and D409 in Fet3p provide a FeII binding site that actually favors ferric iron; this site thus reduces the reduction potential of the bound FeII in comparison to that of aqueous ferrous iron, providing a thermodynamically more robust driving force for electron transfer. In addition, E185 and D409 constitute parts of the electron-transfer pathway from the bound FeII to the protein's type 1 CuII. This electronic matrix coupling relies on H-bonds from the carboxylate OD2 atom of each residue to the NE2 NH group of the two histidine ligands at the type 1 Cu site. These two acidic residues and this H-bond network appear to distinguish a fungal ferroxidase from a fungal laccase since the specificity that Fet3p has for FeII is completely lost in a Fet3pE185A/D409A mutant. Indeed, this double mutant functions kinetically better as a laccase, albeit a relatively inefficient one.